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ABSTRACT BOOK
HERSONISSOS-CRETE, GREECE, 1-5 NOVEMBER 2010
CRETA MARIS HOTEL
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CONGRESS ORGANISERS
CONGRESS SECRETARIAT
15, MARKOY BOTSARI, KIFISSIA, ATHENS, GREECE
T. (++30) 210 6231305 - F. (++30) 210 6233809
E. [email protected]
we mind your own business
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DEAR COLLEAGUES,
We are pleased to present the comprehensive scientific program of the 14th International Conference
on Harmful Algae, with state-of-the-art lectures by world-renowned scientists, oral and poster
presentations from many research institutes around the globe, a true international forum.
We have been very ambitious quite from the start of the organization of this venue, to provide an
interesting as well as challenging conference that would address all issues related to the causes
and effects of marine and freshwater harmful micro- and macro-algae and serve as a forum for all
stakeholders to exchange research outcomes and ideas.
This book contains the abstracts of all the work that is presented in the 14th International Conference
on Harmful Algae. More than 570 abstracts were submitted out of which 154 were selected for
oral presentation and 400 for posters.
The abstracts from the plenary sessions are presented separate, as the first part of this book. All
other abstracts are presented according to the announced order of subjects of the conference.
- Population dynamics of Harmful Algae Blooms
- Time series of HAB events: Climatic and anthropogenic induced impacts
- Impact of HABs on marine food webs and ecosystem structure and function
- Biological interactions: allelopathy, mixotrophy, parasitism, symbiosis, bacteria and viruses
- New regional HAB events
- Introduction of alien species and HABs
- Cyanobacterial ecology, physiology and bioactive compounds
- Genomics and genetic diversity of HABs
- Toxins: chemical structure and synthesis, detection and analytical methods
- Mechanisms of toxicity
- Novel sensor technologies for bio-sensing applications in HAB research and monitoring
- Management, mitigation and public outreach for HABs
- Health and HABs
Oral presentations abstracts precede abstracts from posters. A list of contents will help you easily
find the abstracts of your interest. An author index at the end of the book is also provided.
The Organizing Committee of this conference hopes to have fulfilled the expectations of all
participants and to have successfully served the common goal to discuss some of the most urgent
and pressing problems of our time.
DR KALLIOPI PAGOU
Chair of the Local Organizing Committee of the 14th International Conference on Harmful Algae
03
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INTERNATIONAL SCIENTIFIC COMMITTEE
Donald M. Anderson: Woods Hole Oceanographic Institution, USA
Stephen Bates: Gulf Fisheries Centre, Canada
JoAnn Burkholder: N. Carolina State University, USA
Jose Bustillos-Guzman: Northwest Center for Biological Research, Mexico
Henrik Enevoldsen: IOC
Yasuwo Fukuyo: University of Tokyo, Japan
Patricia M. Glibert: Horn Point Laboratory, USA
Edna Graneli: University of Kalmar, Sweden
Gustaaf Hallegraeff: ISSHA Vice President, University of Tasmania, Australia
Kin Chung Ho: ISSHA council, Open University of Hong Kong, China
Rita Horner: ISSHA council, USA
Anke Kremp: ISSHA council, Finland
Lincoln MacKenzie: ISSHA council, New Zealand
Kalliopi Pagou: Chair of the Local Organizing Committee, HCMR, Greece
Grant Pitcher: Marine and Coastal Management, Cape Town, South Africa
Beatriz Reguera: ISSHA President, Instituto Espanol de Oceanografia, Spain
Sandra Shumway: University of Connecticut, USA
Takeshi Yasumoto: Okinawa CREATE, JST, Japan
Mingjiang Zhou: Chinese Academy of Sciences, China
LOCAL ORGANIZING COMMITTEE
Kalliopi Pagou: Chair, Institute of Oceanography, HCMR
Epaminondas Christou: Institute of Oceanography, HCMR
Manos Dassenakis: University of Athens, President of Greek Oceanographers’ Association
Athina Economou-Amilli: Department of Biology, University of Athens
Antonia Giannakourou: Institute of Oceanography, HCMR
Louisa Giannoudi: Institute of Oceanography, HCMR
Olympia Gotsis-Skretas: Institute of Oceanography, HCMR
Christos Katsaros: University of Athens, President of Hellenic Phycological Society
Michael Karydis: Department of Marine Sciences, Aegean University
Maria Moustaka: Department of Biology, University of Thessaloniki
Michael Papadakis: Head of Cretaquarium, HCMR
Evangelos Papathanassiou: Institute of Oceanography, HCMR
Sofia Reizopoulou: Institute of Oceanography, HCMR
Ioanna Varkitzi: Institute of Oceanography, HCMR
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TABLE OF CONTENTS
08
PLENARY TALKS
12
POPULATION DYNAMICS OF HARMFUL ALGAE BLOOMS
62
TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
83
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
98
BIOLOGICAL INTERACTIONS: ALLELOPATHY, MIXOTROPHY,
PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
124
NEW REGIONAL HAB EVENTS
149
INLAND SEAS AND HABS
153
INTRODUCTION OF ALIEN SPECIES AND HABS
158
CYANOBACTERIAL ECOLOGY,
PHYSIOLOGY AND BIOACTIVE COMPOUNDS
177
GENOMICS AND GENETIC DIVERSITY OF HABS
07
204
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
239
MECHANISMS OF TOXICITY
257
NOVEL SENSOR TECHNOLOGIES FOR BIO-SENSING
APPLICATIONS IN HAB RESEARCH AND MONITORING
278
HARMFUL ALGAE CULTURE COLLECTIONS
285
MANAGEMENT, MITIGATION AND PUBLIC
OUTREACH FOR HABS
304
HABS TO HEALTH - SCIENCE TO ACTION
312
AUTHORS’ INDEX
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PLENARY TALKS
POPULATION DYNAMICS
OF ALEXANDRIUM FUNDYENSE OF THE GULF OF MAINE:
A SYNTHESIS AND OUTLOOK FOR IMPROVED
MANAGEMENT AND FORECASTING
Donald M. Anderson
Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. E-mail: [email protected]
Paralytic shellfish poisoning (PSP) is arguably the most widespread of all HAB poisoning syndromes. One region with a long history of
PSP is the Gulf of Maine. As a result of sustained research funding, considerable progress has been made in elucidating the population
dynamics of Alexandrium fundyense within the region. This presentation will synthesize that knowledge, highlighting what we have learned,
what we still do not understand, and plans for future improvements to monitoring and management capabilities.
Several habitats or hydrographic systems have been identified that support A. fundyense populations. These include: 1) areas where in
situ germination produces localized blooms that subsequently reseed those sites with new cysts, leading to recurrent, annual outbreaks;
and 2) large-scale advective blooms originating from cysts in regional seedbeds, with the resulting vegetative populations transported
hundreds of kilometers by coastal currents and wind patterns.
Transitions between life history stages clearly play a major role in A. fundyense dynamics. The relative importance of the stages varies
between habitats; in some locations, resting cyst abundance is a major determinant of the magnitude of subsequent blooms, whereas
in other areas, bloom size is more dependent upon conditions for vegetative growth. Complicating the emergence of cells from cysts
are multiple controls on dormancy and quiescence, including temperature, an endogenous annual clock, burial, and germination inhi08
bition by anoxia, all of which regulate where, when, and how many cells inoculate blooms. Significant questions remain as to the size
of the excystment inoculum, the depth within the sediments from which cysts germinate and emerge into the water column, the relative importance of resuspended cysts, and the survival rate of cysts that germinate in deep waters.
Another major area of uncertainty is bloom termination. We have observed an apparent inverse relationship between the magnitude
of regional blooms and the size of the cyst population determined later that year, suggesting an imbalance between excystment
and encystment. Over the eight years that large-scale cyst mapping surveys have been conducted, the total abundance of A.
fundyense cysts in surface sediments has fluctuated more than 10-fold, with significant retractions and extensions in spatial
distribution evident as well. We do not yet understand the relative importance of losses due to encystment, grazing, parasites,
viruses, or advection.
Despite these and other gaps in our understanding, we have formulated conceptual models for A. fundyense bloom dynamics
that are consistent with observations and have developed numerical models that are used to provide hindcast analyses of
past blooms, as well as nowcasts and forecasts of short-term bloom distribution, and seasonal forecasts issued months
before the bloom season. Plans for the deployment of multiple Environmental Sampling Processors (ESPs) to provide
real-time data on cell abundance that can be assimilated into the model to improve forecast accuracy will be discussed,
as will plans to couple our model to a sediment transport model, thereby allowing us to simulate the formation of cysts
and their deposition in seedbeds.
The information that is now available on the many mechanisms underlying the blooms of A. fundyense in the Gulf of
Maine region is remarkable in its scope, and bears testimony to the value of sustained research funding. Equally remarkable are the many of important processes that remain poorly understood.
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HABS AND HEALTH:
FORGING PUBLIC
HEALTH POLICY
Lorraine C. Backer
National Center for Environmental Health, 4770 Buford Highway NE, MS F-57,
Chamblee, GA 30341, USA. E-mail: [email protected]
The potential negative health and environmental effects from harmful algal blooms (HABs) are many -HABs
threaten beaches, recreation venues, and drinking water sources. In November 2009, an unusually large algal
bloom 80 feet deep surrounded the U.S. Virgin Islands. During that same month on the U.S. west coast, an
unusual bloom of a common marine phytoplankton Ashikawa sanquinea caused extensive seabird mortalities and
was associated with a range of acute health complaints from surfers. Ongoing modeling of offshore populations of
Alexandrium fundyense by the Woods Hole Oceanographic Institute in Massachusetts predicted the presence of
high concentrations of this saxitoxin-producing organism near shellfish beds off the Northeastern coast. The 2010
harvest is expected to be severely limited. Mounting evidence indicates global climate changes support increased frequency and geographic extent of HABs. Moreover, each year our desire to live at lakeside and our reliance on large
surface waters for recreation and drinking water put more people and animals at risk for exposure to HABs and the
toxins they produce. Despite these documented events, we do not have a good sense of either the extent of exposure
to algal toxins or the resulting public health impacts. The challenges facing exposure and disease risk assessment include
the characteristics of the toxins themselves, environmental exposures that are barely detectable with current analytic methods, and biological effects occurring at the molecular level. Limited guidance is available for those tasked to protect public
health. To support public health decision-making, we need creative new means to assess exposure to algal toxins and to
evaluate the consequences from these exposures.
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PLENARY TALKS
OCEAN CLIMATE CHANGE
AND HARMFUL ALGAL BLOOMS:
A FORMIDABLE PREDICTIVE CHALLENGE
Gustaaf M. Hallegraeff
Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia.
E-mail: [email protected]
Prediction of the impact of global climate change on marine harmful algal blooms is fraught with difficulties. However, we can learn
important lessons from the fossil record of dinoflagellate cysts, long-term monitoring programmes such as the Continuous Plankton
Recorder surveys and short-term phytoplankton community responses to El Niño-Southern Oscillation and North Atlantic Oscillation
episodes. Increasing temperature, enhanced surface stratification, alteration of ocean currents, intensification or weakening of local
nutrient upwelling, stimulation of photosynthesis by elevated CO2, reduced calcification through ocean acidification (“the other CO2
problem”), heavy precipitation and storm events causing changes in land runoff and micronutrient availability, may all produce contradictory species- or even strain-specific responses. Complex factor interactions exist and simulated ecophysiological laboratory experiments rarely allow for sufficient acclimation and rarely take into account physiological plasticity and genetic strain diversity. We can
expect: (1) Range expansion of warm-water species at the expense of cold-water species which are driven pole wards; (2) Speciesspecific changes in the abundance and seasonal window of growth of HAB taxa; (3) Earlier timing of peak production of some phytoplankton; (4) Secondary effects for marine food webs, notably when individual zooplankton and fish grazers are differentially
impacted (“match-mismatch”) by climate change. The greatest problems for human society will be caused by being unprepared for
significant range expansions or the increase of algal biotoxin problems in currently poorly monitored areas, thus calling for increased
vigilance in seafood biotoxin and HAB monitoring programmes. Changes in phytoplankton communities provide a sensitive early
10
warning for climate-driven perturbations to marine ecosystems.
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EVOLUTION OF THE DINOFLAGELLATES:
FROM ORIGIN OF THE GROUP
TO THEIR GENES
Linda K. Medlin
Marine Biological Association of the UK, the Citadel, Plymouth PL1 2PB, UK.
E-mail: [email protected]
Dinoflagellates have often been regarded as bizarre examples of evolution. They belong to one of the most
strongly supported macrolineages among the protists: the superphylum/kindgom Alveolata containing three
main phyla: the Dinoflagellata, the Apicomplexa and the Ciliata. These organisms share the features of
cortical alveoli and micropores. Until the early 1990s, dinoflagellate classification of both living and fossil taxa
relied almost exclusively on morphological characters, such as plate tabulation. Molecular data have supported
many morphological groups, whereas others have been shown to be paraphyletic. Phylogenetic relationships
within the dinoflagellates have changed as more taxa and more genes have been added. There is also much
variation with the algorithm used to analyse the data. Dinoflagellates have become notorious for the variety of
plastid types that they have acquired by secondary and even tertiary symbiosis. They possess the most diverse
array of plastids of any eukaryotic lineage and they are truly the kings of symbioses. Genome rearrangements
have taken place as the plastid has evolved. The genes that have been moved to the nucleus in the peridinin plastid
dinoflagellates are different from all other eukaryotes, as well as the genes left in the plastid, being arranged in mini
circles. As tertiary endosymbiosis has taken place, the plastid genome has become rearranged again. At the species
level, some, but not many, cryptic species have been uncovered. However, below the species level, microsatellites
have shown immense spatial fragmentation in dinoflagellate populations. EST libraries have been constructed for several species. Initial annotation results indicated that a low percentage of the genes could be annotated. Recent advances
have pushed this level to nearly 20% of the ESTs. Gene expression has been studied in relationship to stress conditions
for several species. Some common responses to stress conditions have been noted. Only in EST libraries that have
been screened by 454 sequencing (Illumina) have toxin genes been identified. The complete genome of Heterocapsa
circulisquama and its virus have now been completed and preliminary data will be shown.
WHAT WE KNOW AND DON’T KNOW
ABOUT CIGUATERA AND RELATED TOXINS
Takeshi Yasumoto
Okinawa Science and Technology Promotion Center, 12-75 Suzaki, Uruma-shi, Okinawa, 904-2234, Japan.
E-mail: [email protected]
We first identified the benthic dinoflagellate Gambierdiscus toxicus as the origin of ciguatoxins (CTXs) in fish in1977. Nearly a
decade passed before we determined the planar structures of major two toxins (CTX1B, CTX4B) in 1989. After another decade,
the planar or stereochemical structures of 23 congeners were determined using submicrogram samples in 2001. On top of the
difficulty of preparing toxins from fish or algal cultures, the extremely low concentration and complex profiles of toxins to be posed
a serious obstacle to development of a analytical methods. Recently, we made a breakthrough in which nearly all major toxins in
fish and G. toxicus were determined by LC-MS/MS analysis, revealing distinct regional and generic variations of toxin profiles in
fish. The sensitivity and accuracy of the method are enough to replace the MBA.
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POPULATION
DYNAMICS
OF HARMFUL ALGAE
BLOOMS
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ORAL PRESENTATIONS
ECOLOGY OF OSTREOPSIS OVATA BLOOMS
IN THE NORTHERN ADRIATIC SEA (SUMMER 2009)
Stefano Accoroni1, Tiziana Romagnoli1, Federica Colombo1, Emellina Cucchiari1, Antonella Penna 2,
Cecilia Battocchi 2, Ernesto Fattorusso 3, Patrizia Ciminiello 3, Cecilia Totti1
1
Dip. Scienze del Mare, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy.
E-mail: [email protected]
2
Dip. Scienze Biomolecolari, Università di Urbino, viale Trieste 296, 61100 Pesaro, Italy
3
Dip. Chimica delle Sostanze Naturali, Università di Napoli “Federico II”, via D. Montesano 49, 80131, Napoli, Italy
Since 2006, intense blooms of the toxic benthic dinoflagellate Ostreopsis ovata regularly occur in the rocky coast of the northern Adriatic
Sea. Such blooms are associated with noxious effects on human health and mortality of benthic organisms, due to the production of palytoxin-like compounds. We investigated the O. ovata bloom in two areas along the Conero Riviera (March-November 2009), and the
role of environmental factors and substratum on the bloom dynamics. O. ovata developed from August to November, with the highest
abundances at mid-September (6.4x104 cells cm-2, i.e. 1.3x106 cells g-1fw). Abundances were significantly higher on rocks than on seaweeds. Hydrodynamism plays a major role in the bloom dynamics: significantly higher abundances were observed in sheltered sites than
in exposed ones. The morphometric analysis showed a marked variability (DV:17.5-75 μm; W:12.5-60 μm), and a relationship between
cell dimensions and bloom phases was shown. In the bloom the presence of the single O. ovata genotype was confirmed by the PCR
assay, excluding the co-occurrence of O. siamensis. Other toxic benthic dinoflagellates (Coolia monotis, Prorocentrum lima, Amphidinium
cf. carterae) co-occurred with abundances of 3-4 orders of magnitude lower. Differently from what observed in other Mediterranean
areas, bloom developed when temperatures were decreasing. Nutrient concentrations did not seem to affect the bloom dynamics. Toxin
analysis, performed by liquid chromatography coupled to mass spectrometry (LC/MS), revealed high ovatoxin-a content (14.8 pg cell-1).
The presence of cysts was detected in sediment samples collected after the bloom decline through molecular PCR methods.
14
IMPLICATION OF CASPASE 3-LIKE PROTEASES IN THE
DEATH OF KARENIA BREVIS AND KARENIA MIKIMOTOI
Josée Nina Bouchard, Duncan A. Purdie
National Oceanography Centre, University of Southampton, Waterfront Campus, European Way, Southampton, SO14 3ZH,
United Kingdom. E-mail: [email protected]
To obtain a better understanding of cell death processes in harmful dinoflagellates, culture-grown Karenia brevis and Karenia mikimotoi
were monitored over a 56-d period. The cellular abundance, Fv/Fm, the percentage of dead cells (sytox-positive cells) and the variation
in caspase 3-like activity were monitored every week. The presence of degraded DNA was also assessed in 50-d old cells using a
TUNEL assay. Karenia brevis reached higher cell numbers than K. mikimotoi and K. brevis cells generally maintained a good physiological
state (Fv/Fm >0.60). The decline in cellular abundance observed for K. brevis after 21 days of growth coincided with the 16%-increase
in the percentage of dead cells (day 21) and the subsequent increase in caspase 3-like activity (day 28). Subsequently, cellular abundance
kept declining to reach its lowest value on day 56 when the caspase-like activity also peaked. During the two first weeks of the K.
mikimotoi experiment, cellular abundance remained low and Fv/Fm ~ 0.50. The 10%-increase in the percentage of dead cells on day
14 and the sustained activation of caspase-3 like enzymes during this time can either be explained by the mechanical stress encountered
during initial subculture, or by the inoculation of old cells. Subsequent growth was accompanied by higher Fv/Fm values (~ 0.70), low
percentages of dead cells, and low caspase 3-like activity. From day 42 the cellular abundance of K. mikimotoi started declining to reach
its lowest value on day 56 when the percentage of dead cells was above 40% and the caspase 3-like activity increasing again. Using
the TUNEL assay, DNA degradation was confirmed in both species of Karenia. Overall, our results suggest the implication of caspase3 like proteases in the prevention of growth and death of Karenia spp.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
INTERANNUAL VARIABILITY IN ALEXANDRIUM
CYST DENSITIES IN CORK HARBOUR, IRELAND
AND ITS RELATION TO BLOOM INTENSITY
Sarah Cosgrove, Paul O’Connor, Aoife Ní Rathaille, Nicolas Touzet, Isobel Evans, Robin Raine
The Martin Ryan Institute, National University of Ireland, Galway, University Road, Co. Galway, Ireland.
E-mail: [email protected]
The distribution of Alexandrium cysts in the sediments of Cork Harbour, Ireland, have been investigated since 2003 in order to better understand the dynamics of Alexandrium spp. in this region. Cyst densities of A. minutum and A. tamarense were consistently higher in the
North Channel than elsewhere in the estuary, and it is in this region where blooms of toxin producing Alexandrium initiate. During each
survey, both horizontal and vertical distributions of cysts were investigated during the winter months. The means and variances of the
horizontal cyst distribution in the top 1 cm of sediment in the North Channel were compared between years, and the data were also
analysed in conjunction with the maximum observed vegetative cell densities in subsequent summers. The variability in the cyst distribution
is discussed in relation to the hydrodynamics of the North Channel, in particular coincidence of highest cyst densities with the location of
tidally induced eddies. Nevertheless, the results show a statistically significant decreasing trend in cyst density since 2003. An analogous
decreasing trend in the maximum observed bloom cell density was also apparent since 2004 when an exceptional Alexandrium bloom
(5*105 cells l-1) occurred. The potential of the winter cyst density in controlling the intensity of summer blooms is discussed.
15
A MODELLING STUDY OF THE ROLE OF RESTING CYSTS
IN ALEXANDRIUM MINUTUM POPULATION DYNAMICS
Marta Estrada, Jordi Solé, Silvia Anglès, Esther Garcés
Institut de Ciències del Mar, CSIC, Pg. Marítim de la Barceloneta, 37- 49, 08003, Barcelona, Spain.
E-mail: [email protected]
We implemented a numerical model to simulate the population dynamics of Alexandriumm minutum in a Mediterranean semi-enclosed
water body (Arenys de Mar Harbor, NW Mediterranean). The effect of encystment and excystment rates, and length of the cyst dormancy
period on intensity and duration of blooms of vegetative cells was tested by means of a series of scenarios, constrained when possible
by experimentally-measured parameters. Excystment fluxes could substantially increase the population size of vegetative cells in initial
phases of the bloom and when net growth rates of the vegetative population were low or negative, but had negligible effect during periods
of exponential growth. Encystment rates not exceeding the maximum values determined in the study area (0.01 d-1) contributed to reduce
the peak concentration of vegetative cells but did not have a dominant effect on bloom termination. Increasing the dormancy times of the
resting cysts in the sediment resulted in greater losses of sediment cysts and reduced excystment fluxes. Our simulations highlight the
importance, regarding A. minutum population dynamics, of considering the magnitude and variability not only of growth and life-cycle
transition rates, but also of loss rates of both vegetative cells and cysts.
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PHASED CELL DIVISION AND GROWTH OF DINOPHYSIS
POPULATIONS OFF THE SOUTH COAST OF IRELAND
Hazel Farrell1, Robin Raine1, Lourdes Velo-Suarez2,3, Beatriz Reguera2
1
The Martin Ryan Institute, National University of Ireland, Galway, Ireland. E-mail: [email protected]
Instituto Español de Oceanografia, Aptdo 1552, 36200 Vigo, Spain
3
current address: IFREMER, Centre de Brest, BP 70, 29280 Plouzane, France
2
The proportions of viable cells of Dinophysis spp. that were paired (dividing) and recently divided cells were measured on populations of
D. acuta and D. acuminata observed off the south coast of Ireland in July 2007 and July 2009. Measurements were made regularly
through 24 hours, and clearly showed in phase division with maximum frequencies of both paired and recently divided cells observed in
the morning between 08:00-10:00h (GMT). The results were used to generate estimates of specific division rates, μ, for these species.
Values of μ were compared to estimates derived in similar fashion from observations on Dinophysis populations in northwestern Spain
and other parts of the world. The question of whether phased cell division always occurs in Dinophysis populations, and the incorporation
of the specific division rate into models of Dinophysis growth are discussed. Comprehensive field data sets demonstrate the impact of
the results on the coherence of Dinophysis populations during their transport along the Irish coast in jet-like flows towards sites of intensive
shellfish culture.
16
ANTHROPOGENIC HABITATS AS THE PRIMARY DRIVING
FORCE IN THE INCREASE OF COASTAL INSHORE
MICROALGAL BLOOMS IN THE MEDITERRANEAN SEA
Esther Garces, Jordi Camp
Departament de Biologia Marina i Oceanografia, Institut de Ciencies del Mar, CSIC, Passeig Maritim de la Barceloneta 37-49,
E08003 Barcelona, España. E-mail: [email protected]
Harmful algal blooms (HABs) are recurring events in coastal waters that can affect either large or smaller, localized areas. Several explanations have been proposed for the increase of HABs, including improved study and knowledge, the enhancing effects of anthropogenic
eutrophication, and broader geographical dispersion. An integrated perspective on HABs in the Mediterranean Sea (MS) suggests that
an additional explanation is crucial to our understanding of these blooms in tideless seas characterized by intense human settlement,
such as the MS: the development in coastal inshore waters of microhabitats with modified hydrodynamic regimes (higher nutrient input,
water stability, and water residence time), such as harbors and protected beaches. In these coastal inshore areas, small-scale physical,
chemical, and biological events converge that favor HAB occurrence and persistence. These events include greater water stability and
nutrient availability as well as the nature of the HA species inoculum, which frequently comprises resting stages. Our contemporary conceptual model accounts for the frequent small-scale coastal HABs documented in the MS. These blooms are diverse, highly localized,
and either recur annually or emerge in a seemingly arbitrary manner. They may be of short (2 or 3 weeks) or prolonged (up to 2 months)
duration and are not necessarily restricted by seasonality. While some blooms are predictable - since the benthic-pelagic coupling of a
species determines its bloom dynamics - others remain unpredictable. An improvement in the control of HABs in the MS relies on a
better understanding of bloom dynamics but also on a more-informed use of the coastal environment.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
ORAL PRESENTATIONS
GENETIC STRUCTURE IN THE PLANKTONIC
AND BENTHIC HABITAT - HOW ARE ALGAL
BLOOMS SEEDED?
Anna Godhe, Karolina Härnström
Dept of Marine Ecology, University of Gothenburg, Box 461, SE 405 30, Göteborg, Sweden. E-mail: [email protected]
Dormant life stages are important strategies for many bloom-forming microalgal species. The formation of resting stages will provide a
refuge from unfavourable conditions in the water column, and the successive accumulation in the benthos will provide a genetic reservoir
for future planktonic populations. We are using Skeletonema marinoi as a model organism, as it constitutes an important primary producer
but is also known to be harmful, often causing red tides or dense blooms. We have determined the genetic structure of S. marinoi, in the
sediment and the plankton, in Gullmar Fjord on the Swedish west coast, during spring bloom, summer and autumn for two subsequent
years (2007-09). Eight polymorphic microsatellite loci were used to assess the level of genetic differentiation and the gene diversity of
the two different habitats. We also determined the degree of genetic differentiation between the seed banks inside the fjord and the
open sea. The results indicate that Gullmar Fjord has one dominant endogenous population of S. marinoi, which is genetically differentiated
from the open sea population. This fjord population is encountered in both plankton and sediment. Shifts from the dominant population
can occur, and in our study two genetically differentiated plankton populations with reduced genetic diversity, were observed in September
2007 and 2008. Based on our results we suggest that sill fjords maintain local long-lived and well-adapted protist populations, which
continuously shift between the planktonic and benthic habitats. Intermittently, short-lived and mainly asexually reproducing populations
can replace the dominant population in the water column, without influencing the genetic structure of the benthic seed bank.
17
THE PHOTOBIOLOGY OF HETEROSIGMA AKASHIWO:
PHOTOACCLIMATION, DIURNAL PERIODICITY,
AND THE ABILITY TO EXPLOIT RAPID EXPOSURE
TO HIGH LIGHT
Sebastian Hennige, Kathryn Coyne, Mark Warner
University of Delaware, College of Earth, Ocean, and Environment, 700 Pilottown Rd., Lewes, USA. Email: [email protected]
Periodic and seasonal exposure to high light is a common occurrence for many near-shore estuarine phytoplankton. Rapid acclimatization
to shifts in light may provide an axis by which some species of harmful algae can outcompete other microalgae. Patterns of photoacclimation and photosynthetic capacity of the raphidophyte Heterosigma akashiwo isolated from Delaware, were followed in continuous cultures at low and high light, followed by reciprocal shifts to the opposite light level. The maximum quantum yield of photosystem II (Fv/Fm)
as well as the effective absorption cross section (σPSII) was higher in high light cultures compared to low light cultures. Despite growing
under static light, significant diurnal variability in photochemistry and photoprotection was noted at both light levels, and high light acclimated
cultures displayed greater variability in photoprotective pathways as well as PSII efficiency in the light. When shifted from low to high light,
there was only a slight and temporary decline in maximum quantum yield, while cellular growth nearly doubled within 24 hours. Rapid acclimation to high light was facilitated by short-term photoprotection (non-photochemical quenching), reduced connectivity and electron
transport. By 48 hours, photochemistry of cultures shifted from low to high light resembled long-term high light acclimated cultures. This
isolate of H. akashiwo appears to be well poised to exploit rapid shifts in light by using unique cellular adjustments in light harvesting and
photochemistry. This will be contrasted with other measures of photoacclimation and photoprotection (photosynthetic vs. photoprotective
pigments) noted in other algal species.
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SEASONAL CHANGE IN ABUNDANCE OF THE BENTHIC
DINOFLAGELLATE GAMBIERDISCUS SP.
IN TEMPERATE COASTAL WATERS OF JAPAN
Akira Ishikawa1, Yumi Takeichi1, Setsuko Sakamoto2, Mineo Yamaguchi2
1
Graduate School of Bioresources, Mie University, Kurima-Machiya, Tsu, Mie 514-8507, Japan.
E-mail: [email protected]
2
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi,
Hatsukaichi, Hiroshima 739-0452, Japan
The benthic dinoflagellate Gambierdiscus spp. is the primary causative organism of ciguatera fish poisoning (CFP) which occurs in tropical
and subtropical regions. In the last decade, several incidents of ciguatera were reported even in temperate coastal waters of Japan. Recently we have found the cells of Gambierdiscus, attaching to the macroalgae, in Ago Bay located in temperate part of Japan. The morphology of the cells is similar to that of G. toxicus. However, phylogenetic analysis utilizing 18S rDNA as well as 28S rDNA sequence
indicated that this species is not classified to any other Gambierdiscus species reported so far, although close to G. caribaeus. We here
present the seasonal change in abundance (density of the cells attached to macroalgae) of Gambierdiscus sp. in Ago Bay. The cell abundance has been monitored monthly since November 2008 in a seaweed bed of the bay. Gambierdiscus sp. showed an obvious seasonal
trend in abundance: cell density was very low, sometimes under the detection limit, from January to July but relatively high in September
and October. Maximum density, 32 cells g-1 (of macroalga wet weight) was recorded in the brown alga Sargassum thunbergii (Phaeophyceae) in October 2009. This maximum density is comparable to that recorded for G. toxicus in Okinawa Islands located in subtropical
part of Japan, where CFP have often been reported, suggesting that further studies on ecology and physiology, including toxicology, of
Gambierdiscus sp. are certainly needed to evaluate future risks of CFP even in temperate waters of Japan.
18
DINOFLAGELLATE VERTICAL MIGRATION
IS DEPENDENT ON HALO- AND THERMOCLINE
STRENGTHS
Therese Jephson, Per Carlsson, Tony Fagerberg, Karine Bresolin
Department of Ecology, Limnology, Ecology Building, S-22362 Lund, Sweden. E-mail: [email protected]
Increasing precipitation and surface water temperature due to global change may strengthen stratification in coastal regions which could
influence the behaviour of dinoflagellate migration. We studied diel vertical migration (DVM) behaviour by the dinoflagellates Prorocentrum
minimum and Heterocapsa triquetra using vertically-stratified laboratory columns with 3 different salinity gradients (6, 11 and 16 psu difference) and 2 temperature gradients (10°C and 17°C difference). At surface nutrient-deplete conditions, with nutrients added below a
pycnocline, P. minimum was mainly aggregated in the bottom water, while H. triquetra performed DVM in all treatments. H. triquetra migrated through a salinity difference of 6 and 11 psu but salinity gradient of 16 psu stopped cells from swimming through the gradient and
resulted in an aggregation of cells within the halocline during night. At midday cells were again found in the surface. Also, a temperature
gradient of 17°C stopped H. triquetra cells from passing the gradient. We found an altering DVM behaviour when P. minimum and H.
triquetra were exposed to different salinity- and temperature gradients. The two species responded differently to stratification which may
affect growth and competitive ability by these species also in situ.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
ORAL PRESENTATIONS
POPULATION DYNAMICS OF MARINE DIATOMS
AND DINOFLAGELLATES IN A TEMPERATE EMBAYMENT
Ken-Ichiro Ishii1, Akira Ishikawa2, Ichiro Imai3
1
Laboratory of Marine Environmental Microbiology, Division of Applied Biosciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan. E-mail: [email protected]
2
Graduate School of Bioresources, Mie University, Kurima-Machiya, Tsu, Mie 514-8507, Japan
3
Plankton Laboratory, Division of Marine Bioresource and Environmental Science, Graduate school of Fisheries Sciences,
Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
Diatoms and dinoflagellates are major components of phytoplankton in coastal ecosystems. Coastal planktonic diatoms and dinoflgellates
produce resting stage cells for temporarily avoiding adverse environments. Therefore, investigations on germination/rejuvenation of the resting
stage cells are important to understand the population dynamics and species alterations of phytoplankton in water columns. Quantitative monitorings of in situ germination/rejuvenation was performed on the resting stage cells of both diatoms and dinoflagellates in bottom sediments
in a temperate embayment (Ago Bay), central part of Japan, from July 2006 to July 2008 using a “plankton emergence trap/chamber (PET
Chamber)” which allows us to measure germination/rejuvenation of plankton. The 58 centric diatoms species of 29 genera were identified in
total. The largest germination/rejuvenation flux (>6×105 cells m-2day-1) was recorded for Skeletonema spp. It is notable that almost all detected
species appeared to show no obvious trends in seasonal germination/rejuvenation pattern but somewhat sporadic, suggesting that their
resting stage cells always possess an ability to germinate/rejuvenate. The dinoflagellates of 43 species of 17 genera were identified in total.
The germination/rejuvenation flux (3836 cells m-2day-1) was recorded for the toxic dinoflagellates Alexandrium catenella. The germination flux
of dinoflagellate species always changed with lower values than diatoms. The diatom abundance was always much higher than dinoflagellates
in water columns of sampling point. The light intensities at the sea bottom (6.4 ~ 53.5 μmol photons m-2sec-1) were enough for the germination/rejuvenation of diatoms, leading to the predominance of diatom resting stage cells (prevention of HAB occurrences).
19
VARIABILITY IN PSEUDO-NITZCHIA SPP. ABUNDANCE
AND TOXICITY IN ALABAMA COASTAL WATERS:
A “HOT SPOT” LINKED TO SUBMARINE
GROUNDWATER DISCHARGE
Justin D. Liefer, Hugh L. MacIntyre, A. Robertson, L. Novoveska, W.L. Smith, C.P. Dorsey
Dauphin Island Sea Lab, University of South Alabama, Dauphin Island, 102B Bienville Blvd., AL 36528, USA. E-mail: [email protected]
The potentially toxic diatom Pseudo-nitzschia is common in the northern Gulf of Mexico. Seven sites along the Alabama Gulf Coast have been
monitored weekly to bi-weekly for Pseudo-nitzschia spp., which were detected in 489 of 829 samples (59%) taken between November,
2003, and July, 2008. A cluster analysis of the relative frequency distributions of abundance by site showed that Little Lagoon Pass had a
strong dissimilarity compared to other sites, due to a higher frequency of bloom densities and a lower frequency of absences. Salinities at Little
Lagoon Pass were higher and less variable than at other sites. Pseudo-nitzschia spp. were absent more frequently from sites at the mouths of
Perdido and Mobilo Bays, where salinity was lower and more variable. Freshwater transport from Baldwin County, which lies between these
bays, has previously been shown to be primarily through submarine groundwater discharge into the Gulf of Mexico. Blooms of Pseudo-nitzschia
spp. At Little Lagoon Pass in spring were highly correlated with discharge from the Styx River, a proxy for groundwater discharge. Little Lagoon
Pass may therefore be a hot-spot for blooms of Pseudo-nitzschia spp., because local maxima in discharge result in nutrient availability without
significant reductions in salinity. In anticipation of a peak in Pseudo-nitzschia spp. abundance at Little Lagoon during mid-spring following an
increase in groundwater level, a mooring was conducted within the lagoon from April 1-20, 2008 and toxicity experiments using natural populations were conducted in the spring of 2008 and 2010. The mooring in 2008 coincided with a sustained bloom of Pseudo-nitzschia spp.,
with concentrations ranging from 800,000 to 5,000,000 cells L-1. Pseudo-nitzschia spp. concentrations were similar for the toxicity experiments.
Both the mooring and experiments demonstrate that the production of domoic acid, the toxin associated with the Pseudo-nitzshia genus, is associated with stress and physiological imbalances, with higher toxin production being associated with nutrient-limitation or photoinhibition.
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THIRTY YEARS - ALEXANDRIUM FUNDYENSE CYST,
BLOOM DYNAMICS AND SHELLFISH TOXICITY
IN THE BAY OF FUNDY, EASTERN CANADA
Jennifer L. Martin, Murielle M. LeGresley, Fred H. Page
Fisheries and Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, NB E5B 2L9, Canada.
E-mail: [email protected]
Sediment and water samples were collected for Alexandrium fundyense spatial and temporal distribution and abundance at more than
120 locations throughout the Bay of Fundy during the summers and winters of 1980-1984. These broad surveys have been repeated
at various times through the past 30 years, with annual more intense sampling since 2004. In addition, A. fundyense abundance has
been monitored at several locations within the Bay of Fundy at weekly intervals from April - November and monthly during the remaining
months since 1987. Paralytic Shellfish Poisoning (PSP) toxins in shellfish (including Mya arenaria) have also been monitored at multiple
locations in the Bay of Fundy since 1944. The 30+ year data sets were examined to determine relationships and roles between overwintering resting cysts, bloom initiation, bloom decline, cell dispersal and A. fundyense motile populations and resulting shellfish toxicity
since 1980. Cysts are widely dispersed throughout the Bay of Fundy in the offshore, inshore and intertidal zones with the largest deposits
located in the offshore in silt/clay sediments to the east and north of Grand Manan Island at depths of 60-180 m. Highest concentrations
of cysts (9270 cysts cm3) were observed in1984 and the highest concentrations of A. fundyense motile cells (>3 million cells•L-1) were
observed in 2003. Interannual changes in abundance in A. fundyense populations, resting cysts and the temporal trends in M. arenaria
toxicity will be discussed. Results suggest that the offshore seed beds serve as an important source for the motile cells that form the
major bloom initiations and resulting shellfish toxicity through the Bay of Fundy. Oceanographic influences, environmental and weather
patterns influence A. fundyense bloom intensity and dispersion.
20
SEASONAL VARIABILITY OF BIOLOGICAL CONDITIONS
OF DINOPHYSIS ACUMINATA IN THE RÍA DE PONTEVEDRA
(NW SPAIN)
L. Velo-Suárez1,2, S. González-Gil3, Y. Pazos4, I. Ramilo1, B. Reguera1
1
Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Vigo, Aptdo. 1552, E-36200 Vigo, Spain.
E-mail: [email protected]
2
Institut Français de Recherche pour l’ Exploitation de la Mer (IFREMER), Centre de Brest, DYNECO, Pointe du Diable BP70,
E-29280 Plouzané, France
3
Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Madrid, Orense 58, 7ª planta, E- 28020 Madrid, Spain
4
Instituto Tecnolóxico Para O Control Do Medio Mariño (INTECMAR), Peirao de Vilaxoán,s/n, E-36611 Vilagarcía de Arousa, Spain
The seasonal distribution and physiological condition of the DSP-toxin producer Dinophysis acuminata and its relation with accompanying microplankton
populations is described on the basis of weekly samplings (2 and 5m depth) from March to December 2007 at a fixed station in a Galician ría (NW
Spain). D. acuminata was detected from 18 June to 01 October. A maximum of the phototrophic ciliate Mesodinium rubrum (up to 27,000 cells L-1)
matched up with the beginning of the D. acuminata bloom. Increased vacuolation of Dinophysis (72%) was observed one week after the seasonal
maximum of its potential optimum prey. This M. rubrum peak was short-lived (one week) and declined afterwards to background levels (40 cells L-1).
These data suggest that in situ blooming of Dinophysis is triggered by heterotrophic feeding on a co-occurring peak of M. rubrum. High frequencies
(80-100%) of green fluorescing metabolically-active Dinophysis cells (FDA-treated) were always found at 5m depth. Conversely, the fraction of viable
cells at 2m decreased from ca. 90% to 52% on 09 July, ranged from 50 to 70% on August, and increased to above 90% on 24 September.
Infection (frequency of 1-17%) of Dinophysis by the parasite dinoflagellate Amoebophrya sp. was observed just before the decline of the D. acuminata
bloom, suggesting that parasites may represent a key factor in the population dynamics of D. acuminata. This work sheds new light on the biological
processes involved in the in situ development of D. acuminata blooms and its relation with its potential prey M. rubrum.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
ORAL PRESENTATIONS
ONCE AGAIN ON THE WAY TO GAINING PLASTIDS
IN THE MARINE DINOFLAGELLATE GENUS DINOPHYSIS?
Myung Gil Park, Miran Kim
LOHABE, Department of Oceanography, Chonnam National University, Gwangju 500-757, Republic of Korea.
E-mail: [email protected]
To survive, the marine photosynthetic dinoflagellates Dinophysis spp. must feed on the plastidic ciliate Myrionecta rubra (= Mesodinium
rubrum), itself a consumer of cryptophytes. While ultrastructural and molecular studies and pigment analyses all demonstrate that
photosynthetic Dinophysis species contain plastids of cryptophyte origin, whether the plastids are permanent or periodically derived
kleptoplastids (i.e. stolen plastids from its ciliate prey) has not been confirmed yet. There has been an obvious contradiction between
molecular sequence data and ultrastructural data for the status of the plastid. We addressed this issue here using several lines of
data from feeding/starvation experiments with the established cultures and from field samples. Restriction fragment length polymorphism (RFLP) patterns of the psbA genes, their sequences and phylogenetic analysis, and percentage of food-vacuolated cells from
feeding/starvation experiments will be discussed in the context of the status of Dinophysis plastids.
21
DOES WET DUST DEPOSITION TRIGGER
COCHLODINIUM BLOOMS?
A. Cemal Saydam1, Tim Wyatt2, Sanaz Lakestani1
1
2
Hacettepe University, Beytepe Ankara, Turkey. E-mail: [email protected]
Instituto de Investigaciones Marinas, 36208 Vigo, Spain. E-mail: [email protected]
Major blooms of Cochlodinium polykrikoides took place in the Persian Gulf, Strait of Hormuz, and Gulf of Oman in 2008/09. The
causes of these events are not understood, but the scarcity of riverine inputs eliminates explanations based on anthropogenic eutrophication. Here we propose that wet deposition of desert dust provides iron fertilization leading to bloom conditions 6-7 days later.
The model steps are: i) warm fronts of synoptic scale meteorological events promote long range desert dust transport; ii) during
transport, oxalic acid of soil microorganisms reacts with dust iron in cloud droplets to produce iron oxalate; iii) above a solar radiation
threshold of 250 W m -2, decarboxylation yields reduced iron; iv) wet deposition of ferrous iron promotes algal growth in sea. The
first step, the distribution of atmospheric pressure and wet deposition, governs the spatial and temporal patterns of phytoplankton
blooms in this model, independently of the oceanographic properties of receiving sea areas. The key role of the radiation threshold
in step (iii) may explain ambiguities in the interpretation of C. polykrikoides blooms in the western Pacific and elsewhere. Although
the focus here is on Cochlodinium, we suggest the proposed mechanism is of wider significance in bloom ecology.
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INFLUENCE OF ENVIRONMENTAL FACTORS ON DEVELOPMENT AND TOXICITY OF THE EPIPHYTIC DINOFLAGELLATE
OSTREOPSIS CF. OVATA IN THE NW MEDITERRANEAN SEA
Véronique Sechet1, Rodolphe Lemée2, Manoella Sibat1, Nicolas Chomerat3, Elisabeth Nezan3, Nicolas Ganzin4,
Hubert Grossel4, Aurélie Blanfuné2, Zouher Amzil1
1
IFREMER, Laboratoire Phycotoxines, Rue de l’Ile d’Yeu BP 21105, Nantes Cedex 44311, France. E-mail: [email protected]
2
Laboratoire d’Océanographie de Villefranche, UMR CNRS 7093, BP 28, 06234 Villefranche-sur-mer, France
3
IFREMER, Laboratoire Environnement Ressources Concarneau, 13 Rue de Kérose, 29187 Concarneau, France
4
IFREMER, Laboratoire Environnement Ressources Provence Azur Corse, zone portuaire de Brégaillon, BP 330, 83507
La Seyne-sur-mer, France
Ostreopsis spp. has been first observed in 1972 along Mediterranean coasts, in the bay of Villefranche sur Mer (Taylor 1979). However, over the
past ten years harmful events related to this benthic dinoflagellate have been reported in Italian, Spanish, Greek, French, Tunisian and Algerian
coastal areas. In France, during a hot period in August 2006, cases of dermatitis and respiratory problems were registered in Morgiret (Marseille).
At that time, a link to the proliferation of Ostreopsis was highlighted for the first time in that area. Subsequently, a research project “Medios 2” was
implemented during 2008-2009 to investigate the phenomenon. As part of the project, two strains of Ostreopsis cf. ovata, collected in 2008 from
Villefranche sur Mer and Morgiret coastal waters and grown in culture, were studied to characterize their growth and toxin profile. Liquid chromatography-mass spectrometry (LC-MS) indicated that both strains produced ovatoxin-a (OVTX-a) as the major component (ca. 90%), and traces of
palytoxin (PLTX). Toxin content was determined at the end of exponential growth phase with highest concentrations of 0.5 μg eq.PLT.mL-1. Analysis
of environmental variables in vitro and in situ highlighted the major role of temperature and nutrients on the development of Ostreopsis ovata. The
use of satellite derived Sea Surface Temperature (SST) data combined with in situ data of Ostreopsis spp. concentrations allowed for the identification
of two distinct areas: the more wind-affected, cooler Western part of Provence where Ostreopsis appearances are late in the summer (end of July
- August) and the Eastern part of Liguria-Provence with warmer seas and developments of Ostreopsis as soon as early summer (June).
22
AZADINIUM SPINOSUM IN IRISH WATERS,
AN AZASPIRACID PRODUCING DINOFLAGELLATE
Joe Silke1, Rafael Salas1, Jane Kilcoyne1, Kieran Lyons1, Amanda Burson1, Conor Duffy1, Urban Tillmann2
1
2
Marine Institute, Renville, Oranmore, Co. Galway, Ireland. E-mail: [email protected]
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany
Azaspiracids (AZAs) are a marine toxin group, first discovered in Ireland in 1995. Since this time, AZAs have recurred in Irish waters
and continue to pose a risk management problem for the shellfish industry and regulators from a consumer safety perspective. In
the intervening years a more widespread occurrence of AZAs was shown along the North African and European Coast from Morocco
to Norway. While initial studies suggested Azaspiracid to be originating from a Protoperidinium species, it was not demonstrated
conclusively that the toxin was produced by this species. The absence of a correlation between the occurrence of the Protoperidinium
and the occurrence of AZAs in shellfish suggested heterotrophic feeding behaviour was responsible for the presence of toxins. A recently discovered species, Azadinium spinosum isolated from the North Sea was shown to produce Azaspiracid on an ongoing basis
in experimental culture conditions. This same species was isolated during field trials in Ireland in autumn 2009 and cultures were established. These cultures were shown to produce Azaspiracids, suggesting that Azadinium spinosum is a contributor to the Azaspiracid
presence in Irish shellfish. The toxin production and the toxin profile of the culture will be discussed and compared with the data from
the North Sea cultures. An oceanographic model of affected regions of Ireland was developed and was seeded with Azadinium particles. This was then run prior to the arrival of the Azaspiracid in an attempt to understand if the species is advected from offshore or
is a localised population within the bays.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
ORAL PRESENTATIONS
MOTILITY DURING LIFE-STAGE TRANSITIONS
OF THE HARMFUL ALGA, HETEROSIGMA AKASHIWO:
ASSESSING IMPLICATIONS FOR BLOOM DYNAMICS
Elizabeth D. Tobin, Daniel Grünbaum, Rose Ann Cattolico
School of Oceanography, University of Washington, Seattle, WA 98195-7840, USA. E-mail: [email protected]
In many harmful algal blooming (HAB) species, life-stage transitions between a vegetative, motile stage in the water column and a
dormant, non-motile cyst stage in the sediments strongly impact the timing and location of bloom events. Transitions between pelagic
and benthic habitats often involve significant vertical movements. Swimming is the primary mechanism used by many HAB-species
to control vertical distributions, suggesting that swimming during life-stage transitions may be used to actively influence vertical fluxes,
and hence, subsequent population level distributions. However, swimming behaviors during these transitions are poorly understood.
We used video-based motion analysis to test the hypothesis that cells of the HAB-forming alga Heterosigma akashiwo actively influence their benthic distributions and shift from up-swimming to down-swimming during transition to the benthic cyst stage. We developed an assay based on statistical characteristics of individual swimming paths to assign each tracked cell to one of three
physiological states observed during pelagic-benthic transition. We measured net vertical velocities of individuals in each state and
found no evidence of down-swimming. Even early-transition, motile cells moved downwards at the same speed as late-transition,
non-motile cells. Thus, our results suggest that cells reach the benthos by sinking out of the water, and benthic distributions are controlled primarily by physical mechanisms. We are currently applying these video tracking techniques to quantify H. akashiwo cell
swimming immediately following emergence from the benthos, and to in situ remote sensing of H. akashiwo HABs. Resolving statespecific movement behaviors that influence population-level distributions will greatly improve HAB prediction.
23
IN SITU EMERGENCE FLUX OF ALEXANDRIUM
FROM SURFACE SEDIMENTS IN A TIDAL SALT POND
Emil Vahtera1, David K. Ralston1, Vitalii Sheremet2, Bruce Keafer1, Akira Ishikawa3, Donald M. Anderson1
1
Woods Hole Oceanographic Institution, 266 Woods Hole Rd, Woods Hole, MA 02543, USA. E-mail: [email protected]
University of Rhode Island, 215 South Ferry Rd, Narragansett, RI 02882, USA
3
Mie University, Kurima-Machiya, Tsu, Mie 514-8507, Japan
2
We measured the in situ emergence flux of Alexandrium fundyense cells from natural surface sediments in a tidal salt pond using
Plankton Emergence Traps. Prior to emergence flux measurements, the distribution of resting cysts was mapped on different spatial
scales. We monitored the development of the vegetative population and measured a suite of environmental parameters, including
bottom shear stress, to determine factors that regulate the emergence flux of cells. Distinct cyst accumulation areas were identified
within the pond. The measured emergence flux of cells was subject to large natural variation, which stems from the spatial variation
in cyst concentrations in the surface sediment on smaller spatial scales, as well as differences in the “readiness” of the cysts to germinate. In the latter context, spatial differences were observed in chlorophyll autofluorescence in cysts, which we believe is indicative
of impending germination. The viability of germlings also most likely varies with the age of the cysts. The emergence flux increased
as water temperature and solar irradiation increased. A surge in the emergence flux of cells was observed approximately three weeks
prior to the observed vegetative cell biomass peak that occurred in the beginning of May. The bloom was short and declined after
only one week. Deposition of new cysts was recorded during the bloom by sediment traps and from cyst counts from surface
sediment samples. The spatial distribution of the newly formed cysts conformed to the previously observed distribution.
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1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:23 PM Page 25
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
DIVERSITY OF POTENTIALLY TOXIC BENTHIC
DINOFLAGELLATES IN SOUTHERN EUROPE
Katerina Aligizaki1, Cecillia Battocchi2, Antonella Penna2, Francisco Rodríguez Hernández3, Minas Arsenakis1,
Santiago Fraga3
1
School of Biology, Faculty of Sciences, Aristotle University, 54124 Thessaloniki, Greece. E-mail: [email protected]
Dep. of Biomolecular Sciences, University of Urbino, Viale Trieste 296, 61121 Pesaro, Italy
3
Centro Oceanográfico de Vigo, IEO (Instituto Español de Oceanografía), Subida a Radio Faro 50, 36390 Vigo, Spain
2
In recent years benthic dinoflagellates records are growing in numbers; this increase is mostly apparent in temperate areas where
until a decade ago studies on these dinoflagellates were rare. It is commonly recognized that enhanced records are not always representative due to directed intensive research in some specific fields. A more accurate estimation of the present diversity, the actual
biogeographical distribution and the possible expansion range of some taxa, requires both morphotaxonomy and genetic analyses.
In this context, benthic dinoflagellates of the genera Gambierdiscus, Ostreopsis, Coolia and Prorocentrum collected from the Mediterranean Sea and the Canary Islands were studied using phenotypical and phylogenetic characters. This approach has resulted in the
detection of at least two new Gambierdiscus species, currently being described, one from the Canaries and one from the Mediterranean Sea, as well as a new Ostreopsis genotype dispersed in both areas. Phylogenetic analyses of the genera Gambierdiscus,
Ostreopsis and Coolia showed that the different groups correspond to distinct, described or undescribed, species. However, in the
case of the genus Prorocentrum, its monophyletic or polyphyletic origin has not been yet elucidated, as different molecular markers
have yielded contradictory results; it has yet to be determined whether the two genetic lineages can constitute separate genera. Furthermore, the taxonomy of this genus seems to have been hampered so far by the accumulation of misidentifications, the erection
of too many species and the variability of the classical morphological characters used until recently.
25
SEASONAL PHYTOPLANKTON DYNAMICS AND OCCURRENCE
OF POTENTIALLY TOXIC SPECIES IN EXTREME SOUTHERN
SOUTH AMERICA (BEAGLE CHANNEL, ARGENTINA)
Gastón O. Almandoz1, Marcelo P. Hernando1,2, Martha E. Ferrario1
1
Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n (1900), La Plata, Argentina,
and CONICET, Argentina. [email protected]
2
Universidad de Morón, Cabildo 134 (1708), Buenos Aires, Argentina
The occurrence of HABs is a recognized problem for shellfish aquaculture in the Beagle Channel, where there is an extensive
historical record of PSP and DSP events. Within the framework of a monitoring program carried out in its Eastern zone, we focused
on the phytoplankton and oceanographic dynamics during an annual cycle (July 2006-2007) for the first time in this area. Samples
were examined by light and scanning electron microscopy. The annual cycle was characterized by a sharp contrast between low
density and biomass during the autumn-winter period and a significant increase during spring and summer. The maximum biomass
peak was observed during early spring (8.7 μg Chl α l-1) and was associated with a bloom of the diatom genus Chaetoceros. Towards
the end of spring and during summer diatom density decreased and assemblages were enriched by unidentified tiny phytoflagellates
and dinoflagellates. A total of 10 potentially toxic species were found: Alexandrium catenella, A. ostenfeldii, A. tamarense, Dinophysis
acuminata, D. rotundata, Prorocentrum cordatum, Pseudo-nitzschia australis, P. calliantha, P. fraudulenta and P. cf. seriata. Peak
densities of A. catenella, D. acuminata and P. calliantha were within the ranges of abundances from which toxin accumulation in
shellfish is usually observed in other regions. Likewise, the presence of A. catenella and A. ostenfeldii coincided with PSP detection
in mussels (up to 80 μg STX / 100 g). The finding of specimens of P. seriata with an unusual morphology prompts new questions
about the distribution of this species and its morphological differentiation with P. australis.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:23 PM Page 26
QUANTITATIVE PCR COUPLED WITH MELTING CURVE
ANALYSIS FOR DETECTION OF MICROALGAE
Karl B. Andree1,4, Margarita Fernández-Tejedor1,4, Laurence M. Elandaloussi1,4, Sonia Quijano-Scheggia3,
Nagore Sampedro2,4, Esther Garcés2,4, Jordi Camp2,4, Jorge Diogène1,4
1
IRTA, Crta. Poble Nou, Km 5.5, 43540-Sant Carles de la Rápita, Spain
2
Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CSIC, Passeig Marítim de la Barceloneta,
37-49, E08003 Barcelona, Spain
3
Centro Universitario de Investigaciones Oceanológicas, Universidad de Colima, Carretera Manzanillo-Barra
de Navidad Km 19.5, Colonia El Naranjo, C.P. 28860, Manzanillo, Colima, México
4
Xarxa de Referència de Recerca i Desenvolupament en Aqüicultura, Generalitat de Catalunya, Spain.
E-mail: [email protected]
Along the coast of Catalonia large blooms of Pseudo-nitzschia spp. have been increasing steadily in frequency and magnitude during the past twenty
years of surveillance and are of serious concern due to their toxic potential. Among species from this genus found locally, P. calliantha, P. delicatissima
and P. multistriata have been documented as toxic elsewhere. However rapid assessment, by morphometry, of species identity through optical microscopy is not possible for members of this genus due to the small size of the cells and the presence of cryptic species. Therefore differentiation
of potentially toxic and non-toxic species is also not possible. We describe herein a methodology initiated to attempt a remedy to the inability to adequately differentiate species of the genus Pseudo-nitzschia, and present the results from field testing of this technique. Clonal cultures of locally
isolated Pseudo-nitzschia species were characterized morphologically using scanning electron microscopy and samples of each culture were used
in creating an ITS-5.8S ribosomal DNA sequence database for designing primers specific for the predominant species encountered in Catalan
coastal waters. Melting curve analysis from the resulting qPCR assays allowed differentiation of amplicons and identification of false positives in seawater samples. This methodology was applied to seawater samples collected during a two year period from Alfaques Bay in the south of Catalonia
to assess the changing population structure of Pseudo-nitzschia blooms. Results obtained from this qPCR methodology, which included eight
different species, showed good overall statistical correlation with the optical microscopy method of enumeration at the genus level.
26
SWIMMING AND SINKING IN A MEDITERRANEAN BAY:
SURVIVAL STRATEGIES IN A STRATIFIED AND WEAKLY
TURBULENT WATER COLUMN
Mireia L. Artigas1, Oliver N. Ross2, Ruben Quesada2, Jaume Piera2, Elisa Berdalet1
1
Institut de Ciencies del Mar (CSIC), Pg. Maritim de la Barceloneta 37-49, E-08003 Barcelona, Catalunya, Spain.
E-mail: [email protected], [email protected]
2
Unitat de Tecnologia Marina (CSIC), Pg. Maritim de la Barceloneta 37-49, E-08003 Barcelona, Catalunya, Spain
The mechanisms underlying the population dynamics of phytoplankton species causing harmful blooms are complex. They result
from the interplay of physico-chemical and biological factors to which the organisms respond with a variety of strategies. In this
context, the vertical distribution of the organisms in the water column may be crucial for their success as it determines their access
to light and nutrients and affects the different strategies regarding reproduction, life cycle, and predator avoidance. Some phytoplankton
cells can actively regulate their vertical position through swimming, buoyancy changes and/or sinking provided that they can overcome
the turbulent mixing and advection. We collected a two-year time series of biological and physical (turbulence) measurements in
Alfacs Bay (Ebre Delta, NW Mediterranean), a shallow estuary (max depth is 6m) and active aquaculture site. We found that while
the ichtiotoxic dinoflagellate Karlodinium spp. was mainly located below 4m, the potentially amnesic shellfish toxin producer Pseudonitzschia spp. showed a homogeneous distribution throughout the upper layer of the water column. We used our observations of turbulence intensity to set up a combination of turbulence closure and individual based model (IBM). The model results enable us to
evaluate the extent to which the different physical and physiological mechanisms could be involved in creating the observed differential
vertical distribution of these two harmful phytoplankton species.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:23 PM Page 27
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
A DENSE BLOOM OF DINOFLAGELLATE HETEROCAPSA
TRIQUETRA BELOW THE ICE-WATER INTERFACE
IN A BRACKISH LAKE SHIHWA, KOREA
Seung Ho Baek1, Kai You2, Toshiya Katano3, Jang Seu Ki4, Bum Soo Park4, Kyoungsoon Shin1,
Young Ok Kim1, Myung-Soo Han4
1
Korea Ocean Research and Development Institute / South Sea Institute, Geoje 656-830, Korea. E-mail: [email protected]
Department of Fisheries Science, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
3
Ariake Sea Research Project, Saga University, Honjo-machi 1, Saga 840-8502, Japan
4
Department of Green Life Science, College of Convergence, Sangmyung University, Seoul 110-743, Korea
2
To clarify favorable environmental conditions for the population development of dinoflagellate Heterocapsa triquetra, the seasonal abundance of the species and related environmental factors were investigated from May 2007 to May 2008 in a brackish Lake Shihwa,
Korea. The growth and morphological characteristics of this species was also examined under laboratory conditions. In field, the population
densities of H. triquetra remained low level during the period from late spring to early summer and completely disappeared from August
to November, 2007. Interestingly, a dense bloom of H. triquetra below the ice-water interface was appeared on 17 January. The other
peaks reached 67 X 104 cells L-1 on 28 March 2008, with the water temperatures of 9.1oC. In laboratory experiments, the growth rates
of H. triquetra increased with the temperature increments within a range from 10 to 20oC. The highest growth rate of H. triquetra reached
0.62 d-1 at 20oC with a salinity of 30. At above 25oC, the dinoflagellate was unable to grow at 10 and 15 salinities, and only reached relatively low growth rates (< 0.12 d-1) in other salinity conditions. However, under the continuous cultures at 5oC and 8oC, the H. triquetra
retained growth capability, suggesting that H. triquetra grew very well at such very low temperature conditions. The equivalent spherical
diameter (ESD) in H. triquetra did not change markedly between 10 and 25oC, but the ESD was significantly different at 5oC. Also,
several cells shed the theca but remain covered with the pellicle wall at 5oC culture conditions. These specific characteristics make H. triquetra become an unusual dominant dinoflagellate during cold winter season in the Lake Shihwa.
27
MARINE PHYTOPLANKTON MONITORING IN THE GULF
OF TEHUANTEPEC, SOUTHERN MEXICAN PACIFIC
(2003-2007), WITH EMPHASIS IN HARMFUL SPECIES
Sofia A. Barón-Campis1, David U. Hernández-Becerril2, Casimiro Ramírez-Camarena1
Fishing National Institute, SAGARPA, Pitágoras 1320, Col. Santa Cruz Atoyac, 03310, Mexico D.F.
E-mail: [email protected]
2
Instituto de Ciencias del Mar y Limnología, UNAM, A. P. 70-305, Cd. Universitaria, 04510, Mexico D.F.
1
Marine phytoplankton, especially the harmful phytoplankton species has been monitored since 2003 from coasts of the Gulf of Tehuantepec, southern Mexican Pacific, to prevent events that could affect the fisheries and human health. Monthly samplings by bottle and
net in two locations in that area have been made from March 2003 to August 2007. Various species of the diatom genus Pseudonitzschia have been frequent along the time, but during winter (“nortes”) season, the period of highest mix in the area, some of them
potentially domoic acid producers, have been numerical dominants, with densities reaching 2x105 cells l-1. The thecate dinoflagellate
Alexandrium tamarense, also known as potentially saxitoxin-producer, was detected in lower concentrations (1.5x104 cells l-1); the
cyanobacterium Trichodesmium erythraeum and the silicoflagellate, bloom forming species, Dictyocha fibula were additionally recorded
in low densities. In February 2006 was observed Pyrodinium bahamense var. compressum which was followed in March by Gymnodinium
catenatum, with densities of 1,196 and 2,926 cells l-1, respectively; both dinoflagellates have caused toxic and fatal previous events in
the Mexican Pacific. The presence of “cryptic” species, which are associated to toxins or harmful blooms, was recognized during this
study and they still remain to be revised. Additionally, a review of harmful species with an approach for fisheries is in process.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:23 PM Page 28
OSTREOPSIS CF. SIAMENSIS BLOOMS IN MOROCCAN
ATLANTIC UPWELLING WATERS (2004-2009)
Asmae Bennouna1, Jaouad El Attar1, R. Abouabdellah1, Abdelghani Chafik1, Antonella Penna3, Paulo B. Oliveira2,
Sofia Palma2, Mª Teresa Moita2
1
National Institute of Marine Research (INRH) Regional Center of Agadir, Aghsdis, Nouveau Port, Agadir, Morocco.
E-mail: [email protected]
2
INRB-IPIMAR, Avenida de Brasília, 1449-006 Lisbon, Portugal. E-mail: [email protected]
3
Sez. Environmental Biology, Dep. of Bimolecular Sciences, University of Urbino, Vle. Trieste 296, 61121 Pesaro, Italy.
E-mail: [email protected]
In 2004, Ostreopsis siamensis Schmidt blooms were detected for the first time on the central upwelling coast of Morocco by the HAB
and phycotoxins monitoring program. The species identification was confirmed by genetic studies developed by sequencing the 5.8S
–ITS and LSU ribosomal genes. In October 2004, O. siamensis blooms reached 3.7x103 cells L-1 in seawater samples of the cape
Ghir. In the following years, under surface temperatures of 20-24°C, the blooms became recurrent, longer and increased in concentration
reaching 9.8x103 to 1.2x104 in 2008, with a maximum of 105 cells l-1 observed in August 2009. The detection of toxins in mussels
collected from the same area, by mouse bioassay, evidenced the presence of neurotoxins. At cape Ghir, an important upwelling center
on the Morrocan coast, the cell maxima occurred in rocky areas well exposed to winds and waves, and not in wind sheltered areas
south of the cape, highlighting the importance of hydrodynamism on the re-suspension of this epiphytic species in the water column.
Satellite images, atmospheric re-analysis data and operational hydrodynamic model results, for the periods preceding the bloom events,
showed that in the first two years (2004, 2005) the blooms occurred during periods of upwelling relaxation. Since then, the species
seems to be established in the region and its concentration is not directly related to upwelling intermittency / intensity. During the bloom
events, the model solutions for the surface circulation suggest that cape Ghir area was affected by waters from the Safi coast flowing
southward and turning westwards at the cape.
28
KINETICS OF NITROGEN UPTAKE AND TRANSIENT
AMMONIUM UPTAKE RESPONSE BY THE TOXIGENIC
DIATOM PSEUDO-NITZSCHIA TURGIDULA
Brian D. Bill1,2, William P. Cochlan2, Vera L. Trainer1
1
2
NOAA Fisheries, Marine Biotoxins Program, 2725 Montlake Blvd E, 98112, Seattle, USA. E-mail: [email protected]
Romberg Tiburon Center for Environmental Studies, San Francisco State University, 3152 Paradise Drive, 94920,
Tiburon, USA
Nitrogen uptake kinetic parameters and transient ammonium uptake capabilities of Pseudo-nitzschia turgidula were investigated using
the N-15 isotopic tracer technique using unialgal batch cultures grown under saturating light conditions. Maximum specific uptake rates
and affinity values for three nitrogen substrates: nitrate, ammonium and urea were estimated for this primarily oceanic species for the
first time. Transient, or ‘surge’, ammonium uptake capabilities were also investigated on nitrogen-starved batch cultures. P. turgidula
demonstrated a nitrogen uptake preference of the order: ammonium > nitrate > urea, and mean maximum uptake rates (Vmax) of 96.3,
48.5, and 12.6x10-3 h-1, respectively. Substrate affinity at low nitrogen concentrations was determined by the initial slope (α) of the
Michaelis-Menten curve (or equivalent), and nitrogen preference followed the same order as determined with maximum uptake rates
and elevated nitrogen concentrations. Values for the initial slope were 36.7, 29.4 and 0.9x10-3 h-1/(μg-at N L-1), respectively for the
ammonium, nitrate and urea substrates. P. turgidula demonstrated a capacity for transient ‘surge’ uptake of ammonium during the first
3-5 minutes and this capacity declined within 0.5 h to a relatively constant rate. These are the first results to demonstrate that an
oceanic isolate of Pseudo-nitzschia can utilize both inorganic and organic nitrogen for assimilation and has the capacity for surge uptake
of ammonium. These results will be useful for managing anthropogenic inputs into marine systems and understanding the potential
contributions of P. turgidula to phytoplankton bloom development and its toxigenic capacity in both coastal and oceanic waters.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:23 PM Page 29
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
STUDIES ON CULTURES OF OSTREOPSIS CF. OVATA:
LIFE CYCLE OBSERVATIONS
Isabel Bravo1, Magda Vila2, Susana Magadan1, Pilar Rial1, Francisco Rodriguez1, Santiago Fraga1, Jose M. Franco1,
Pilar Riobo1, M. Montserrat Sala2
1
Ιnstituto Espanol de Oceanografia (IEO). Subida a Radio Faro 50, Aptado 1552, 36200 Vigo, Spain.
E-mail: [email protected]
2
Institut de Ciencies del Mar (ICM-CSIC), Pg. Maritim de la Barceloneta 37-49, 08003 Barcelona, Spain
Blooms of the epiphytic palytoxin-producer dinoflagellates Ostreopsis spp. have been reported over the last few years to become established in some Mediterranean coastal areas. One of these sites is Llavaneras beach (Catalonia, NE of Spain), where during summer, the
blooms form a conspicuous, thick, brownish mucilage layer covering benthic macroalgae. Asexual and sexual reproduction were studied
in cultures of Ostreopsis cf. ovata isolated from several locations in the Mediterranean Sea. Asexual division took place in the motile stage
by sharing of the theca (desmoschisis). By means of that process, culture gradually became rich of small clumps of cells joined by threads
of mucus. Two types of cells were observed in culture during late exponential phase: 1) dark medium/large size cells (40-70 μm) and 2)
light small cells (30-40 μm). These cells differed in cellular content and physiological behaviour. Diel pattern of the cellular DNA content
showed that maximum cell division occurred 3 hours after the beginning of dark period. In middle stationary phase, fusing gamete pairs
were observed even in clones (homothallism). However, complete gamete fusion was never observed and planozygotes were unproven.
Most of the gamete pairs isolated divided before the fusion was completed. Two types of cysts were detected in culture: 1) shortdormancy pellicle cysts formed by ecdysis, and 2) putative resting cysts which, so far, showed no signal of germination.
29
AN INTEGRATED APPROACH FOR THE ASSESSMENT
OF HAB DYNAMICS IN TWO NW MEDITERRANEAN
BAYS FROM A GEOHAB PERSPECTIVE
Julia A. Busch1, Allan D. Cembella2, Margarita Fernández-Tejedor3, Jorge Diogène3, Oliver Zielinski1
1
Institute for Marine Resources, Bussestrasse 27, 27570 Bremerhaven, Germany. E-mail: [email protected]
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany
3
Ιnstitut de Recerca i Tecnologia Agroalimentaries, Ctra. Poble Nou Km 5,5, 43540 Sant Carles de la Rápita,Tarragona, Spain
2
Alfacs and Fangar Bay in the Ebro Delta, NW Mediterranean are the major sites in Catalonia for cultivation of shellfish, e.g. the mussel
Mytilus galloprovincialis. These bays are subject to occasional closures shellfish harvesting due to the presence of phycotoxins and
fish kills have also been associated with harmful algal blooms. The comparison of phytoplankton dynamics in both bays therefore
offers the opportunity to reveal differences in bloom patterns of species known to be harmful for the ecosystem and aquaculture activities. Field research is underway under the GEOHAB framework within the Core Research Project on HABs in Fjords and Coastal
Embayments. The overall objective of this study is to improve understanding of HAB biogeographical patterns, and key elements
driving bloom dynamics in time and space within these semi-constrained embayments. Via the comparative approach we aim to
improve the prediction for monitoring purposes, with a focus on Karlodinium spp. associated with massive kills of aquaculture species.
This objective is addressed by incorporating long-term time series of phytoplankton identification and enumeration with the first results
of recent field work in both bays. The latter includes the application of optical sensors, to yield a complementary view with enhanced
spatial and temporal resolution of bloom phenomena.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 30
AN OVERVIEW OF THE OCCURRENCE OF TOXIC ALGAE
BLOOMS IN THE RIO DE LA PLATA AND ITS MARITIME FRONT
Jose Carreto1, Silvia Mendez2
1
Instituto Nacional de Investigacion y Desarrollo Pesquero, Paseo Victoria Ocampo 1, Escollera Norte,
B7602HSA-Mar del Plata, Argentina. E-mail: [email protected]
2
Direccion Nacional de Recursos Acuaticos, Constituyente 1497, (CP11200) Montevideo, Uruguay.
E-mail: [email protected]
The Rio de la Plata and its Maritime Front (RPMF) is one of the places to have recurrent blooms of species which caused harm to
humans and the marine ecosystem, especially of toxic species producing paralytic shellfish toxins (PST), amnesic shellfish poisoning
(AST), diarrhetic shellfish poisoning (DST) and hepatotoxic cyanobacterial toxins (HCT). In this work, we review the occurrence, distribution and toxicology of these toxic algal species. Thus far, the increased reports of human health and economic impacts had been
caused by the increased in intensity and geographic spread of Alexandrium tamarense and Gymnodinium catenatum blooms. In this
review, the observed northern spread of A. tamarense and the inter-annual variability in A. tamarense and G. catenatum blooms are
related to the oceanographic patterns in order to discuss the factors controlling the occurrence, distribution and toxicity of these toxic
dinoflagellates along the region.
30
SPATIAL AND SEASONAL VARIATIONS OF ALGAL
PROLIFERATION IN A DAM-CONTROLLED SUBTROPICAL
RIVER: EVIDENCE FROM δ13C AND δ15N
OF PARTICULATE ORGANIC MATTER
Fajin Chen1,2, Guodong Jia1, Jianfang Chen2, Haisheng Zhang2
1
CAS Key Laboratory of Margional Sea Geology, Guangzhou Institute of Geochemistry,
Chinese Academy of Sciences, Guangzhou 510640, China. E-mail: [email protected]
2
Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography,
State Oceanic Administration, Hangzhou 310012, China
Artificial dams constructed on numerous rivers around the world may exert significant impacts on river biogeochemistry, change the quality of
OM delivered to the oceans, and has the potential to induce algal proliferation. Therefore, understanding the biogeochemical processes occurring
in the dam-controlled rivers is important for water resource management. In this work, δ13C and δ15N signatures of particulate organic matter
(POM) in a dam-controlled subtropical river, Beijiang River, in south China are reported for their spatial and seasonal distributions. POM affected
by reservoirs is lighter in δ13C and heavier in δ15N relative to unaffected POM. In April, POMδ13C and δ15N values show less spatial variation
in the main stem, and suggest relatively greater contributions of terrestrial organic matter (OM) to POM. This could be related to the onset of
summer monsoon that caused an abrupt increase in terrestrial input to the river by the monsoon-induced enhancement of rainfall and runoff.
In August and December, however, POM isotopic values for the sites affected by the Feilaixia dam reservoir in the middle of the river show
marked changes, suggesting aquatic plankton proliferation in the reservoir during the times. Upstream from the reservoirs, POM isotopes are
seasonally less varied and suggest mainly terrestrial origin. However, the isotopic signals of aquatic plankton proliferation in the reservoir in
August and December is imprinted on the POM isotopic compositions downstream the reservoir, indicating far-reaching influences of the
reservoir on the downstream water environment.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 31
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
SPATIAL AND TEMPORAL VARIABILITY OF HARMFUL
PHYTOPLANKTON IN THE FIRTH OF LORNE, SCOTLAND
Keith Davidson, Sarah Swan, Eleanor Martin, Sharon McNeill
Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll, PA37 1QA, UK.
E-mail: [email protected]
The fjordic Scottish west coast is an area with extensive shellfish and finfish aquaculture activity. Using data from the EU Shellfish Hygiene
Directive based regulatory monitoring of biotoxin-producing phytoplankton, and the EU Northern Periphery Programme project ‘WATER’,
we have conducted both qualitative and multivariate statistical analysis of the spatial and temporal variability in phytoplankton at a number
of stations in the important shellfish harvesting area of the Firth of Lorne. Water samples were collected during regular ship board cruises
to the sampling sites, and also obtained from bi-annual transects across the open firth, from Loch Spelve in the south to Loch Creran in
the north. Cell counts were studied in relation to potential environmental drivers including temperature and salinity, which were recorded
during these cruises. The relationship between harmful organisms and the environment is discussed, along with the potential to use any
such relationship to develop models that could provide early warning of harmful blooms.
31
OBSERVATION OF ECOLOGICAL FEATURES OF ALGAL
POPULATION IN THE PALIASTOMI LAGOON
OF THE BLACK SEA
Manana Devidze
Sokhumi State University. E-mail: [email protected]
There are many eutrophicated areas in the coastal zone of the Black Sea. But largest water body in this area is Paliastomi which has been
connected to the Black Sea with artificial channel since 1934. At the same time since 2000 Paliastomi has been declared as part of the
Kolkheti National Park. Since 1964 has begun scientific researches of phytoplankton composition in the lagoon. Periodically there were
recorded harmful algal bloom events in the lagoon. Dominant species were Nodularia spumigena f. litorea, Anabaena elenkini. Researches
of following years and dynamic of algal population show process of succession and radical changes of phytoplankton composition. In spring
of 2010 phytoplankton samples of Paliastomi were taken from three places: first from river mouth, second from sea mouth and third from
the middle of lagoon. Phytoplankton species analyses have shown 124 algae taxons: Cyanoprocariota – 5, Euglenophyta – 10, Dinophyta
– 3, Bacillariophyta -96, Xanthophyta – 1, Chlorophyta – 1. In comparison with previous years phytoplankton taxons composition in recent
researches was completely different and there have not been detected the dominant species which bloomed previous years. Among the different taxons the species of Bacillariophyta have been in majority. Also, it was detected existence of euryhalinic forms of algae [Cyclotella
sp., Coconeis placentula Ehrenb, Amphora coffeaeformis (Agardh) Kütz. var angularis V.H.] despite of places from where the samples had
been taken, in mouth of river, in mouth of sea or in middle of lagoon with very changeable salinity. Also, according to the researches of the
last year diversity of algae species has been increased and algal bloom events have not been recorded which may be explained with reducing
the fertilizers from agricultural fields as lagoon is located in the protected area.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 32
SPATIAL AND TEMPORAL VARIABILITY
OF DINOFLAGELLATE CYSTS IN RECENT MARINE
SEDIMENTS FROM CHILEAN INLAND SEAS
Patricio Díaz1, Carlos Molinet1, Miriam Seguel2, Manuel Díaz1, Gisella Labra2
1
2
Instituto de Acuicultura, Universidad Austral de Chile, Los Pinos s/n, Puerto Montt, Chile. E-mail: [email protected]
Lacoratorio Ceram, Universidad Austral de Chile, Los Pinos s/n, Puerto Montt, Chile
The distribution and abundance of dinoflagellates cysts were investigated between March and December 2009 in recent sediments of
two areas of Chilean inland seas: Ovalada Island and Low Bay. A total of 31 cysts types were recorded from 288 sediment samples,
representing 7 genera (Alexandrium, Protoceratium, Scripssiella, Pentapharsodinium, Protoperidinium, Polykrikos and Diplopelta). Total
cyst abundance ranged from 0-164 cyst ml-1 wet sediment in Low Bay and 3-165 cyst ml-1 wet sediment in Ovalada Island. Cysts of
two toxic dinoflagellates -Alexandrium catenella and Protoceratium reticulatum- were recorded. Cysts maximum abundances of A.
catenella were observed during June in Ovalada Island (51 cysts ml-1 wet sediment) and Low Bay (128 cysts ml-1 wet sediment) decreasing
in October to less than 10 cysts ml-1 wet sediment in both areas. Maximum abundance of the P. reticulatum cyst was 88 and 8 cysts
ml-1 wet sediment in Ovalada Island and Low Bay, respectively. The abundance of A. catenella cysts was probably associated with the
bloom of this species occurred during summer 2009. However, the spatial and temporal variability recorded could be associated with environmental variables, such as, the circulation pattern and the high environmental heterogeneity in southern Chilean. Grant: MR07I1007
FONDEF - CONICYT.
32
THE UPTAKE AND GROWTH DYNAMICS OF TOXIC
AND NON TOXIC RIBOTYPES OF THE HARMFU
DINOFLAGELLATE ALEXANDRIUM TAMARENSE
Lisa Eckford-Soper1, Keith Davidson1, Eileen Bresnan2, Grigorios Moschonas1
1
2
SAMS, Scottish Marine Institute, Oban, Argyll, PA37 1QA, UK. E-mail: [email protected]
Marine Scotland Marine Laboratory, P.O. Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK.
E-mail: [email protected]
One of the most prominent harmful dinoflagellates is Alexandrium tamarense, that produces bio-toxins associated with paralytic shellfish
poisoning (PSP). A. tamarense can be highly toxic requiring the presence of relatively low cell densities (<1000 cells l-1) to result in
shellfish fishery closures. Historically Scottish waters were though to be dominated by the toxic group I ‘North American’ Strain but in
recent years there has been a shift towards the non-toxic group III ‘Western European’ Strain with both strains potentially co-occurring
at the same location. A set of batch culture, time course growth and nutrient uptake experiments were carried out using both toxic and
non-toxic strains of A. tamarense. Cells were cultured in low phosphate media (5μM) so cells experienced the phosphate limitation that
is thought to promote toxicity. Differences in growth rates were noted between toxic and non-toxic strains. Phosphate concentrations
quickly declined as cultures became phosphate limited. POP concentrations increased over time up until the stationary phase, with POP
per cell declining over time. These early results appear to suggest that there is a difference in growth dynamics between toxic and nontoxic strains. The uptake experiments followed the Michaelis-Menten expression with a hyperbola describing the relationship between
the concentration and the uptake of the nutrient. The toxic strain took up nutrients faster at the lowest concentrations. Results therefore
indicate that there is a difference in nutrient uptake and the competitive dynamics between different strains of A. tamarense strains at
low and high cell concentrations which may influence their environmental niche.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 33
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
A COMPARISON OF INTEGRATED AND DISCRETE DEPTH
SAMPLING FOR MONITORING DINOPHYSIS
Laura Escalera1, Yolanda Pazos2, M. Dolores Doval2, Beatriz Reguera1
Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Aptdo 1552, 36200 Vigo, Spain.
E-mail: [email protected]
2
Instituto Tecnolóxico para o Control do Medio Mariño, Conselleria de Pesca e Asuntos Marítimos, Peirao de Vilaxoán s/n,
36600 Vilagarcía de Arousa, Pontevedra, Spain
1
Dinophysis acuminata and D. acuta are the main producers of lipophilic shellfish toxins in the Galician Rías (NW Spain), the group of toxins
that cause more shellfish harvesting closures in a region of intensive mussel cultivation (up to 3x105 t y-1). They often exhibit very patchy distributions; sampling with conventional methods may miss their presence, in high concentrations, restricted to thin layers in the water column.
For all these reasons, since 1986, integrated hose samplers have been recommended by an ICES group of experts as a reliable sampling
method in HAB monitoring programmes. In 2006, a calibration experiment was carried out by performing weekly high vertical resolution
sampling with bottles (from 0 to 10 m, at 1m intervals), and comparing Dinophysis concentration estimates with those obtained with a hose
sampler during the whole “Dinophysis season” (June to November). The two species of Dinophysis presented conspicuous subsurface
maxima, both under stratified and mixed conditions. Concentration estimates from dividable hose samples were comparable to those from
bottle samples at some depths, but their maxima were 1-2 orders of magnitude lower than those from bottle samples under thin-layering
conditions. These results add new information on the heterogeneous vertical distribution of Dinophysis spp. throughout their growing season
and should be considered when: i) Comparisons are made between Dinophysis cell maxima from different locations; ii) Cell maxima estimates
are needed for species-specific models, and iii) “Trigger levels” of Dinophysis cell concentrations are used to guide precautionary harvesting
closures and initiation/intensification of toxin tests.
33
TEN YEARS OF HARMFUL ALGAL BLOOM TAXA
IN THREE ESTUARINE RIVERS OF NORTH CAROLINA, USA
E. E. Fensin1, B. W. Touchette2
1
2
NC Division of Water Quality, Raleigh, NC 27607, USA. E-mail: [email protected]
Center for Environmental Studies, Elon University, Elon, NC 27244, USA
A decadal study considering the interactions between phytoplankton populations and photic-zone physiochemical parameters (e.g.,
nutrients, chlorophyll a, pH, dissolved O2, and temperature) was conducted on three coastal rivers (Neuse, Pamlico, and New Rivers).
Over the ten year period, often dynamic inter-annual fluctuations in cell densities were observed in three harmful algal taxa - the
cyanobacteria Cylindrospermopsis raciborskii and the dinoflagellates Karlodinium micrum and Prorocentrum minimum. C. raciborskii
tended to have a strong temperature response where it frequently bloomed during mid- to late summer (July to September) between
2005 and 2009. In contrast, P. mimimum favored the cooler temperatures of winter and early spring (December to April), especially
in 2006 and 2007. K. micrum, however, was prevalent throughout most of the study during all seasons. Nevertheless, peak abundances of K. micrum did occur between June 2001 and February 2002, March through October in 2006, and January 2008 through
June 2009. To better understand these temporal variations in phytoplankton populations, we employed multivariate analyses to
compare cell densities with ambient physiochemical parameters. The interactions between these environmental components and the
three harmful algal taxa will be discussed.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 34
SPATIAL DISTRIBUTION AND ABUNDANCE OF DINOFLAGELLATE CYSTS IN SURFACE SEDIMENTS OF COASTAL
WATERS OF NORTHERN AND CENTRAL PHILIPPINES
Elsa F. Furio1, Kazumi Matsuoka2, Valeriano M. Borja1, Iris U. Baula3, Dusit Srivilai4, Kian Weng Chan5,
Rhodora V. Azanzan3, Yasuwo Fukuyo6,7
1
NFRDI, Quezon City 1101, Philippines. E-mail: [email protected]
2
Graduate School of Science and Technology, Nagasaki University, Nagasaki, Japan
3
MSI, University of the Philippines, Philippines
4
Rajamangala University of Technology Tawan-Ok, Chanthaburi, Thailand
5
Kolej Universiti Sains san Teknologi, Malaysia
6
Institute for East China Sea Research, Nagasaki University, Japan
7
The University of Tokyo, Japan
Sediment cores were collected from northern Philippines in Masinloc and Subic Bays, Zambales and; from central Philippines in Western Samar Bays
from November 21-25, 2005. The surface sediment cores were collected from each station of all sampling areas to analyze their dinoflagellate cyst
content and characterize their species assemblage, distribution and abundance with emphasis on toxic dinoflagellate species Pyrodinium bahamense var.
compressum. The species assemblag es, diversities, abundances and distributions of dinoflagellate cysts are broader and vary among different features
of surface sediments of different areas. Masinloc Bay and Western Samar Bays have shown relatively high total concentrations of various dinoflagellate
cysts though their distribution patterns were apparently sporadic. While scarce distribution of few species of dinoflagellate cysts were consistently observed
in Subic Bay. Hence, it appears that the surface sediments in Subic Bay may harbor less cyst density. A total of 33 dinoflagellate cyst taxa were identified,
which belonged to orders Gonyaulacales, Gymnodiniales and Peridiniales including Calciodinoloides and Diplopsaloides. Cysts assemblages in Masinloc
Bay, and Western Samar Bays were characterized with marginally high proportion of heterotrophic species and low proportion of autotrophic species. The
cysts of toxic species P. bahamense were widely distributed and most dominant in all stations in Masinloc and Western Samar Bays while they were rarely
found in nominal counts in Subic Bay. In general, the distribution and abundance of dinoflagellate cysts particularly the toxic species P.bahamense var.
compressum are influenced by the hydrographic and geographic conditions, and the sediment features of the sampling areas.
34
MICROBIAL TROPHIC INTERACTIONS DURING
KARLODINIUM BLOOM IN CANNING RIVER,
WESTERN AUSTRALIA
Alice I. Gedaria, Matthew R. Hipsey
School of Earth and Environment, University of Western Australia, M004, 35 Stirling Highway, Crawley WA 6009, Australia. Email: [email protected]
The dynamics of algal bloom formation are the result of complex interactions between various physical, biogeochemical and microbial
processes. An understanding of how these interactions play a role in bloom formation is essential, yet the role of biological interactions
in this context are often less well understood. This study reports on the mixotrophic dinoflagellate Karlodinium veneficium in the SwanCanning estuary, Western Australia, which has been observed to form nuisance blooms regularly over the past few years. During a
recent bloom in the Canning River in May 2009, intensive field sampling was conducted to gain a better understanding of the patterns
of distribution and interactions of various microbial populations, including pico-, nano- and net- phytoplankton, bacteria and virus like
particles, in relation to Karlodinium bloom dynamics. The microbial populations were analysed using a combination of flow cytometric
and microscopic techniques. The distributions were compared with the physico-chemical properties in the water column (salinity, temperature, dissolved oxygen and nutrients), which together were monitored over the initiation, persistence and decline of the bloom at
different stations along the estuary. The results demonstrate the microbial changes that occurred during bloom conditions in response
to the environmental changes, and the subsequent decline of the bloom due to physical processes brought about by increased flow
following a rain event. The approach has highlighted the spatio-temporal variability and interactions between different microbial groups
relative to the Karlodinium bloom.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
NATURAL AND ATHROPOGENIC PRESSURES TRIGGERING
HABS AND THEIR EFFECTS AT THE ROMANIAN BLACK
SEA COAST: PAST AND PERSPECTIVES
Marian-Traian Gomoiu1, Laura Boicenco2
1
National Institute for Marine Geology and Geoecology “GeoEcoMar”, Constanta Branch, 304 Mamaia Bvd., 900581,
Constanta, Romania. E-mail: [email protected] or [email protected]
2
National Institute for Marine Research and Development “Grigore Antipa”, 300 Mamaia Bvd., 900581 Constanta, Romania.
E-mail: [email protected] or [email protected]
Romanian coastal waters of the Black Sea were known in the 1960s as a productive marine system thanks to the fertilizing role of the
Danube River, where the process of naturally-induced eutrophication is the result of the permanent discharge of river-borne nutrients.
Starting with the ’70s until ’90s this productive system underwent negative changes originating in anthropogenic causes. The processes
in this period are compared to a chain-reaction triggered by the increase in nutrient quantities and other substances discharged into the
sea. The reaction goes through a series of phenomena linked by the excessive development of phytoplankton and ending in benthos
obliteration. Presenting the chronicle of algal blooms and their effects, the authors mention that 49 monospecific blooms were produced
between 1981-1990; after 1990 the number of blooms decreased. Consequently, the mass species number with high abundance diminished and the quantities of phytoplankton decreased. After 2000 a recovery trend was observed, diatoms prevailing. The tendency
towards the improvement of general ecological conditions remains, however, fragile as nutrients still have higher concentrations than
before the eutrophication period. There are uncertainties and the coastal ecosystem is still vulnerable.
35
ROLE OF CYST GERMINATION IN THE BLOOM INITIATION
OF HARMFUL ALGAL SPECIES IN KOREAN WATERS
Myung-Soo Han
Department of Life Science, Hanyang University, Seoul 133-791, Korea. E-mail: [email protected]
Harmful algal species can be present in high numbers for a short period in polar, boreal and temperate latitudes, where seasonal
variations in environmental conditions are large. Nuisance algal blooms are particularly important due to their deleterious effect on fishing
industries, public health and drinking water. To clarify the role of cyst germination in the bloom initiation of the harmful algal species
isolation and germination experiments of cyst were carried out biweekly or monthly. The bimodal nature of Alexandrium tamarense
blooms, a large bloom in spring and a much smaller bloom in fall, was observed. Germination maxima (80 to 90%) were observed
during the period of decreasing water temperature in December and November, while little or no germination occurred in summer. The
seasonal germination exhibited an opposing pattern with temperature. In case of Scrippsiella trochoidea, vegetative cell numbers peaked
in June to July, when the surface water temperature increased to over 18oC. Mass encystments were detected in the water column in
July and August, when the vegetative population flourished. Active germination was observed during the period of decreasing water
temperature in September and October, after a 2-month dormancy period following the mass encystments. The vegetative population
of Anabaena flos-aquae flourished from August to November, with Anabaena verrucosa and Aphnizomenon flos-aquae observed as
the dominant species. The akinetes in the sediment has a high density in the winter. The most active akinete germination occurred from
the end of March to early April. The cyst abundance of Peridinium bipes in sediment samples fluctuated from 4 to 427 cells g-1, and did
not show season-dependent variation. Active germination success revealed in November. However, bloom period of Peridinium bipes
was found differently in each season and locality in lake. From these results, types of germination pattern showed that the temporal discrepancy between the peak of germination success and bloom of vegetative population was found. It means that active germination will
be not a direct trigger to form the blooms of HABs.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 36
POTENTIALLY TOXIC DINOPHYSIS IN SOUTHERN BRAZIL
Lumi Haraguchi, Clarisse Odebrecht
Rio Grande Federal University, Institute of Oceanography, Rio Grande, 96201-900, Brazil. E-mail: [email protected]
We present data on the occurrence and distribution of potentially toxic Dinophysis and their influencing factors in southern Brazil. We
identified six potentially toxic species, D. acuminata, D. caudata, D. fortii, D. mitra, D. rotundata and D. tripos in coastal and oceanic
waters off Santa Marta Grande Cape (SMGC; 28°40’S) and Albardão-Chuí (AC; 34°S). Water samples (surface and depths; Niskin
bottles) and plankton vertical hauls (plankton net 20 μm porosity) were collected on board R. V. Atlântico Sul at 66 oceanographic
stations in austral Winter 2005 (August/September) and Summer 2007 (February). D. caudata was the most abundant in both periods
off AC, and in winter off SMGC, while the wide distribution of D. acuminata in summer near the coast off SMGC (max. 808 cells L-1)
coincided with the influence of South Atlantic Central Water. D. fortii was observed off SMGC in winter and in both locations in summer
always under the influence of Subtropical Shelf Water and Tropical Water, with higher temperature and salinity. In summer samples,
the lower frequency and concentration of D. acuminata and the absence of D. tripos off AC, may be related with lower salinity due to
the influence of La Plata River and Patos Lagoon outflow.
36
SEASONAL AND SPATIAL VARIABILITY OF
PHYTOPLANKTON COMMUNITY STRUCTURE IN XIAMEN
WESTERN BAY BASED ON PIGMENT ANALYSIS
Xue-Jia He, Xing-Yue Peng
Research Center for Harmful Algae and Aquatic Environment, College of Life Science and Technology, Jinan University, China.
E-mail: [email protected]
The spatial and seasonal variations of phytoplankton community in Xiamen Western Bay were investigated based on HPLC analyzing photosynthetic pigments in algal cells and CHEMTAX processing pigment data on Cruise I (June), II (October), and III (November) in 2001.
Chla concentration reached 18.9 μg/L on Cruise I and declined to 0.28-2.17 and 0.578-1.87 μg/L on Cuise II and III, respectively,
consistent to the observation of algal bloom of diatom during Cruise I. Among accessory pigments, fucoxanthin was consistently the most
abundant, and it ranged from 0.172 to 8.46 μg/L during Cruise I, while the maximum concentrations were 0.787 or 0.540 μg /L on Cruise
II or III. 19’-hexfucoxanthin and alloxanthin, as the other two abundant pigments during Cruise I and II, varied in the range of 0.0448-2.86
μg/ L or 0.163-2.46 μg/L during Cruise I, and average level of both were well above 0.1 μg/L during Cruise II. Besides, 19’-butanoyloxyfucoxanthin or peridinin seemed also important on Cruise II or III. CHEMTAX processing revealed the dominance of diatom for all cruises in
phytoplankton assemblage with its contribution of 14.6-52.5% to total Chla. Cryptophytes and Haptophytes were the other two important
groups, with average contribution of 16.9-31.4% and 12.1%-26.3%, respectively. On the contrary, Dinoflagellates, Chlorophytes and the
group of others were minor groups, but the former was present sporadically with high contribution. The distinctive seasonal variation in phytoplankton community was shown, and the seasonal changes in Chla or biomass of most of groups may be coupled with the changes in
temperature and dissolved oxygen, suggested by the strong positive correlations between biomass and temperature or concentration of
dissolved oxygen. However, spatial trend was generally lacked probably because of rather homogeneity in the surface water of the bay.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 37
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
MODELLING THE LIFE CYCLE OF HARMFUL ALGAE
Inga Hense1, Alexandra Kroll1, Anke Kremp2
1
IHF, KlimaCampus, University of Hamburg, Grosse Elbstrasse 133, 22767 Hamburg, Germany.
E-mail: [email protected]
2
Finnish Environment Institute (SYKE), Marine Research Centre, P.O. Box 140, 00251 Helsinki, Finland
Observations indicate that the life cycle of harmful algae plays an important role in their bloom dynamics. Hence, models used to study
HABs need to include life cycle processes like en- and excystment. We present a general framework of how this can be realized in
Eulerian ecosystem models. Growing and resting stages, with their specific nutrient requirements and motility patterns can be mathematically represented by subcompartments. In a first application, we show results of a life cycle model of harmful, nitrogen-fixing cyanobacteria. Coupled to an ecosystem model and a vertically resolving water column model, the bloom characteristics are very well reproduced.
In a second application with realistic settings, the life cycle of dinoflagellates is studied. Overall, the results indicate that much can be
gained if the life cycle is taken into account: bloom duration and beginning of the bloom are better represented with the advanced
compared to traditional model approaches.
37
ENVIRONMENTAL CONDITIONS AND DYNAMICS OF A RED
TIDE IN THE CENTRAL MEXICAN PACIFIC (MARCH, 2009)
David U. Hernández-Becerril1, Sofía A. Barón-Campis2, Gerardo Ceballos-Corona3,
Alejandro Morales-Blake4, Mónica Rodríguez-Palacio5
1
Instituto de Ciencias del Mar y Limnología Universidad Nacional Autónoma de México,
Ciudad Universitaria, Coyoacán, México D.F. 04510, México. E-mail: [email protected]
2
Instituto Nacional de Pesca, SAGARPA, México D.F. 03310, México
3
Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México
4
Facultad de Ciencias Marinas, Universidad de Colima, Manzanillo, Colima, México
5
Dpto. de Hidrobiología, Universidad Autónoma Metropolitana-Iztapalapa, México D.F., México
We carried out an oceanographic cruise (“MareaR I”) in the central Mexican Pacific coasts, 25 March to 4 April, 2009, to investigate the environmental conditions associated to red tides and the structure of the phytoplankton communities. Cast and moored CTD data, and discrete
bottle samples for nutrients, chlorophyll α, phytoplankton and pigments, were obtained. Additionally ocean color satellite imagery in near realtime was used during the cruise, to find high concentrations of chlorophyll zones and the presence of red tides. Oceanographic conditions
showed no evidence for upwellings; the thermoclines were well defined at subsurface (9-20 m) from Cabo Corrientes to Manzanillo, and
coincided with maxima concentrations of chlorophyll α. Phytoplankton cell densities were higher at surface and subsurface, coinciding with depth
of the main thermocline and the maxima chlorophyll layers. Leptocylindrus danicus and Leptocylindrus minimus were the most abundant species
in Cabo Corrientes, with maxima of 153,992 cells l-1. A red tide outside Manzanillo bays was detected, with chlorophyll α values from 3-8.5 mg
l-1 (fluorometer) and 21.8-29.2 mg l-1 (discrete samples) at surface, decreasing immediately at higher depths. Low concentrations (1.3-3.5 μM)
of NO3+ NO2 were found at surface (0-5 m), and much higher (9.3-15.2 μM) at 10 and 20 m. Dominances in cells were due to the ciliate Myrionecta rubra, with a maximum density of 278,600 cells l-1, and to the potentially toxic dinoflagellate Gymnodinium catenatum (maximum density
of 229,650 cells l-1), followed by Akashiwo sanguinea (6,600 cells l-1), total phytoplankton densities were 384,866 cells l-1.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 38
THE SIGNIFICANCE OF SI:N:P RATIO TO HARMFUL
ALGAL BLOOM IN TOLO HARBOUR, HONG KONG
Kin-Chung Ho
School of Science & Technology, The Open University of Hong Kong. E-mail: [email protected]
The water quality and harmful algal bloom (HAB) data of Tolo Harbour, Hong Kong for the past 30 years were reviewed. Recognizing
the significance of N:P ratio to the outbreak of HAB there, it was found that the level of silicate, which is crucial to the growth of most
diatoms, also played an important role. Statistical analysis of water quality data showed that the growth of the diatom species favored for
Si:N (atomic) ratio 16. The variations in Si:N:P ratio were closely associated with the ecological succession in phytoplankton community
in marine and estuarine environments. Particularly, the prominence of diatoms would depress the growth of dinoflagellates and other
micro-algae. On the other hand, the dinoflagellate-related HAB was often triggered by the suppression of diatoms in response to the
change of climatic conditions and nutrients supply. Relevant conclusions were supported by experimental results of algal assay: bottle
tests with the use of 8 species of diatoms and dinoflagellates as studied subjects.
38
ANNUAL CYCLE OF PSEUDO-NITZSCHIA SPECIES
IN THE OUTER OSLOFJORD, NORWAY
Vladyslava Hostyeva1, Wenche Eikrem1,2, Bente Edvardsen1
1
2
Department of Biology, University of Oslo, Blindernveien 21, 0316 Oslo, Norway. E-mail: [email protected].
Norwegian Institute for Water Research, Gaustad Alléen 31, 0349 Oslo, Norway
Monitoring of microalgae, including toxic species like Pseudo-nitzschia, is based on light microscopy; nevertheless precise identification
of Pseudo-nitzschia at the species level requires verification by electron microscopy and/or molecular biological tools. Earlier investigations
from Norwegian waters have shown a considerable variation in the species composition of the genus Pseudo-nitzschia, both geographically
and seasonally. The genus contains more than 30 species and many of them are found in Norwegian waters (e.g. P. delicatissima, P.
fraudulenta, P. granii, P. heimii, P. pungens, P. multiseries, P. calliantha, P. seriata and P. americana). Several species of Pseudonitzschia have been shown to be toxic, including P. seriata, P. callianta, P. delicatissima, P. multiseries and P. fraudulenta, which all may
occur in Norwegian waters. Vertical net tows (0-20 m), quantitative water samples and hydrographical data have been collected monthly
over one year, from June 2009 to June 2010, in the outer Oslofjord, southern Norway. The succession of Pseudo-nitzschia species
have been recorded from acid cleaned samples viewed in the scanning electron microscope and transmission electron microscope. The
abundance of Pseudo-nitzschia species has been determined and related to hydrographical conditions. The ability to produce domoic
acid varies between species, thus a precise identification and knowledge about their geographical and seasonal occurrence at the species
level is important to early warning systems serving the aquaculture industry.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 39
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
GROWTH AND TOXIN PRODUCTION OF AZADINIUM
SPINOSUM IN BATCH AND CONTINUOUS CULTURE
Thierry Jauffrais1, Veronique Sechet1, Christine Herrenknecht2, Urban Tillman3, Zouher Amzil1, Philipp Hess1
1
IFREMER, Centre de Nantes, Departement Environnement Microbiologie Phycotoxine, Rue de l'Ile d'Yeu BP 21105,
44311 Nantes Cedex 3, France. E-mail: [email protected]
2
Nantes Atlantique Universites, EA 2160 Mer-Molecules-Sante, UFR de Sciences Pharmaceutiques et Biologiques,
1 Rue Gaston Veil BP 53508, 44035 Nantes Cedex 1, France
3
Alfred Wegener Institute, Am Handelshafen 12, D-27570, Bremerhaven, Germany
Azaspiracids are lipophilic marine biotoxins causing gastrointestinal symptoms similar to DSP toxins. Since 1995, azaspiracids have been encountered in Europe, Africa and more recently in South America and Japan. The biological primary producer remained undiscovered during
many years and has now been identified as Azadinium spinosum. After transfer from the Alfred Wegner Institute, A. spinosum underwent a
period of adaptation of several weeks. The organism was grown using K and L1 modified medium, at 18°C with a PFD of 150 μmol.m-2.s-1 and
a photoperiod of 16L/8D, in batch and continuous culture. Batch cultures were carried out using 10 liter flasks, while continuous cultures were
produced in 2.5 L lab scale and 100 L chemostats. Cells were recovered using centrifugation or filtration. Different extraction solvents and procedures as well as evaporation modes were evaluated for yield. Quantitation was carried out using LC-MS-MS. A. spinosum had a maximum
growth rate of 0.57d-1 with K modified medium, and reached maximum cell concentration of 300000 cells.mL-1. Toxins were mostly intracellular,
with 5 to 10% toxin in the culture medium. Analogues detected included AZA1, -2 and the methyl esters of AZA1 and -2, AZA1 being the predominant toxin. Toxin content varied over a large range, from 10 to 300 fg.cell-1, according to culture conditions. Cell volume could be estimated
via particle size or pellet weight to range from 380 to 1000 μm3. Azaspiracid content per biomass in A. spinosum appeared thus to be of similar
height of order as for other dinoflagellates, e.g. Dinophysis.
39
CHANGES IN THE OCCURRENCE OF DIFFERENT TOXIC
ALGAE OVER THE LAST DECADES IN WATERS ALONG
THE SWEDISH WEST COAST
Marie Johansen, Johan Kronsell
SMHI, Sven Kallfelts gata 15, 42671, Vastra Frolunda, Sweden. E-mail: [email protected]
Farming of mussels along the Swedish west coast has been going on since the beginning of the 1970s. One drawback for the industry
has been that toxic algae started to appear in the 1980s making the mussels occasionally toxic and subsequently hampering the business
economically. The monitoring of the occurrence of toxic algae has however been a valuable tool to foresee closure of farms preventing
that toxic mussels are harvested. The dataset on the occurrence of toxic algae along the coast is now quite extensive. The long time
series enables the analyses of trends in the occurrence of different toxic algae. Several changes in the occurrence of different toxic algae
have been found and are hear presented. The occurrence of the potent diarrheic shellfish toxin producing Dinophysis acuta has for
example diminished during the last years. The recent identified azaspiracid producing species, Azadinium spinosum, has on the other
hand started to appear on the Swedish west coast in recent years. The use of time series analyses is hear presented as a possible tool
for assisting mussel farmers in there work.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 40
RELATING THE PHYTOPLANKTON COMMUNITY
STRUCTURE TO ENVIRONMENTAL PARAMETERS
ON THE WEST FLORIDA CONTINENTAL SHELF
DURING BLOOM AND NON-BLOOM YEARS
Gary Kirkpatrick1, Terence Evens2, Anamari Boyes1, Valeriy Palukok1, Emily Hall1, Ari Nissanka1, Kellie Dixon1
1
2
Mote Marine Laboratory, 1600 Ken Thompson Parkway, 34236, Sarasota, Florida, USA. E-mail: [email protected]
U.S. Department of Agriculture, Agricultural Research Service, 2001 S. Rock Rd, 34945, Ft Pierce, Florida, USA
Over the past 13 years the phytoplankton taxonomic community structure has been examined along the central west coast of Florida.
During that time span four large-area blooms of Karenia brevis were sampled as well as four lesser blooms and four minor blooms.
Detailed water quality measurements have been collected along with the community structure data for the past ten years. These data
were collected during a varied of water sampling cruises with varying sampling periods from as short as two weeks to as long as one
year. One regular transect line has been maintained for all 13 years with a sampling period of one month. Taxonomic community composition was determined from HPLC pigment analyses and chemotaxonmic analyses using ChemTax™ software.
Subsets of these datasets have been analyzed in the past with general findings that the variability in community structure was correlated
to salinity and nitrogen concentration. This paper incorporates a new approach to determining major nutrient availability by accounting for
ion solution balance. Incorporating these nutrient availability findings the more complete dataset is subjected to multi-parameter, multi-dimensional space analyses using Primer-E™ software to reveal significant correlations between the community structure and environmental
variables during and between K. brevis blooms.
40
THE EFFECT OF NITROGEN, PHOSPHORUS
AND THEIR RATIOS ON HARMFUL DINOFLAGELLATE
PROROCENTRUM MINIMUM IN THE CHESAPEAKE BAY,
OBSERVATIONS AND MODELING
Ji Li, Patricia M. Glibert
University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge,
Maryland 21613, USA. E-mail: [email protected]
A decadal data set of algae bloom events, with biweekly sampling in the Chesapeake Bay area, was analyzed to characterize the influence of
ambient nutrient factors and related variables to the Prorocentrum minimum blooms in the Chesapeake Bay. N, P and their ratios were important factors correlated with the bloom development. To better understand the progression of bloom development, nutrient limitation models were developed to simulate the P. minimum blooms tributaries of Chesapeake Bay which have frequent blooms (Potomac, Patuxent and
Choptank rivers). First, both Monod’s equation and Droop’s equation were tested to simulate the growth of P. minimum. The Monod’s equation uses the parameters maximum growth rate (μmax) and half saturation constant (Ks); Droop’s equation uses the parameters minimum
cell quota (Qmin), N specific uptake rates (V) and assimilation rates (A). To get the parameters for the growth simulation, laboratory experiments were conducted at different nutrient condition (from P limitation to N limitation) and light intensities in both batch culture and turbidostat, a continuous culture system. 15N tracer techniques were used to measure the V and A. Then, the growth model, which was developed
based on laboratory experiment results, was coupled with a box model in the high frequent P. minimum bloom tributaries. Field data, including ambient nutrients and river flow rates, were processed in the box model to simulate the P. minimum bloom in these areas.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 41
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
TOXIC DIATOMS AND DINOFLAGELLATES IN THE
SOUTHERN GULF OF MEXICO: DATA BASE (1979-2008)
Sergio Licea1, Maria Eugenia Zamudio-Resendis1, Ruth Luna1, Felipe Vazquez1, Casimiro Ramirez-Camarena2
1
Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.,
Apartado postal 70-305, Mexico. E-mail: [email protected]
2
Instituto Nacional de Pesca, SAGARPA, Pitagoras 1320, Col. Santa Cruz Atoyac, 03310 Mexico D.F., Mexico
For the purpose of this study our attention is focused on 30 toxic species (Alexandrium catenella, Amphidinium carterae, A. operculatum,
Chaetonella sp., Dinophysis acuta, D. caudata, D. diegensis, D. fortii, D. mitra, D. rapa, D. rotundata, D. tripos, Gonyaulax spinifera,
Gymnodinium catenatum, Karenia brevis, K. mikimotoi, K. papilionacea, Lingulodinium polyedrum, Pseudo-nitzschia multiseries, P. pseudodelicatissima, P. pungens, P. subfraudulenta, Prorocentrum lima, P. mexicanum, P. minimum, Protoperidinium crassipes, P. curtipes,
P. depressum, P. oblongum, Pyrodinium bahamense var. bahamense) of a data-base of 25,801 entries obtained during 15 oceanographic
surveys. The selected material consisted of water and net samples collected at about 508 sites between June 1979 and May 2008. In
addition, material was obtained from 4 recent cruises in November-December 2005; October 2007; May 2008 and May 2006 at 10 m
depth. Species identifications were made mostly on water mounts; Transmission electron microscope was useful for Pseudo-nitzschia
identifications. The most frequent species were: Pseudo-nitzschia pungens, P. pseudodelicatissima and P. subfraudulenta; and the dinoflagellates: Dinophysis caudata, Karenia brevis and Prorocentrum minimum. The species of the genus Karenia were found commonly
along the coast between Terminos and Carmen-Machona. Lagoons. K. mikimotoi had 9,258 cells l-1 near oil platforms in May 2008.
Blooms of Karenia brevis were observed along the southern coast on October 1996 and 1997; December 1999, November and December 2001; and from May to July 2004. Particularly on November 2005 there was 5’477,843 cells l-1 near the river plume of the
river-system Grijalva-Usumacinta. Data on distribution and relative abundance are discussed.
41
SHORT-TERM VARIATION OF PSEUDO-NITZSCHIA
SPP. AND DOMOIC ACID IN SE KOREAN COASTAL WATERS
Weol-Ae Lim1, Tae-Gyu Park1, Young-Sik Lee1, Ka-Jeong Lee2, Jong-Gyu Park3, Eung-Kwon Kim3
1
National Fisheries Research & Development Institute, 650-943, Tongyeong, Korea. E-mail: [email protected]
National Fisheries Research & Development Institute, 619-705, Busan, Korea
3
Department of Oceanography, Kunsan National University, Korea
2
Recent climatic warming trends in Korea appear to be affecting phytoplankton community structure, including increased abundances of
Pseudo-nitzschia spp., especially in summer-autumn following periods of heavy rainfall. Abundance and composition of Pseudo-nitzschia
spp. and concentrations of DA were determined during 2008 and 2010 around Tongyeong, Korea, an area noted for shellfish aquaculture.
We also analyzed phytoplankton composition, physical and chemical water properties, and meteorological data during the same sampling
periods. Fourteen species were observed, including P. americana, P. brasiliana, P. caciantha, P. calliantha, P. cuspidata, P. delicatissima,
P. frandulanta, P. micropora, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens, P. subfraudulenta and P. subpacifica.
Of 14 species, 8 Pseudo-nitzschia species were cultured including P. americana, P. brasiliana, P. caciantha, P. delicatissima, P. frandulanta, P. multiseries, P. multistriata and P. pungens. Three of the strains, including P. multiseries, P. multistriata and P. calliantha,
were confirmed to produce domoic acid. For rapid detection of toxic species in the field, species specific real-time PCR probes were also
developed against the internal transcribed spacer 2 ribosomal DNA region. The assay specificity was confirmed by testing against related
species and was used to investigate the abundance of toxic species.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 42
ENVIRONMENTAL CONDITIONS DURING
THE PRESENCE OF RAPHIDOPHYCEAE IN BAHIA
DE LA PAZ, GULF OF CALIFORNIA
D. J. López-Cortés1, C. J. Band-Schmidt2, I. Gárate-Lizárraga2, J. J. Bustillos-Guzmán1,
F. E. Hernández-Sandoval1, E. J. Nuñez-Vázquez1
1
Centro de Investigaciones Biológicas del Noroeste, Apdo. Postal 128, La Paz, B.C.S. 23000, Mexico.
E-mail: [email protected]
2
Departamento de Plancton y Ecología Marina, Centro Interdisciplinario de Ciencias Marinas-Instituto Politécnico Nacional,
Apdo. Postal 592, La Paz, B.C.S. 23000, Mexico
Blooms of Raphidophyceae have a serious impact in aquaculture and public health. Some species are producers of brevetoxins, hemolysins, hemaglutinins, and reactive oxygen substance. From March to May 2009, three species of Raphidophyceae were recorded in
Bahía de La Paz. Here, we describe the physical and chemical conditions associated with these flagellates. Samples were collected
weekly at three stations to measure temperature, dissolved oxygen, inorganic nutrients, and photosynthetic pigments. Live and fixed
phytoplankton samples were obtained for counting and identifying the species. Water column temperature indicated a quasi-homogeneous
water column during March and April (20.8 to 21.5 °C in March and 21.2 to 22.0 °C in April). In May, stratification of water column was
established with a temperature difference of 2°C between the surface and 20 m. Nitrates varied from 0.47 to 8.0 μM; orthophosphates
from 0.2 to 1.5 μM; chlorophyll α at the surface was 2.5 mg m-3 and 1.6 to 5.0 mg m-3 at the subsurface in March. Raphidophyceae
species were Chattonella marina var. marina, C. marina var. ovata, and Fibrocapsa japonica. Variety ovata was the most abundant 37,000
cells L-1 at the surface. Variety marina and F. japonica were scarce and only observed in net samples. The toxic dinoflagellate Gymnodinium
catenatum was also found. We conclude that high concentrations of C. marina var. ovata were linked to the reduction of the mixing
processes in the bay.
42
THE PHYSIOLOGICAL RESPONSE OF THE MARINE
DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM
TO OCEAN ACIDIFICATION
Christos M. Loukas, Debora M. Iglesias-Rodriguez
National Oceanography Centre, University of Southampton, Waterfront Campus, European Way, Southampton SO14 3ZH, UK.
E-mail: [email protected]
Ocean acidification is at the centre of debate in the oceanographic community and has been identified as a potential threat to ecosystems. Within the last 220 years, atmospheric CO2 concentration has increased from 280 p.p.m.V (parts per million volume) to approximately current levels of 390 p.p.m.V, and it is predicted to reach as much as 700 p.p.m.V by 2100, with seawater pH falling
from 8.2 to 7.8. Extensive work has been undertaken regarding the response of various phytoplankton groups to ocean acidification,
including coccolithophores, diatoms, and marine cyanobacteria. The response by dinoflagellates, a group of highly complex eukaryotes
comprising autotrophs, heterotrophs, toxin producing and bioluminescent representatives, has received little attention. We conducted
experiments to test the short term physiological response of the harmful marine dinoflagellate Lingulodinium polyedrum to elevated
CO2 concentrations. We bubbled cultures for 10 days with air, containing 385, 750, 1200, and 1500 p.p.m.V CO2, and we measured
parameters including cell size, cell growth rate, bioluminescence, CO2:O2 ratio, pH, alkalinity, and nutrient concentrations. We discuss
the results in an ecological context and provide the first results on the effect of ocean acidification on the physiology and bioluminescence
of a free-living marine dinoflagellate.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 43
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
RELATED FACTORS DURING MODERATE BLOOMS
OF PYRODINIUM BAHAMENSE VAR. BAHAMENSE
IN TWO COASTAL LAGOONS FROM THE SOUTHERN
GULF OF CALIFORNIA
Lourdes Morquecho1, Rosalba Alonso-Rodríguez2, José A. Arreola-Lizárraga1, Amada Reyes-Salinas1
1
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Sta. Rita,
La Paz, B.C.S. 23096, México. E-mail: [email protected]
2
Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Unidad Académica Mazatlán,
Apdo. Postal 811, Mazatlán, Sinaloa 82040, México
The vegetative stage of Pyrodinium bahamense var. bahamense occurs in the southern Gulf of California. Toxin production in some strains
has increased interest in its bloom dynamics. We examined environmental and biological factors of summer blooms, focusing on the ecosystem
at the lagoon at the southwestern corner of Isla San José (24.52°N, 110.33°W) on the western side of the gulf from July to October 2008,
and to a limited extent, in Laguna Yavaros (26.41°N, 109.00°W) on the eastern side of the gulf. During September in both areas, moderate
blooms occurred inside shallow coastal lagoons bordered by mangroves during the warmest month and peak of the rainy season. At San
José, the vegetative stage peaked at 63×103 cells L–1. At Yavaros, the maximum was 110×103 cells L–1. During October, production of
cysts in the most protected part of the San José lagoon peaked at 8.5×103 g–1 (dry weight). Spearman correlation coefficient showed that
P. bahamense var. bahamense from San José exhibited a moderate positive correlation with salinity, seawater temperature, silicates, ammonia,
and a moderate negative correlation with dissolved oxygen (P<0.05). For the southern Gulf of California this dinoflagellate occurred in shallow
and protected areas where the environmental window that favors development of blooms is short-term (August and September) and influenced
by seawater temperature (25-32°C), salinity (31-36), and relatively high nutrients concentrations of ammonia and phosphates. Cysts appear
to be more prevalent as blooms decline with declining temperature at the end of the summer.
43
POTENTIAL HARMFUL DINOFLAGELLATES (DINOPHYCEAE)
FROM THE COAST OF PAKISTAN
Sonia Munir1, Zaib-un-nisa Burhan1, P. J. A. Siddiqui1, Steve L. Morton2
1
2
Centre of Excellence in Marine Biology, University of Karachi, Karachi 75270, Pakistan. E-mail: [email protected]
National Oceanic and Atmospheric Administrations, Marine Biotoxins Program, 219 Fort Johnson Road, Charleston,
SC 29412, USA
Harmful algal blooms (HABs) and red tide events frequently appear in northern Arabian Sea and along the coast of Pakistan, however
data on the causative species are generally lacking from Pakistani waters. Plankton survey (May 2002-July 2003) was initiated in two
separate sites along the Manora Channel, Karachi, Pakistan. Utilizing both light and electron microscopy about 97 species were identified,
most were first records for the Northern Indian Ocean. Ecologically, dinoflagellates constitute 3-95% of the total phytoplankton population
with highest cell concentrations occurring during periods of temperature (30oC) and salinity (35psu). Potentially harmful, bloom forming
nontoxic dinoflagllate genera include Prorocentrum (10 species), Ceratium (20 species), Scrippsiella (2 species), Gyrodinium (2 species),
Katodinium (1 species), Gymnodinium (2 species), Akashiwo (1 species), Protoperdinium (13 species). A number of known toxin producers
were also observed including the ichthyotoxic dinoflagellate Cochlodinium (1 species), the okadaic acid producing dinoflagellates Dinophysis
(8 species), the saxitoxin producing dinoflagllates Alexandrium (5 species), yesotoxin producing Gonyaulax (5 species) and Gymnodinium
(1 species). A number of typically benthic dinoflagellates were also observed including the ciguatoxin producing Gambierdiscus (3 species)
and okadaic acid producing Prorocentrum (2 species) and the palytoxin producer Ostreopsis (1 species). Most observations are first
reports of specific potential HAB species along the coast of Pakistan and highlight the need for further ecological studies and monitoring
of shellfish and finfish for potential toxicity.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 44
FIRST REPORT ON ATHECATE DINOFLAGELLATE
COCHLODINIUM POLYKRIKOIDE (GYMNODINALES)
FROM PAKISTAN
Sonia Munir1, Zaib-un-nisa Burhan1, Tahira Naz1, Steve L. Morton2, P. J. A. Siddiqui1
1
2
Centre of Excellence in Marine Biology, University of Karachi, Karachi 75270, Pakistan. E-mail: [email protected]
National Oceanic and Atmospheric Administrations, 219 Fort Johnson Road, Charleston, SC 29412, USA
Cochlodinium polykrikoides is a red tide producing dinoflagellate which is common in both the Atlantic and Pacific Oceans and also
reported from Indian Ocean. The chain forming C. polykrikoides is an athecate dinoflagellate observed in concentration between
>20 to 926 cells/L during the period May 2002 - July 2003 from coastal waters in the northern Arabian Sea bordering Karachi,
Pakistan. The identification was based on morphological characters of cell ornamentation, displacement of sulcus and cingulum,
position of nucleus and spiral shaped gridle view, shape of chloroplasts and size of the cells. The taxonomical characters were
studied using epifluorescence and scanning electron microscopy. The occurrence of Cochlodinium on Pakistani coast is of great
concern from fisheries view point, since this species has been implicated in considerable economic losses worldwide.
44
THE ECOLOGY AND TAXONOMY OF THE FAMILY
PROROCENTROID (DINOPHYCEAE) FROM
THE NORTHERN ARABIAN SEA
Sonia Munir1, P. J. A. Siddiqui1, Zaib-un-nisa Burhan1, Tahira Naz1, Steve L. Morton2
1
2
Centre of Excellence in Marine Biology, University of Karachi, Karachi 75270, Pakistan. E-mail: [email protected]
National Oceanic and Atmospheric Administrations, 219 Fort Johnson Road, Charleston, SC 29412, USA
Numerous species of the Prorocentrales have been investigated for first time from coastal waters of Karachi, Pakistan. A total of
13 species were identified including both planktonic and benthic/epiphytic species using both light microscopy and scanning electron
microscopy. Of these species, P. gracile, P. sigmoides, P. arcuatum, P. scutellum, P. donghaiense, P. emarginatum, P. lima, P.
faustiae, P. balticum, P. minimum and Mesoporos perfortus constitute new records for the Northern Indian Ocean. Only three
species, P. micans, P. compressum and P. dentatum, were previously reported species from Sindh coast of Pakistan. This is first
description of the family Prorocentrales from the northern Indian Ocean based on SEM examination differentiating morphtypes
based on the cell shapes, size, ornamentation of thecal plates, architecture of apical platelets, apical pore area, marginal pores, intercalary band. Most of these characters are difficult to assess under the light microscope. The dominant species P. micans, P.
gracile and P. cf. minimum observed throughout the year along the near shore area’s St. A and mouth of Manora Channel St. B,
Karachi, Pakistan. In contrast to the occurrence of benthic/epiphytic species observed only at the St. A with low abundance level
known to produce phycotoxins and diarrheic shellfish poisoning may be harm to fisheries in Pakistan.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 45
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
POTENTIALLY TOXIC SPECIES OF THE GENUS
DINOPHYSIS IN OUALIDIA LAGOON (MOROCCO)
Elmaslouhia Nafil1, Btissam Ennaffah2, Mohammed Chair1, Abdelghani Chafik2
1
2
Faculty of Science, University Chouaib Doukkali, 24000 Eljadida, Morocco. E-mail: [email protected]
Laboratoire des Efflorescences Nuisibles, Institut National de Recherche Halieutique, (I.N.R.H.), 2 Rue de Tiznit,
Casablanca, Morocco
Analysis of samples of seawater collected from the Oualidia lagoon (Morocco) in 2009 has been able to reveal the presence of seven
species of genus Dinophysis, which six are known for their ability to produce toxin diarrheic (DSP Diarrheic Shellfish Poison): Dinophysis
acuminata, D. acuta, D. caudata, D. fortii, D. rotandata, D. sacculus, and D. hastata whose toxicity has not been confirmed not yet and
has been described for the first time at the Moroccan Atlantic coast. These species were observed at four sampling sites (pass, parc7,
parc1 and former park Marost). The presence of these species presents a potential danger to the oyster (Crassostrea gigas), which is
cultivated in aquaculture farms at Oualidia lagoon, especially the species of the genus Dinophysis were carried toxic even at low concentrations. In this study, the cell density of these species did not exceed 2X102 cells/liter and their presence was noted during the period
between March and July, then, no contamination of shellfish stock by diarrheic toxin (DSP) associated with the presence of Dinophysis
has been declared, while during the year 2007 has registered a concentration of the order of 6700 cells/liter.
45
PHYLOGENY AND DISTRIBUTION OF GAMBIERDISCUS
SPP. IN COASTAL AREAS OF JAPAN
Tomohiro Nishimura1, Shinya Sato2, Keita Uehara1, Hiroshi Sakanari1, Haruo Yamaguchi1, Masao Adachi1
1
Laboratory of Aquatic Environmental Science, Faculty of Agriculture, Kochi University, Otsu-200, Monobe, Nankoku,
783-8502 Kochi, Japan. E-mail: [email protected]
2
Royal Botanic Garden Edinburgh, 20 Inverleith Row, EH3 5LR Edinburgh, UK
The primary causative agent of Ciguatera fish poisoning (CFP) is the epiphytic and benthic dinoflagellate genus Gambierdiscus. A previous
phylogeographic study of Japanese Gambierdiscus spp. revealed the existence of two distinct ribotypes: the type 1 from the subtropical
area and the type 2 from the temperate area. In the present study, the phylogeny and the distribution of Gambierdiscus spp. were further
investigated based on the samples mainly collected from southern part of Japan where CFP have increasingly been reported. Approximately
80 clones of Gambierdiscus spp. were established from 12 sites during the years 2007-2010. In addition to the types 1 and 2, which
were recovered as robust monophylies in the 18S rDNA phylogeny, we also found two more types -the types 3 and 4. The distributions
of the types 1, 2 and 3 were widespread from temperate to subtropical areas; on the other hand, that of the type 4 was restricted to the
subtropical area. The type 1 was sister to a clade comprised G. belizeanus/G. pacificus/G. toxicus. The type 2 diverged in a clade comprised G. caribaeus/G. carpenteri, although the topology of the clade was not fully resolved. The type 3 was nested within the clade of
G. australes. The type 4 was closely related to G. yasumotoi/G. ruetzleri. Given the molecular phylogeny together with the SEM observations, it is reasonable to assume that the type 1 and 2 are hitherto undescribed species.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 46
EFFECTS OF WATER TEMPERATURE, SALINITY
AND IRRADIANCE ON THE GROWTH OF COCHLODINIUM
POLYKRIKOIDES MARGALEF FROM SOUTH SEA OF KOREA
Seok Jin Oh1, Yang Ho Yoon2, Chang-Hoon Kim1
1
2
Pukyong National University, Daeyeon-dong, Nam-gu, Busan 608-737, Korea. E-mail: [email protected]
Chonnam National University, Dundeok-dong, Yeosu 550-749, Korea
The effects of temperature, salinity and irradiance on the growth of the harmful red tide dinoflagellate Cochlodinium polykrikoides were
examined in the laboratory. Their growth were tested for conditions of temperature and salinity combinations; 15, 20, 25, 30oC and 15,
20, 25, 30, 35 psu at a constant irradiance of 180 μmol/m2/s. At temperature of 15oC, growth was not observed at salinities of 15 and
20 psu. Moderate growth rates of approximately more than 0.30/day were obtained at 25oC in the salinities 25-35 psu. These values
are closely slimier to observed for this species in situ. The maximum growth rate of 0.35/day was obtained at 25oC and 30 psu. In light
experiments, cell growth of C. polykrikoides was carried out at 20oC, 30 psu and different light photon flux densities (PFD) of 10, 25,
50, 70, 100, 150, 250 and 350 μmol/m2/s. C. polykrikoides did not grow at 10 μmol/m2/s. Cell growth was observed at irradiance
values of 25 μmol/m2/s and above. The irradiance-growth curve was described as Î=0.30(I-15.27)/(I+27.22), (r=0.99). This suggests
a compensation PFD of 15.27 μmol/m2/s and maximum growth rate of 0.30/day. In conclusion, C. polykrikoides prefers high salinity,
temperature and irradiance in summer of Korea. These results provide important information for understanding the mechanism of C.
polykrikoides blooms and developing the technology to predict blooms of this organism in the field.
46
MICROBIAL GANGS ARE TAKING OVER
THE WATER COLUMN OF A RECONSTRUCTED LAKE
Antreas Oikonomou1, Matina Katsiapi2, Panagiotis Berillis1, Maria Moustaka-Gouni2, Konstantinos Ar. Kormas1
1
Department of Ichthyology & Aquatic Environment, School of Agricultural Sciences, University of Thessaly,
384 46 Nea Ionia, Greece. E-mail: [email protected]
2
Department of Botany, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
Lake Karla, central Greece, used to cover ca. 180 Km2 but it was dried up in the beginning of the 1960s to expand agricultural land. In
the 1990s it was decided to refill with water an area of ca. 38 Km2 of the old lake. After building a peripheral dam, the lake’s actual filling
started in 2009 by inflowing water from the River Pinios. Here we present the first results of the lake’s nano- and microeukaryotic communities of the water column from spring and early summer 2010. We used light microscopy in fresh and fixed samples (with Lugol and
formaldehyde) along with scanning electron microscopy (SEM) observation and construction of 18S rRNA and cyanobacterial 16S rRNA
gene clone libraries. In spring, the community was dominated by the species Prymnesium parvum, Pfiesteria piscicida, Cyclotella cf.
meneghiniana, Euglena spp., Nitzschia acicularis, Pseudoanabaena limnetica and Planktothrix sp. while in early summer an almost
cyanobacterial dominance of Anabaenopsis cf. elenkinii and Pseudoanabaena limnetica was observed. Species richness was high with
more than 55 morphological species of algae and cyanobacteria whereas 40 nano- and microeukaryote phylotypes were revealed. The
most dominant (>50%) phylotypes were affiliated to Cyclotella meneghiniana, Scenedesmus communis, Paulsenella vonstoschii, Ebria
tripartite, Protaspis sp. and Prymnesium parvum. Each size-fraction had several singleton phylotypes indicating a non-specialised community, which belonged to chlorophytes, fungi, dinoflagellates and euglenoids, with several of them having no cultivated close relatives
and some of them being related to parasitic or toxic organisms.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 47
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
TEMPERATURE AND NUTRIENT DEPLETION EFFECTS
ON THE DEVELOPMENT AND VIABILITY OF PELLICLE
CYSTS OF PYRODINIUM BAHAMENSE VAR. COMPRESSUM
Deo Florence L. Onda, Rhodora V. Azanza, Arturo O. Lluisma
The Marine Science Institute, Velasquez St., University of the Philippines, Diliman, 1101 Quezon City, Philippines.
E-mail: [email protected]
Dinoflagellates have been observed to form pellicle cysts in culture and in their natural environment but little are known about the environmental conditions that induce their formation. We investigated the role of temperature- and nutrient-mediated stress as factors that
induce pellicle cyst formation in batch cultures of Pyrodinium bahamense var. compressum. On the last day of the observation (day 10),
cultures maintained at 13°C and 30°C were completely dominated by pellicle cysts regardless of nutrient condition, suggesting that temperature is a strong inducer of temporary encystment. The total number of cells however decreased in cultures maintained at 30°C
resulting from the lysis of both vegetative cells and cysts. Cysts that formed at the low temperature (13°C) treatments remained viable
even after 28 days. On the other hand, low nitrogen and low total nutrient conditions had relatively minor though still observable effect on
encystment. At normal culture temperature (24°C), a major fraction of the cell population in the low nitrogen and low total nutrient treatments remained vegetative although encystment in the latter treatment was observed to occur at a relatively greater degree. Effects of
the treatments on the pellicle morphology, structure, size and shape, and stages of development that have not been reported previously
are also described.
47
AN UNUSUAL ASSEMBLAGE OF GYMNODINIUM
CATENATUM AND DINOPHYSIS IN THE SPANISH
GALICIAN RÍAS IN 2009
Yolanda Pazos1, Fabiola Arévalo1, Jorge Correa1, Eric Desmond Barton2
Instituto Tecnolóxico para o Control do Medio Mariño de Galicia, 36611 Peirao de Vilaxoán, Vilagarcía de Arousa, Spain.
E-mail: [email protected]
2
Instituto de Investigaciones Marinas, CSIC, 36208, Eduardo Cabello 6, Vigo, Spain
1
After having been detected as short chains and in low concentrations during the summer, at the start of October plankton samples
indicated the presence of long chains of Gymnodinium catenatum in the Rías, where intensive raft cultivation of mussels takes place. INTECMAR immediately carried out a precautionary closure that was effective on confirming the accumulation of PSP toxins in the molluscs.
The oceanographic conditions at the time were of downwelling of surface water of oceanic origin. We expected a long period of closures,
as in earlier years, when G. catenatum appeared with Dinophysis acuta and there was an accumulation of lipophilic toxins in the molluscs.
However, in autumn 2009, D. tripos and not D. acuta flourished, reaching cellular concentrations of 4200 cells L-1, but no lipophilic
toxins were detected in the molluscs. The greatest concentration of toxins detected was of 11400 μg SXT/Kg in mussels and the maximum concentration of G. catenatum was 6720 cells L-1. After 9 November, G. catenatum was no longer detected in the plankton
samples and at the end of that month all the production zones were re-opened. D. tripos had not appeared in water samples of the Rías
for several years before this outbreak. The unusual substitution of D. tripos for D. acuta in this downwelling situation resulted in the red
tides being much less harmful than otherwise. The reason for this replacement of species within the same genus Dinophysis is unknown,
but the implications for the management of the episode are important.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 48
TOXIN CONTENT OF DIFFERENT LIFE STAGES
OF ALEXANDRIUM FUNDYENSE
Agneta Persson1, Barry C. Smith2, Jennifer H. Alix2, Christina Senft3, Gary H. Wikfors2
1
Department of Marine Ecology, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden.
E-mail: [email protected]
2
National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center,
Milford Laboratory, Milford, Connecticut 06460, USA
3
Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340, USA
Different life stages of two compatible clones of the PSP-producing dinoflagellate Alexandrium fundyense (BF-2 and 38-3) were separated
using a combination of techniques; culturing and sampling techniques were used to separate vegetative cells and gametes, and sorting
flow cytometry was used to separate zygotes. PSP toxin profiles were significantly different between life stages; the two gonyautoxins
GTX 1 and 2 were present in vegetative cells and in senescent cells, but absent from gametes and zygotes. Toxin profile changes were
shown to occur very quickly in both strains. Temporary cysts that were produced from exponentially-growing, vegetative cells (by shaking
four minutes on vortex, and rinsing with seawater onto a 20-μm screen) lost GTX 1 and 2 completely. Rapid toxin epimerization is one
possible explanation (GTX1 to GTX4 and GTX2 to GTX3) for the observations, although the biological advantage of this remains unclear.
Another possible explanation could be that, during the mating phase of a bloom when cells are assembled in a layer, toxins are released
into the surrounding water as a “defensive chemical cloud” protecting gametes from grazing and competition.
48
INFLUENCE OF THE ENVIRONMENTAL CONDITIONS
ON OSTREOPSIS OVATA GROWTH AND TOXICITY
Rossella Pistocchi1, Laura Pezzolesi1, Franca Guerrini1, Silvana Vanucci2, Patrizia Ciminiello3,
Carmela Dell’Aversano3, Emma Dello Iacovo3, Luciana Tartaglione3, Ernesto Fattorusso3
1
Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Università di Bologna, Via S’Alberto 163, 48123 Ravenna, Italy.
E-mail: [email protected]
2
Dipartimento di Biologia Animale ed Ecologia Marina, Università di Messina, Salita Sperone 31, 98166 S. Agata, Messina, Italy
3
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
Since the end of the ’90s blooms of the benthic dinoflagellates Ostreopsis spp. are spreading in many tropical and temperate regions
worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. O. ovata has been recently found to produce
six palytoxin-like compounds, a class of highly potent toxins. Since O. ovata cells are easily re-suspended in the water column, the important
role of hydrodynamism in bloom development and decline has been highlighted; moreover, higher cell concentrations of Ostreopsis spp.
are usually recorded during warm climate conditions, characterized by high temperature and salinity and low hydrodinamism. Therefore,
the environmental conditions appear to be one of the main factors determining the proliferation on these species. A few studies have been
performed on the effects of environmental conditions on the growth and toxin production of benthic dinoflagellates. In this study we investigated the effects of temperature, salinity and nutrients on the growth and toxicity of an Adriatic (Ancona, Italy) O. ovata isolate. Some
comparisons were made also with strains isolated from different Italian coastal sites. All the toxins were quantified by high resolution LCMS analyses. Results showed that the growth rates of the Adriatic strain were higher when grown at 20°C than at 25 and 30°C, in accordance with data obtained from natural field blooms, at salinity values of 36 and 40 and in nutrient replete conditions. Toxicity resulted
to be influenced by environmental conditions and, in particular, a significant decrease was observed at both N- and P-depleted conditions.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 49
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
COCHLODINIUM GEMINATUM BLOOMS AT THE ESTUARY
OF PEARL RIVER, GUANGDONG, CHINA
Yuzao Qi, Linjian Ou, Yong Pang, Songhui Lu
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510632, China.
E-mail: [email protected]
In recent years, the blooms of Cochlodinium geminatum occur continuously at the estuary of the Pearl River, Guangdong, China, since
2006. This is the first record of this species bloom in China. The outbreak of these algal blooms often appears in the fall and winter in
the eutrophicated sea area. The bloom in 2006 turned up in October and November, moreover in 2009 there was the same C. geminatum
bloom arised also in this period. Both of these two blooms showed some identical characteristics. In 2006, the bloom emerged at the
temperature ranging from 23.4 to 27.5oC, and salinity from 15.49 to 17.92 psu respectively. It reached the highest density of 1.6 x 106
cells l-1. During the bloom occurrence, the ambient DIN and PO4-P concentrations were between 22.2-88.9 and 0.2-1.4 μmol l-1 respectively. High biomass appeared at the site where DIN and PO4-P concentrations were 40.3 and 0.6 respectively. The bloom of C.
geminatum in 2009 showed a similar tendency with that in 2006. The bloom covered about 300 km2 and lasted almost a month until
the late fall. The average sea water temperature was 24.89oC and the salinity was 21.42 psu. The ambient DIN and PO4-P concentrations
were between 2.64-132.36 and 0.3-17.7 μmol l-1 respectively. The results of the principal component analysis indicated that nutrients,
salinity and temperature were the most important factors for the outbreak of the C. geminatum bloom in the Pearl River Estuary in 2009.
The bloom in 2009 was terminated by temperature decrease and succeeded by another bloom, the Phaeocystis globosa bloom.
This work was supported by grant of the National Basic Research Programme (2010CB428702).
49
SEASONAL DIVERSITY OF PSEUDO-NITZSCHIA SP.
IN THE CENTRAL ZONE OF THE MEXICAN TROPICAL
PACIFIC, MANZANILLO, COLIMA
Sonia Quijano-Scheggia1, Maria Rivera-Vilarelle2, Esther Garcés3, Aramis Olivos-Ortiz1, Juan Gaviño-Rodriguez1,
Karen Zepeda-Borja2, Marcos Galicia Perez1, Manuel Patiño Barragan1
1
Centro Universitario de Investigaciones Oceanológicas, Universidad de Colima, Carr.
Manzanillo-Barra Navidad Km 19.5, 28860 Manzanillo, México. E-mail: [email protected]
2
Facultad Ciencias Marinas, Universidad de Colima, 28860 Manzanillo, México
3
Inst Ciencies Mar, Dept Biol Marina & Oceanog, CGIC E-08003, Barcelona, Spain
Seasonal diversity of potentially toxic species of the diatom genus Pseudo-nitzschia were studied (February 2009 - February 2010) at six
coastal and offshore stations in the central zone of the Mexican Tropical Pacific, Manzanillo, Colima. Identification of Pseudo-nitzschia at the
species level required the use of Scanning Electron Microscopy (SEM). For phytoplankton identification integrated water samples of the water
column were monthly collected but weekly when there was an algal bloom. Subsamples for total phytoplankton enumeration were examined
using inverted light microscopy (LM). Identification of the species among the genus Pseudo-nitzschia in the light microscopy was attempted on
the basis of the length and shape of the cells. When observations in the light microscope revealed a Pseudo-nitzschia spp. population exceeding
104 cells L-1, Lugol-fixed samples were subjected to SEM observation. Species of Pseudo-nitzschia were identified: P. australis, P. brasiliana,
P. delicatissima, P. fraudulenta, P. linea, P. multistriata, P. cf. subcurvata, P. pungens, P pseudodelicatissima and P. micropora. During the
studied period, the highest abundance of phytoplankton was observed in March forming a diatom dominated bloom. In the genus Pseudonitzschia, P. pseudodelicatissima was dominant in spring and P. australis was observed in summer and autumn. We observed a higher abundance
of diatoms in offshore stations than in costal stations. This study reports the first detection P. brasiliana, P. cf. subcurvata, P. linea and P.
micropora in the Mexican Tropical Pacific.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 50
BEQUALM PROFICIENCY TESTING SCHEME:
PHYTOPLANKTON IDENTIFICATION
AND ENUMERATION EXERCISE
Rafael Salas, Tara Chamberlain, Josephine Lyons, Paula Hynes, Joe Silke
Marine Institute, Rinville, Oranmore, Co. Galway, Ireland. E-mail: [email protected]
Biological effects measurements are increasingly being incorporated into national and international environmental monitoring programmes
to supplement chemical measurements. The Biological Effects Quality Assurance in Monitoring Programmes (BEQUALM) project, funded
by the European Union through the Standards, Measurements and Testing programme of the European Commission, was initiated in
1998. This was in direct response to the requirements of OSPAR to establish a European infrastructure for biological effects QA/QC, in
order that laboratories contributing to national and international marine monitoring programmes could attain defined quality standards.
The Marine Institute, Galway, Ireland, has conducted a Phytoplankton Enumeration and Identification ring trial, under the auspices of BEQUALM annually since 2005. The purpose of this exercises are to compare the performance of laboratories engaged in national
official/non-official phytoplankton monitoring programmes and other labs working in the area of phytoplankton analysis. The participation
in proficiency testing schemes is becoming an essential requirement for national phytoplankton monitoring laboratories in order to achieve
accreditation for their methodologies. The BEQUALM phytoplankton scheme is rapidly becoming a recognized scheme by phytoplankton
monitoring laboratories in Europe.
50
EFFECTS OF IRRADIANCE AND INORGANIC NUTRIENTS
ON THE GROWTH OF THE BLOOM-FORMING
DINOFLAGELLATE PROROCENTRUM BELIZEANUM
Asterio Sánchez-Mirón, Juan J. Gallardo-Rodríguez, Francisco García-Camacho, Emilio Molina-Grima
University of Almería, 04120 Almería, Spain. E-mail: [email protected]
The development of red tides is related with some complex natural and human-linked processes. Environmental factors such as water
temperature, salinity and light are believed to be the most important factors for the outbreaks of red tide organisms. Laboratory studies
constitute an unvaluable tool for clarifying the role of those environmental factors on the blooms of harmful algal species. Benthic dinoflagellates of the genus Prorocentrum are common in tropical and subtropical waters and several species produce toxins involved in
human toxic outbreaks of the diarrethic shellfish poisoning (DSP) syndrome. P. belizeanum that frequently appear attached to detritus
in mucilaginous matrix has been found integrating blooms in the coral-reef-mangrove ecosystems. The effects of temperature and
nutrients on the growth of Prorocentrum belizeanum were examined in the laboratory. The optimum irradiance for growth was >200
mmol photons m-2 s-1 and photoinhibition occurred at 500 photons mmol m-2 s-1 (the maximum irradiance in the experiments). Similar
values to optimum irradiance are found in nature in the subsurface layer with water depth of 3-15 m. The biomass production was also
found to be importantly conditioned by the nitrate and phosphate content. Acknowledgements: This research was supported by the
Ministerio de Ciencia e Innovación de España (CTQ2008-06754-C04-02/PPQ) and the Consejería de Innovación, Ciencia y Empresa
de Andalucia (P09-TEP-5375).
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 51
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
PHYLOGEOGRAPHY OF OSTREOPSIS REVEALED
A NOVEL CLADE FROM JAPANESE COAST
Shinya Sato1, Tomohiro Nishimura2, Keita Uehara2, Haruo Yamaguchi2, Kirsty Smith3, Lesley Rhodes3, Masao Adachi2
1
Royal Botanic Garden Edinburgh, 20 Inverleith Row, EH3 5LR Edinburgh, UK
Kochi University, Otsu-200, Monobe, Nankoku, 783-8502 Kochi, Japan. E-mail: [email protected]
3
Cawthron, Private Bag 2, Nelson, New Zealand
2
A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin (or its derivatives) which is among the most potent nonproteinaceous compounds reported so far. In the present study the phylogeography of Ostreopsis spp. was investigated based on the
samples mainly collected from southern part of Japan where putative palytoxin poisonings have increasingly been reported. We first obtained the sequences of the ITS and 5.8S rDNA for 20 clones. A phylogenetic analysis revealed that 6 of them formed a novel clade
sister to O. ovata, while the other clones fell into several clades being grouped with previously reported sequences. We then conducted
finer scale sampling to see the detailed distribution of Ostreopsis spp. using the D8/D10 region of the LSU rDNA. As a consequence
199 clones of Ostreopsis were sequenced, of which 109 belonged to the novel clade, 72 in O. ovata, 3 in O. siamensis, 3 in O. cf.
labens and 12 in O. cf. lenticularis. The clones belonging to the novel clade were most widely distributed, and often sympatrically found
with the O. ovata. So far we have detected no morphological difference between the two clades. Toxicity in the crude cell extracts was
also tested with mouse bioassay for a representative of each clade. The strongest toxicity was observed in the novel clade. The results
strongly suggested the urgent need for the establishment of the early detection/warning systems of the Ostreopsis spp. in Japanese
coast, particularly for the widely distributed toxic novel clade.
51
SPATIAL AND TEMPORAL DYNAMIC OF PSP IN NATURAL
BEDS OF VENUS ANTIQUA IN SOUTHERN CHILE
Miriam Seguel1, Carlos Molinet2,4, Patricio Díaz2, Manuel Díaz2, Edwin Niklitschek4, Daniel Varela3, Andrea Sfeir1,
Marcia Gangas1
1
Laboratorio Ceram, Universidad Austral de Chile, Puerto Montt, Chile. E-mail: mseguel@uach
Instituto de Acuicultura, Universidad Austral de Chile, Los Pinos s/n, Puerto Montt, Chile
3
Centro Imar, Universidad de Los Lagos, Camino Chinquihue km 6, Puerto Montt, Chile
4
Centro Trapananda, Universidad Austral de Chile, Portales 73, Coyhaique, Chile
2
Harmful algal blooms (HABs) have been a recurrent problem in southern Chile. Most concern has been focused on Alexandrium catenella,
which is associated with paralytic shellfish poison (PSP). Despite efforts made through monitoring programs, few of them have considered
explicitly spatial arrangement of PSP concentration trends in bivalves, particularly in small spatial scales, such as natural beds. We studied
the spatial and temporal dynamic of PSP concentration in two commercial beds of the clam Venus antiqua, which were affected by a
toxic bloom of A. catenella observed during summer 2009. We identified patches of V. antiqua within each bed, and monitored them for
a year, measuring the following variables: toxin concentration, individual density, cysts abundance, temperature, salinity, phytoplankton
and chlorophyll-a. Data were analyzed using a generalized linear models approach in order to identify those variables that had significant
effects upon PSP concentration. In order to obtain quantitative descriptors for accumulation and detoxification dynamics, we included the
selected variables in a simplified one-box model, defined by two parameters: the proportionality constant between A. catenella concentration
and PSP and the instantaneous PSP decay rate. Results showed significant variability on the spatial and temporal dynamic of PSP concentration, within and between beds, associated to the presence of A. catenella. It was also observed a decreasing rate of detoxification
as concentrations approached the permissible limit for human consumption. Grant: MR07I1007 FONDEF - CONICYT.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 52
BLOOM AND TOXIN DYNAMICS OF ALEXANDRIUM
OSTENFELDII IN A SHALLOW EMBAYMENT AT
THE SW COAST OF FINLAND, NORTHERN BALTIC SEA
Päivi Sillman1, Anke Kremp1, Johan Franzen2, Helene Franzen2, Katrin Erler3
1
Marine Research Centre, Finnish Environment Institute, P.O. Box 140, 00561 Helsinki, Finland.
E-mail: [email protected]
2
Bänö, 22710 Föglö, Finland
3
Department of Food Chemistry, Institute of Nutrition, University of Jena, Dornburger Strasse 25, D-07743 Jena, Germany
In the past years, some coastal areas in the central and northern Baltic Sea have experienced recurrent blooms of Alexandrium ostenfeldii
(Paulsen) Balech and Tangen. Although generally considered as a background species, in the Baltic Sea A. ostenfeldii can have remarkably
high cell concentrations, reaching even up to 106 cells L-1. During the summer of 2009, A. ostenfeldii cell concentrations, the phytoplankton
community, PSP toxins and environmental factors were monitored weekly at two stations in a sheltered embayment of the Föglö archipelago in Åland, northern Baltic Sea. A. ostenfeldii cells were present in the water column from late May to early September, but proliferated
to bloom concentrations (104-105 cells L-1) only at water temperatures > 20°C. Abundance peaks occurred at both sampling stations in
late July and early August, coinciding with inorganic nutrient pulses that promoted growth of a diverse phytoplankton community. This
study provides the first evidence of dinoflagellate blooms causing PSP toxicity in the northern Baltic Sea: PST dynamics correlated with
the progression of the A. ostenfeldii bloom with toxin peaks mirroring the species abundance peaks. The highest PSP toxin concentration
measured was 2,327 μg L-1. Toxin analysis detected GTX2, GTX3 and STX from both stations, with GTX3 being the dominant variant.
This is in accordance with the toxin profiles of the strains isolated earlier from the study site.
52
THE SIGNIFICANCE OF NUTRIENT AND METAL LIMITATION
ON THE GROWTH OF PSEUDO-NITZSCHIA DELICATISSIMA
Sofie Spatharis1, Eleni Palamafti1, Nicolas P. Dolapsakis2, Daniel B. Danielidis2
1
2
University of the Aegean, Department of Marine Sciences, Mytilini, Greece. E-mail: [email protected]
University of Athens, Faculty of Biology, Department of Ecology and Systematics, Athens, Greece
The potentially harmful diatom Pseudo-nitzschia delicatissima is one of the best studied Pseudonitzschia species both in the field and in
laboratory experiments due to its cosmopolitan presence and easiness of culture. However, little is yet known on the role of inorganic nutrients and trace metals on the growth of this species. The aim of the present study was to assess the growth of P. delicatissima under
different nutrient and metal concentrations in order to infer to potential factors affecting the occurrence and blooming of this species in
coastal ecosystems. The species was cultured in triplicates using f/2 medium altered for one of its constituents, as follows: medium f/2
with no alteration (no limitation), NO3/4 (nitrate limitation), PO4/4 (phosphate limitation), biotin/20 & thiamine/20 (vitamin limitation),
and metals+EDTA/10 (metal limitation). Results showed maximum growth of the species under the f/2 and vitamin limitation treatments
whereas the NO3 and PO4 limitations resulted to an identical and significant growth decrease expressed both as chl a and cell number.
No growth however was observed for the metal limitation treatment. In order to further assess the role of specific metals on growth, six
altered f/2 treatments were carried out: Zn/10, Co/10, Mo/10, Mn/10, Cu/10, Fe+EDTA/10. No growth was observed for the Mn
and Fe+EDTA treatments whereas the growth rate for Zn was much slower than the Co, Mo, and Cu treatments. The results of the
present study indicate that P. delicatissima growth is equally limited by NO3 and PO4 whereas Mn and Fe can have a major role on bloom
formation of this species in the field.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 53
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
DEVELOPMENT OF CHLOROPHYLL α ALGORITHMS
FROM AN OCEAN COLOUR SENSOR FOR OPTICALLY
COMPLEX WATERS AND APPLICATION DURING
AN UPWELLING CYCLE
Evangelos Spyrakos1, Luis González Vilas1, Jesús M. Torres Palenzuela1, Eric Desmond Barton2
1
Department of Applied Physics, Faculty of Marine Sciences, Universidad de Vigo, Lagoas-Marcosende, E-36200, Vigo, Spain.
E-mail: [email protected]
2
Instituto de Investigaciones Marinas, C/Eduardo Cabello, 6, E-36208, Vigo, Spain
This study takes advantage of the neural networks (NNs) technology and the characteristics of Medium Resolution Imaging Spectrometer
(MERIS) in order to deliver accurate, detailed chlorophyll α (chlα) maps of optically complex (Case 2) coastal waters. MERIS FR chlα concentrations and in-situ data were obtained in three Galician rias (NW Spain), an area of extensive mussel culture that experiences frequently
harmful algal events. Fifteen MERIS FR cloud-free images paired with in situ chlα data for the time period 2002-2008 were used for the
development and validation of the Multilayer Perceptron (MLP) Artificial NNs. Fuzzy c-means clustering techniques (FCM) were applied
to the satellite derived data to determine different spectral classes. All the NNs developed in this study showed high R2 and low root
mean square error (RMSE) values and detected accurately the peaks of chla in both training and validation sets. The algorithms were applied to six MERIS images delivered from the area at July 2008, in order to create chlα maps. The transport of high phytoplankton
biomass areas during the upwelling cycle was clearly captured in the images. Relatively high biomass “patches” were detected in detail
inside the rias. There was a significant variation in the timing and the extent of the chlα peak areas related to the winds and surface
currents. A local-based algorithm for the chlα retrieval from an ocean colour sensor with the characteristics of MERIS can be a great
support in quantitative monitoring of chlα and study of harmful algal events in Galician rias.
53
BLOOM DYNAMIC OF PSEUDO-NITZSCHIA SPECIES
(BACILLARIOPHYCEAE) IN TROPICAL ESTUARINE WATER
S. N.-P. Su1, H.-C. Lim1, S.-Z. Kamarudin1, Y. Kotaki2, C.-P. Leaw3, P.-T. Lim1
1
Department of Aquatic Science, Universiti Malaysia Sarawak, Sarawak, Malaysia.
E-mail: [email protected], [email protected]
2
School of Fisheries Science, Kitasato University, Japan
3
Institute of Biodiversity and Conservation, Universiti Malaysia Sarawak, Sarawak, Malaysia
Amnesic shellfish poisoning (ASP) is a type of seafood poisoning due to human consumption of shellfish mollusks contaminated with algal
toxin, domoic acid (DA) derived from the diatom Pseudo-nitzschia. However, blooms dynamic of this diatom in the tropic was poorly understood
due to lack of long term study on the organism. In this three-years study, sampling of Pseudo-nitzschia species was carried out at Santubong
and Samariang estuarine waters, Kuching, Sarawak. The estuaries were characterized by shallow water with semi-diurnal tidal cycle. Cell
abundance was determined by microscopic enumeration. Temperature, pH, salinity and macronutrients at the sampling sites were determined
at each sampling occasions. Two Pseudo-nitzschia species, i.e., P. pungens and P. brasiliana were identified under scanning and transmission
electron microscopy. Morphological characters of P. pungens and P. brasiliana observed in this study fit well with the original description of
the species. Highest cell density of Pseudo-nitzshia spp. was recorded at 8.0 X 103 cells L-1 in Santubong in April 2009 and P. brasiliana
cells were found highest in October 2007 (2 X 104 cells L-1). Increase in cell abundance was coincided with high salinity and low precipitation
rate. Water temperature and pH showed insignificant influence on the abundance of Pseudo-nitzschia spp. Toxicity analysis of clonal cultures
of the two species using HPLC however showed no detectable level of DA production. The result obtained in this study provided fundamental
understanding on bloom dynamic of potential harmful Pseudo-nitzschia species in the tropical estuarine waters.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 54
RED TIDE OF THE MIXOTROPHIC DINOFLAGELLATE
HETEROCAPSA TRIQUETRA IN A EUTROPHIC ESTUARY,
GOLDEN HORN (TURKEY)
Seyfettin Tas
Institute of Marine Sciences and Management, University of Istanbul, Muskule Sok, No: 1, 34134 Vefa-Istanbul, Turkey.
E-mail: [email protected]
Red tide of the dinoflagellate Heterocapsa triquetra (Ehrenberg) Stein was detected between January and April 2007 in the Golden Horn
Estuary (GHE). During the red tide the surface water discolored to orange-brownish. This event was evaluated together with concurrent
physical and chemical variables. The red tide started in mid-January and lasted to the late of April in the shallow uppermost of the GHE.
In April, the H. triquetra cell density exceeded 19,000 cells ml-1 and the chlorophyll a concentrations locally reached to 104 μg l-1 in upper
part where low inorganic nitrogen and phosphorus, and high TN:TP ratios were found. The surface water temperature was 13.4oC and
salinity was 16.6 psu in April. In addition, dissolved oxygen (DO) increased to 20.4 mg l-1 (super saturation) at this time. The trophic index
(TRIX) values obtained in April ranged between 6.01 and 8.17, indicating to a eutrophic environment. As a result, a mesohaline frontal
zone and weak hydrodynamic conditions in this eutrophic area was favorable for the H. triquetra growth.
54
MICROSATELLITE AND ITS PATTERN INHERITANCE
ACROSS SEXUAL REPRODUCTION EVENTS IN THE TOXIC
DIATOM PSEUDO-NITZSCHIA MULTISTRIATA
Sylvie Tesson, Christophe Legrand, Marina Montresor, Gabriele Procaccini, Wiebe Kooistra
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy. E-mail: [email protected]
Pseudo-nitzschia multistriata is a toxic diatom able to produce the neurotoxin domoic acid responsible for Amnesic Shellfish Poisoning.
This species is present in the Gulf of Naples (Mediterranean Sea) in summer and/or autumn. Strains sampled in the Gulf of Naples
exhibit ITS-type A, or markedly distinct ITS-type B or they possess both types (A/B). Results of microsatellite screening of these strains
revealed high population genetic diversity within blooms and evidence for the existence of two sympatric populations. Mating experiments
were conducted to assess the mating compatibility between individuals of the same population and between the two populations as
defined by microsatellite patterns. We also explored the mode of inheritance of ITS- and microsatellite markers in the F1 generation and
used the results to assess the presence of null alleles in microsatellite loci. Crosses conducted between strains of opposite mating types
(=gender) were successful irrespective of their ITS-types (A, B or A/B), their population assignment inferred from the microsatellites, or
the season or year in which they were sampled. Most of the F1-strains possessed microsatellite patterns expected according to Mendelian
inheritance rules. Patterns that deviated from these rules could be explained because of the existence of null-alleles, i.e. mutations or
chromosome non-separation during meiosis.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 55
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
STUDY OF THE GROWTH KINETICS OF P. DELICATISSIMA
ON NITRATE AND PHOSPHATE IN A BATCH CULTURE
USING A SIMULATION MODEL
George Tsirtsis, Ioannis Tsakalakis, Sofie Spatharis
University of the Aegean, Department of Marine Sciences, Mytilini, Greece. E-mail: [email protected]
Batch cultures of the potentially harmful diatom Pseudo-nitzschia delicatissima were carried out in the laboratory in triplicates using f/2
medium. The growth kinetics of the species on nitrate and phosphate were studied using a simulation model. The state variables of the
model were phytoplankton biomass (estimated from the cell number), nitrate and phosphate concentrations. The growth of the diatom
on nitrate and phosphate was limited by a Michaelis-Menten mechanism, and mortality due to senescence and other factors was also
taken into account. The model was calibrated with the experimental data and the goodness-of-fit was good. The half-saturation constant
of the species growth on nitrate was almost fourfold compared to that for phosphate, implying the higher requirements of Pseudonitzschia delicatissima for nitrate. However due to the higher concentration of nitrate than phosphate in the culturing medium, both of
them seem to limit the diatom growth throughout the experiment. The model was further used to test the effects of various combinations
of concentrations of nitrate and phosphate for the growth of Pseudo-nitzschia delicatissima. It was estimated by the model that the decrease of phosphate or nitrate or both does not significantly change the growth rate of the diatom, however results to the decrease of
the biomass developed in the culture, an outcome also confirmed experimentally. In conclusion, the combination of the growth of potentially
harmful algal species in the lab under controlled conditions and the application of simulation techniques for data processing can shed light
on the dynamics of harmful algal blooms and reveal the controlling factors or mechanisms.
55
SPATIO-TEMPORAL DISTRIBUTION OF DINOPHYSIS SPP.
IN RELATION TO ORGANIC MATTER AND OTHER
PARAMETERS IN THERMAIKOS GULF, GREECE
(EASTERN MEDITERRANEAN)
Ioanna Varkitzi1, Kalliopi Pagou1, Edna Graneli2, Ioannis Hatzianestis1, Evangelia Krasakopoulou1,
Alexandra Pavlidou1
1
Hellenic Centre for Marine Research, PO Box 712, Anavyssos, 19013, Athens, Greece. E-mail: [email protected]
2
Marine Ecology Department, Linnaeus University, 39182 Kalmar, Sweden
The most frequent HABs in Greece occur in Thermaikos Gulf, which holds important bivalve cultures and natural stocks at national and European level. Dinoflagellates of “Dinophysis acuminata complex” (D. cf. acuminata and D. cf. ovum), producing mostly the Diarrheic Shellfish
Poisoning (DSP) toxin okadaic acid, are mainly responsible. The aim of this study was to investigate Dinophysis populations in relation to the
availability of organic matter and other parameters in Thermaikos Gulf. A network of stations was sampled from November 2002 to March
2004 and the spatio-temporal distribution of Dinophysis spp., chlorophyll α, inorganic nitrogen and phosphorus nutrients, particulate organic
carbon and nitrogen were studied. D. cf. acuminata abundance ranged at high levels in general, with maximum values in a surface layer (2
to 5m depth). D. cf. acuminata was dispersed in the Gulf area but maximum abundances were found in the northern part, where eutrophication
is most severe. D. cf. acuminata reached maximum abundance and toxicity levels (10.7 X 103 cells L-1 and 63.06 pg OA cell-1) during or right
after the spring phytoplankton bloom, forming HABs in most of the Gulf area. The spatial and temporal distributions of D. cf. acuminata were
significantly correlated with particulate organic carbon and nitrogen (ANOVA, p<0.05, Pearson coefficient 0.56-0.66) but not with chlorophyll
α or inorganic nitrogen and phosphorus. Blooms of D. cf. acuminata in Thermaikos during or after the spring phytoplankton bloom appear to
be favored by high organic matter availability at that time, given the mixotrophic character of this dinoflagellate.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 56
INFLUENCE OF ENVIRONMENTAL VARIABLES
ON ALEXANDRIUM CATENELLA MOTILE STAGE
AND OTHER HARMFUL TAXA IN SOUTHERN CHILE
(43°- 55° S.L.) (JAN 2009 - DEC 2009)
Gastón Vidal1, Leonardo Guzmán1, Ximena Vivanco1, Victoria Arenas1, Luis Iriarte2,
Susana Mercado2, Cesar Alarcón3, Hernán Pacheco3
1
Instituto de Fomento Pesquero, Balmaceda 252, Puerto Montt, Chile. E-mail: [email protected]
Instituto de Fomento Pesquero, Camino Ensenada Baja s/n, Puerto Chacabuco, Chile
3
Instituto de Fomento Pesquero, Enrique Abello 0552, Punta Arenas, Chile
2
The monitoring of twenty seven biological, toxicological, chemical, hydrological and meteorological variables using an almost monthly sampling
frequency at 151 sites located in Southern Chile, was made during 2009. Data were sorted, converted into logarithm base 10 and analyzed
using principal components analysis (PCA) to identify the most important environmental variables related to A. catenella vegetative phase
and other harmful taxa, as well as, to PSP, DSP and ASP results. Information was analyzed according to previous defined geographic
areas. In the studied geographic areas, the total explained variance summarizes only between 38 - 54 % in the first two components of the
data variability. Meteorological variables as air temperature and cloudiness are strongly related to water temperature, salinity and transparency
and these in turn are associated with increases of phytoplankton species number and relative abundance of harmful species, especially A.
catenella, Dinophysis acuta, D. acuminata, Pseudo-nitzschia cf. australis and P. cf. pseudodelicatissima, in addition to increases in PSP
levels. The correlation between DSP and ASP results with other environmental variables are not so clear. Although PCA helps to describe
in a synthetic way, the structure and interrelationships of the original variables, allowing also to rank and to weigh the variables important to
explain A. catenella blooms, since the analyzed information does not explain an acceptable proportion of the global variance, is not yet
possible to build a mathematical model to anticipate the blooms occurrence and to obtain a better comprehension of these events.
56
QUANTITATIVE PHYTOPLANKTON ASSEMBLAGES
ASSOCIATED TO ALEXANDRIUM CATENELLA BLOOMS
IN SOUTHERN CHILE
Gastón Vidal1, Leonardo Guzmán1, Heraldo Contreras1, Victoria Arenas1, Susana Mercado2, Hernán Pacheco3
1
Instituto de Fomento Pesquero, 252 Balmaceda, Puerto Montt, Chile. E-mail: [email protected]
Instituto de Fomento Pesquero, Camino Ensenada Baja, Puerto Aysén, Chile
3
Instituto de Fomento Pesquero, 0552 Enrique Abello, Punta Arenas, Chile
2
Primary productivity in marine environments is a community property of complex modelling based on the regulation of multiple factors, among
them, nutrient concentration and light intensity, and also species - specific differences of phytoplankton referred to cellular structure, chemical
composition, trophic state and surface/volume ratio are basic features to establish different phytoplankton assemblages in coastal environments.
This contribution is oriented to define the phytoplankton assemblage structure associated to Alexandrium catenella blooms. Quantitative samples
were collected during 2009 from surface layer to 10 meters and between 10 to 20 meters depth, using an almost monthly sampling frequency
from 61 and 48 sampling sites located at the region of Aysén and region of Magellan, respectively. The structure and phytoplankton community
associations were analyzed using multivariate statistics (i.e. Cluster, MDS, Simper and ANOSIM). The results showed significant differences between sampling sites, although the geographical proximity of the sites favours the formation of homogeneous groups, in both studied areas.
During cluster analysis, sampling sites were analyzed considering and excluding A. catenella abundance. These analyses suggest that in places
with the greatest abundance of A. catenella, the phytoplankton structure assemblage were more dissimilar and significantly different from those
places with lower abundance of this microalga (P<0.001). In the region of Aysén, the dissimilarity was given mainly by species such as Eucampia
zodiacus, Skeletonema costatum, Thalassionema nitzschioides and Thalassiosira subtilis, among others. In the Magellan region, these dissimilarities
were given mainly by species such as Chaetoceros socialis, Chaetoceros didymus, Chaetoceros radicans and Guinardia delicatula, among others.
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 57
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
A THREE-YEAR TIME SERIES OF TOXIC OSTREOPSIS
BLOOMING IN A NW MEDITERRANEAN COASTAL SITE:
PRELIMINARY RESULTS
Magda Vila1, Pilar Riobó2, Isabel Bravo3, Mercedes Masó1, Antonella Penna4, Albert Reñé1, M. Montserrat Sala1,
Cecilia Battocchi4, Santiago Fraga3, Francisco Rodriguez3, José M. Franco2
1
Institut de Ciències del Mar (ICM-CSIC), Pg. Marítim de la Barceloneta 37-42, 08003 Barcelona, Spain.
E-mail: [email protected]
2
Instituto Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, 36208 Vigo, Spain
3
Instituto Español de Oceanografía (IEO), Subida a Radio Faro 50, 36390 Vigo, Spain
4
Biomolecular Science Department, University of Urbino, Viale Trieste 296, 61100 Pesaro, Italy
Epiphytic dinoflagellates of the genus Ostreopsis have been related to harmful episodes in many Mediterranean coastal areas since 1998:
respiratory difficulties, fever, and skin irritations affected people exposed to marine aerosols by recreational activities on the beach. Although
this genus is widespread in most of the Mediterranean coasts, harmful events have been recorded only in some localities and periods. One
of those sites has been studied for 3 years in a high frequency monitoring (EBITOX project) in order to elucidate the relationship between the
microalgal community and their toxicity. 1) Bloom dynamics: the epiphytic Ostreopsis bloom starts in late May-early July, and few weeks later
is detected in the water, where it reaches maximum concentrations of 104-105 cells·l-1 in July-August; maximum epiphytic concentrations
(above 106 cells·g-1 FW) are detected from mid July to mid November. Between December and April, Ostreopsis is not detected in the water,
but low epiphytic concentrations are detected sporadically. 2) Species composition: two Ostreopsis species have been identified to bloom together by a molecular PCR-based assay; Ostreopsis cf. siamensis and O. cf. ovata. 3) Toxicity: the bloom is toxic; palytoxin, analyzed by
haemolysis assay and HPLC-FLD is detected in epiphytic samples taken during bloom-period. 4) Resting stages: short-dormancy pellicle
and thin-walled cysts were observed in bloom-incubated samples, which germinated even after 6 months. Resting cysts have not been
detected during the sampling of macroalgae and sediment during no-bloom season.
57
TEMPERATURE EFFECTS ON GROWTH, COLONY
DEVELOPMENT AND CARBON PARTITIONING
IN THREE PHAEOCYSTIS SPECIES
Xiaodong Wang1, Kam W. Tang2, Yan Wang3, Walker O. Smith Jr.2
1
College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China.
E-mail: [email protected]
2
Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, USA
3
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510632, China
Phaeocystis is an ecologically important marine phytoplankton genus that is globally distributed. We examined the effects of temperature on
the 3 most common species: P. globosa, P. antarctica, and P. pouchetii, which grew at 16-32, 0-6, and 4-8°C, respectively. P. pouchetii
did not form colonies; P. globosa formed colonies at 16, 20, and 24°C, and P. antarctica colonies were observed at all temperatures. More
cells were partitioned into the colonial form at lower temperatures than at higher temperatures for P. globosa and P. antarctica. P. globosa
colony size decreased with temperature, whereas P. antarctica colony size showed no distinct response to temperature. Numbers of cells per
unit of colony surface area of P. globosa and P. antarctica were lowest at temperatures where highest growth rates and colonial abundances
were observed; more organic carbon was partitioned into solitary cell biomass at higher temperatures, whereas the carbon concentration of
colonies was not affected by temperature. Maximum quantum yield of P. antarctica and P. globosa exhibited subtle responses to temperature,
whereas that of P. pouchetii was relatively invariant within the growth temperature range. Future changes in sea surface temperature may
dramatically alter the ecology and biogeochemical cycles of systems dominated by Phaeocystis spp. and result in further degradation, via
oxygen depletion and altered food web structure.
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MESOZOOPLANKTON COMMUNITY SUCCESSION
AND HARMFUL ALGAL BLOOMS IN THE YANGTZE
RIVER ESTUARY AND ITS ADJACENT OFFSHORE AREA
Zhao-Li Xu, Dong Zhang
East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Science, No. 300 Jun Gong Road, 200090,
Shanghai, China. E-mail: [email protected]
It is puzzling why major harmful algal blooms (HABs) only occur in the spring in the Yangtze River estuary and its adjacent offshore area.
We examined the mesozooplankton collected in the spring (May, 2002), summer (August, 2002), autumn (November, 2002) and winter
(March, 2003), and those collected in the 8 surveys carried out from March to June of 2005 to test the hypothesis that mesozooplankton
control the initiation of HAB in the area. Our study suggests that copepod Calanus sinicus is the key grazer of the harmful algae in the
Yangtze River estuary and its adjacent offshore area. Abrupt temperature change during early and mid May resulted in sudden change
of mesozooplankton community from temperate-water species to warm-water species. The crash of temperate-water species, C. sinicus,
resulted in reduction of grazing pressure on phytoplankton and may initiate HABs.
58
VARIATION OF THE PHOSPHATASE ACTIVITIES IN FIELD
POPULATIONS OF HARMFUL ALGAE IN THE COASTAL
ENVIRONMENT, JAPAN
Haruo Yamaguchi, Hiroshi Arisaka, Takamichi Yoshimatsu, Naoki Otsuka, Masao Adachi
Laboratory of Aquatic Environmental Science, Faculty of Agriculture, Kochi University, Otsu-200, Monobe, Nankoku,
783-8502 Kochi, Japan. E-mail: [email protected]
Some species of cultured marine algae possessing the enzymes, alkaline phosphatase (APase) and phosphodiesterase (PDEase),
can utilize various compounds of dissolved organic phosphorus as phosphorus sources. In this study, we examined the variation of
the phosphatase activities in field populations of harmful algae in a coastal environment and discussed the roles of the phosphatases
in algal-bloom dynamics. Surface seawaters of Uranouchi Inlet, Western Japan were collected from July 2008 to Aug 2009. Nitrate
(N), phosphate (Pi), N+P and distilled water were individually added into flasks containing the seawaters. The flasks were incubated
at 25 C and 12hr:12hr LD cycle. APase and PDEase in algal cells and in the flasks and sampled seawaters were detected by using
the specific substrates for each enzyme. In natural seawaters, many algal-cells including harmful species such as Karenia mikimotoi
and Rhizosolenia spp. expressed APase activity during spring-autumn periods but not during winter. In laboratory incubation, the
enzyme activities detected by using a single-cell assay generally appeared to be induced strongly in the phosphate-limited waters (cα.
40% for total algal-cells) but not in nitrate-limited (0.08 ~ 13%). PDEase activities were sometimes increased as well as APase in
the phosphate-limited treated waters. No APase activities in Chattonella marina and a few species were not found in phosphatelimited conditions. These results showed that field populations of harmful algae in the coast of Japan expressed inducible phosphatases
in phosphate-limited environments.
O
1. ABSTRACT BOOK (12-61)_Layout 1 10/22/10 2:24 PM Page 59
POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
MASS DEPOSITION OF CHATTONELLA CYST BY
THE BLOOM IN THE YATSUSHIRO SEA, WESTERN JAPAN
Mineo Yamaguchi1, Setsuko Sakamoto1, Kiyonari Sakurada2, Hiromi Nishi3, Yoshio Tahara3
1
National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi, Hatsukaichi,
Hiroshima 739-0452, Japan. E-mail: [email protected]
2
Kumamoto Prefectural Fisheries Research Center, 2450-2 Oyano, Kamiamakusa, Kumamoto 869-3603, Japan
3
Kagoshima Prefectural Fisheries Technology and Development Center, 160-10 Iwamoto, Ibusuki,
Kagoshima 891-0315, Japan
The Yatsushiro Sea is one of the “hot spot” of Chattonella bloom in Japan, where recurrent blooms of the organism have occurred since
the early 1990s. In the summer of 2009, a massive bloom of Chattonella occurred over almost the entire area of the Sea and caused
serious fishery damages. In this study, we determined the number of the cyst before and after the bloom to examine the change in the
abundance and the distribution of the cyst by the bloom. Sediment samples were collected in May (before the bloom), September,
October and November (after the bloom) and the cysts were enumerated by the direct-count method using an epifluorescence microscope.
Before the bloom, mean abundance of the cysts was 15 cysts/cm3 and the distribution was limited in northern part of the Sea. On the
other hand, the cysts were found at all the stations examined and the mean density increased to 378 cysts/cm3 after the bloom. This indicated that the bloom had supplied a large number of newly-formed cysts onto the bottom sediment. To evaluate the impact of the cyst
deposition on the bloom dynamics, we extrapolated the inoculation of the vegetative cells into the water column through the germination.
The calculated cell density (203 cells/L) is high enough for the population to reach the warning cell level of 10 cells/ml within a week, if
the organism proliferates under the suitable conditions. Thus there is the possibility of continuing occurrence of Chattonella bloom and
careful monitoring is necessary in the area.
59
FURTHER EVIDENCE OF HAB SUCCESSION
IN CHANGJIANG ESTUARY AND ITS ADJACENT
SEA AREAS – SUMMARY OF A CAS INNOVATION
PROJECT CRUISE IN 2007
Tian Yan, Mingjiang Zhou
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China. E-mail: [email protected]
Studies on the ecology and oceanography of HABs in China (CEOHAB) have been carried out during 2002-2006 under the support of
the National Basic Research Priority Program (national “973 Program”). Via a multidisciplinary approach, this CEOHAB project have
achieved important results, including understanding of the mechanism for the large-scale occurrences and the impacts of the HABs
caused by Prorocentrum donghaiense in the East China Sea. Furthermore, it is also found that there is a clear succession shift of HAB
dominant species of large scale HABs, from diatom type to dinoflagellates type. In spring of 2007, a CAS Innovation Project cruise has
been carried out in the same area, the investigation showed further evidence of the succession of HABs in Changjiang estuary and its
adjacent sea areas. The results of biological, chemical and physical Oceanography process during the HAB succession in 2007 are
summarized in this report. And the roles of long-term variation pattern of nutrient concentration and composition in this region, as well as
the different nutrient-utilizing characteristics of dinoflagellates and diatoms in the HAB succession are discussed.
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CAPTURING THE SPATIALLY-EXPLICIT LIFE HISTORY
DYNAMICS OF PYRODINIUM BAHAMENSE VAR.
COMPRESSUM IN AN INDIVIDUAL-BASED MODEL
Aletta T. Yñiguez, Cristopher Olavario, Cesar L. Villanoy, Edward Bornilla, Joseph Dominic Palermo,
Irene Alabia, Ian Fernandez, Rhodora V. Azanza, Garry A. Benico, Fernando P. Siringan
Velasquez St., Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines.
E-mail: [email protected], [email protected]
Harmful algal blooms are a recurrent, expensive and at times fatal problem plaguing the Philippines. In particular, Sorsogon Bay in the
Bicol region has experienced prolonged shellfish bans due to blooms by the Paralytic Shellfish Poisoning causative species Pyrodinium
bahamense var. compressum (Pbc). A shellfish ban was in effect for more than three years in the bay and was lifted only March this
year. Results from field studies in the past year showed that Pbc blooms developed a few months after the onset of the rains, relatively
lower temperature, and stratification of the water column. In order to help explore and understand the population dynamics of Pbc in Sorsogon Bay in relation to environmental factors, an individual-based model has been developed. This model accounts for key life history
processes (reproduction, mortality, encystment, excystment) in a spatially-explicit setting. Vegetative cells and resting cysts are separately
represented. Movement is due to advection rates from a 3D hydrodynamic model, as well as sinking rates. Asexual reproduction (population
growth) occurs in response to light, temperature, and nutrient conditions where the cells are spatially situated. Encystment and excystment
processes are also spatially-explicit and based on known biological information and rates on this species. Grazing effects on blooms are
also being explored in the model. This model will be used to investigate the conditions leading to blooms and their declines, specifically
the role of transport, stratification, encystment, excystment, and grazing on Pbc blooms in Sorsogon Bay.
60
THE EFFECT OF SOME ENVIRONMENTAL FACTORS
ON THE RESTING CELL FORMATION
OF PLEUROCHRYSIS CARTERAE
Chengxu Zhou, Xiaojun Yan, Ying Jiang, Jiao Zhan
Ningbo University, 818, Fenghua Road, Ningbo, Zhejiang, China. E-mail: [email protected]
In Pleurochrysis carterae (Prymnesiophyceae, Haptophyta) life cycle, apart from the unicellular coccolith-bearing cells, benthic branched
filamentous of non-coccolith-bearing resting cells exit. The heteromorphic phases supplied an excellent opportunity for the study of the
factors that trigger the resting cell formation of P. carterae. In this paper, the effects of several environmental factors were investigated.
The factors included: nitrogen or phosphate stress, calcification inhibition, low temperature, and extracellular α-ketoglutaric acid. The
results showed that: nitrogen deficiency or nitrogen limitation significantly triggered the early formation of the resting cells. It appears that
phosphate deficiency or limitation had no significant effect on filamentous forming during the early period. When coccolith-bearing cells
were moved into the calcium-deficiency medium, coccolith detached from the cells gradually. However, no filamentous colonies occurred.
Under low temperature stress, coccolith-bearing swimming cells were unable to divide and no filamentous colony occurred. Extracellular
α-ketoglutaric acid had concentration dependent effects on the resting cell formations. The induction of filamentous occurrence was
suppressed when α-ketoglutaric acid concentration was lower than 1.2mM. In contrast, it has the positive effects when concentration
was higher than 1.2mM.
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POPULATION DYNAMICS
OF HARMFUL ALGAE BLOOMS
POSTER PRESENTATIONS
EXPRESSION PROFILING OF MITOTIC CYCLIN
IN TOXIC DINOFLAGELLATE, ALEXANDRIUM FUNDYENSE
Yunyun Zhuang, Huan Zhang, Senjie Lin
Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, PC 06340, Groton, CT, USA.
E-mail: [email protected]
Cell cycle regulation is of particularly relevance to phytoplankton population dynamics since cell division in these unicellular organisms
directly leads to population growth. Central among the regulators are cyclins. In particular, mitotic cyclin, which is highly accumulated
during the late G2 and M phase of the cell cycle, can be a marker for in situ growth rate studies on harmful algal blooms. In this study,
full-length cDNA of a mitotic cyclin was isolated from toxic dinoflagellate Alexandrium fundyense (Afcyc1), which displays 78% similarity
to that from Lingulodinium polyedrum and clusters with cyclin B from other eukaryotic counterparts in phylogenetic trees. The conserved
‘cyclin box’ and ‘destruction box’ were identified within the sequence. Species-specific primers were designed to study the dynamics of
Afcyc1 expression throughout the growth period and the cell cycle using real-time qPCR. In parallel, expression patterns of GAPDH,
FSD, and 18S rRNA throughout cell cycle and growth stages in A. fundyense were examined to identify the most suitable reference
gene for studying gene expression in this species. Afcyc1 showed differential expression between different growth phases and cell cycle
phases. Despite the unusual nuclear characteristics of dinoflagellates, our results suggest 1) the mitotic cyclin is conserved in Gonyaulacoid
dinoflagellates during evolution; 2) Afcyc1 is a promising growth marker for monitoring the development of A. fundyense blooms.
61
WHY A DINOPHYSIS HAB-SPOT OFF AVEIRO, PORTUGAL?
Mª Teresa Moita1, Sofia Palma1, Alexandra Silva1, Rita Nolasco2, Jesus Dubert2, Paulo B. Oliveira1
1
Instituto Nacional de Recursos Biologicos (INRB-IPIMAR), Avenida de Brasilia, s/n, 1449-006 Lisboa, Portugal.
E-mail: [email protected]
2
Centro de Estudos do Ambiente e do Mar (CESAM), Departmento Fysica, Universidade de Aveiro, 3810 193 Aveiro, Portugal
Twenty years of monitoring of HABs along the upwelling coast of Portugal indicate that the region offshore Aveiro is a spot of annual
recurrent outbreaks of Dinophysis blooms. This region is characterized by a low frontal probability on the outer shelf, suggesting the
presence of a warmer stratified pool with weak offshore advection, retentive for biological particles. The HABSPOT project aims at
the identification of the main processes in that region and their role on bloom dynamics, with focus on: (i) shelf retention and dispersion
of cells, (ii) HAB population growth, (iii) onshore and alongshore transport during upwelling relaxation events. In September 2006,
after three weeks of upwelling relaxation, an oceanographic cruise was carried out off Aveiro during a Dinophysis acuta bloom. The
temperature distribution showed that the outer-shelf was occupied by warm surface waters extending in the northeast direction, separated from the colder inner-shelf waters by a weak thermal front. The species was mainly distributed at shallow depths along the
inshore side of the front, reaching a maximum concentration of 9x103cells L-1 at 40º 26'N, where the warmer surface water was
closer to the coast. Growth rate estimates revealed very low in situ division rates with a maximum (μmin= 0.08) observed at 09h30a.m
during a 24h cycle. Results from a hydrodynamic numerical model show complex patterns of alongshore flows and counter flows,
varying at short space and time scales. The dispersion resulting from these conditions, are further analyzed to identify the structures
favoring the accumulation of D. acuta cells nearshore.
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2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 63
TIME SERIES
OF HARMFUL ALGAE
BLOOM EVENTS:
CLIMATE AND
ANTHROPOGENIC
INDUCED IMPACTS
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 64
ORAL PRESENTATIONS
WHAT CAN THE FOSSIL RECORD OF DINOFLAGELLATES
TELL US ABOUT CLIMATE CHANGE AND HABS?
Barrie Dale
Department of Geosciences, University of Oslo, PB 1047 Blindern, 0316 Oslo, Norway. E-mail: [email protected]
There is a more or less continuous record of fossil dinoflagellate cysts from the Late Triassic (around 200 million years ago) to the present
day. Prior to this, organic geochemistry has revealed the presence of marker species of compounds (dinosterols) believed to be uniquely
produced by dinoflagellates at least as far back as the Cambrian, suggesting the presence of dinoflagellates well before 500 million years
ago. The geological record is punctuated throughout by climatic variation, dinoflagellates account for many of the different forms of HABs
today, and their fossil record would thus seem to offer a potential long term record of HABs and climatic change.
There is no unequivocal direct evidence of HABs in the geological record. Harmful effects are difficult to identify in ancient times from
species with no living representatives, and even in more recent geological times where fossil cysts of known HAB species persist into
the record, it is virtually impossible to establish if the species was harmful previously (e.g. some living species have both toxic and nontoxic forms). These limitations must be recognized, but since HAB species generally only differ from others by being harmful to human
interests, any evidence of algal response to climate variation may be of interest to HAB research. The geological record of dinoflagellate
cysts contains such information, and this talk will discuss some examples.
64
ASSESSING THE HISTORICAL RELATIONSHIP BETWEEN
ALEXANDRIUM CATENELLA AND CLIMATE WITH
A CYST RECORD FROM SEQUIM BAY IN PUGET SOUND,
WASHINGTON
Kirsten M. Feifel, Rita A. Horner
School of Oceanography, University of Washington, Box 357940, Seattle, WA 98195, USA. E-mail: [email protected]
A general lack of long time series of HAB occurrences has limited statistical assessments of potential relationships with climate in Washington,
USA. After a bloom, some species of harmful algae (i.e., the dinoflagellates) can produce resting cysts that settle onto bottom sediments.
Concentrations of these cysts preserved in sediments can be useful for reconstructing historical changes in blooms and have been shown to
reflect changes in the environment. In the Puget Sound of Washington, USA, blooms of the harmful dinoflagellate A. catenella have apparently
increased in frequency and spatial extent since the 1950s. This increase has been linked to large-scale climate variability such as the Pacific
Decadal Oscillation (PDO) and ENSO, and local variables such as air temperature, stream flow, and sea surface temperature. In this study,
we examined the relationship between historical climate variability and profiles of A. catenella cysts in a sediment core from Sequim Bay in
northern Puget Sound to better determine the influence of climate on A. catenella populations; cysts were identified using epifluorescence
microscopy and genetic samples have been collected for additional confirmation. Cysts were present down-core to a depth of at least 100
cm, equating to the year 1878. Local air temperature and sea surface temperature were positively and significantly correlated with cyst abundances from 1878-2005. No significant relationships were found for PDO or ENSO and cyst abundances. This may highlight the importance
of local environmental variability over large-scale climate variations in determining A. catenella population dynamics and toxic events.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
TWO DECADES OF HAB’S MONITORING IN
THE COASTAL EMBAYMENTS OF THE EBRO DELTA,
NW MEDITERRANEAN: EXPLORING ENVIRONMENTAL
CONDITIONS AND PHYTOPLANKTON BLOOMS
Margarita Fernández-Tejedor1, Maximino Delgado1,2, Esther Garcés2, Jordi Camp2, Jorge Diogène1
1
2
IRTA, Carretera del Poblenou, km 5.5, 43540 Sant Carles de la Ràpita, Spain. E-mail: [email protected]
Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CSIC, P. Marítim de la Barceloneta,
37-43, E08003 Barcelona, Spain
Phytoplankton and environmental parameters in Alfacs and Fangar Bays are monitored every week since 1990. Blooms of Alexandrium
minutum, Dinophysis sacculus, Pseudo-nitzschia spp., Karlodinium veneficum and K. armiger are recurrent in these bays showing a seasonal pattern. A. minutum (104 cells/L) and D. sacculus (104 cells/L) blooms occur in winter/spring, while Pseudo-nitzschia spp. (107
cells/L) blooms occur in summer/fall. A shift in the timing of occurrence of Karlodinium spp. (107 cells/L) blooms from autumn/winter
to spring/summer has been observed during the last 8 years in Alfacs Bay. An increase of the Pseudo-nitzschia spp. abundances has
been observed in Alfacs Bay although in Fangar Bay Pseudo-nitzschia spp. blooms do not show the increase in abundance. Other HA
species such as Dinophysis caudata, Dinophysis acuta and Protoceratium reticulatum are also present in lower densities; Alexandrium
catenella bloomed only in the year 2001. From 1990 to 2009 the average seasonal temperatures range from 11ºC in winter to 26ºC
in summer. The lowest average seasonal salinity is attained in fall and winter ranging from 32 in autumn to 35.5 in spring. From 1990
to 2009 an increase in salinity is observed in Alfacs Bay while a decrease is observed in Fangar Bay due to local conditions related to the
management of irrigation channels. The role of other parameters such as inorganic nutrients concentration has also been assessed.
65
ANNUAL AND SEASONAL VARIABILITY
OF DINOFLAGELLATE BLOOMS IN MONTEREY BAY,
CALIFORNIA (USA)
Andrew Fischer1, John Ryan2
1
National Center for Marine Conservation and Resource Sustainability, University of Tasmania, Locked Bag 1370,
Launceston, Tasmania 7277, Australia. E-mail: [email protected]
2
Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California, 95039, USA
Dinoflagellate blooms in Monterey Bay, California (USA) have recently increased in frequency and intensity with a noted a shift toward
dinoflagellate predominance. Eight years of satellite imagery from the Moderate Resolution Imaging Spectrometer (MODIS) is used to
describe the annual and seasonal variability of this new bloom activity within the Bay. Three classes of MODIS algorithms, empirical
(OC3M), semi-analytical (Carder) and fluorescence line height (FLH) were correlated against in situ chlorophyll measurements to find
the most robust and reliable predictor of bloom activity. The FLH algorithm provided the most robust estimate of bloom activity especially
in conditions when chlorophyll concentrations were high (~50mg/l). Elevated concentrations of phytoplankton were evident during the
months of August-November, a period during which increased occurrences of dinoflagellate blooms have been observed in situ. Seasonal
patterns of FLH show the on- and offshore movement of areas of high phytoplankton biomass between oceanographic seasons. This
suggests an increased cyclonic circulation throughout the Bay during the upwelling period causing blooms to form alongside the upwelling
fronts which sweep from the north across the mouth of the Bay. Higher concentrations of phytoplankton are also evident in the vicinity
of the land-based nutrient sources, and outflows and the cyclonic bay-wide circulation can transport these nutrients to the northern Bay
bloom incubation region.
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EUTROPHICATION AND BLOOMS
IN COASTAL EMBAYMENTS
Patricia M. Glibert1, Cynthia A. Heil2, Catherine S. Wazniak3
1
University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge, MD 21613, USA.
E-mail: [email protected]
2
Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 Eighth Ave. S.,
St. Petersburg, FL 33701, USA
3
Maryland Department of Natural Resources, Tidewater Ecosystem Assessment Division, 580 Taylor Ave.,
D2, Annapolis, MD 21401, USA
Coastal lagoons and embayments are shallow, highly enclosed estuaries with low riverine input and reduced exchange with the ocean
and which tend to be retentive environments for nutrients. In coastal lagoons large spring diatom blooms are rare, but picoplankton
blooms, “ecosystem disruptive algal blooms” (EDABs), are more common. In this talk, the long term trends in nutrients and blooms in
two coastal lagoons of the US East Coast, Chincoteague Bay, Maryland, and Florida Bay, will be highlighted. In both systems ammonium
and organic nitrogen concentrations are many-fold higher than those of nitrate. The blooms that are supported are Aureocococcus
anophagefferens in Chincoteague Bay, and Synechococcus spp. in Florida Bay. These picoplankton blooms have physiological characteristics that make them well suited to compete effectively for reduced forms of nitrogen. Strong coupling to benthic or water column regeneration is thus important to maintain the required nitrogen to sustain the blooms. Grazing by zooplankton on these blooms is reduced,
and shellfish mortality is often linked with these blooms. Mixotrophic dinoflagellates and raphidophytes, having the capability of grazing
picoplankton, as well as using dissolved nitrogen sources directly, are able to be sustained once picoplankton blooms become established
and may be the primary grazers of these blooms.
66
EFFECTS OF INCREASED TEMPERATURE
AND ACIDIFICATION ON SPRING AND SUMMER BALTIC
SEA PHYTOPLANKTON COMMUNITIES
Edna Granéli, Claudia Romero-Oliva, Paulo S. Salomon
School of Natural Sciences, Marine Ecology Department, Linnaeus University, SE-39182, Kalmar, Sweden.
E-mail: [email protected]
Mesocosm experiments were used to test the phytoplankton communities’ responses to increased temperature and acidification predicted
for year 2100, and their combination. In both experiments acidification and higher temperature significantly increased phytoplankton biomass above present conditions, and to even higher levels when these two drivers were combined. Higher temperature leads to an acceleration of the blooms by ca. 1 day °C-1. In the spring, diatoms dominated in all but the combined higher temperature and acidification
treatments, where a shift from diatoms to haptophytes and dinoflagellates developed. In the summer experiment, diatoms were stimulated
by present conditions, picocyanobacteriae by acidification and cyanobacteriae (mainly the toxin-producer Nodularia spumigena) by the
combined higher temperature and acidification treatments. In the summer, the highest phytoplankton diversities were found in the present
conditions and combined higher temperature and acidification treatments, whereas acidification alone lead to the lowest phytoplankton
diversity found in all treatments. If seawater temperature continues to rise and pH to decrease as predicted by global climate change
models, shifts in the phytoplankton community will occur, the size of the spring and the cyanobacteriae summer blooms will be bigger
than already are now, but will occur some days earlier. Also sedimentation of these blooms to deeper waters will increase the area of
oxygen depletion in these waters. The ultimate consequence will be less available edible algae to sustain the trophic food webs and a decrease in the quality of the marine habitat.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
ORAL PRESENTATIONS
IS THERE A LINK BETWEEN N:P RATIOS
AND RED TIDES IN TOLO HARBOUR?
P. J. Harrison1, K. Yin2, J. Xu1, H. B. Liu1, J. H. W. Lee1, D. M. Anderson3, I. J. Hodgkiss4
1
Hong Kong University of Science & Technology, Hong Kong. E-mail: [email protected]
Australian Rivers Institute, Griffith University, Nathan, GLD 4111, Australia
3
Dept Biology, Woods Hole Oceanographic Institution, Woods Hole MA, USA
4
258 Poulton Rd, Fleetwood, Lancs, FY7 7BT, UK
2
A study conducted in Tolo Harbour over a 7 year period (1982-89) suggested that the growth of most red tide species in Hong Kong is
optimized at low molar N:P ratios between 6 and 15. It was concluded that the decreasing N:P molar ratios were a key factor responsible
for the increase in the occurrence of red tides and the shift in the dominant red tide species. When we extended this time series for
another 19 years (1990 to 2008), we found no significant relationship between ambient N:P ratio and the occurrence of red tides. Our
laboratory growth experiments revealed that there is no significant difference in the growth rate and cellular N:P ratios for phytoplankton
growing on three different N:P molar ratios of 4:1, 16:1 and 64:1 when nutrients are saturating. Furthermore, the yield of algal biomass
depended on the concentration of the potential limiting nutrient. The most common red tide species in Hong Kong is Noctiluca scintillans.
Since it is heterotrophic, Noctiluca is only indirectly connected to nutrients through its preferred food supply of diatoms. The winter and
early spring blooms of Noctiluca are more likely due to surface aggregation induced by monsoon winds. Although there is a gradient in
N:P ratios from 10:1 to 80:1 across Hong Kong waters, diatoms are generally the dominant group of phytoplankton suggesting that
N:P ratios are not a key factor regulating the community structure of phytoplankton. Hence, it is suggested that an increase in the number
of red tides at the early stage (prior to 1990s) in Tolo Harbour was more likely due to the increase in sewage loading into the harbour
and not the reduction in N:P ratios.
67
PHENOLOGY AND MULTI DECADAL DISTRIBUTION
OF HARMFUL ALGAL BLOOM SPECIES IN NE
ATLANTIC WATERS, 1960-2007
Stephanie L. Hinder1,2, Graeme C. Hays2, Martin Edwards3,4, Antony W. Walne3, Mike B. Gravenor1
1
Institute of Life Science, Swansea University, Singleton Park, Swansea, SA2 8PP, UK. E-mail: [email protected]
Institute of Environmental Sustainability, Swansea University, Singleton Park, Swansea SA2 8PP, UK
3
SAHFOS, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK
4
Marine Institute, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, UK
2
There has been the suggestion that Harmful Algal Blooms (HABs) are increasing in abundance, in association with changing climatic
conditions, which could have significant affects on human health, economics, and environment. We used Continuous Plankton Recorder
(CPR) survey data to investigate seasonal and spatial abundances of five key HAB taxa and, for comparison, an abundant wide ranging
diatom species, in the North Atlantic and the North Sea from 1960-2007. We found that while some HABs are increasing in abundance,
this certainly does not apply to all taxa. Since the 1960s, for three out of the five HAB taxa the seasonal timing of maximum abundance
has advanced significantly: Dinophysis spp., by 14 days, Protoperidinium spp., by 28 days and the autumn bloom of Pseudo-nitzschia
seriata by 11 days. Over the five decades, pronounced changes in abundance in the North Sea were observed, with the most significant
increases being restricted to the coast of Norway. Noctiluca scintillans has become more widespread during 2000s, for the first time
nearly occupying the whole of the southern region of the North Sea. Potential environmental factors driving these changes have yet to
be identified. In a set of time series analyses, no strong correlations with the North Atlantic Oscillation index were found. The change in
these species phenology and spatial abundance could lead to the emergence of a new successional regime in phytoplankton, causing a
mismatch in successive trophic levels.
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GAMBIERDISCUS PERMISSIVE HABITAT:
TEMPERATURE, LIGHT AND SALINITY REQUIREMENTS
Steven R. Kibler, Patricia A. Tester, William C. Holland, Mark W. Vandersea, R. Wayne Litaker
National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA. E-mail: [email protected]
Ciguatera fish poisoning (CFP) is a circumtropical disease caused by ingestion of a variety of fish that bioaccumulate algal toxins. Distribution and abundance of the organisms that produce these toxins, chiefly dinoflagellates of the genus Gambierdiscus, are reported to
correlate positively with water temperature. Consequently, there is growing concern that higher temperatures associated with climate
change could increase the incidence of CFP. This concern prompted laboratory experiments on the growth rates of six Gambierdiscus
species at temperatures between 18oC -35oC. It also prompted examination of sea surface temperatures in the Caribbean and West
Indies for areas that could sustain Gambierdiscus populations year round and support rapid growth rates. Experimental results showed
_ 29oC. Growth experiments testing the effect of irradiance
that the thermal optimum for five of the six Gambierdiscus species tested was >
-2 -1
(4-664 μE m s ) on seven Gambierdiscus species and of salinity (15-41) on four Gambierdiscus species were also conducted. Intraspecific differences in light requirements were examined using 6 strains each of G. caribaeus, a widely distributed species, and G. carolinianus, a species first collected at ~ 40 meters depth.
68
DO EXTREME CLIMATIC EVENTS HAVE
AN IMPACT ON THE DEVELOPMENT
OF CYANOBACTERIA IN LAKE GENEVA?
Nicole Gallina1, Orlane Anneville2, Martin Beniston1
1
Climate Change and Climate Impacts Research Group, Institute for Environmental Sciences (ISE), University of Geneva,
7 rte de Drize, CH-1227 Carouge, Switzerland. E-mail: [email protected]
2
INRA, UMR CARRTEL, Stn Hydrobiol Lacustre, 75 Ave Corzent, F-4203 Thonon Les Bains, France
Lake Geneva is the largest lake in Western Europe and therefore plays an important role as freshwater ecosystem and its use in drinking
water supply. It has been forecast that the temperature of the epilimnion in Lake Geneva will increase by as much as 3.8°C as a result
of global warming. Cyanobacteria are of a particular interest in freshwater ecosystems due to their ability to form toxic blooms that can
result in numerous negative impacts on the environment and on public health. Even though Cyanobacteria blooms still remain minor in
Lake Geneva, their outbreaks could become more important under warmer climatic conditions as projected for the coming decades. The
aim of this study is to analyze whether, as hypothesized, more Cyanobacteria blooms will occur in Lake Geneva under the global warming
scenario. In this study, air temperature extremes under current climate were used as a proxy for mean temperatures in a future climate
forced by enhanced greenhouse gases, since many climate models suggest that today’s extremes may become the norm by 2100. We
investigated the evolution of Cyanobacteria in response to extreme air temperature events which occurred in Lake Geneva from 2001 to
2008, and compared their behaviour to a 30-year reference period. The results indicate that under extreme seasonal temperature events,
Cyanobacteria become relatively more important and are able to alter the common species composition.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
ORAL PRESENTATIONS
COASTAL PLANKTON COMMUNITIES APPEAR
RESILIENT TO YEAR 2100 CO2 AND pH CHANGES:
EVIDENCE FROM MICROCOSM STUDIES
Lasse Tor Nielsen1, Hans Henrik Jakobsen2, Gustaaf Hallegraeff3, Simon W. Wright4, Per Juel Hansen1
Marine Biological Laboratory, Strandpromenaden 5, 3000 Helsingør, Denmark. E-mail: [email protected]
DTU-AQUA, Kavalergården 6, 2920 Charlottenlund, Denmark
3
Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia
4
Australian Government Antarctic Division, Channel Highway, Kingston, 7050 Tasmania, Australia
1
2
The atmospheric CO2 concentration is rising, and models predict that by the end of the century it will have increased to twice the amount
seen at any given time during the last 15 my. This will cause a decrease in average surface water pH of 0.4 units and planktonic protists
are supposedly among the first organisms to be affected by this change. We tested whether reduced pH (and increased free CO2) would
affect three different plankton communities over incubation periods of 14 days. Two treatments with reduced pH (both within 21st century
predicted levels) were compared to an unaltered control in 2.5L microcosm experiments in the laboratory using natural plankton communities from Øresund, Denmark and from the River Derwent estuary, Australia. Measured parameters included community photosynthesis,
nutrient take-up and biomass build-up as well as enumeration of 25 protist taxa, including ichthyotoxic Karlodinium spp., DSP producing
Dinophysis spp. and yessotoxin producing Protoceratium reticulatum. No effects were found of year 2100 pH and CO2 changes, and
all three plankton communities were thus highly resistant to 21st century predicted pH and CO2 changes. This has been found before for
other coastal plankton assemblages, and we suggest that high pH tolerance is a necessity for protist species living in coastal waters with
large pH fluctuations.
69
DINOPHYSIS ACUTA TIME SERIES AND THERMAL
STRATIFICATION IN NORTHWESTERN IBERIAN WATERS
Beatriz Reguera1, Laura Escalera1, Lourdes Velo1,2, Yolanda Pazos3, Manuel Ruiz-Villarreal4
1
Instituto Español de Oceanografía, Subida a Radiofaro 50, 36390 Vigo, Spain. E-mail: [email protected]
Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), Centre de Brest, DYNECO,
Pointe du Diable BP70, 29280 Plouzané, France
3
Instituto Tecnolóxico Para o Control do Medio Mariño (INTECMAR), Peirao de Vilaxoán s/n,
36611 Vilagarcía de Arousa, Spain
4
Instituto Español de Oceanografía, Muelle de las Ánimas s/n, 15001 A Coruña, Spain
2
Dinophysis acuta is a seasonal (late summer) lipophilic toxin producer in European Atlantic coastal waters, its blooms associated with
thermal stratification. This species typically co-occurs with and follows Dinophysis acuminata. The highest concentrations of D. acuta in
the NW Iberian upwelling region (Cape Finisterre to Cape Carvoeiro) are usually located off Aveiro (Portugal). At the northern limit of this
region, the Galician Rías, D. acuta replaces D. acuminata during very hot summers with marked and deeper thermoclines. Nevertheless,
several conditions seem to be required for a bloom of this species to build up in late summer (August): i) The occurrence of inoculum
populations in the area since early summer; ii) The maintenance of stable stratified conditions through July; iii) The attainment of critical
density gradients at the appropriate depth. Here we examine a 24-y (1986-2009) time series of Dinophysis acuta from samples collected
with the same method (hose sampler) at the hot spot of DSP outbreaks in the region. Our main objective is to identify the climate patterns
that promoted the exceptional blooms of D. acuta during the summer-autumn of 1989 and 1990.
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CHANGES IN PHYTOPLANKTON AND HAB DATA
FROM THE CONTINUOUS PLANKTON RECORDER
SURVEY LINKED TO HYDROCLIMATIC VARIABILITY
IN THE NORTH ATLANTIC
Philip C. Reid1,2, Martin Edwards1,2
1
Sir Alister Hardy Foundation for Ocean Science, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK. E-mail: [email protected]
Marine Institute, University of Plymouth, Plymouth, UK
2
Results from the Continuous Plankton Recorder survey show that rapid changes have occurred in ‘chlorophyll’ concentration (Phytoplankton Colour index) and the composition, abundance and timing of phytoplankton in the North Atlantic in recent decades.
Harmful algal bloom (HAB) taxa show similar regional changes, especially in their frequency of occurrence and distribution. Many of
these changes have been linked to rising temperatures that for European shelf seas have been correlated with Northern Hemisphere
temperature and thus linked to global warming. Hydroclimatic variability and regional warming appear to modulate long-term changes
in phytoplankton assemblages and biomass in the North Atlantic, especially at the scale of sampling of the CPR (integrated samples
over 10 nautical miles). Given these links it can be expected that as global temperatures rise patterns of HAB events will show a
clear response to climate change, with possible additive effects from an increasing human population. New developments in the
CPR ‘ship of opportunity’ programme that has sampled over 5 million miles of the ocean since it started in 1931 and routinely
identifies >500 taxa of phytoplankton and zooplankton will be presented that include: plans for a global expansion of the survey, application of molecular analysis to HAB taxa preserved in formalin, results from instrumented CPRs and the potential of a new water
sampler that is deployed with the CPR for HAB studies.
70
DECIPHERING THE ROLE OF CLIMATE CHANGE
AND EUTROPHICATION IN THE HAB OCCURRENCES
IN JUAG LAGOON, SORSOGON,
FROM THE SEDIMENT RECORD
Joan Reotita1, Fernando Siringan1, Ma. Yvainne Sta. Maria1, Rhodora Azanza1, Carlo Primo David2
1
The Marine Science Institute, Velasquez St., University of the Philippines, Diliman, Quezon City, Philippines. E-mail:
[email protected]
2
National Institute of Geological Sciences, Velasquez St., University of the Philippines, Diliman, Quezon City, Philippines
Worldwide proliferation of specific types of harmful algae has been associated with eutrophication and climate variability. Lead-210
dated sediment record in Juag lagoon, Sorsogon, Philippines is used to reconstruct the temporal variation in the dinoflagellate cyst
assemblage, nutrient and sedimentological parameters during the past 100 years. The sediment record demonstrates longer harmful
algal history than previously reported for the area and shows an overall increasing cyst density and a similar trend for the toxic species
particularly Pyrodinium bahamense var. compressum (Pbc). This general trend is concurrent with an increase in total nitrogen which
may be due to restriction of flushing caused by spit growth that presently bounds Juag Lagoon from the outer sea. The influence of
increasing sea surface temperature (SST) to the changes of dinoflagellate assemblage is indicated by overall increasing trends of
species that thrive in warm waters. P. bahamense, which is the most dominant species in the past 30 years, shows affinities to both
eutrophication and warming events. However, P. bahamense cyst density appears to correlate closer to temperature fluctuations.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
ORAL PRESENTATIONS
THE ROLES OF INFLOW AND SALINITY ON PRYMNESIUM
PARVUM POPULATION DYNAMICS AND SYSTEM-WIDE,
FISH-KILLING BLOOMS IN SOUTH-CENTRAL USA
Daniel L. Roelke, James P. Grover, Bryan W. Brooks
Texas A&M University, University of Texas at Arlington, Baylor University, USA. E-mail: [email protected]
Fish-killing Prymnesium parvum blooms have occurred in south-central USA for at least ~30 years, with the last decade experiencing
recurrent blooms of large magnitude. In the systems reported here, Lakes Possum Kingdom, Granbury and Whitney (Texas), P. parvum
blooms were winter phenomena developing under conditions far from the growth optimum. Bloom thresholds of 10 x 106 cells L-1 were
observed as a function of inflow and salinity for the period 2000-2009. In Lake Possum Kingdom, blooms occurred only when 7-day
accumulated inflows were <10 x 106 m3 and salinities were >1.5 psu. For Lakes Granbury and Whitney, blooms occurred when 7-day
accumulated inflows were <20 x 106 m3 and <40 x 106 m3, respectively, and salinities were >0.5 psu. Inflow to these lakes exceeded
thresholds during the spring and early-summer months in eight (Lake Possum Kingdom), seven (Lake Granbury) and six (Lake Whitney)
of the ten years analyzed. Salinities typically exceeded these thresholds during the period of study prior to the spring of 2007. The spring
of 2007 was a period of high precipitation, after which salinities were typically below thresholds. The linkage between incidence of P.
parvum blooms, inflows and salinity is of concern because combined effects from human population increase and climate change could
lead to periods of decreased inflow and increased salinity, which may then increase the frequency and magnitude of P. parvum blooms.
71
LONG-TERM TRENDS IN REGIONAL DOMOIC ACID
EVENTS ASSOCIATED WITH LARGE-SCALE VARIABILITY
IN THE CALIFORNIA CURRENT SYSTEM
Emily Sekula-Wood1, Claudia-Benitez Nelson1, Steven Bograd2, Clarissa Anderson1,3
1
Department of Earth and Ocean Sciences, University of South Carolina, EWS 408, 701 Sumter Ave.,
Columbia, SC 29208, USA
2
National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Southwest Fisheries Science Center,
Environmental Research Division, 1352 Lighthouse Ave., Pacific Grove, CA 93950, USA
3
Department of Ocean Sciences, University of California, Santa Cruz, 1156 High St., Santa Cruz, CA, 95064, USA.
E-mail: [email protected]
The widespread domoic acid (DA) poisoning event in 1998 along the US west coast marked a pivotal moment for Pseudo-nitzschia
bloom ecology. Rather than an isolated event, it appeared to signify an upward trend in the seasonal occurrence of large Pseudo-nitzschia
blooms over the following decade, particularly in regional hotspots like the Santa Barbara Channel, CA. In the absence of HAB monitoring
programs in the 1990s, the increase in blooms was partly attributed to increased monitoring post-1998. Recent analysis of archived
sediment trap samples collected at 540 m depth from 1993-2007 in the Santa Barbara Basin, however, reveals an abrupt shift towards
an increased frequency and magnitude of DA events over the last decade compared to that previously. DA flux to sediment traps has increased by an average of 18 ± 5 μg/m2/d, with 1 large event (>5 μg/m2/d) from 1993-1999 versus 16 large DA events from 20002007. We relate this shift to a change in the composition and magnitude of upwelled waters associated with shifting biogeochemical
profiles in the California Undercurrent throughout the Southern California Bight since 1998, the transport of which into the Santa Barbara
Basin may be further enhanced by a coincident switching of the North Pacific Gyre Oscillation mode.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
OUTBREAK OF COCHLODINUM POLYKRIKOIDES
FOR THE FIRST TIME IN THE COASTAL WATER OF OMAN
Adnan Al-Azri1, Sergey Piontkovski1, Khalid Al-Hashmi1, Hamed Al-Gheilani2
1
Sultan Qaboos University, P.Box 34 Al-Khoudh, PC 123, College of Agricultural and Marine Sciences, Muscat,
Sultanate of Oman. E-mail: [email protected], [email protected]
2
Ministry of Fisheries Wealth, Sultanate of Oman
The diversity of harmful algal blooms (HABs) and their impacts present a significant challenge to those responsible for the management
of coastal waters resources. Here we present an unusual emergence of the red tide forming dinoflagellate Cochlodinium polykrikoides
along the coastal water of Oman in 2008. Following satellite images, blooms were initially concentrated in the Northern part of the country
and caused massive fish kill in cages and gradually moved towards the capital area where the first patches were observed from October
2008 and ending up in the Southern coast of Oman (Masira Island). The occurrence of the blooms accompanied water discoloration to
red, strong odor and large quantities of a combination of white and yellow foams. For the first two weeks, Cochlodinium polykrikoides
abundance revealed a near monospecific bloom, with cell densities varied between the minimum of 4.6 x 103 cells.L-1 and the highest
cell density of 9 x 106 cells.L-1. The highest density coincided with high nutrient concentrations. High chlorophyll α (78 μg.L-1) and
dissolved oxygen (10 mg.L-1) were also recorded during the blooms. It may be hypothesized that changes in environmental conditions
driven by large scale events such as mesoscale eddies contributed to the outbreak of the dinoflagellate Cochlodinium polykrikoides in the
coastal water of Oman. The significance of these results is discussed within the context of the ecological changes.
73
SEASONAL VARIATION IN PSEUDO-NITZSCHIA SPP.
AND DOMOIC ACID PRODUCTION IN THE WESTERN
ENGLISH CHANNEL
Naomi Downes-Tettmar1,2, Steve Rowland2, Peter Miller1, Carole Llewellyn1
1
2
Plymouth Marine Laboratory, Prospect Place, Plymouth, PL1 3DH, UK. E-mail: [email protected]
University of Plymouth, Drake Circus, Plymouth, PL4 8AA, UK
The widely studied chain forming diatom, Pseudo-nitzschia spp., is considered to be a cosmopolitan plankter due to its occurrence
globally in marine environments. The genus comprises 30 species, of which 10 have been found to produce a potent neurotoxin,
domoic acid (DA), responsible for amnesic shellfish poisoning (ASP) in sea birds, marine mammals, and humans. The mechanisms
that control the growth of Pseudo-nitzschia spp. and the production of DA are intricately linked to the external environment and remain
largely unclear. High temporal resolution samples were collected weekly between June 2009 to June 2010 from the research station
L4 (50˚15’N, 4˚13’W), which is a long term monitoring site for Plymouth Marine Laboratory, situated approximately ten nautical miles
south west of Plymouth in the English Channel. Water samples were analysed for particulate DA using liquid chromatography mass
spectrometry (LC/MS), data were combined with phytoplankton assemblage (microscopy and chlorophyll-a) and environmental data
(nutrients, temperature, and salinity) to determine the conditions that promote toxin production. This study describes the seasonal
variation of Pseudo-nitzschia species in the Western English Channel, and assesses the environmental factors that influence species
growth and toxin production.
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ASSESSMENT OF NUISANCE MACROPERIPHYTON
AND CYANOBACTERIAL BLOOMS IN THE DAMMED
SYSTEM OF THE DAUGAVA RIVER, LATVIA
Ivars Druvietis
Faculty of Biology, University of Latvia, 4 - Kronvalda Blvd., Riga, LV-1010, Latvia. E-mail: [email protected]
The Daugava River (in the territory of Russia and Belarus - Zapadnaja Dvina) as the largest river of Latvia has the total length of 1005
km (lower part - 352 km of the Daugava River belongs to Latvia). Three dammed HPP reservoirs creates “serial discontinuity” in the
Daugava River ecosystem, especially it reflects in phytoplankton and periphyton communities taxonomic and abundance changes in
running and standing Daugava River waters. During summer low-flow periods the massive growths of filamentous periphytic green algae
(dominated by Cladophora glomerata, Stigeoclonium sp.) is common in reaches on the limestone bottom substrata of rhitral part of
Daugava River. It supports up to 100% Cladophora coverage at 0.5-1.5 m depth during midsummer. Drifting mass of C. glomerata
mats is characteristic for Daugava from August to September. In the lower potamal part of river and in dammed reservoirs it caused
decay and depletion in oxygen content in water near the bottom. High productivity of the phytoplankton increases in all three Daugava
River HPP reservoirs. The highest cyanobacteria (mainly Microcystis aeruginosa) biomass ever recorded in any reservoir in Daugava River
was 85 mgl-1. It was recorded in August 1999, in connection with observations of enhanced water temperatures above 27°C during
both July and August. Last decade (2000-2010) was characterised by more lower summer blooms (biomass 7-12 mgl-1) of cyanobacteria
in all three Daugava River dammed reservoirs.
74
KARENIA BREVIS’ RESPONSE TO INCREASED pCO2
AND SEA SURFACE TEMPERATURES
Reagan M. Errera1, John Kessler1, Shari Yvon-Lewis1, Lisa Campbell1,2
1
2
Texas A&M University, Department of Oceanography, 3146 TAMU, College Station, Texas, USA. E-mail: [email protected]
Texas A&M University, Department of Biology, 3146 TAMU, College Station, Texas, USA
Changes in global climate are expected to impact oceanic ecosystems and have been suggested as one potential contributor for increases
in harmful algal blooms. Our study focuses on Karenia brevis, the major harmful algal species in the Gulf of Mexico, and its response to
global climate change. Carbon dioxide concentrations currently present in the Gulf of Mexico may actually hinder phytoplankton growth
by limiting phytoplankton’s ability to fix carbon. Preliminary data for K. brevis culture (Wilson clone) indicates a change in specific growth
rate from 0.24 d-1 ± 0.002 at an atmospheric CO2 level of 390 ppm (current conditions) to 0.34 d-1 ± 0.4 at a CO2 concentration of 700
ppm, an increase of greater then 25 percent. While it is hypothesized that ocean acidification could influence toxin production, our results
indicate brevetoxin and brevenal concentrations per cell were constant under different CO2 regimes. Thus, we suggest that brevetoxin
and brevenal production are not correlated with growth rate. Our results suggest that future climate conditions may produce larger K.
brevis blooms with similar brevetoxin concentrations, thereby, having an even greater impact on ecosystem and human health. Our initial
data have also indicated an increase in carbon concentration per cell in K. brevis under increased CO2 concentrations, suggesting increase
sequestration of available carbon. Supporting data showing changes in growth rate, photosynthetic rate, brevetoxin and brevenal production
across a CO2 and temperature gradient for a variety clones will also be presented.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
NUTRIENT LEVELS AND HARMFUL ALGAE IN
THE PHYTOPLANKTON OF MANILA BAY,
PHILIPPINES, DURING THE NE MONSOON
Ma. Teresa Escobar, Lara Patricia Sotto, Gil Jacinto, Maria Lourdes San Diego-McGlone, Rhodora Azanza
Marine Science Institute, University of the Philippines, Diliman, 1101, Quezon City, Philippines. E-mail: [email protected]
Manila Bay is a eutrophic marine environment where blooms of Pyrodinium bahamense var. compressum and Noctiluca scintillans have
been observed since 1988. A recent bay-wide study conducted from 11-13 February 2010 in the bay during the northeast monsoon,
which coincided with an El Niño episode, revealed high concentrations of nitrate (16.75 μM), nitrite (6.61 μM), ammonia (13.75 μM)
and phosphate (2.00 μM) found near bottom depths in the midsection of the bay. These nutrient concentrations exceeded the allowable
limits for the ASEAN Region. The corresponding dissolved oxygen concentrations were lowest where nutrients are high with values >2
mg/L. Levels of chlorophyll-a were high in the northwestern and eastern part of the bay. Phytoplankton analysis showed that 53.91%
of the phytoplankton composition are diatoms and 45.20% are dinoflagellates. High densities of the Paralytic Shellfish Poisoning (PSP)
causative organism Gymnodinium catenatum (60-24,100 cells/L) were observed near the surface in the northwest side of the bay while
another harmful species Noctiluca scintillans (20-180 cells/L) had lower counts but were widespread. High nutrient concentrations may
continue to support the subsistence of harmful algal blooms in the bay whose death and decomposition could lead to hypoxia in the
bottom waters.
75
THE BALTIC SEA PHYTOPLANKTON COMMUNITY AT THE
BEGINNING AND END OF THE 20TH CENTURY- A COMPARISON OF HISTORICAL AND MODERN SPECIES DATA
Heidi Hällfors1,2, Hermanni Backer3, Juha-Markku Leppänen1, Seija Hällfors1, Guy Hällfors2,4
1
Marine Research Centre, Finnish Environment Institute, P.O. Box 140, FI-00251 Helsinki, Finland. E-mail: [email protected]
2
Tvärminne Zoological Station, University of Helsinki, J.A. Palménin tie 260, FI-10900 Hanko, Finland
3
HELCOM (Baltic Marine Environment Protection Commission) Secretariat, Katajanokanlaituri 6 B, FI-00160 Helsinki, Finland
4
Haltiantie 8 C 20, FI-01600 Vantaa, Finland
Changes in the phytoplankton community may provoke a cascade of alterations in the ecosystem owing to food web interlinkages. A potential
consequence is increased harmful algal blooms, which can have substantial socioeconomic impacts in the affected areas. Despite more
than 100 years of phytoplankton research in the Baltic Sea, little is known about what has happened to the species composition in the transition from the pre-eutrophication era to the heavily eutrophied system of today, as few studies comparing the results of the early pioneers
to modern data have been published. Also, the majority of the existing phytoplankton data have been collected only after the effects of eutrophication have been apparent. However, assessments of what the Baltic Sea used to be like, before being severely impacted by anthropogenic degradation, are needed to facilitate effective environmental protection measures required by Baltic Sea area regional
intergovernmental commitments and the European Marine Strategy Framework Directive. In this study we investigated whether, and how,
the open sea phytoplankton community of the early 1900’s differs from the present day community. We compared the spring, summer and
autumn communities in the western Gulf of Finland and the northern Baltic proper utilising data collected in 1903-1911 and 1993-2005.
We found e.g. that the toxin-producing dinoflagellates Dinophysis acuminata, D. norvegica, and D. rotundata occur considerably more frequently today, while the bloom-forming cyanophytes Aphanizomenon flos-aquae and Nodularia spumigena occurred commonly in open sea
areas already 100 years ago. We discuss the potential factors causing the differences in the phytoplankton communities.
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 76
HYPOXIA AND DIATOM BLOOM ASSOCIATED
WITH A FISH KILL IN A FISH FARMING AREA IN ANDA,
PANGASINAN (NW PHILIPPINES)
Gil S. Jacinto, Lara Patricia A. Sotto, Ma. Teresa L. Escobar, Maria Lourdes San Diego-McGlone,
Garry A. Benico, Rhodora V. Azanza
The Marine Science Institute, University of the Philippines - Diliman, Velasquez Street, University of the Philippines,
Diliman, Quezon City 1100, Philippines. E-mail: [email protected]
A massive fish kill event occurred in June 2010 in the waters of Anda, Pangasinan, located on the western side of Lingayen Gulf, NW
of the Philippines, affecting principally farmed milkfish (Chanos chanos) kept in cages and pens with an estimated loss of 1.06 M USD
(50 M PHP). A bloom of Skeletonema costatum was associated with the fish kill with cell counts reaching 1.2 x 105 cells L-1 in the
affected areas. Diatoms comprised >98% of the phytoplankton community. The diatom bloom was preceded a month earlier by the
bloom of the PSP-causing Alexandrium sp. with counts up to 2.9 x 105 cells L-1. However, the direct cause of the fish kill was the decline
of dissolved oxygen to <1 mg L-1 from the surface down to depths of 10 m in the areas with the highest fish mortalities. The stress on
the fish caused by extremely low water column DO levels could have been exacerbated by the presence of fish-killing phytoplankton
species (e.g., Chatonella sp.), the bloom of Skeletonema costatum, the dominance of Protoperidinium spp. and Prorocentrum micans,
the toxic levels of ammonia in the water column (up to 25 μM), and the likely release of H2S from the bottom and the sediments amidst
near anoxic conditions. Environmental conditions in the area have worsened steadily due to the proliferation of fish pens and cages and
the constriction of narrow channels by the fish farming structures which also increase water residence times.
76
RELATIONSHIP BETWEEN THE RECURRENCE
OF GYMNODINIUM CATENATUM GRAHAM AND
THE SHELLFISH TOXICITY IN SHELF OF OUEST
MEDITERRANEAN SHORES OF MOROCCO
Laila Tahri Joutei
Institut National de Recherche Halieutique 2, Rue de Tiznit, B.P 21000, Casablanca, Morocco.
E-mail: [email protected]; [email protected]
Since 1989, the Mediterranean Moroccan coasts were affected by shellfish toxicity. The high levels of PSP toxins were caused by proliferation of the toxic dinoflagellate Gymnodinium catenatum. G. catenatum cysts research in sediments surface layers had shown that
the origin of shellfish contamination is probably due to the vegetative cells developing in the layers less than 10m during the periods out
of the seasonal blooms. It has been confirmed by the toxins profiles analysis established at the same time on Phytoplankton and shellfish
extracts. Thus it is established that the recurrence of G. catenatum blooms in the Ouest Mediterranean shores of Morocco is a local phenomenon due to exystement of the species cysts but it must be a regional event.
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 77
TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
A MULTI-YEAR COMPARISON OF SPIROLIDE PROFILES
IN PLANKTONIC FIELD SAMPLES FROM THE NORTH SEA
AND ADJACENT WATERS
Bernd Krock1, Urban Tillmann1, Tilman Alpermann2, Rafael Salas3, Allan D. Cembella1
1
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany.
E-mail: [email protected]
2
LOEWE Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, 60325 Frankfurt A.M., Germany
3
Marine Institute, Rinville, Oranmore, Galway, Ireland
Surveys of toxigenic phytoplankton were carried out over three years in the North Sea and adjacent waters between 2007 and 2009. In
subsequent years, toxin profiles were also determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
from the Danish west and north coasts (2008), and the Scottish east and west coasts, Irish Sea and Celtic Sea (2009). Spirolide profiles
were measured in the 20-50 μm plankton size-fractions at more than 100 stations. In addition, spirolide profiles of field populations were
compared to those of two strains of Alexandrium ostenfeldii, the only known producer of spirolides, isolated from the Scottish east coast
and Irish west coast, respectively. Alexandrium ostenfeldii isolates from distinct geographical locations showed almost identical profiles,
primarily consisting of 20-methyl spirolide G (20-meG). Whereas the Scottish isolate produces only this variant, the Irish isolate additionally
yields slight amounts of 13-desmethyl spirolide C (13-desmeC). These profiles were also reflected in the field data, where 20-meG was
the most abundant spirolide throughout all samples and years. Even though 13-desmeC was also detected in the North Sea, the highest
13-desmeC/20-meG ratios were found Irish waters. Spirolide A was barely detected in 2007 and 2008, in contrast to relatively high
concentrations found in 2009 in the North Sea and western Scottish waters, but not along the Irish coast. There is no known source of
spirolide A in Europe, but field surveys based upon LC-MS/MS profiling provide a plausible means of identifying new toxin sources and
their biogeographical distribution.
77
RELATIONSHIP BETWEEN VARIATIONS OF DINOFLAGELLATES SPECIES COMPOSITION AND CLIMATE CHANGES
AROUND JEJU ISLAND, KOREA
Joon-Baek Lee1, Seung-Hyun Kim3, Han-Sik Chung1, Hyeung-Sin Kim2
1
Department of Earth and Ocean Sciences, Jeju National University, Jeju, 690-756 Korea. E-mail: [email protected]
Marine Living Resources Research Department, KORD, Ansan, 425-600 Korea
3
Research Institute for Basic Science, Jeju National University, Jeju, 690-756 Korea
2
Jeju Island is located in the center of the East China Sea, which has bounders of Japan, Korea and China and shows characteristics of
temperate region. However some tropical fishes and macrophytes have been found during the last decade by acceleration of global
warming. We surveyed one of possible evidences from variations of plankton community. Some tropical (warm-water) dinoflagellates occurred frequently around Jeju Island, not observed before. These species might be introduced into Jeju waters by Tsushima Warm Current
which is a branch from Kuroshio Current. The tropical species shows seasonal and local variations by strength of the warm current. The
warm current is stronger in winter than in summer, and the tropical species occur more frequently in winter. In locality, the tropical species
are more frequently found in the eastern and western coast of Jeju Island rather than in northern and southern coast, which mean the
species are existed fallowing the route of the warm current. We believe that the tropical species occurrence is highly related to a flow
pattern of Tsushima Warm Current which affect stronger than in the past by climate changes such as global warming.
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 78
THE PHYSICAL MEDIATION OF HAB PATCH FORMATION
ON THE WEST COAST OF SOUTH AFRICA
Andrew J. Lucas1, Grant C. Pitcher1, Raphael Kudela2, Trevor Probyn1
1
Marine and Coastal Management, Sea Point Research Aquarium, Beach Road, Cape Town, South Africa.
E-mail: [email protected]
2
Ocean Sciences Department, University of California, Santa Cruz, CA, 95064, USA
The Namaqua shelf region of the wind-swept west coast of South Africa is subjected to severe HAB events, which lead to closures of
local shellfish fisheries and massive mortalities in the inshore rock lobster population. In the austral summer of 2010, we deployed a
cross-shore mooring array of wave-driven autonomous profiling vehicles equipped with CTDs, fluorometers, and, on one mooring, a dissolved oxygen sensor. A total of ~35,000 profiles were collected over ~55 d. The vertical resolution of the observations was on the order
of centimetres and profiles were collected approximately once every five minutes. Superimposed over the signature of the upwellingreversal cycle, strong near-inertial (~1 d-1) variability in both water column structure and shelf currents was reflected in large and rapid
variation in both the vertically-integrated concentration of mixed layer chlorophyll and the depth distribution of chlorophyll. Vertical gradients
in chlorophyll exceeded 10 μg L-1 m-1 on occasion. High concentrations of phytoplankton (>80 μg chl α L-1) were distributed both as subsurface chlorophyll maxima and near surface assemblages at various times. Diurnal vertical movement of the phytoplankton, presumably
associated with behavior, could be observed in the decoupling between vertical isopycnal displacement and the distribution of fluorescence.
The blooms were also extremely patchy horizontally. Near-inertial waves associated with diurnal variability in alongshore windstress drove
frontal formation in HAB patches, which, based on current records and temporal changes in near-surface fluorescence, were characterized
by very strong horizontal gradients in chlorophyll (0.25 μg chl α L-1 m-1). Finally, the impact of HAB patches on near-surface dissolved
oxygen concentrations was evident, with LOW formation occurring below the dense phytoplankton blooms.
78
WHAT ARE THE LIKELY EFFECTS OF CLIMATE CHANGE,
HUMAN POPULATION GROWTH AND OCEAN
ACIDIFICATION ON FUTURE SCENARIOS FOR HAB
DEVELOPMENT TO 2100?
Philip C. Reid1,2
1
Sir Alister Hardy Foundation for Ocean Science, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK.
E-mail: [email protected]
2
Marine Institute, University of Plymouth, Plymouth, UK
Globally coastal seas and even the open oceans have witnessed a deterioration of ecosystems from anthropogenic impacts due to an increasing and exploitative human population. Impacts include: HABs, overfishing, non-native species, global warming and ocean acidification
among others. The occurrence, intensity and impact of HABs appear to have increased, although this may be due to a greater interest
and recognition of the problem. Whatever the reason, HABs have a major economic impact especially on aquaculture and there is concern
that their frequency and effects may increase in the future. In the past, formation of many HABs was linked to eutrophication, but it is
now recognised that many, if not all, blooms are linked to hydroclimatic variability. Given this link it can be expected that as global temperatures rise patterns of HAB events will show a clear response to climate change, with possible additive effects from human population
increase. Some HAB taxa have calcareous body parts during part of their life cycle and may be especially vulnerable to ocean acidification
although all taxa may potentially be affected. The future regional and global development of HABs, within the context of climate change
and human population growth, will be evaluated against past records of HABs and the factors considered to have given rise to their formation. Interactions with impacts from ocean acidification and other impacts will also be considered. Larger scale patterns will be addressed
using information on HAB from the long time series (1931 to present) of the Continuous Plankton Recorder survey.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
THE IMPACTS OF HYPOXIC ENVIRONMENTS
ON CALCAREOUS DINOFLAGELLATE CYSTS
IN SEDIMENTS
Hyeon Ho Shin, Seung Won Jung, Eun Sun Lee, Young-Ok Kim
Korea Ocean Research and Development Institute, Geoje 656-830, Republic of Korea. E-mail: [email protected]
To investigate the effect of hypoxic environments on the resting stage of planktonic organisms, a calcareous cyst population (Scrippsiella
trochoidea) was monthly monitored in two different sites, hypoxic (G1) and normoxic zone (G2) of Gamak Bay from February 2009 to
February 2010. Hypoxia indicating a lower level of dissolved oxygen (< 2.0 mg/L) was developed in the bottom waters of G1 in August
however was not occurred at G2. Morphological and excystment characteristics of S. trochoidea cysts isolated from the natural sediment
samples in the sites were compared. Two morphotypes of S. trochoidea cysts, one typical type with short calcareous spines (calcareous
cyst) and another transparent type without the calcareous outer wall (organic-walled cyst), were simultaneously detected in the surface
sediments. Most of the cyst distributed at G2 was the calcareous cyst while organic-walled cysts were abundant at G1. The abundances
of organic-walled cysts were relatively high in the winter and spring (January to June) when the vegetative population was not detected
or occurred in lower abundances. On the contrary, higher abundances of calcareous cysts were in the summer (July to September),
which is coincided with flourishing of vegetative cells in the water column. The excystment capability of S. trochoidea cysts exhibited over
the excystment ratio of 35% from March to May and the maximum ratio (> 70%) at G1, however there was no difference in the seasonal
germination pattern between two sites. As the outer wall of S. trochoidea cysts is composed of calcium carbonate, the cyst surface can
theoretically be solved in the acidic environments produced by the fermentation process in hypoxic bottom waters. We confirmed that the
spines of S. trochoidea cyst are dissolved below pH 7.07, using CO2 incubator. This suggests that the hypoxic environments cause acidification in the bottom layer and sediments and are contributed to break out the morphological change of S. trochoidea cysts.
79
THE EFFECT OF BIOTIC AND ABIOTIC FACTORS ON THE
DYNAMICS AND BLOOMING OF MARINE HAB SPECIES
Sofie Spatharis1, Nicolas P. Dolapsakis2, Athena Economou-Amilli2, Daniel B. Danielidis2
1
2
University of the Aegean, Department of Marine Sciences, Mytilini, Greece. E-mail: [email protected]
University of Athens, Faculty of Biology, Department of Ecology and Systematics, Athens, Greece
Monitoring the dynamics and proliferation of HAB species, and their possible relation to environmental factors, is an important issue for
coastal ecosystems. In this study the temporal and spatial dynamics of potentially harmful microalgae were investigated in the semi-enclosed gulf of Kalloni (Lesvos Island, Greece), which is affected by terrestrial runoff during winter. Temporal and spatial variability of HAB
species abundance was investigated along a seaward gradient, from August ’04 through March ’06, in relation to physicochemical variables. Analysis showed that cell densities of all potentially harmful species were strongly related to the nutrient-rich freshwater inputs in
the gulf during winter. The potentially harmful diatom Pseudonitzschia calliantha was present throughout the year (high sample occupancy),
developing extremely high abundances only in the interior part of the gulf, triggered by terrestrial loadings during February. Cyst-forming
species such as Alexandrium insuetum and Scrippsiella trochoidea, remained dormant throughout the year (low sample occupancy) and
bloomed only during a narrow time-window when conditions were favorable. The dinoflagellate Karlodinium veneficum was highly correlated
with densities of Cryptomonas sp. (Pearson, r=0.75, p<0.001) in support to recent field and laboratory studies on the phagotrophic preference of this HAB species. The diversity of potentially harmful dinoflagellates in the Kalloni gulf is substantial and density often exceeding
the threshold set by the local authorities. However, it seems that the diatom P. calliantha is by far the most successful species, having a
year-round presence and forming massive winter blooms by readily exploiting nutrient loadings.
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 80
GENETIC EVALUATION OF HARMFUL ALGAL BLOOM
SPECIES FROM ARCHIVED SAMPLES
Rowena Fay Stern1, Jixen Chen2, Declan C. Schroeder2, Martin Edwards1
1
Sir Alistair Hardy Foundation for Ocean Science, The Laboratory, Citadel Hill, The Hoe, Plymouth, PL1 2PB, UK.
E-mail: [email protected]
2
Marine Biological Association of the United Kingdom, Citadel Hill, Plymouth, PL1 2PB, UK
DNA extraction from preserved, archival material is a powerful resource to reconstruct past events but often preserving methods and potential for contamination cast doubt on results. Sir Alistair Hardy Foundation for Ocean Sciences has been recording phytoplankton
species from plankton tow samples using Continuous Plankton Recorders (CPR) globally on 250 commercial shipping vessels since
1931. These archival samples could yield important data on distribution of known and unknown harmful algal bloom species that can be
related to an array of parallel biotic and abiotic data. However, CPR samples have been preserved in Formalin and some suffer fungal
contamination. Furthermore, many recorded HAB species are difficult to identify. In this study we evaluate the quality and utility of genetic
data recovered from CPR “silks” and compare morphological species evaluations with genetic assays for several HAB species. We also
discuss how HABS could be routinely monitored using genetic methods from CPR surveys in future.
80
SHORT-TERM STUDY ON THE EFFECTS OF OCEAN
ACIDIFICATION IN THE GROWTH AND TOXIN PRODUCTION
OF ALEXANDRIUM MINUTUM 1V
Rhett Simon Tabbada1, Raymond Francois Obelhensi2, Jean-Louis Teyssie2, Beatriz Reguera-Ramirez3,
Olivier Thomas4, Elvira Sombrito1, Florence Boisson2
1
Philippine Nuclear Research Institute, Diliman, Quezon City, Philippines
2
IAEA Marine Environment Laboratories, Principality of Monaco. E-mail: [email protected]
3
Instituto Español de Oceanografía, Vigo, Spain
4
University of Nice, Nice, France
Impact of ocean acidification caused by increasing absorption rates of atmospheric carbon dioxide resulting from anthropogenic activities
is a major concern for marine organisms. In this study, the short-term effects of the decrease in pH on the growth and on the Paralytic
Shellfish Toxin (PST) production of the dinoflagellate Alexandrium minutum 1V were evaluated. Laboratory cultures of this toxin producer
in seawater enriched with f/2 media were exposed to three pH conditions, 8.1 (normal), 7.85 (intermediate) and 7.60 (end of century
predicted value by Orr et al., Nature, 2005), for 14 days at constant temperature. The desired pH was obtained through appropriate
carbon dioxide bubbling. Results showed that the decrease in pH down to 7.8 led to increased growth rate of the algae, but lower final
yield. Dinoflagellates cultures exposed to the lowest pH values had a similar rate as those under middle values but reached a significantly
lower final yield. Changes in toxin profile and content per cell with pH were also observed using an HPLC-ECOS system. A. minutum
(Al 1V) produces gonyautoxin (GTX) analogues dominated by GTX 1. The decrease in pH down to 7.8 favoured its production that
became more than 90% of the total toxin against the 70% obtained under normal pH conditions. Toxin content (fmole/cell) was also
significantly increased when exposed to a lower pH. On the other hand, cultures at pH 7.60 had toxin concentrations lower than detection
limit which tends to indicate that under the predicted acidified conditions, production of the toxin is being hindered.
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TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
TRAVELLING THROUGH TIME: MICROALGAL DIVERSITY
IN NEW SOUTH WALES, AUSTRALIA
Penelope Ajani1, Leanne Armand1, Shauna Murray2
1
Climate Futures at Macquarie, Department of Biological Sciences, Macquarie University North Ryde, NSW 2109, Australia.
E-mail: [email protected]
2
School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia
With over half a century of physical and chemical data from south-eastern coastal waters of Australia now under review, climate-related
trends in water chemistry, temperature and physical circulation are emerging, with consequences predicted for phytoplankton ecology.
This PhD study aims to investigate the spatial and temporal patterns of phytoplankton along the New South Wales coastline in light of
these climate-related variations. Changes in species composition and abundance, frequency and type of reported microalgal blooms,
tropical species incidence and duration, timing of spring bloom onset, climate change indicator species and the presence and enumeration
of toxic/nuisance taxa will be investigated in light of associated physicochemical data. Toxic phytoplankton data collected from thirty oyster-growing estuaries will also be investigated for spatial trends in toxic episodes. One of the most troubling genera in these waters,
Pseudo-nitzschia (producer of domoic acid), will be examined for molecular analysis/genetics with the aim of resolving the taxonomy of
this group within Australia.
81
EVALUATING THE RIVER DE-EUTROPHICATION GAIN
ON THE MAGNITUDE OF PHAEOCYSTIS BLOOMS IN
THE SOUTHERN NORTH SEA BETWEEN 1985 AND 2005:
A MODEL STUDY
Nathan Terseleer Lillo, Nathalie Gypens, Christiane Lancelot
Université Libre de Bruxelles, Ecologie des Systèmes Aquatiques CP 221, Bvd du Triomphe, B-1050, Brussels, Belgium.
E-mail: [email protected]
High-biomass blooms of undesirable Phaeocystis colonies occur each spring in the eutrophied eastern English Channel and Southern
North Sea area as a result of excess river nutrient loads. Over the last 60 years, nitrogen (N) and phosphorus (P) delivery to the coastal
sea has shown up and down variations related to human activity. After 1950, an accelerated increase of nutrient loads was reported due
to a combination of increased human population, socio-economic development and intensive agriculture that cumulated in mid-1985
(eutrophication period). After this period, nutrient reduction measures were slowly implemented (de-eutrophication period), leading in particular to an important decrease in P loads subsequent to the removal of phosphates in washing powders. Today the P delivery has decreased to earlier values of 1960 while the N decrease is less important leading to an imbalanced N:P enrichment of the receiving coastal
waters yet characterized by elevated spring blooms of Phaeocystis colonies. In order to understand the link between Phaeocystis colony
magnitude and the decrease of nutrient loads to the coastal area, the existing MIRO model describing diatom/Phaeocystis blooms and
related nutrient cycles in Phaeocystis-dominated ecosystems is implemented in the eastern English Channel and Southern North Sea
over the 1985-2005 period.
2. ABSTRACT BOOK (62-82)_Layout 1 10/22/10 2:26 PM Page 82
TIME SERIES OF HARMFUL ALGAE BLOOM EVENTS:
CLIMATE AND ANTHROPOGENIC INDUCED IMPACTS
POSTER PRESENTATIONS
ARTIFICIAL NEURAL NETWORK MODELS
FOR PREDICTING PSEUDO-NITZSCHIA SPP.
ABUNDANCE IN THE GALICIAN RIAS (NW SPAIN)
Jesus M. Torres Palenzuela, Luis González Vilas, Evangelos Spyrakos
Department of Applied Physics, Faculty of Marine Sciences, Universidade de Vigo, Lagoas-Marcosende, E-36200, Vigo, Spain.
E-mail: [email protected]
In the highly primary productive upwelling estuarine systems of the Galician Rias, transient increases of the diatom Pseudo-nitzschia spp.
are considered a frequent phenomenon that contribute to shrinking the mussel production and are potential harmful to human health. In
this study, time-series data (1992-2000) of Pseudo-nitzschia spp. provided by “Technological Institute for the Control of the Marine Environment of Galicia (INTECMAR)” coupled with oceanographic and meteorological data were used to develop and test a prediction
system for Pseudo-nitzschia spp. in Galician Rias. A Multilayer Perceptron (MLP) artificial Neural Network (NN) was implemented to
model the presence/absence of Pseudo-nitzschia spp. and a second MLP to predict the abundance. Water temperature and salinity,
the evolution of the upwelling, daylight time and spatial data regarding the concentration of Pseudo-nitzschia spp. in the different Rias
one and two weeks before events of high Pseudo-nitzschia spp. (>100,000 cells/l) were used as input variables. The results of the
presence/absence model in the training and validation data set showed precision values of 83.24% and kappa=0.63. The performance
parameters of the NN for the prediction of the Pseudo-nitzschia spp. abundance in the training set were R2=0.79, root mean square
error (RMSE) =0.47 cells/l and in the validation set R2=0.48, RMSE=0.71 cells/l. 81% of the Pseudo-nitzschia spp. high abundance
events (>100,000 cells/l) were predicted correctly by the model showing the potential of the method as a tool for the prediction of this
diatom abundance. The limitations and possible improvements of the models are also discussed.
82
IMPACT OF TEMPERATURE AND NUTRIENTS
ON THE GROWTH AND TOXICITY OF OSTREOPSIS OVATA
Nayani K. Vidyarathna, Edna Granéli
Marine Ecology Department, School of Natural Sciences, Linnaeus University, 391 82 Kalmar, Sweden.
E-mail: [email protected]
The benthic dinoflagellate Ostreopsis ovata Fukuyo occurs worldwide in tropical and sub-tropical waters. On the last 10 years toxic
blooms of O. ovata have been reported for Brazilian and French-Italian Riviera waters. O. ovata cells produce palytoxin, a very
potent toxin, only second in toxicity to botulism. Laboratory experiments were performed with O. ovata, isolated from the Tyrranean
Sea to determine the effects of water temperature (20oC and 30oC) and N and P deficient versus sufficient conditions on the cells
growth and toxicity. Irrespective of N and P conditions the toxicities in the cells grown at 20oC were significantly higher than those
at 30oC. However, at 20oC under N deficiency cell toxicity was significantly higher than in the P deficient and NP sufficient treatments. Contrarily, independent of nutrient treatment algae biomass were higher at 30°C than at 20°C, except in the P deficient
treatment. The lowest biomass was measured at 20°C and N deficiency. Our results show that lower temperature (20oC) and N
deficiency increase O. ovata toxicity but decrease their biomass accumulation. Although NP sufficiency induces lower toxicity per
cell but a much higher O. ovata biomass is produced specially at 30°C, toxin concentrations per water volume are higher. Therefore,
an increase in seawater temperatures as a consequence of global warming will induce higher biomass O. ovata blooms with stronger
negative impacts on the environment and human health than they are now.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 83
IMPACT OF HABS
ON MARINE
FOOD WEBS
AND ECOSYSTEM
STRUCTURE
AND FUNCTION
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 84
ORAL PRESENTATIONS
PYRODINIUM BAHAMENSE VAR. COMPRESSUM TOXIC
BLOOMS IN SORSOGON BAY, PHILIPPINES
Rhodora Azanza, Garry Benico, Iris Baula
Marine Science Institute, C.S., University of the Philippines, Diliman Quezon City 1101, Philippines.
E-mail: [email protected]
Pyrodinium bahamense var. compressum bloom was first recorded in the Philippines in 1983 at Samar-Leyte-Sorsogon area, when it
caused Paralytic Shellfish (PSP) cases and deaths. Studies on Pyrodinium bloom events and accompanying toxicities in Sorsogon area
have been few and unpublished. As part of an on-going R&D program on Philippine Harmful Algal Blooms (PhilHABs), Sorsogon bay
has been a focus of several studies beginning 2009 to help local government manage the phenomenon. Pyrodinium bahamense was
found at the stations near the mouth during Trade winds (April) Season at a mean cell density of 158 cells/L. During mid-southwest
monsoon (SW) the mean cell density rose from 857 cells/L (in August) to about 5,500 cells/L (September) when almost the entire bay
had Pbc cells as shown by the bay-wide samplings. During mid-northeast (NE) monsoon (December) no cells were encountered in the
area but in late NE monsoon some cells (55 cells/L) were encountered. The seasonality of the organism in the bay is compared with its
seasonality in Manila Bay where it has a record of yearly blooms during the SW monsoon from 1987 to 1998.
84
THE GEOHAB CORE RESEARCH PROJECT ON HABS
IN BENTHIC SYSTEMS
Paul Bienfang1, Jacqueline Evans2, Patricia Tester3, Adriana Zingone4
1
University of Hawaii-Manoa, Oceanography Dept. MSB#205, Honolulu, HI 96822, USA. E-mail: [email protected]
Te Ipukarea Society, Inc., Cook Islands
3
NOAA, 101 Pivers Island Road, Beaufort, NC 28516, USA
4
Stazione Zoologica Anton Dohrn, Villa Comunale, Naples 80121, Italy
2
The Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) program is an international activity to coordinate national
and regional research on HABs. GEOHAB conducts much of its work through its Core Research Projects (CRPs). A new CRP, on
HABs in Benthic Systems, is under development. An open science meeting on HABs in Benthic Systems, held in June 2010, brought
together scientists working on Gambierdiscus, Ostreopsis, Prorocentrum, and other benthic HAB species to identify research needed to
understand how these organisms proliferate and cause harm. This presentation will report on research priorities regarding the taxonomy,
distribution and ecology of benthic HABs. Some evidence indicates that distributions have increased; if so, why? Are benthic HABs ubiquitously present in low background numbers? What triggers transitions to harmful population levels? How many types (i.e., phenotypic,
genotypic, morphologic) exist, and how do these map to each other and to toxicity? What are optimal nutrient requirements of benthic
HABs? Does mixotrophy play a key role in their nutrition? What roles do nutrients play on toxicity? How might decline of coral reefs
related to increase of benthic HABs? Are benthic HABs benthic for their entire life cycles? Do their life cycles include cysts? A training
workshop held in conjunction with the meeting focused on sharing methods for collecting and processing benthic HAB samples.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 85
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
OBSERVATIONS ON THE INTERACTION BETWEEN
THE DINOFLAGELLATES NOCTILUCA SCINTILLANS
AND GYMNODINIUM CATENATUM: GRAZING ACTIVITY
AND GROWTH
Jose Bustillos-Guzmán1, C. J. Band-Schmidt2, D. López-Cortés1, F. E. Hernández-Sandoval1,
E. Nuñez-Vázquez1, I. Gárate-Lizárraga2
1
2
CIBNOR, Apdo. Postal 128, La Paz, B.C.S. 23000, Mexico. E-mail: [email protected]
CICIMAR-IPN, Apdo. Postal 592, La Paz, B.C.S. 23000, Mexico
During a bloom of N. scintillans and G. catenatum in Bahía de La Paz of the Gulf of California, we followed the physical and chemical
conditions and percentage of cells of N. scintillans that engulfed Gymnodinium catenatum. Additionally, live N. scintillans cells were
collected to determine under laboratory conditions the ingestion rate during a 5-day period when fed the paralytic toxin producer Gymnodinium catenatum. Three treatments were tested (concentration of G. catenatum at 312, 625, and 1015 cells mL-1) on ~0.53 N.
scintillans mL-1. Sea surface sea temperature clearly shows a transitional period from colder to warmer waters at moderate nutrient values.
Under natural conditions, 30% of N. scintillans cells contained G. catenatum cells in their vacuoles, this value increased, up to 80%,
under laboratory conditions. Ingestion rates were high but variable, with no clear tendencies among treatments; however, a general decrease in consumption occurred by the end of the experiments. These results clearly show that G. catenatum is highly consumed by N.
scintillans, leading to doubling of the population of N. scintillans. However, lower ingestion rates and mature trophonts that shed their
crust (cytoplasmic wall) that occurred at the end of the experiments suggest that a longer period of ingestion could have a negative effect
on the predator. The possible use of N. scintillans as a biocontrol is discussed.
85
HARMFUL COCHLODINIUM POLYKRIKOIDES BLOOMS:
GLOBAL EXPANSION AND ECOLOGICAL STRATEGIES
FACILITATING BLOOM FORMATION
Christopher J. Gobler, Ying Zhong Tang, Xiaodong Jiang, Darcy Lonsdale
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA.
E-mail: [email protected]
The past decade has witnessed a substantial expansion in the reported occurrences of HABs caused by the dinoflagellate Cochlodinium
polykrikoides. Prior to 2000, blooms had been primarily reported in Southeast Asia and were particularly destructive in South Korea
where they caused more than $100M USD in annual fisheries losses during the 1990s. Since this time blooms have been reported on
the east and west coasts of North America, in Central America, as well as in China, Malaysia, the Philippines, the Arabian Gulf, and the
Black Sea. We will review this recent expansion of C. polykrikoides blooms and present a comprehensive characterization of ecological
strategies utilized by North American C. polykrikoides populations to form HABs. We will specifically present data from five years of novel
laboratory and field experiments which demonstrate C. polykrikoides blooms are promoted and sustained by: 1) potent allelopathic inhibition
of a broad range of competing phytoplankton including natural populations and ten species from five algal classes; 2) inhibition of grazing
by inducing rapid mortality in a diverse set of predators including copepods, microzooplankton, larval bivalves, and planktivorous fish; 3)
chain formation as a grazer-induced defense mechanism; and 4) a flexible nutrient acquisition strategy which permits the efficient growth
on, and utilization of, multiple organic and inorganic forms of nitrogen. Finally, we will compare some of these strategies among strains
of C. polykrikoides isolated from the USA, Mexico, Japan, and Korea, permitting global conclusions to be drawn.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 86
THALASSORHEOLOGY, ALGAL BLOOMS
AND VERTICAL FLUX
Ian R. Jenkinson
Chinese Academy of Sciences, Institute of Oceanology, 7 Nanhai Road, 266071 Qingdao, P.R. China.
E-mail: [email protected]
Thalassorheology is the study of the deformation properties of seawater and other natural waters. In the Yellow Sea (YS), algal blooms
occur, particularly in deep chlorophyll maxima (DCMs) in generally stratified water. Primary production is high. This gives the beneficial
effects that fish landings among the highest in the world. Also, sinking organic aggregates probably fuel a powerful beneficial carbon
pump sequestering a portion of the CO2 fixed by phytoplankton in the photic zone. Harmful effects in the YS, however, are increasing
zones of hypoxia, and the occurrence of HAB plankton species, as well as thick floating mats of Enteromorpha, all probably favoured by
increasing nutrient input. Seawater viscosity consists of two components, one contributed by water and salt, the other mainly by phytoplankton exopolymeric substances (EPS). The EPS component may by changing pycnocline dynamics and water deformation around
organisms, including gill ventilation in fish, so the YS is a promising place for thalassorheological research. Furthermore, the rheological
properties of marine organic aggregates (MOAs), together with flocculation dynamics, determine size distributions of MOAs and their
sinking characteristics. Our project is to measure the rheological properties of MOAs, and we are determining their dependence on length
scale using capillary tubes of different diameters. At the Hong Kong Harmful Algae meeting in 2008, Jenkinson suggested a need for
rheological expertise in research teams on HABs. In Qingdao, an interdisciplinary team is now being formed with expertise in plankton,
rheology, chemical engineering and surface science.
86
THE COMPARATIVE METHODS FOR THE STUDY
OF HARMFUL ALGAE: RECENT ADVANCES FROM
THE GEOHAB PROGRAMME
Raphael M. Kudela1, Ken Furuya2, Robin Raine3, Suzanne Roy4
1
University of California Santa Cruz, 1156 High Street, Santa Cruz, CA, USA. E-mail: [email protected]
Department of Aquatic Bioscience, University of Tokyo, Japan
3
Martin Ryan Institute, National University of Ireland, Galway, Ireland
4
Institut des Sciences de la Mer, Université du Québec à Rimouski, 310 Allée des Ursulines, Rimouski, QC, G5L 3A, Canada
2
The Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) programme is an international effort sponsored by IOC and
SCOR that coordinates and builds on related national, regional and international efforts in HAB research within an ecological and oceanographic context. A central component of GEOHAB is to identify areas where multidisciplinary and comparative approaches can contribute
to the advancement our understanding of HAB dynamics. This is achieved through establishment of Core Research Projects (CRPs) and
Regional Programmes. Publication of Project Reports from these groups provides a synthesis of knowledge and needs, as well as a roadmap
for the international community. GEOHAB currently supports five Core Research Projects and coordinates with GEOHAB Asia, a Regional
Programme. We are pleased to announce that three new reports have been released in 2010, from the Fjords & Coastal Embayments and
Stratified Systems CRPs, and from GEOHAB Asia (all available at http://www.geohab.info). This presentation will report on research accomplishments and priorities regarding key aspects of biogeography, biodiversity, adaptive strategies of target species, eutrophication,
physical forcing, modeling, and observations for these projects. Finally, we will discuss the role of GEOHAB as we approach the final years
of this international endeavor. A separate presentation will report on the development of the newest CRP, on HABs in Benthic Systems.
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IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
ORAL PRESENTATIONS
RED TIDES AND ANOXIA: AN EXAMPLE FROM
THE SOUTHERN BENGUELA CURRENT SYSTEM
Grant Pitcher, Trevor Probyn
Fisheries Management, Private Bag X2, Rogge Bay 8012, Cape Town, South Africa. E-mail: [email protected]
The expansion of hypoxia and anoxia represent major perturbations to the diversity, structure and functioning of marine ecosystems. It is
recognized that dissolved oxygen is a property of the ocean that has changed dramatically during recent decades, with oxygen deficiencies
having increased in frequency, duration and severity. Anthropogenic influences such as eutrophication are considered important causal
factors for this increase, and warming associated with climate change is likely to further intensify shelf hypoxia and anoxia. Of the world’s
four major eastern boundary current systems, low oxygen waters are best known in the Humbolt and Benguela, although declining oxygen
concentrations have recently been reported in the California current system. This study reports on the scales of variability of dissolved oxygen
on the inner shelf of the southern Benguela. The oxygen regime is examined specifically in relation to the development of a high biomass
bloom of the dinoflagellate Ceratium balechii and an anoxia-induced mass mortality. A time series of bottom water oxygen exhibited pronounced variation with nearshore hypoxia driven by the upwelling of oxygen depleted waters onto the shelf, while bottom water oxygenation
episodes were observed in association with downwelling events and winter mixing. These remotely forced advection events ensure a close
coupling of dissolved oxygen concentration and water temperature. However there is a decoupling of the oxygen-temperature relationship
during the mortality event, attributable to local oxygen depletion triggered by the decay of the C. balechii bloom and consequent rise in respiratory demand. Oxygen consumption was sufficient to cause anoxia throughout the water column.
87
TOXIN ACCUMULATION IN BENTHIC POPULATIONS
UNDER BLOOMS OF DIFFERENT HARMFUL ALGAL
SPECIES
Sofia Reizopoulou1, Evangelia Strogyloudi1, Antonia Giannakourou1, Edna Graneli2, Kalliopi Pagou1
1
Institute of Oceanography, Hellenic Centre for Marine Research, P.O. Box 712, 19013 Anavyssos, Attica, Greece.
E-mail: [email protected]
2
Marine Sciences Department, University of Kalmar, S-39182 Kalmar, Sweden
Although the direct effects of HAB toxins on humans are known, it is virtually unknown what pathways different HAB toxins take in the food
web during and after a HAB event. One of the scientific objectives of the European project FATE was to investigate how HAB toxins are
accumulated in benthic populations. The present study summarizes the results of toxin accumulation in macroinvertebrates during Dinophysis
acuminata and Pseudo-nitzschia sp. blooms, in Thermaikos Gulf and Ria de Vigo, respectively. Toxin accumulation was investigated in
cultured mussels and harvested natural benthic species sampled from the soft bottom sediments. Okadaic acid (OA) contamination in Thermaikos Gulf was higher in Mytilus galloprovincialis. Among the natural populations the highest amounts of OA have been found in Modiolus
barbatus, while scallops, clams, and polychaetes contained only traces or undetectable values of OA. Domoic acid (DA) contaminated all
the natural populations in Ria de Vigo, while M. galloprovincialis showed undetectable or minimal toxicity. Among the benthic populations
Hinia reticulata presented the highest toxin concentration, while high amounts of DA have also been detected in Cucumaria sp. and Aporrhais
pespelecani. Marine toxins generally can enter the benthic food web, among which DA is an especially potent food web transferred toxin
entering the benthic compartment. The high DA accumulation in benthic populations seems to be related to the enhanced delivery of the
toxin to the benthic environments. Zoobenthos can then act as a vector for transferring DA to higher trophic levels.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 88
ORAL PRESENTATIONS
UNDERSTANDING THE POTENTIAL IMPACTS
OF GREEN TIDES IN THE YELLOW SEA, CHINA:
AN EXPERIMENTAL APPROACH
Chao Wang, Rencheng Yu, Mingjiang Zhou
The Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology,
Chinese Academy of Sciences, 7, Nanhai Road, Qingdao, 266071, P.R. China. E-mail: [email protected]
In the summer of 2007, a large-scale bloom of green alga Ulva (Enteromorpha) prolifera (the so-called “green tide”) first occurred in the
Yellow Sea, China. In 2008, green tide appeared again and huge amount of green algae accumulated along the coast of Qingdao, leading
to the mass mortality of cultured animals and a deep concern on the potential impacts of green tides on marine ecosystems. To understand
the impacts of green tides, the effects of green alga U. prolifera on the growth of 5 microalgal species, the survival and reproduction of
misid shrimp Neomysis awatschensis, the egg hatching and development of scallop Chlamys farreri and oyster Crassostrea gigas, and the
survival of abalone Haliotis discus hannai and sea cucumber Apostichopus japonicus, were studied. Based on the experimental results, it
can be suggested that: (1) the rapid proliferation of green algae can inhibit the growth of phytoplankton through the effects of both allelopathy
and resource competition. And the long-lasting green tides may threat the survival and reproduction of zooplankton; (2) decomposition of
the green algae will consume dissolved oxygen in seawater and release sulfide and ammonium, which might lead to the toxic effects to
marine organisms and promote the formation of red tides; (3) animals cultured in the ponds are more vulnerable to the occurrence of green
tides due to the low exchange rate of seawater in the ponds. However, more investigations and researches are still needed to elucidate the
potential impacts of green tides on marine ecosystems.
88
MASSIVE ACUTE FINFISH MORTALITY DUE TO THE
TOXIC RAPHIDOPHYTE MICROALGA CHATTONELLA SP.
INFESTATION IN GREEK COASTAL WATERS
Mary Yiagnisis1,3, Kalliopi Pagou1, Athanasios Prapas2, George Rigos1, Ioanna Varkitzi1,
Chrysanthi Nikoloudaki1, Fotini Athanassopoulou3
1
Hellenic Centre for Marine Research, P.O. Box 712, PC 19013, Attica, Greece. E-mail: [email protected]
Fish Pathology Department, Centre of Athens Veterinary Institutions, Ministry of Rural Development and Food,
Agia Paraskevi, Athens, Greece
3
Laboratory of Ichthyology & Aquatic Animal Health, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece
2
Rhaphidophyte species of Chattonella sp. were involved in severe harmful algal blooms phenomena in Greece. The highly toxic alga Chattonella sp. effectively chokes fish within a few hours by forming thick mucus or other toxic compounds damaging their gills and/or neurotoxins,
which affect the nervous system. On late February 2009, dead wild and farmed marine fish were reported from all sides of the Maliakos
Gulf, in central Greece. The phenomenon was lasted till May and the result was many tons of dead fish. Examination of moribund dead fish
revealed inflated swim bladder, thick mucus on their gills and phytoplankton in the gills that was identified as Chatonella sp. and was the
reason of the mass kill. Also examination of dead Mugilidae fish from Thermaikos Gulf revealed infestation by Chatonella sp. during the
same period. A lot of different species of fish were infested in both areas, like Mugilidae, Pagellus erythrinus, Sparus aurata, Dicentrarchus
labrax, Umbrina cirroca, Pagellus erythrinus.
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IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
POSTER PRESENTATIONS
EFFECTS OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM
MINUTUM AND THE TOXIC DIATOM PSEUDO-NITZSCHIA
MULTISERIES ON THE COPEPOD ACARTIA SPP.
Soultana Zervoudaki1, Isabel Maneiro2, Ioanna Varkitzi1, Castor Guisande3,
Epaminondas D. Christou1, Kalliopi Pagou1
1
Hellenic Centre for Marine Research, P.O. Box 712, Anavyssos, 19013 Athens, Greece. E-mail: [email protected]
Facultad de Ciencias, Universidad de Vigo, 36200 Vigo, Spain. Current address:
Área Ecología, Facultad de Ciencias, A Zapateira, Universidad de A Coruña, 15170 A Coruña, Spain
3
Facultad de Ciencias, Universidad de Vigo, 36200 Vigo, Spain
2
The harmful effects of toxic microalgae on zooplankton grazers and the fate of toxins through the food webs are key issues for Harmful Algal
Blooms research. Two experimental studies were carried out in Vigo (Spain) and Athens (Greece) in order to determine the effects of Amnesic
Shellfish Poisoning toxins (Domoic Acid: DA by Pseudo-nitzschia multiseries) and Paralytic Shellfish Poisoning toxins (Gonyautoxins: GTXs by
Alexandrium minutum) on feeding behaviour and reproductive success of two co generic species of Acartia (A. clausi and A. tonsa). In Vigo, the
copepod A. clausi was fed with single and mixed diets of the toxic diatom P. multiseries and the non-toxic diatom Pseudo-nitzschia delicatissima.
Ingestion rate was similar for the toxic and non-toxic Pseudo-nitzschia species, indicating no selective feeding behaviour against P. multiseries.
The DA ingested by the copepods did not affect feeding behaviour, egg production or hatching success. In Athens, the copepod A. tonsa was
fed with mixed diets of the toxic dinoflagellate A. minutum and the non-toxic dinoflagellate Prorocentrum micans. We found a positive correlation
between A. minutum concentration and copepod ingestion, whereas egg production and hatching success decreased with increasing food
toxicity. Our results indicate that DA and GTXs do not affect the feeding behaviour of copepods but have different effects on egg production and
hatching of the above copepods. The different responses to toxins reflect a complicated pattern of copepod vital rates against HABs.
89
SODIUM CHANNEL VARIANT IN THE SOFTSHELL
CLAM MYA ARENARIA OFFERS NATURAL RESISTANCE
TO TOXIC ALGAL BLOOMS
Laurie Connell1, V. Monica Bricelj2, Jennifer Phillips1
1
School of Marine Sciences, University of Maine, Orono, Maine 04469, USA.
E-mail: [email protected], [email protected]
2
Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, New Jersey 08904, USA
Paralytic shellfish poisoning (PSP) caused by human consumption of bivalve molluscs that feed on toxic dinoflagellates Alexandrium spp. is
a recurrent problem along the coast of the Gulf of Maine. The softshell clam Mya arenaria is a species native to that area that is commonly
affected by paralytic shellfish toxins, necessitating extensive annual closures of shellfish beds and resulting in significant economic losses.
A variant of the Na+ channel gene (Domain II pore region) in certain M. arenaria individuals dramatically decreases affinity at the saxitoxinbinding site, allowing these resistant clams to achieve much higher toxin levels than those of sensitive clams without this variant. Prior work
has discovered that the incidence of this Na+ channel variant in clam populations is higher in locations that experience frequent, intense
blooms of Alexandrium spp., compared to those that do not experience such blooms. This study surveyed M. arenaria populations from locations along the eastern seaboard and the northeast coast of North America that varied in their history of PSP. Domain II of the Na+
channel pore region was sequenced and the populations were compared for percent of homozygous (resistant or sensitive) and heterozygous
individuals. These data were then related to the clams’ PSP history and to spat planting practices. Results of this study revealed that the resistant genotype occurs in many clam populations including those that are geographically distant, thus suggesting that this variant may have
occurred multiple times in separate populations.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 90
THE INFLUENCE OF NATURALLY OCCURRING
HARMFUL ALGAL TOXINS ON KEY NORTH ATLANTIC
COPEPOD SPECIES
Kathryn Cook, Ana Amorim, Shona Kinnear, Jean-Pierre Lacaze, Eileen Bresnan, Elizabeth Turrell
Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB, UK. E-mail: [email protected]
Understanding the ecosystem, its components and how they interact is fundamental for the future management of our seas. Copepods are
a critical link in marine food webs between phytoplankton and higher trophic levels, and many harmful algal toxins can affect copepod
feeding, mortality and recruitment rates. There are two primary methods by which algae can transmit their toxins to copepods: by active
release into the surrounding medium (dissolved toxins), or by passive release during cell damage through grazing (ingested toxins). This
study aims to investigate the impact of naturally occurring harmful algal toxins on the key copepod species found at the Stonehaven inshore
ecosystem monitoring site in the western North Sea (http://www.frs-scotland.gov.uk/). Analysis of historic data identified five copepod
species potentially vulnerable to influences from toxin producing phytoplankton genera. Direct exposure to dissolved okadaic acid, domoic
acid and saxitoxin, at ecologically relevant and elevated concentrations, had no effect on the survival of any of the copepod species studied.
Copepod feeding behaviour on toxin producing Alexandrium tamarense (Group I strain) was species specific. Temora longicornis and Centropages spp. fed on all strains of A. tamarense regardless of toxicity whilst Acartia spp. and Pseudocalanus elongatus avoided all A.
tamarense strains. Calanus spp. seem able to distinguish and avoid toxic A. tamarense but ingested non-toxic strains. These differences in
feeding behaviour could affect copepod growth rates, and therefore have implications for the pelagic ecosystem productivity.
90
ACCUMULATION AND METABOLIC EFFECTS OF PST
TOXINS PRODUCED BY ALEXANDRIUM OSTENFELDII
ON BLUE MUSSELS DURING A BLOOM IN ÅLAND
ARCHIPELAGO, SW FINLAND
Sari Lehtinen1, Outi Setälä2, Anke Kremp3, Lehtonen Kari3, Katrin Erler4
1
Tvärminne Zoological Station, FI-10900, Hanko, Finland
2
Marine Biology, Dept. Environmental Sciences, P.O. Box 65, University of Helsinki, FI-00014, Finland.
E-mail: [email protected]
3
Finnish Environment Institute, Marine Research Centre, FI-00251, Helsinki, Finland
4
Institute of Nutrition, University of Jena, Dornburger Str. 25, D-07743, Jena, Germany
Dense blooms of the dinoflagellate Alexandrium ostenfeldii have become a regular phenomenon in shallow coastal waters of Åland archipelago in SW
Finland. The species produces neurotoxins known to cause paralytic shellfish poisoning around the world. We conducted the first study in Finland to
measure accumulation and effects of toxins in the blue mussel Mytilus edulis both in the field and under laboratory conditions. In the field experiment
clean mussels fed with diatoms were placed in cages at the bloom site and sampled twice during the season, in August (bloom peak) and again in
September. Plankton samples were taken weekly to follow the bloom dynamics. The highest concentrations of PSTs were found in mussels during
the bloom peak in mid-August: 128 μg GTX3, 64 μg GTX2 and 36 μg STX /100 g dried mussel tissue, approaching food safety limits of Nordic
countries. In September when the bloom had passed mussels still contained 60-90% of maximum toxin values. A strain of A. ostenfeldii isolated from
the bloom site was fed to blue mussels in two concentrations for two weeks in an aquarium experiment. With the exception of STX the maximum
PST levels were achieved after one week. After two weeks of feeding on A. ostenfeldii the ratios of different toxins were altered in the mussels. GTX3
concentrations were much higher in mussels compared to other toxins, possibly indicating conversion of toxins in mussels. Enzyme activities in mussels
were measured to monitor effects of exposure to PSTs. Glutathione-S-transferase levels remained constant whereas catalase seemed to correlate
with overall food concentration. Acetylcholinesterase levels were adversely affected by toxin levels, indicating a neurotoxic effect.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 91
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
POSTER PRESENTATIONS
DINOFLAGELLATE TOXINS IN TWO WARM-TEMPERATE
ESTUARIES; RANGAUNU AND PARENGARENGA HARBOURS, NORTHLAND, NEW ZEALAND
Lincoln A. MacKenzie, Andrew Selwood, Lesley Rhodes
Cawthron Institute, 98 Halifax Street, Nelson, New Zealand 7010. E-mail: [email protected]
The analysis of a variety of environmental samples, and the installation of passive solid phase adsorption devices (SPATT) in Rangaunu and
Parengarenga Harbours revealed the pervasive influence of bioactive polyether compounds secreted by benthic dinoflagellates within the
mangrove and sea-grass habitats. Pinnatoxins (PnTx) analogues PnTx-E and PnTx-F and okadaic acid (OA) and its esters were the most
abundant, though traces of other polyether compounds (dinophysistoxins, pectenotoxin, spirolides), were also detected. In sediments, macroalgal mats and micro-algal films, the parent compound PnTx-F was predominate whereas in bivalves and gastropods PnTx-E and PnTx-F
were either present in equivalent amounts or there was a predominance of the former. Esterified OA was the predominant form of this toxin
in the common sea hare Bursatella leachii however OA-toxins were only about 14% as abundant as the combined PnTx analogues in this
animal. Conversely levels of PnTxs accumulated within the SPATT bags were only 50% of total-OA, about 10% of which was in the form
of OA-esters. The levels of total-PnTxs in C. gigas were about 8% of that observed in B. leachii (201 and 2,579 μg/kg respectively).
Although there is an abundance of soluble OA in this environment, neither OA nor OA-esters were observed in the cultured oyster Crassotrea
gigas, presumably because they are rarely exposed to Prorocentrum lima cells in the water column.
91
FATE OF AZADINIUM SPINOSUM AND ASSOCIATED
AZASPIRACIDS IN MARINE PLANKTONIC FOOD WEBS
Philip Messtorff, Urban Tillmann, Bernd Krock, Allan Cembella
Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany. E-mail: [email protected]
Azaspiracids (AZAs) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents
of shellfish poisoning. A biological source was recently unambiguously identified as a small photosynthetic dinoflagellate, described as Azadinium spinosum Elbrächter & Tillmann. In spite of detailed knowledge on structural analogues of AZAs, very little is known about the fate
of the toxin and its possible transfer pathways through the marine food web. Azaspiracids measured in unicellular heterotrophs such as the
dinoflagellates Protoperidinium crassipes and the tintinnid Favella ehrenbergii collected from field samples indicate protistan grazing to be
an important toxin-transfer mechanism. We are addressing the current lack of experimental evidence linking Azadinium to higher trophic
levels and dynamics of azaspiracid accumulation and metabolism with cultures of A. spinosum experimentally exposed to various plankton
assemblages. These studies include both the qualitative response of potential grazers after inoculation of natural North Sea plankton with
cultured A. spinosum and the estimation of growth and grazing rates of selected protistan grazers supplied with pure A. spinosum and/or
mixed algal cultures. We report on the differential species-specific grazing upon Azadinium.
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IMPACT OF CYANOBACTERIA ON ZOOPLANKTON
DYNAMICS IN THE SENEGAL RIVER HYDROSYSTEM
Juana Mireya Mendoza-Vera1, Samba Ka2,3, Marc Bouvy4, Corinne Cuoc5,
Xavier Moreau5, Marc Pagano1, Nicolas Pech5
1
UR 167, IRD - Laboratoire d’Oceanographie Physique et Biogeochimique (LOPB), UMR 6535, Centre d’Oceanologie
de Marseille, Campus Luminy Case 901, 13288 Marseille, Cedex 9, France. E-mail: [email protected],
[email protected]
2
Institut of Marine Biology, National Taiwan Ocean University, Keelung 202, Taiwan
3
Departement de Biologie Animale, Universite Cheikh Anta Diop, BP 5005, Dakar, Senegal
4
UMR 5119, Universite Montpellier II - CNRS-IRD-Ifremer, CC 093 - Universite Montpellier II Place Eugene Bataillon,
34095 Montpellier, Cedex 5, France
5
UMR 6116, Universite de Provence, 3 Place Victor Hugo, 13001 Marseille, France
The cyanobacterial blooms of potentially toxic cyanobacteria (Cylindrospermopsis raciborskii, Anabaena solitaria, Anabaena flos-aquae and Microcystis aeruginosa) on the lower delta of the Senegal river pose a serious threat to the water resource of the riparian countries. A study was carried
out to analyze the effects of these cyanobacteria on zooplankton communities with a coupling of in situ and experimental approaches. An in situ
survey explored the space-time distribution and dynamics of the phyto- and zooplankton communities in 13 representative sites (reservoirs, ponds,
irrigation channels, etc.), at three seasons in the Senegal River lower delta. A Multiple Factor Analysis (MFA) of these data illustrated negative relationships between the cyanobacteria abundance and the main zooplankton groups (rotifers, cyclopids and cladocerans). Grazing experiments
with copepods, cladocerans and rotifers showed that the colonial cyanobacterium, M. aeruginosa, was not consumed, while the filamentous
cyanobacteria, Cylindrospermopsis raciborskii and Anabaena spp., were ingested and/or split with a variable efficiency according to the grazer
species. Toxicity experiments clearly indicate negative effects of the cyanobacteria on survival and growth of cladocerans and copepods. Variable
responses were observed according to the zooplankton species. Muscular and intestinal degradation as well as modification of the acetylcholinesterase activity were highlighted for copepods incubated with C. raciborskii or A. flos aquae. However, several resultants suggest these
effects are probably not related to classical cyanotoxins, but to secondary metabolites excreted. Thus evidence compiled in our study highlights
the potential negative impact of cyanobacterial blooms on the zooplankton communities in the water bodies of the Senegal River hydrosystem.
92
IMPACT OF OSTREOPSIS OVATA ON MARINE
BENTHIC COMMUNITIES: ACCUMULATION OF
PALYTOXINS IN SUFFERING MUSSELS, SEA URCHINS
AND OCTOPUSES FROM ITALY
Anna Milandri1, Alfiero Ceredi1, Elena Riccardi1, Laura Gasperetti2, Francesca Susini2,
Monica Casotti3, Luciana Faiman4, Silvia Pigozzi1
1
Fondazione Centro Ricerche Marine, V.le A. Vespucci 2, PC 47042, Cesenatico (FC), Italy.
E-mail: [email protected]
2
Istituto Zooprofilattico Lazio e Toscana, S.S. Abetone e Brennero 4, PC 56123, Pisa, Italy
3
ARPA Toscana, Dipartimento di Massa Carrara, Via del Patriota 2, PC 54100, Massa, Italy
4
Servizio Igiene degli Alimenti di Origine Animale, ASL1 Massa e Carrara, Zona delle Apuane,
Viale Democrazia 44, PC 54100, Massa, Italy
Since 1998 blooms of the potentially toxic benthic dinoflagellate Ostreopsis ovata have interested sheltered marine areas along the coast of
Tuscany (Italy). These phenomena caused stress signals in the benthic community, affecting both sessile and free-moving organisms. Sea urchins
were observed to lose their spines, whilst sea stars showed an anomalous position of the arms. In July-August 2008, the area was impacted by
a new massive bloom of O. ovata, which concentration reached two maximum peaks: 88,760 cells/L on July 24th and 95,200 cells/L on August
26th. Marine organisms (mussels, sea urchins and octopuses) suffering from the event, were taken and tested for the presence of palytoxin-group
toxins (PlTXs) using both LC-MS/MS and the haemolysis assay. Both techniques confirmed that all samples were contaminated by PlTXs. Ovatoxin-a was the dominant compound according to highly specific LC-MS/MS analyses. The accumulation of toxins along the food web might
have played a role in the observed extensive ecosystem suffering (e.g. octopuses were found stranded). On the other hand, it is recognized that
blooms of O. ovata typically occur in shallow water sheltered marine areas, where high temperatures and anoxia represent adverse conditions for
the benthic community. The contamination of edible fauna by PlTXs poses a serious risk for the consumers and should be further investigated.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 93
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
POSTER PRESENTATIONS
SELECTIVE FEEDING OF THE MUSSELS MYTILUS
GALLOPROVINCIALIS AND PINNA NOBILIS
ON NATURAL PHYTOPLANKTON COMMUNITY
IN THE MALI STON BAY (CROATIA)
Živana Ninčević-Gladan, Ivona Marasović, Ana Jelačić, Sanda Skejić, Melita Peharda, Daria Ezgeta-Balić
Institute of Oceanography and Fisheries. E-mail: [email protected]
Since, large rates of mortality are not usually observed in cultivated bivalves during harmful algal blooms outbreaks we assumed that
marine bivalves used ingested toxins for protection against their own predators. Because of bivalves ability to sort particles based on size
and quality we examined the ability of mussels Mytilus galloprovincialis and Pinna nobilis to preferentially ingest or reject toxic phytoplankton
in situ by comparing the phytoplankton composition of water and bivalve gut contents. This in situ experiment were done in Mali Son Bay
which is the most important shellfish cultivate area in Croatia in a period from May 2009 to May 2010. Mussel M. galloprovincialis preferentially selects dinoflagellate from natural phytoplankton community. Among dinoflagellate they prefer Dinophysis and Prorocentrum in
relation to Ceratium and Protoperidinium, which were abundant in the phytoplankton community. During the extensive bloom of Dinophysis
fortii significant difference in ingestion of D. fortii species between small and large mussels P. nobilis have been observed. These
differences could be attributed to differences in digestive capability of small and large individuals.
93
STUDY OF NUTRITION OF PHYTOPHAGOUS FISH FED
COCHLODINIUM POLYKRIKOIDES PHYTOPLANKTON
(RED TIDE) AND CLINICAL SYNDROME ON THE PUBLIC
HEALTH IN THE TIDAL NORTHWEST PERSIAN GULF (IRAN)
N. Nassirabady
Department of Microbiology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. E-mail: [email protected]
Red tides in the Persian Gulf are a marine phenomenon in which water is stained a red, brown, or yellowish color because of the temporary
abundance of a particular species of pigmented dinoflagellates (these events are known as "blooms"). Cochlodinium polykrikoides, a planktonic
algal species and a common ‘red water’ bloom species (sometimes to millions of cells per litre) species in the Persian Gulf and Oman Sea. The
purpose of this research is to understand environmental conditions and effects where C. polykrikoides proliferate in a shallow coastal located
in the Northwest Coast of the Persian Gulf (Iran). An observation plane was chartered to overfly the coast in the reportedly affected area on
Summer 2008. The three stations were chosen for their ecological difference of Bushehr Port with latitude 28°55’N and longitude 50°55’E.
Samples required for the study were collected by falcon bottle sterile (50cc) for phytoplankton determination from different depths (5m, 10m
and 15m) from three stations (Station Ι and Station II: Tidal & Subtidal Bushehr Ports; Station III: Open Sea Persian gulf). At each of three
stations, one 2-min surface phytoplankton haul (plankton net) was carried out over an estimated distance of 120 meters, with 150 μm mesh
size. The results show that shellfish and phytophagous fish, are unsafe to consume due to contamination from C. polykrikoides. These small
animals were eaten by small and large fish (phytophagous and carnivorous), which also accumulated the toxic chemical and then poisoned and
other organisms including fish-eating cormorants and coast birds that died in large numbers. A large-scale fish mortality in different coastal
villages and cities and hospitalization of over 50 school children due to illness (diarrhea and nausea) fish was reported. Red tides caused by C.
polykrikoides have severe impact on the ecosystem organisms (zooplankton, shellfish, fish, birds) and public health in these local waters.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:27 PM Page 94
MESOZOOPLANKTON GRAZING ACTIVITY DURING
A BLOOM DOMINATED BY PYRODINIUM BAHAMENSE
VAR. COMPRESSUM IN SORSOGON BAY, PHILIPPINES
Joseph Dominic H. Palermo, Aletta T. Yñiguez, Marianne Camoying, Christine Barrera, Aldwin Almo,
Christopher Mendoza, Lourdes J. Cruz, Rhodora V. Azanza
The Marine Science Institute, University of the Philippines, Diliman, 1101 Quezon City, Philippines.
E-mail: [email protected]
Studies have mostly dealt with physico-chemical interactions leading to the formation and decline of harmful algal blooms in the Philippines
while information on the role of grazers and their ability to transfer toxins is limited. We have conducted grazing experiments in six stations
in Sorsogon Bay to elucidate the possible roles of mesozooplankton during a bloom of Pyrodinium bahamense var. compressum. Gut
Pigment Content (GPC), Gut Passage Time (GPT), Ingestion Rate (IR), and Paralytic Shellfish Toxins (PST) of Lucifer hanseni and two
size classes, >200 and >500 μm, of Calanoida were measured using gut fluorescence method, 240 min starvation experiment, and
pre-oxidation HPLC analysis, respectively. We found that the most prominent mesozooplankton grazer was the > 200 μm Calanoida
(GPC 41±28 ng chlα μg body C-1, GPT 130±71 min, IR 34±38 ng chlα μg body C-1 hr-1, and PST 122 ng mg body C-1). While the >
500 μm Calanoida was an intermediate grazer (GPC 19±31 ng chlα μg body C-1, GPT 160±48 min, IR 9±17 ng chlα μg body C-1hr-1,
and PST 12 ng mg body C-1), L. hanseni was classified as relatively the least significant mesozooplankton grazer (GPC 4±3 ng chlα μg
body C-1, GPT 190±149 min, IR 5±7 ng chlα μg body C-1hr-1, PST 12 ng mg body C-1). Therefore, mesozooplankton grazers are shown
to be vectors in transferring PST to higher trophic levels during the P. bahamense var. compressum bloom in Sorsogon Bay, and could
be contributing to bloom decline through ingestion of vegetative cells.
94
EFFECTS OF THE FILAMENTOUS CYANOBACTERIUM
NODULARIA ON FITNESS AND FEEDING BEHAVIOR
OF YOUNG-OF-THE-YEAR (YOY) EURASIAN
PERCH (PERCA FLUVIATILIS)
Karl-Johan Persson, Patrik Stenroth, Catherine Legrand
School of Natural Sciences, Marine Science Center, Linnaeus University, S-39182 Kalmar, Sweden.
E-mail: [email protected]
During the 1990’s a reduction in recruitment of young-of-the-year (YOY) perch (Perca fluviatilis) occurred along the Swedish East coast
of the Baltic Sea. Habitat loss due to eutrophication and cascading food web effects due to overfishing of cod (Gadus morhua), are
factors suggested to have affected recruitment of coastal fishes. Concurrently, large blooms of filamentous cyanobacteria, also shown
to influence fish fitness, have increased in both the Baltic Proper and in coastal waters. This study examined whether extended exposure
to toxic and non-toxic filamentous cyanobacterium Nodularia affect YOY perch growth and feeding behavior (attack and feeding rates)
under simulated bloom conditions (30 days at 50 μg chl a L-1). Specific growth rate (SGR), the somatic condition index (SCI) and the lipid
content of YOY perch (10-12 weeks old) were significantly lower in perch exposed to Nodularia compared to fed controls (no Nodularia)
but higher than in food limited controls. YOY perch exposed to non-toxic Nodularia displayed a higher attack rate than perch living in
Nodularia free controls in 2 out of 3 trials. This study reveals that both cyanobacterial toxicity and turbidity have the potential to reduce
the growth and energy storage of YOY perch and thereby influence survival rates.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:28 PM Page 95
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
POSTER PRESENTATIONS
SPATIO-TEMPORAL DISTRIBUTION AND TROPHIC TRANSFER
OF CARIBBEAN CIGUATOXINS, PRECURSORS, AND RELATED
POLYETHER COMPOUNDS IN THE U.S. VIRGIN ISLANDS
Alison Robertson1, H. Ray Granade1, Ann Abraham1, Jennifer I. Hooe-Rollman1, Mindy L. Richlen2, Tyler B. Smith3,
Donald M. Anderson2, Steven M. Plakas1, Robert W. Dickey1
1
Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, Dauphin Island, Alabama 36528, USA.
E-mail: [email protected]
2
Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA
3
University of the Virgin Islands, St. Thomas, U.S. Virgin Islands 00802, USA
Ciguatoxins (CTXs) are cyclic polyether toxins derived from benthic marine dinoflagellates such as Gambierdiscus spp. Ciguatera fish poisoning
(CFP) results from consumption of CTX-contaminated finfish and is characterized by neurological, gastrointestinal, and cardiovascular symptoms.
The Virgin Islands represent a ciguatera “hot spot”, with a long history and high incidence of CFP. This has had a significant impact on human
health and artisanal fisheries in the region. As part of an ongoing collaborative study, this research aims to identify precursors and toxin congeners
present in epiphytic dinoflagellate assemblages, and characterize their trophic transfer and biotransformation in herbivorous and predatory fish
species. Several sampling sites were selected in the waters surrounding St. Thomas, USVI, based on previous surveys showing high prevalence
of potential ciguatoxigenic dinoflagellate species. Phytoplankton and a selection of herbivorous fish species were collected from these areas
and subsequently extracted for CTX related analyses. A two-tiered approach for sample analysis was undertaken to estimate composite “toxicity”
and “toxin”. This included an optimized neuro-2A cytotoxicity assay for screening ion channel activity and LC-MS/MS for chemical profiling.
Results from these studies revealed unique chemical profiles related to CTX-like activity in dinoflagellate samples and fish collected during
regular intervals over the sampling period. Based on these data, we discuss the spatio-temporal distribution of Caribbean CTXs, gambiertoxins,
and related polyether congeners, and evaluate the relationships between profiles observed at various trophic levels. We will also present methods
of analysis for the detection of Caribbean CTX congeners, chemical isolation, and purification strategies.
95
FEEDING MYTILUS GALLOPROVINCIALIS
AND PARACENTROTUS LIVIDUS WITH OSTREOPSIS OVATA:
TOXICITY AND PATHOLOGICAL ASPECTS
Angela Sardo1, Rachele Rossi2, Vittorio Soprano2, Patrizia Ciminiello3, Ernesto Fattorusso3,
Paola Cirino1, Adriana Zingone1
1
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected]
Istituto Zooprofilattico Sperimentale per il Mezzogiorno, Via Salute 3, 80051 Portici, Naples, Italy
3
Dipartimento di Chimica delle Sostanze Naturali, Via Montesano 49, I-80131 Naples, Italy
2
Around the turn of the century, blooms of Ostreopsis ovata have become very popular among scientists and the general public. Besides
pathological problems in beach-goers, signals of stress or mortality have been detected in benthic organisms during blooms, while toxins
have been detected in marine animals used for human consumption. To elucidate the trophic relationship and impact of these toxic microalgae,
the sea-urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis were fed cultures and natural material of O. ovata from the Gulf
of Naples (Mediterranean Sea). Toxicity in the animals was tested with the mouse bioassay and with LC/-MS analysis. Sea urchins feeding
over 5-10 days on the red alga Asparagopsis taxiformis colonised by O. ovata generally did not manifest pathological reactions and became
toxic in some cases. However, the exposure of sea urchins to liquid cultures of O. ovata caused the loss of the spines and even mortality,
depending on microalgal cell concentrations. As for mussels, toxicity was detected in animals maintained in O. ovata cultures (106 cells ml-1)
for one day. In two-three days, however, mussels exposed to the same cell concentrations died and were highly toxic. In naturally toxic
mussels maintained in running 20μm-filtered seawater, toxin levels remained high for two weeks, declining after 17 days. Results of our experiments demonstrate that both sea urchins and mussels can feed on Ostreopsis ovata and can become toxic. However, the prolonged exposure to high cell concentrations of O. ovata produces permanent damages and mortality in the animals.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:28 PM Page 96
DOMOIC ACID ACCUMULATION IN MYTILUS
GALLOPROVINCIALIS: LABORATORY EXPERIMENTS
WITH PSEUDO-NITZSCHIA MULTISERIES
Evangelia Strogyloudi1, Antonia Giannakourou1, Ioanna Varkitzi1, Isabel Maneiro2, Anika Petermann3,
Castor Guisande2, Edna Graneli4, Kalliopi Pagou1
1
Hellenic Centre for Marine Research, Institute of Oceanography, P.O. Box 712, 19013, Attica, Greece.
E-mail: [email protected]
2
University of Vigo, Dept. of Ecology, Lagoas-Marcosende, 36310, Vigo, Spain
3
University of Jena, Dept. of Food Chemistry, Dornburger Strasse 25, D-07743, Jena, Germany
4
Linnaeus University, Faculty of Science and Engineering, S-39182, Kalmar, Sweden
The fate and transfer of domoic acid (DA) by the mussel Mytilus galloprovincialis was investigated in order to assess the possible pathways
of the toxin in these filter feeder organisms. Domoic acid accumulation was studied in cultured mussels collected from rafts in Ria de Vigo,
Spain. Mussels were fed on Pseudo-nitzschia multiseries cultures (1900 cells/ml-expI, 3800 cells/ml-expII, 6000 cells/ml-expIII) and P.
multiseries blooming in a phosphorus limited mesocosm (-P mes-expIV), during short-term experiments (6h). Toxin was measured in mussel
whole tissue and produced fecal material. Mean values of DA in mussel whole tissue, in all exposure experiments, were lower than the regulatory limit for public health. Toxin concentration in the mussel whole tissue was proportional to the toxin offered in food (r=0.79, p<0.05,
Spearman rank correlation) except for the lowest Pseudo-nitzschia cell concentration experiment (expI). In that experiment no toxin was detected in the mussel whole tissue probably due to the most effective digestion of food with nutritional value lower than in experiments III and
IV (6000 cells/ml and -P mes respectively). Toxin budgets presented a lot of variation mostly due to the high variability of DA content in fecal
material, although differences among experiments were not statistically significant (analysis of variance Kruskal Wallis, p<0.05, Tukey test).
Except for the lowest cell concentration (expI), mussels rejected part of the excess organic carbon of the ingested food as pseudofaeces enriched in DA. However the large percentage of missing ingested DA was assumed to be due to toxin metabolism within mussel body.
96
CONCURRENT EXPOSURE OF BOTTLENOSE DOLPHINS
(TURSIOPS TRUNCATUS) TO MULTIPLE ALGAL TOXINS
IN SARASOTA BAY, USA
Michael Twiner1,2, Spencer Fire1, Lori Schwacke3, Leigh Davidson1, Zhihong Wang1,
Steve Morton1, Steve Roth4, Brian Balmer5, Teri Rowles6, Randy Wells5
1
Marine Biotoxins Program, NOAA/National Ocean Service, 219 Fort Johnson Rd, Charleston, SC 29412, USA
2
Department of Natural Sciences, University of Michigan - Dearborn, 4901 Evergreen Rd, Dearborn, MI 48128, USA.
E-mail: [email protected]
3
Cooperative Center for Marine Animal Health, NOAA/NOS, Hollings Marine Laboratory, 217 Fort Johnson Rd, Charleston, SC29412, USA
4
NOAA/National Ocean Service, Coastal Ecology Program, 219 Fort Johnson Rd, Charleston, SC 29412, USA
5
Chicago Zoological Society, c/o Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA
6
NOAA Fisheries, Cooperative Center for Marine Animal Health, 1315 East-West Highway, Silver Spring, MD 20910, USA
Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to exposure to marine algal
toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population is frequently exposed to harmful algal blooms
of Karenia brevis and the neurotoxic brevetoxins (PbTx) produced by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested positive for two HAB toxins; PbTx and domoic acid (DA). We have determined over a ten-year study
period (2000-2009) that bottlenose dolphins are exposed to PbTx and/or DA on a nearly annual basis with 36% of all animals testing positive for
PbTx (n=118) and 53% positive for DA (n=83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/fluid. To
date there have been no previously published reports of DA in southwestern Florida marine mammals, however the May 2008 health assessment
coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of DA observed in seawater and live dolphin samples. Concurrently, both DA and PbTx were observed in potential prey fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was
identified in seawater, fish, sediment, snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters
including total white blood cell count (p=0.001) and eosinophil count (p<0.001). Our findings demonstrate that dolphins within Sarasota Bay are
commonly exposed to two algal toxins, and provide the impetus to further explore the potential long-term impacts on bottlenose dolphin health.
3. ABSTRACT BOOK (83-97)_Layout 1 10/22/10 2:28 PM Page 97
IMPACT OF HABS ON MARINE FOOD WEBS
AND ECOSYSTEM STRUCTURE AND FUNCTION
POSTER PRESENTATIONS
COPEPOD EXPERIENCING A MULTI-STRESS
SITUATION IN A FUTURE SEA
Anu Vehmaa1, Andreas Brutemark1, Jonna Engström-Öst1, Elena Gorokhova2, Hedvig Hogfors2, Towe Holmborn2
ARONIA Coastal Zone Research Team, Novia University of Applied Sciences & Åbo Akademi University,
Raseborgsväg 9, 10600, Ekenäs, Finland. E-mail: [email protected]
2
Department of Systems Ecology, Stockholm University, Svante Arrhenius väg 21A, SE-10691, Stockholm, Sweden
1
Cyanobacteria blooms are a natural phenomenon in the Baltic Sea, but bloom frequency has increased due to anthropogenic eutrophication. The co-occurring calanoid copepods have to some extent developed tolerance for blooms; the blooms are not lethal, but
can still lower the reproductive success. Global climate change is predicted to further increase the surface water temperature and decrease pH, as well as accelerate eutrophication in the Baltic Sea. All these factors alone, and especially in combination, challenge the
ability of copepods to adapt. In our experiment we studied copepod reproductive success in multi-stress situations mimicking the
future sea in the presence of toxic cyanobacterium Nodularia spumigena. We incubated the copepods with and without nodularin
producing N. spumigena, in present temperature and pH conditions, and in higher temperature and lower pH conditions as predicted
for year 2100. In each treatment, copepods were fed with a chlorophyte Brachiomonas submarina. We monitored copepod survival
and measured reproduction parameters: egg viability, egg production and egg hatching success. The physiological response of the
copepods was addressed by oxidative stress measurements. The results highlight the importance of considering multiple stress factors
when estimating future fitness of aquatic organisms.
97
SUBTLE BIOLOGICAL RESPONSES TO INCREASED CO2
CONCENTRATIONS BY PHAEOCYSTIS GLOBOSA
SCHERFFEL, A HARMFUL ALGAL BLOOM SPECIES
Yan Wang1, Walker O. Smith Jr.2, Xiaodong Wang3, Shao Shan Li4
1
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510632, China.
E-mail: [email protected]
2
Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, USA
3
College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China
4
College of Life Sciences, South China Normal University, Guangzhou 510631, China
Recent investigations into the role of carbon dioxide on phytoplankton growth and composition have clearly shown differential effects
among species and assemblages, suggesting that increases in oceanic CO2 may play a critical role in structuring lower trophic levels of
marine systems in the future. Furthermore, alarming increases in the occurrence of harmful algal blooms (HABs) in coastal waters have
been observed, and while not uniform among systems, appear in some manner to be linked to human impacts (eutrophication) on coastal
systems. Models of HABs are in their infancy and do not at present include sophisticated biological effects or their environmental controls.
Here we show that subtle biological responses occur in the HAB species Phaeocystis globosa Scherffel as a result of CO2 enrichment
induced by gentle bubbling. The alga, which has a polymorphic life history involving the formation of both colonies and solitary cells,
exhibited altered growth rates of colonial and solitary forms at [CO2] of 750 ppm, as well as increased colony formation. In addition, substantial modifications of elemental and photosynthetic constituents of the cells (C cell-1, N cell-1, potential quantum yield, chl a cell-1) occurred under elevated CO2 concentrations compared to those found at present CO2 levels. In contrast, other individual and population
variables (e.g., colony diameter, total chlorophyll concentration, carbon/nitrogen ratio) were unaffected by increased CO2. Our results
suggest that predictions of the future impacts of Phaeocystis blooms on coastal ecosystems and local biogeochemistry need to carefully
examine the subtle biological responses of this alga in addition to community and ecosystem effects.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 98
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 99
BIOLOGICAL INTERACTIONS:
ALLELOPATHY, MIXOTROPHY,
PARASITISM, SYMBIOSIS,
BACTERIA AND VIRUSES
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 100
ORAL PRESENTATIONS
ALGICIDAL ACTIVITY OF A MARINE BACTERIUM
MURICAUDA SP. KB05 AGAINST HARMFUL BLOOM
CAUSING ALGA COCHLODINIUM POLYKRIKOIDES
Bandamaravuri Kishore Babu, Si-Wouk Kim
Pioneer Research Center for Controlling of Harmful Algal Blooms (HABs), Chosun University, 375 Seosuk-dong,
Dong-gu, Gwangju 501-759, South Korea. E-mail: [email protected]
Cochlodinium polykrikoides is one of the major harmful dinoflagellate species, occurred almost every year in the southern coast of Korea.
In the present study, an algicidal bacterium against C. polykrikoides was isolated and identified as Muricauda sp. on the basis of morphological, biochemical and 16S rDNA sequences analysis. The culture filtrate of Muricauda sp. KB05 has showed 100% algicidal activity
against C. polykrikoides. Moreover the culture filtrate showed no impact on the growth of green algae such as Chlorella sp. and Dunaliella
salina. The algicidal activity was stable under broad range of pH and temperatures. Our results suggest that Muricauda sp. KB05 could
be used as ideal and potential biocontrol agent to control growth and reoccurrence of C. polykrikoides blooms. Further identification and
characterization of the bioactive compound from Muricauda sp. was under the process.
100
A HARMFUL MICROALGA THAT IMMOBILIZES
AND INGESTS COPEPODS
Terje Berge1, Louise K. Poulsen2, Morten Moldrup3, Niels Daugbjerg1, Per Juel Hansen3
1
Section for Evolution and Ecology of Aquatic Organisms, Dept of Biology, University of Copenhagen,
Ø. Farimagsgade 2D, 1353 Copenhagen K, Denmark
DTU-Aqua, National Institute of Aquatic Resources, Technical University of Denmark, Jægerborg Allé 1,
2920 Charlottenlund, Denmark
3
Section for Marine Biology, Dept of Biology, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark.
E-mail: [email protected]
2
The photosynthetic dinoflagellate Karlodinium is known to form massive blooms worldwide and often these are associated with fish kills.
Here we show that Karlodinum armiger can reverse the traditional trophic pathway from primary producers to copepods, by attacking,
immobilizing and engulfing the much larger metazoan grazers. Copepod immobilisation is fast but dependent on cell density of K. armiger,
suggesting the presence of a potent paralytic toxin. Karlodinium armiger immobilises copepods by direct cell-contact, before ingesting
parts of the copepod through a feeding tube. The common copepod Acartia tonsa was immobilised within a few hours and died 12 hours
after exposure to ecologically relevant bloom cell densities in the laboratory. Karlodinium armiger increases its growth rate when exposed
to copepods and most cells contain large visible food vacuoles following engulfment. Our results show a novel trophic pathway at the
base of planktonic food web which reverses the typical flux of organic matter. Behavioural observations of K. armiger have also revealed
a novel mechanism for faunal kills by this phototrophic microalga.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 101
BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
DINOFLAGELLATE-BACTERIAL INTERACTIONS:
A TWO-WAY STREET
Christopher Bolch1, Shady Amin2, Maria Albinsson1, Thaila Bejoy1, Carl Carrano2, Masako Matsumoto1,
Susan Blackburn3, Andrew Negri4, David Green5
1
National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania,
Locked Bag 1370, Launceston 7250, Tasmania, Australia. E-mail: [email protected]
2
Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Dr., San Diego,
California 92182-1030, USA
3
CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, 7001, Tasmania, Australia
4
Australian Institute of Marine Science, PMB 3, Townsville MC, Townsville 4810, Queensland, Australia
5
Scottish Association for Marine Science, Oban, Argyll, PA37 1QA, UK
Marine algal cells coexist and grow in the presence of a complex microbial community and are subject to stimulatory and/or inhibitory interactions that alter the physiology and behavior of both the algal and bacterial partners. Over several years we have been investigating growth
stimulation of the dinoflagellates Gymnodinium catenatum, Scrippsiella trochoidea and Lingulodinium polyedrum using dinoflagellate-bacteria
experimental models containing simplified bacterial communities. While the bacterial partners benefit from using dinoflagellate-derived organic
carbon, our experiments show that the algal cells also benefit from their interaction with particular marine bacteria. The interactions are
essential for growth of some dinoflagellates species and can be highly species-specific. Our data also indicate a complex link between that
algal genotype and the bacterial community composition that may be mediated through subtle differences in organic carbon exuded by
different dinoflagellate genotypes. Studies of bacterial interactions using Scrippsiella as a model indicate that some growth stimulating bacteria
produce iron-binding siderophores that increase availability and uptake of iron by dinoflagellate cells. However, work with G. catenatum suggests other mutualistic interactions and mechanisms may be equally important in maintaining dinoflagellate-bacteria partnerships.
101
CHARACTERIZATION OF THE ALGICIDAL ACTIVITY
OF SHEWANELLA IRI-160
Kathryn J. Coyne, Mark E. Warner
University of Delaware College of Earth, Ocean and Environment, 700 Pilottown Rd., Lewes, DE 19958, USA.
E-mail: [email protected]
Algicidal bacteria are thought to regulate the abundance of algal species in the environment with varying specificity. A bacterium isolated
from Delaware’s inland bays, DE, USA, was shown to inhibit the growth of dinoflagellates, while having no negative impact on other algal
species (Hare et al., 2004). We investigated the algicidal activity of this bacterium, designated Shewanella sp. IRI-160, to determine its
mode of action. Our results demonstrate that an algicidal compound is secreted by the bacterium into seawater medium, and that direct
contact between the bacterium and the dinoflagellate is not required. To further characterize the algicidal agent and its activity against dinoflagellates, the bacteria were incubated in seawater medium and filtered to provide a bacteria-free filtrate for use in several experiments.
The activity of the algicide was significantly greater when bacteria were incubated at 30°C than at 25°C or 18°C before filtration. Further
investigation demonstrated that the algicidal agent is a hydrophilic molecule that retains its activity after boiling or freezing. Microscopic
analysis of treated algal cultures showed that the cells are not directly lysed by the algicide, but significant changes in cell morphology
were apparent after 24 hours incubation with the algicide. Although physiological investigation suggests little impact on algal photosynthesis
over 72 hours incubation with the algicide, measurement of hydrogen peroxide in treated cultures indicated that the cells were undergoing
oxidative stress. Potential mechanisms for inducing dinoflagellate mortality by the algicide will be discussed.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 102
ANTI-PREDATOR DEFENSE MECHANISMS
OF THE TOXIC RAPHIDOPHYTE ALGA HETEROSIGMA
AKASHIWO AGAINST POTENTIAL HETEROTROPHIC
PROTIST PREDATORS
Elizabeth L. Harvey, Susanne Menden-Deuer
Graduate School of Oceanography/URI, 215 South Ferry Rd. Narragansett, RI 02882, USA. E-mail: [email protected]
The mechanisms involved in the formation of harmful algal blooms (HAB) are not well understood. We hypothesize that some HAB
species may experience decreased grazing pressure, allowing for the formation and maintenance of HABs. We observed the interactions
of the toxic raphiophyte alga Heterosigma akashiwo and two potential heterotrophic protist predators, marine ciliate Favella ehrenbergii
and dinoflagellate Oxyrrhis marina by simultaneously quantifying population distributions and 3D movements. To test the effect of salinity
on predator-prey interactions, we observed organisms in a linear salinity gradient of 0-30 psu. During the 11-h experiment, H. akashiwo
and O. marina distributed throughout a 30 cm/1 L tank without preference for a specific salinity. In contrast, the predator F. ehrenbergii
avoided low salinities; no cells were found above 15 psu. Due to these different salinity tolerances, 50% of the H. akashiwo were inaccessible to F. ehrenbergii. Moreover, the ciliate had a five-fold higher mortality rate in the presence of H. akashiwo. Heterosigma akashiwo
exhibited avoidance behaviors in response to F. ehrenbergii, by modulating swimming behaviors as a function of predator presence and
ultimately aggregating in low salinity areas avoided by the predator. These results indicate that for some HAB algae, defense mechanisms
such as halo-tolerance, toxicity, and avoidance behaviors can decrease the potential for grazing interactions. Predictions of HAB dynamics
may underestimate population abundance if they fail to incorporate predator-prey interactions in addition to algal growth/loss rates.
Defense mechanisms identified here may aid in understanding the puzzling success of H. akashiwo and other HAB species in the
formation and persistence of blooms.
102
IMPACT OF BACTERIAL PRESENCE ON GROWTH
AND METABOLISM OF ALEXANDRIUM TAMARENSE
Cecile Jauzein1, Andrew Evans2, Deana Erdner1, Ahmed Moustafa3, David Kulis4,
Debashish Bhattacharya5, Donald Anderson4
1
University of Texas, Marine Science Institute, TX 78373, Port Aransas, USA. E-mail: [email protected]
2
National Institute of Child Health and Development, National Institutes of Health, MD 20892, Bethesda, USA
3
Microbial and Environmental Genomics, J. Craig Venter Institute, CA 92121, San Diego, USA
4
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
5
Department of Ecology, Evolution, and Natural Resources, Institute of Marine and Coastal Sciences,
Rutgers University, NJ 08901, New Brunswick, USA
Interactions between bacteria and phytoplankton have been shown to influence various metabolic pathways of phytoplankton cells. For harmful algal
species, such interactions may significantly affect bloom dynamics by influencing growth, cyst formation or toxin production. Prior results from the dinoflagellate Alexandrium tamarense show that the presence of bacteria significantly alters the transcription of several genes associated with critical
processes. Specifically, transcripts of genes involved in carbon metabolism (Rubisco, carbonic anhydrase), bioluminescence (luciferase), and cellular
stress (hsp70, peroxidase) are less abundant in xenic cultures. New experiments have been conducted to further characterize bacteria-A. tamarense
interactions, with a focus on carbon flux and bioluminescence. A single strain of A. tamarense has been maintained for several months in three
different culture conditions: without bacteria (axenic clone, CCMP 1598), with bacteria (xenic clone, CCMP 1493) and with introduction of bacteria
from a xenic culture filtrate into an axenic host culture. Cultures were compared based on growth rate, chlorophyll α concentrations, H13CO3 uptake
rate, bioluminescence, cell biovolume, and bacterial diversity. Xenic cultures had higher biovolumes and lower inorganic carbon fixation than axenic
cultures, suggesting that metabolism of cells associated with bacteria may rely significantly on mixotrophic growth. Introduction of bacteria in axenic
host cultures induced a quick response in terms of A. tamarense cell size, but a transition phase of several weeks was needed to restore growth efficiency. These results highlight the critical role of the mutualistic bacteria-phytoplankton relationship in the growth of these toxic microalgae.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 103
BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
ORAL PRESENTATIONS
THE ECOLOGICAL AND EVOLUTIONARY CONCEPT
BEHIND ALLELOCHEMICAL INTERACTIONS FOR
ALEXANDRIUM POPULATIONS
Uwe John1, Urban Tillmann1, Jennifer Hülsköter1, Allan Cembella1, Tilman Alpermann2
1
2
Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven, Germany. E-mail: [email protected]
LOEWE Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 Frankfurt A.M., Germany
Alexandrium tamarense is a prominent harmful algal bloom forming species known to produce paralytic shellfish poisoning toxins (PSTs),
as well as bioactive substances acting as allelochemicals. These specific allelochemical substances have not yet been structurally elucidated, but they have strong negative effects on a broad array of species, including many auto- and heterotrophic microplankton. The impact, however, of allelochemicals on actual bloom formation is currently under debate. Here we show the potential effect of allelochemicals
on reducing grazing pressure on A. tamarense, while additionally demonstrating positive effects on the intra-population level. We established
an allele-specific quantitative PCR (asqPCR) assay based on two A. tamarense microsatellite markers. The application of asqPCR distinguishes among three different strains of A. tamarense (A2, A4 and A5) in a mixed assemblage. Strain A2 expressed strong allelochemical activity, while A4 was only moderately lytic against targets and A5 was not lytic at all. All strains were grown individually, mixed
either pairwise with each other, and/or pairwise together with a common protistan grazer, the dinoflagellate Polykrikos koifidii. When
offered individual Alexandrium strains, the predator clearly grazed upon A5, moderate on A4 yet not upon A2. In pairwise mixtures,
however, no prey strain-selection was observed. Finally, we show that the ecological concept of facilitation, invoking positive species interactions (here intra-specific), is a plausible explanation for the role of allelochemicals for natural populations of Alexandrium and their
success in bloom formation.
103
ISOLATION AND CHARACTERIZATION
OF A NEW ALGICIDAL VIRUS INFECTING A HARMFUL
DINOFLAGELLATE, PROROCENTRUM MINIMUM
JinJoo Kim, Chang-Hoon Kim
Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, Korea. E-mail: [email protected]
Aquatic viruses are known to play an ecological significant role in regulating host population dynamics. Over 50 different viruses infecting
marine eukaryotic algae have been isolated and characterized for the last two decades. To date, virus infection has been reported in
chlorophytes, prasinophytes, raphidophytes, bacillariophytes, dinophytes, pelagophytes, phaeophytes, and haptophytes. Here, we report
the isolation and partial characterization of a new giant double-stranded DNA(dsDNA) algal virus from the family Phycodnaviridae that infects the harmful dinoflagellate Prorocentrum minimum from Korean coastal water. The Prorocentrum minimum virus (PmV01) is icosahedral, lacks a tail, and is ca. 175-185nm in diameter. The latent period of PmV01 was estimated to be about 80 hours. This is the
second isolated dinoflagellate infecting virus and its detailed characterization is now under way.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 104
BENTHIC MACROALGAE AS SUBSTRATA FOR GAMBIERDISCUS:
TOP-DOWN AND BOTTOM-UP CONTROLS
Brian Lapointe, Brad Bedford
Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 US 1 North, Ft. Pierce, FL 34946, USA.
E-mail: [email protected]
Ciguatera fish poisoning (CFP) afflicts up to 500,000 individuals annually, making this the most prevalent form of toxin-borne seafood
poisoning in the world. Gambierdiscus, a benthic dinoflagellate and causative agent of CFP, has been associated with a wide variety of
benthic macroalgae in tropical, coral reef environments. Previous studies have suggested that this association provides important benefits
to Gambierdiscus, which include protection from herbivory (top-down controls) and increased availability of dissolved nutrients (bottomup controls). High cell densities of Gambierdiscus have occurred on a variety of red, green and brown macroalgae (Chaetomorpha,
Dictyota, Jania, Acanthophora, Heterosiphonia, Spyridia) that are generally palatable and consumed by herbivores. Many of these filamentous and/or branched genera have high surface areas for epiphytic growth of Gambierdiscus and grow abundantly in protected
back-reef environments. These observations support the hypothesis that benthic macroalgae provide nutrient-enriched microhabitats
that enhance growth of Gambierdiscus. Studies showing increased toxicity of Gambierdiscus with elevated N:P ratios helps explain
ciguatera outbreaks in offshore, low-lying, carbonate-rich environments where high N:P ratios can occur. Different nutrient dynamics
of macroalgae and ciguatera toxicity could occur on volcanic islands due to geologic differences that decrease N:P ratios and Plimitation. Land-based runoff from limestone islands, especially those experiencing human activities, can increase N:P ratios, contributing
to greater toxicity of Gambierdiscus. Considering that macroalgae are replacing corals as dominant reef biota in many tropical regions
around the world, the incidence of CFP could increase, particularly if growth and toxicity of Gambierdiscus are strongly dependant on
macroalgae with high N:P ratios.
104
IMPACT OF ALGAL ALLELOCHEMICALS
IN ALGAL-BACTERIA INTERACTIONS
Catherine Legrand1,2, Karin Holmfeldt1, Astrid Weissbach1, Urban Tillmann2, Allan Cembella2
1
2
Marine Science Center, Linnaeus University, SE-39182, Kalmar, Sweden. E-mail: [email protected]
Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany
Trophic interactions between bacteria and phytoplankton in aquatic systems are well documented in the literature, as the microbial loop
plays a critical role in important processes such as carbon flux and nutrient regeneration. In contrast, chemical interactions between
bacteria and harmful/toxic phytoplankton and their potential impact on the production of biotoxins and population dynamics have received
increasingly, yet still relatively little attention. While bacteria can play a role in the production of algal biotoxins or bloom termination, phytoplankton can produce biologically active compounds, i.e. allelochemicals that affect bacterial diversity. In recent projects, we have
quantified the antibiotic properties of allelochemicals in model algal-bacterial communities by combining physiological, chemical and
molecular approaches. Depending on the trophic composition of microbial food webs, algal allelochemicals can temporary affect bacterial
growth and production, either direct inhibition or indirectly by decreasing bacterivory or providing DOM through lysis of protists. Our
results focuses especially on interactions of HABs and bacteria at the cellular and population level, which is crucial for revealing microscale processes that have meta scale consequences, e.g. in coastal waters, open ocean, polar regions.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 105
BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
ORAL PRESENTATIONS
STUDY OF ALLELOPATHIC EFFECTS OF THE TOXIC
DINOFLAGELLATE ALEXANDRIUM MINUTUM ON
THE DIATOM CHAETOCEROS NEOGRACILE USING
FLOW CYTOMETRY
Aurélie Lelong, Hansy Haberkorn, Nelly Le Goïc, Hélène Hégaret, Philippe Soudant
Laboratoire des sciences de l’environnement marin (LEMAR), UMR6539, Institut Universitaire Européen de la Mer (IUEM),
Place Nicolas Copernic, 29280 Plouzané, France. E-mail: [email protected]
Allelopathic activities of Alexandrium minutum, a toxic dinoflagellate, on Chaetoceros neogracile were evaluated using flow cytometry.
Natural red fluorescence of cells (linearly related to cellular chlorophyll content), FSC (Forward scatter, small angle scatter signal) and
SSC (Side scatter, 90o angle scatter signal) significantly decreased in C. neogracile exposed to A. minutum. These effects were intensified
with A. minutum age and concentration. The supernatant of A. minutum contained the majority of the allelopathic activities, suggesting
that the allelopathic agent(s) is (are) excreted in the culture medium. Based on effects of the allelopathic compound(s) on photosynthetic
capacities of C. neogracile, we suggest that photosystem, probably PS-II, is targeted. The dinoflagellate A. minutum exhibited allelopathic
activity on C. neogracile, independently of the presence of bacteria in the cultures. Results also demonstrated that the allelopathic agent
is not produced by bacteria, the latter were not necessary for A. minutum cultures to affect C. neogracile. The biochemical composition
of this compound remains unknown.
105
INFLUENCE OF ALTERED LIGHT CONDITIONS
AND GRAZERS ON SCRIPPSIELLA TROCHOIDEA
(DINOPHYCEAE) CYST PRODUCTION
Veronica Lundgren, Edna Granéli
School of Natural Sciences, Marine Ecology Department, Linnaeus University, SE-39182, Kalmar, Sweden.
E-mail: [email protected]
Scrippsiella trochoidea occurs around the world and is one of the causative species of HABs. We have studied if the presence of Acartia
tonsa and different light conditions induce temporary cyst formation in S. trochoidea. S. trochoidea was exposed to A. tonsa directly and
indirectly (grazer filtrate), in light and dark conditions. Acartia tonsa fecal and egg production when feeding on S. trochoidea vegetative
cells and temporary cysts were estimated. Neither direct nor indirect exposure to A. tonsa had any effect on S. trochoidea cyst production
in neither light nor dark conditions. In light, controls and A. tonsa treatments had around 20%, whereas in darkness around 80% temporary
cysts. Thus, darkness had a strong effect on temporary cyst formation. Feeding on temporary cysts compared to vegetative cells decreased
fecal and egg production. These results suggest that induction of temporary cysts in response to A. tonsa could be a favorable survival
strategy; however this is not the case, as S. trochoidea doesn’t produce temporary cysts when exposed to A. tonsa. Rather, induction of
temporary cysts seems to be controlled by decreased light intensity, which is a favorable trait for this species when pushed to water
depths where light is scarce.
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PARASITISM BY AMOEBOPHRYA IN DINOFLAGELLATE
HAB SPECIES FROM MONTEREY BAY, CALIFORNIA,
AND ITS TOP-DOWN CONTROL ROLE
Fernanda Mazzillo, Mary Silver
Department of Ocean Sciences, University of California Santa Cruz, 1156 High Street, Santa Cruz, California 95064, USA.
E-mail: [email protected]
The marine parasite Amoebophrya can infect and kill dinoflagellates and may act as a controlling agent of some dinoflagellates responsible
for harmful algal blooms (HABs). Amoebophrya was detected in 5 dinoflagellate species (Alexandrium catenella, Akashiwo sanguinea,
Ceratium furca, Dinophysis sp., and Prorocentrum micans) from Monterey Bay, California, USA, in surface water samples collected
weekly during a 5-year HAB monitoring time series (2006-2010). Infections were detected in live samples by observing Amoebophrya
green autofluorescence during its 3 known life stages (i.e. trophont, dinospores and vermiform stages) or in preserved samples by
using DAPI (a general DNA stain) and/or FISH (species-specific) probes. Transmission electron micrographs reveal ultrastructure characteristics of Amoebophrya in A. sanguinea. Additionally, quantification of A. sanguinea density and percentage of infected cells (detection
limit of 0.5 - 1%) from 2006 and 2008 samples suggests that host abundance can be controlled by parasitism. However, during the
fall of 2007 A. sanguinea cells were not infected and cell densities (106 cells L-1) of A. sanguinea were the highest recorded. It is
possible that in 2007 A. sanguinea ‘escaped’ Amoebophrya top down control. Explanations for such an ‘escape’ are currently under
investigation and include (i) the presence of a new strain of A. sanguinea immune to Amoebophrya sp. infections or (ii) a faster growth
rate of the host relative to the parasite during 2009. Our study highlights the importance of Amoebophrya parasitism in dynamics of dinoflagellate HABs as well as its frequent presence in several HAB dinoflagellate species from the west coast of North America.
106
THE ECO-PHYSIOLOGICAL RELATIONSHIPS
OF BACTERIAL EPIBIONTS AND THE MARINE
DIATOM PSEUDO-NITZSCHIA
Marilou Sison-Mangus, Kevin Tran, Sunny Jiang
University of California Irvine, Department of Civil and Environmental Engineering, E4130 Engineering Gateway,
Irvine 92697-2175, California, USA. E-mail: [email protected]
Not all species of the marine diatom Pseudo-nitzschia can produce domoic acid, a toxin that poses a health risk to humans and marine
animals when contaminated shellfish is consumed. The reason behind this is not fully understood, but previous studies have shown
that epibiont bacteria play a role on the production of domoic acid by the diatom. A diverse suite of epibiont bacteria are known to be
associated with Pseudo-nitzschia but the specific physiological interactions of these epibionts with the diatom is not fully characterized.
In this study, we isolated and identified the bacteria that adhere to the surface of several strains of toxic and non-toxic Pseudo-nitzschia
using 16s rDNA sequencing. Bacterial epibionts that are not amenable to culturing were molecularly screened using metagenomic
approach in order to compare the bacterial populations of the two Pseudo-nitzschia types. Based on culture approach alone, four
distinct classes of bacteria (comprising of nine genera that are members of a-proteobacteria, g-proteobacteria, Firmicutes and Bacteroidetes) have been identified from four Pseudo-nitzschia strains. Some of these bacteria are potentially algicidal, while other epibiont
isolates can putatively promote the growth and influence the production of domoic acid of Pseudo-nitzschia. In our presentation, we
will illustrate the physiological interactions of each bacterial isolate and Pseudo-nitzschia and aim to describe the influence of the associated bacteria on the toxigenesis of the algae.
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
ORAL PRESENTATIONS
ALLELOPATHIC EFFECT OF THE MACROALGA
ULVA LACTUCA ON SEVEN HARMFUL ALGAL
BLOOM SPECIES FROM THE US
Ying Zhong Tang, Christopher J. Gobler
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA.
E-mail: [email protected]
We report on experiments examining the effects of macroalga Ulva lactuca, collected from estuaries of Long Island, NY, on the growth
of HAB species commonly observed on the east and west coasts of the US including Karenia brevis, Pseudo-Nitzschia multiseries,
Cochlodinium polykrikoides, Karlodinium veneficum, Prorocentrum minimum, Aureococcus anophagefferens, and Chattonella marina.
Fresh thalli of the U. lactuca added at environmentally realistic levels (mg L-1) were capable of lysing or strongly inhibiting the growth
of all species in a dose dependent manner within controlled laboratory experiment during which high nutrient levels and common pH
levels among treatments and controls were maintained. The dramatic allelopathic effects of dried and powdered U. lactuca after heat
treatment on all HAB species demonstrated unambiguously that U. lactuca produces heat-stable allelochemicals. The addition of live
U. lactuca thalli during bottle and mesocosm experiments conducted in the field during the blooms of A. anophagefferens (‘brown
tide’ >105 cells ml-1) consistently yielded a significant (p<0.05) and often large (>50%) reduction in cell densities in ~48h. Our findings
combined with well-known nutrient removal capacity of seaweeds collectively suggest that use of U. lactuca and perhaps other macroalgae may be promising mitigation strategy for HABs in coastal ecosystems.
107
CAN OCHROMONAS SP., A MIXOTROPHIC
CHRYSOPHYTE, CONTROL TOXIC
AND COLONY-FORMING CYANOBACTERIA?
Ellen van Donk
NIOO-KNAW, Netherlands Institute of Ecology, Rijksstraatweg 6, 3631AC Nieuwersluis, The Netherlands.
E-mail: [email protected]
Grazing experiments with Ochromonas, a mixotrophic chrysophyte, were performed in the laboratory to test whether this flagellate can
control cyanobacteria. We investigated three Microcystis aeruginosa strains that varied in toxicity and morphology and also one filamentous cyanobacterium, Pseudoanabaena. Furthermore, we analyzed the co-occurrence of Ochromonas and Microcystis in natural
systems. Ochromonas was able to feed on all four cyanobacteria tested, and showed high growth rates on all of them by reducing the
net growth rates of the cyanobacteria significantly. Further, we observed that after four days of incubation with Ochromonas the total
microcystin content in the Microcystis strains was reduced by more than 90% compared with the controls. Analysis of the field data
from 460 Norwegian lakes showed that Ochromonas occurred in almost all lakes, while the distribution of Microcystis was linked to
higher TN, TP, temperature and pH. Ochromonas occured in 99% of the lake samples, while Microcystis was found in only 7%.
Ochromonas co-occurred in 94% of the samples in which Microcystis was present and the interaction of both species might therefore
be important in natural systems.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 108
EFFECT OF NITROGEN SOURCE AND ENRICHMENT LEVEL
ON A TWO SPECIES FOOD WEB CONTAINING A TOXIC
CYANOBACTERIUM AND A MIXOTROPHIC PREDATOR
Susanne Wilken1, 2, Suzanne Wiezer1, Ellen Van Donk1,3, Jef Huisman2
1
Department of Aquatic Ecology, Netherlands Institute of Ecology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands.
E-mail: [email protected]
2
Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam,
Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands
3
Palaeoecology, Institute of Environmental Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
We investigated a nitrogen limited food web consisting of the mixotrophic flagellate Ochromonas sp. preying on the toxic cyanobacterium
Microcystis aeruginosa. Because the mixotroph cannot use nitrate directly, the system resembles an ordinary predator prey system
when nitrate is the only nitrogen source present. When ammonium is offered instead the mixotroph is also competing for dissolved nitrogen with its prey and therefore acts as an intraguild predator. The population dynamics of these two food web structures were compared in chemostat experiments at three levels of enrichment. We furthermore investigated the role of microcystins in the interaction
of these two organisms in batch experiments. The mixotroph profited most from the presence of ammonium at low nitrogen concentrations using ammonium and the prey as substitutable nitrogen sources. However, it did not reduce its prey to lower densities for high
levels of enrichment as expected from intraguild predation theory. We suggest intraspecific interference within the mixotroph or an induced defense in M. aeruginosa to explain this deviation from theoretical expectations. Since the mixotroph showed very similar
functional and numerical responses on the microcystin-producing strain M. aeruginosa PCC7806 and its microcystin-deficient mutant
and grazing by the flagellate did not induce an increased microcystin production in M. aeruginosa, a possible induced defense would
most likely be caused by peptides other than microcystins.
108
TRANSCRIPTOME ANALYSIS REVEALS
NUCLEAR-ENCODED PROTEINS FOR
THE MAINTENANCE OF TEMPORARY PLASTIDS
IN THE DINOFLAGELLATE DINOPHYSIS ACUMINATA
Jennifer H. Wisecaver1, Jeremiah D. Hackett2
1
Department of Ecology and Evolutionary Biology, University of Arizona, P.O. Box 210088, Tucson, AZ 85721, USA.
E-mail: [email protected]
2
Department of Ecology and Evolutionary Biology, University of Arizona, P.O. Box 210088, Tucson, AZ 85721, USA
Dinophysis is exceptional among dinoflagellates, possessing plastids derived from cryptophyte algae. Although Dinophysis can be maintained
in pure culture for several months, the genus is mixotrophic and needs to feed either to acquire plastids (a process known as kleptoplastidy)
or obtain growth factors necessary for plastid maintenance. Dinophysis does not feed directly on cryptophyte algae, but rather on a ciliate
(Myrionecta rubra) that has consumed the cryptophytes and retained their plastids. Despite the apparent absence of cryptophyte nuclear
genes required for plastid function, Dinophysis can retain cryptophyte plastids for months without feeding. To determine if this dinoflagellate
has nuclear-encoded genes for plastid function, we sequenced cDNA from Dinophysis acuminata, its ciliate prey M. rubra, and the cryptophyte
source of the plastid Geminigera cryophila. We identified five proteins complete with plastid-targeting peptides encoded in the nuclear genome
of D. acuminata that function in photosystem stabilization and metabolite transport. Phylogenetic analyses show that the genes are derived
from multiple algal sources indicating some were acquired through horizontal gene transfer. These findings suggest that D. acuminata has
some functional control of its plastid, and may be able to extend the useful life of the plastid by replacing damaged transporters and protecting
components of the photosystem from stress. However, the dearth of plastid-related genes compared to other fully phototrophic algae suggests
that D. acuminata does not have the nuclear repertoire necessary to maintain the plastid permanently.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 109
BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
ORAL PRESENTATIONS
POSSIBLE IMPACT OF PARASITIC CHYTRID
ON POPULATION DYNAMIC OF THE TOXIC MARINE
DINOFLAGELLATE ALEXANDRIUM TAMARENSE
Mineo Yamaguchi1, Kanae Koike2, Setsuko Sakamoto1
1
National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi, Hatsukaichi,
Hiroshima 739-0452, Japan. E-mail: [email protected]
2
Hiroshima University, 1-3-2 Kagamiyama, Higashihiroshima 739-8511, Japan
Some chytrid fungi are host-specific parasites that have a considerable impact on freshwater phytoplankton dynamics. However there
has been little study of parasitic chytrid of marine phytoplankton. During the germination experiments of the cysts of A. tamarense, we
found that the germinated cells were infected by a kind of parasitic fungus. In this study, we aim to identify the fungus by examining
the morphology and the development with a light microscope, SEM and TEM. Sediment suspensions prepared with the bottom sediments from Mikawa Bay, central Japan, were incubated at 12.5oC in the light. During the incubation, the cells attached with zoospore
were isolated and incubated individually for microscopic observations. The zoospore formed smooth-walled cyst on the surface of the
host cells. A single sporangium developed from the direct enlargement of the cyst. The sporangium was spherical and inoperculated,
with a diameter of ca. 20 μm. The cytoplasm of the sporangium cleaved to form many zoospores that were liberated through discharge
pores. The zoospore had a spherical head of a diameter ca. 2 μm and had only one whiplash flagellum of ca. 16 μm in length. The life
cycle of the fungus was completed within three days after infection. Ultrastructure of the zoospore and 18S rDNA sequences indicate
that the fungus belong to Chytridiales. Our finding is the first report of fungal parasitism on the toxic marine dinoflagellate and suggests
that chytrid fungus may play an important role in marine environment as in freshwater system.
109
STUDIES ON THE PHYGOTROPHIC CHARACTERISTICS
OF KARENIA MIKIMOTOI, A MAJOR HAB CAUSATIVE
SPECIES IN CHINA
Qingchun Zhang1, 2, Rencheng Yu1, Yunfeng Wang1, Tian Yan1, Mingjiang Zhou1
1
2
Institute of Oceanology, Chinese Academy of Sciences, 7, Nanhai Road, Qingdao, 266071, China. E-mail: [email protected]
Graduate University of Chinese Academy of Sciences, 19A, Yunquan Road, Beijing, 100049, China
Karenia mikimotoi is a major harmful algal species in China seas. K. mikimotoi blooms were found many times in China seas. Especially,
an extra large-scale main K. mikimotoi bloom broke out in East China Sea in 2005. Its affected area was more than 15 thousand kilometers. A strain of K. mikimotoi, which was isolated from South China Sea, was used to study the phagotrophic ability and its role in
the growth of K. mikimotoi. The fluorescent microspheres with diameter of 0.5μM and 2.0μM, the bacteria isolated from the culture
of K. mikimotoi, and a marine microalgae Isochrysis galbana were used as preys. It was found that K. mikimotoi cultured in nutrientdepleted conditions under high light intensity conditions could ingest fluorescent microspheres, fluorescence-labeled marine bacteria,
and fluorescence-labeled I. galbana. However, only fluorescent microspheres (diameter 0.5μM) and fluorescence-labeled I. galbana
were ingested by K. mikimotoi cultured under the low light intensity conditions. The phagotrophic behavior was mainly occurred in daytime. K. mikimotoi showed better growth by ingesting living marine bacteria or I. galbana than the control, either in nutrient-depleted
conditions or in nutrient-replete conditions. However, the effects of two preys on the growth of K. mikimotoi are different. The growth
of K. mikimotoi was more remarkable with bacteria as prey in the nutrient-replete conditions, while with I. galbana as prey, the growth
of K. mikimotoi was more obvious in the nutrient-depleted conditions. In conclusion, some marine bacteria and microalgae maybe play
important roles in the process of the occurrence of K. mikimotoi bloom.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 110
ORAL PRESENTATIONS
THE THECATE MIXOTROPHIC DINOFLAGELLATE
FRAGILIDIUM DUPLOCAMPANAEFORME:
FEEDING, PREY SPECIFICITY AND ALLELOPATHY
Miran Kim, Myung Gil Park
LOHABE, Department of Oceanography, Chonnam National University, Gwangju 500-757, Republic of Korea.
E-mail: [email protected]
In summer to autumn of 2008, a recently described thecate mixotrophic dinoflagellate, Fragilidium duplocampanaeforme, occurred in
Masan Bay, Korea, where it frequently contained bright orange fluorescent inclusions. Using cultures of F. duplocampanaeforme isolated
from Masan Bay, we investigated feeding, prey specificity, and allelopathy of this mixotroph. F. duplocampanaeforme fed exclusively on
Dinophysis spp. when offered a variety of prey including dinoflagellates, a raphidophyte, a cryptophyte, a ciliate, and diatoms separately.
In addition, F. duplocampanaeforme had allelopathic effects on other organisms, including cell immobilization/motility decrease (in D.
acuminata, D. caudata, D. fortii, D. infundibulus, Gonyaulax polygramma, Heterocapsa triquetra, Prorocentrum triestinum), breaking of
cell chains (in Cochlodinium polykrikoides), cell death (in P. minimum), and temporary cyst formation (in Scrippsiella trochoidea). F. duplocampanaeforme engulfed whole Dinophysis cells through the sulcus. About 1 h after ingestion, F. duplocampanaeforme became immobile and shed all thecal plates. The ecdysal cyst persisted for about 7 h, during which the ingested prey was gradually digested. These
observations suggest that F. duplocampanaeforme may play an important role in the Dinophysis population dynamics in the field.
110
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
BACTERIAL COMMUNITY INFLUENCE ON SAXITOXIN
PRODUCTION BY GYMNODINIUM CATENATUM
Maria Albinsson1, Susan Blackburn2, Andrew Negri3, Christopher Bolch1
1
National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania,
Locked Bag 1370, Launceston 7250, Tasmania, Australia. E-mail: [email protected]
2
CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, 7001, Tasmania, Australia
3
Australian Institute of Marine Science, PMB 3, Townsville MC, Townsville 4810, Queensland, Australia
Cultures of the toxic dinoflagellate Gymnodinium catenatum show considerable variation in both cellular toxicity and saxitoxin (STX) profile
and preliminary evidence suggests that the bacterial community may be the source of this variation. Here we compare STX production
and profile of equivalent non-clonal uni-bacterial G. catenatum cultures grown with: 1) Marinobacter sp. DG879; 2) Alcanivorax sp.
DG881, and 3) G. catenatum cultures containing mixed bacterial communities. Intracellular STX content and profile at the beginning and
end of logarithmic growth phase was determined using HPLC analysis, and the STX production rate calculated. Toxin content at latelog phase was within the range of previous studies of G. catenatum, but the rate of saxitoxin (STX) production was highly variable among
replicates and we could not detect any statistically significant effect of bacterial community composition. However, the proportion of STX
compounds produced by G. catenatum cultures grown with Marinobacter sp. DG879 exhibited lower proportions of C1+C2 and C3+C4
and greater proportions of dcGTX2+dcGTX3 and GC1+GC2 than G. catenatum grown with either Alcanivorax DG881 or a mixed
bacterial community. The differing STX profile may be due to Marinobacter sp. DG879 influencing toxin synthesis indirectly through its
effect on dinoflagellate physiology, or directly by biotransformation of the PST compounds.
111
DYNAMICS OF AMEOBOPHRYIDEAE PARASITOIDS DURING
A BLOOM OF THE DINOFLAGELLATE PROROCENTRUM
MICANS IN A CHILEAN FJORD
Catharina Alves-de-Souza1,2, José Luis Iriarte3, Humberto González1, Laure Guillou2
1
Instituto de Biología Marina, Universidad Austral de Chile, Campus Isla Teja, P.O. Box 567, Valdivia, Chile.
E-mail: [email protected]
2
CNRS, UMR 7144 & UPMC, Station Biologique de Roscoff, Place Georges Teissier, 29682 Roscoff, France
3
Instituto de Acuicultura, Universidad Austral de Chile, Sede Puerto Montt, Los Pinos S/N, Puerto Montt, Chile
The dynamics of infection by Amoebophryidea parasitoids on the dinoflagellate Prorocentrum micans was evaluated in the Reloncaví fjord
(41ο29’ S; 72ο18’ W) during 08 to 29th March 2009. The sampling started at the maximum cellular concentration of P. micans (~4.5 cells
L-1·104) and lasted during several days after the demise of the bloom. Parasitoids were detected by fluorescence in situ hybridization (FISH)
using a general oligonucleotide probe specific for Amoebophryideae. The maximum of prevalence, i.e. percentage of infected hosts (13%),
was observed the 11th of March followed by a sharp decrease in the P. micans abundances the 14th of March (1.5 cells L-1·103) which
remained stable at low concentration until the end of the sampling period. The free-living stage of Amoebophryidea cells (dinospores) showed
their maximal abundance for the 14th of March and decreased markedly after the 17th March. Two days after the demise of P. micans bloom,
the composition of phytoplankton changed sharply to a dominance of diatoms, mainly Skeletonema spp., which showed its highest abundances
for the 20th March (4.4 cells L-1·107). This Skeletonema spp. bloom followed an increase in the concentrations of silicate. However, no correlation was detected between the decrease of P. micans population and nitrate or phosphate concentrations. It seems that infection by
Amoebophryidae was the main factor hastening the bloom termination of P. micans during this study. Our results suggest that these parasites
could be important biological factors in the demise of dinoflagellate blooms as well as in the competition between major phytoplankton groups.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 112
INGESTION RATES OF THE HETEROTROPHIC
DINOFLAGELLATE NOCTILUCA SCINTILLANS FED
ON THE TOXIC PST PRODUCER ALEXANDRIUM MINUTUM
(HALIM)
Máximo Frangópulos1, Evangelos Spyrakos2, Cástor Guisande2
1
Centro de Estudios del Cuaternario de Fuego-Patagonia (Fundación CEQUA), Av. Bulnes 01890, P.O. Box 6213029,
Punta Arenas, Chile. E-mail: [email protected]
2
Facultad de Ciencias de Mar, Universidad de Vigo, Lagoas-Marcosende, E-36200 Vigo, Spain
In this study, we evaluated the ingestion rates of the heterotrophic dinoflagellate Noctiluca scintillans (Macartney) fed on the toxic microalgae
Alexandrium minutum Halim in laboratory cultures. Specimens of N. scintillans were collected in the Ría de Vigo and subsequently kept
in culture. The results showed that, in both treatments, N. scintillans was actively fed on A. minutum, with maximum ingestion rates of
0.37 μg C ind-1 day-1, showing that there was non-satiated feeding by N. scintillans on A. minutum. The mean cellular toxin content of
A. minutum in the stock culture ranged from 2.57 up to 3.44 fmol cell-1. The HPLC analyses of the extracts from N. scintillans, fed with
4 different abundances of A. minutum, revealed no detectable amounts of toxin. This suggested that, although N. scintillans was able to
ingest into its vacuoles a toxic dinoflagellate such as A. minutum, the ingested toxin was bioconverted or excreted by Noctiluca. Our
findings increase the probability that Noctiluca may inflict grazing pressure on the growth of Paralytic Shellfish Toxin (PST) species in the
field and could, therefore, play an important role as a regulator against PST-producing phytoplankton. This is the first report on grazing
rates of N. scintillans on Alexandrium cells.
112
ALLELOPATHIC EFFECTS OF ARTEMIA SALINA
IN DIFFERENT GROWTH STAGES
ON P. RETICULATUM CULTURES
Juan J. Gallardo-Rodríguez, Asterio Sánchez-Mirón, Francisco García-Camacho, Emilio Molina-Grima
University of Almería, 04120 Almería, Spain. E-mail: [email protected]
There are several works demonstrating the toxicity induction of copepods in dinoflagellates. Copepods-related waterborne chemical cues,
with and without direct presence of these species, have been shown to increase the toxin production in culture up to ten times. Copepods
are able to detect dinoflagellates and to avoid feeding from these toxic species. In contrast, brine shrimps, lacking this ability, feed from
toxic dinoflagellates which result lethal. Whereas there are various examples in literature of detrimental effects of toxic dinoflagellates in
Artemia salina, works dealing with the induction of toxicity in these species are scarce. Effects on growth and induction of toxicity in P.
reticulatum cultures were observed in response to an allelopathic interaction with Artemia salina. The experiments carried out were performed with Artemia salina in its three growth stages: nauplii, metanauplii and adult. Toxin-inducing waterborne cues were sought in
culture medium where the Artemia salina individuals had been kept for 24 hours. In addition, an adult body’s water lysate were used. The
toxin-induction mediums used provoked an increase in cell toxin content. However, the induced effect was considerably lower than that
of copepods. Additionally, a dose-dependent growth inhibition was observed. Acknowledgements: This research was supported by the
Ministerio de Ciencia e Innovación de España (CTQ2008-06754-C04-02/PPQ) and the Consejería de Innovación, Ciencia y Empresa
de Andalucia (P09-TEP-5375).
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
INTERRELATIONS BETWEEN CYANOBACTERIA AND
PLANT-FEEDERS: THE NATURE OF BIOCIDAL EFFECTS
Evgeny Gol'din
Southern Branch of the National University of Biological Resources and Environmental Management - Crimean Agricultural
and Technological University, PB 2223, Simferopol, Crimea, 95043, Ukraine. E-mail: [email protected]
Diverse and multilateral cyanobacteria-arthropod relationships cause different interpretations of these processes. We evaluate cyanobacterial activity by using of terrestrial arthropods as the test objects in model assays. Insects -lackey moth (LM), fall webworm (FW) and
Colorado potato beetle (CPB)- were exposed to cyanobacterial samples (Microcystis aeruginosa - 70.0-100.0%) from Dnieper: leaves
for insect feeding were sprayed by cyanobacteria. Nutrition, growth, metamorphosis and viability were observed, histopathology was examined. Cyanobacteria possessed of general inhibitory effect, which can be specified as biocidal activity with several components.
Deterrent action: larval nutrition was inhibited in dependence on dietary habits, anatomical, biological and ecological characteristics of insects. Cyanobacterial responses were very close to the activity of terrestrial plants producing allelochemicals for their defense. Inhibition
of larval growth: LM - in 50.0%; FW - in 29.2-68.0%; CPB - 48.5-63.5% (larvae), 15.4-16.1% (imago). Disorder of metamorphosis:
imagination fell in 2.0-4.4 times; teratogenesis was observed during pupation and imagination. Metatoxic activity: reproduction and fecundity of the next generation reduced. Toxic action: histopathology was found in digestive tract. Insect mortality was recorded at several
life-history stages, meanwhile its cause consists not only in toxic activity, but also in regression of trophic function and growth, and
cyanobacterial biocidal characteristics can be determined more likely as deterrent than toxic.
113
FIRST STAGES OF POPULATIONS OF DINOPHYSIS
(D. ACUTA, D. CAUDATA) LEADING TO LIPOPHILIC
SHELLFISH TOXIN OUTBREAKS
Sonsoles Gonzalez-Gil1, Lourdes Velo-Suarez2,3, Patrick Gentien3, Christian Bechemin4, Maria Teresa Pérez5, Beatriz Reguera2
Instituto Español de Oceanografía (IEO), Avda de Brasil 31, 28020 Madrid, Spain. E-mail: [email protected]
2
Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Vigo, Aptdo. 1552, 36200 Vigo, Spain
3
Actual address: Institut Français de Recherche pour l’Exploitation de la Mer (Ifremer), Centre de Brest, DYNECO,
Pointe du Diable BP70, 29280 Plouzane, France
4
LERPC, French Institute for the Exploitation of the Sea (Ifremer), Houmeau, France
5
Laboratory of Aquatic Photobiology and Plankton Ecology, Institute of Ecology, University of Innsbruck,
Technikerstrasse 25, 6020 Innsbruck, Austria
1
Dinophysis spp. are usually present in low concentrations (<40 cell L-1), but several coastal species have been associated with lipophilic shellfish toxin
outbreaks at concentrations as low as 100 cell L-1. To follow the early stages of these low-density and scattered populations requires a strategic
sampling to track Dinophysis cells that are practically under detection levels for routine monitoring programmes. Between 20 and 26 August 2003,
4 mini-cruises were carried out in the outer reaches of Ria de Vigo and its adjacent shelf to obtain fine resolution measurements of physical parameters
and phytoplankton. The objectives of this work were: i) to track early stages of Dinophysis acuta and D. caudata during the late upwelling season (April
to October) and ii) to describe the vertical distribution of Dinophysis spp. and accompanying flora (microplankton, phototrophic and heterotrophic
nanoplankton, bacteria) and the environmental conditions associated with them. Hydrographic conditions corresponded to those typical in summer
during mild upwelling pulses. Vertical profiles showed a broad chlorophyll maximum at around 20 m in the pycnocline formed by Pseudo-nitzschia spp.
and large dinoflagellates (Ceratium spp., Protoperidinium spp.). Low density (101- 4·102 cell L-1) but actively dividing (μ = 0.25 - 0.5 d-1) populations
of D. acuta and D. caudata presented a cell maxima in the upper and lower part of the pycnocline respectively; the maximum of their specific division
rate (μmin) by depth (μmin)z coincided with their cell maxima. Laboratory incubations of isolated cells of D. caudata suggested a positive response of
Dinophysis to additions of DOM from different depths in the same station concentrated by ultrafiltration. Here we describe the abiotic and biotic factors
defining the niche of initial populations of two species of Dinophysis that led later to seasonal lipophilic shellfish toxin outbreaks in the Galician Rías.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 114
HOST SWIMMING BEHAVIOR INFLUENCES
AMOEBOPHRYA INFECTION EFFICIENCY
Allen R. Place1, Joseph Katz2, Jian Sheng3
1
Baltimore Marine Biotechnology Laboratory, University of Maryland Center for Environmental Sciences, Baltimore, MD, USA
Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD, USA
3
Aerospace Engineering and Mechanics, University of Minnesota, Minneapolis, MN, USA
2
Prior studies investigating the relationship between different strains of Karlodinium veneficum’s toxicity and parasitism by Amoebophrya sp.
ex. K. veneficum found a positive correlation with host toxicity (i.e. cells with higher toxin cell quotas where parasitized to a greater extent).
However, one highly toxic strain, BM1, was consistently more resistant to infection than other toxic strains. When the swimming behavior
of BM1 was examined recently it was to found to swim in both right-handed and left-handed helices with an average swimming speed of
111.2 ± 55.5 μm/sec. A nontoxic strain, MD5, which was infected poorly also swam in both right-handed and left-handed helices with
an average swimming speed of 81.2 ± 44.9 μm/sec. Two toxic strains (1974 and 2064) which were highly infected only swam in righthanded helices with average speeds of 102.3 ± 56.4 and 80.9 ± 38.0 μm/sec, respectively. When the swimming behavior of the parasite
was examined it swam only in a left-handed helix with an average swimming speed of only 5.8 ± 2.57 μm/sec. We discuss how a 2 μm
cell moving in a left handed helix at such a slow speed might be entrained by a faster 10 μm host moving in a right-handed helix.
114
EXPRESSION AND CHARACTERIZATION OF FAMILY
19 CHITINASE FROM THE CYANOPHAGE MA-LMM01
INFECTING MICROCYSTIS AERUGINOSA
Naohiko Hosoda1, Takashi Yoshida2, Yoshihiko Sako2, Keizo Nagasaki3, Shingo Hiroishi1, Yoichi Kurokawa4
1
Department of Marine Bioscience, Fukui Prefectural University, 1-1 Gakuen-cho, Obama, Fukui 917-0003, Japan.
E-mail: [email protected]
2
Department of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan
3
Harmful Algal Bloom Division, National Research Institute of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi,
Hatsukaichi, Hiroshima 739-0452, Japan
4
Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun,
Fukui 910-1195, Japan
Ma-LMM01 is a cyanophage infecting toxic Microcystis aeruginosa strain NIES298. ORF69 of the genome of Ma-LMM01 shows homology to family 19 chitinase of which catalytic domain have structural similarity with lysozyme. Comparing with the amino acid sequences
of family 19 chitinases, we found putative sugar binding residues Gly209 and Ile213 in ORF69 were replaced to Gln and Lys, respectively.
To study contribution of residues Gly209 and Ile213 to substrate binding, the ORF69 was cloned into an expression vector and expressed
in Escherichia coli and site-directed mutagenesis was carried out. After induction with IPTG, transformants of E. coli with wild-type ORF69
lysed. Recombinant gp69 protein showed both lysozyme and chitinase activities. Mutant enzyme G209Q lost both activities. As compared
with wild-type, the mutant enzyme I213K showed a decrease 70% in lysozyme activity and an increase of 50% chitinase activity by
using pNP-(GlcNAc)3 as substrate. These observations suggest that Ile213 play an important role in substrate binding.
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
THE ALLELOPATHIC INTERACTIONS BETWEEN
PROROCENTRUM MICANS AND SKELETONEMA
COSTATUM, PROROCENTRUM MICANS AND KARENIA
MIKIMOTOI IN LABORATORY CULTURES
Ji Xiaoqing1, Han Xiaotian1, Zheng Li2, Yu Zhiming1, Zou Jingzhong1
1
Key Laboratory of Marine Ecology and Environmental Science Institute of Oceanology,
Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, China. E-mail: [email protected]
2
Marine Ecology Research Center, The First Institute of Oceanography, State Oceanic Administration,
6 Xianxialing Road, Qingdao, China
Algal allelopathy is a manifold ecological/physiological phenomenon, and its research has drawn attention to different facets of interactions
among algae, algal toxins, eutrophications, etc. In this paper, we investigated the allelopathy interactions between Prorocentrum micans
and Skeletonema costatum, Prorocentrum micans and Karenia mikimotoi using bi-algal cultures under stable environmental conditions
(temperature, light and illuminating periodicity, etc.) and sufficient nutrition supply by f/2 enrichment every day. Because the allelochemical
effects on algae were species-specific and size-dependent, we conducted the experiments with different densities. When low cell densities
of P. micans and high cell densities of S. costatum were inoculated into the same medium, growth of P. micans was weakly suppressed,
while growth of S. costatum was notably suppressed. Other experiments indicated that not only in bi-algal cultures with P. micans but
also in enriched filtrate from P. micans, both of S. costatum and K. mikimotoi were strongly inhibited; especially in enriched filtrate, the
S. costatum and K. mikimotoi all died in 3 or 4 days. The allelopathic test proved that P. micans could affect the growth of co-cultured
S. costatum and K. mikimotoi by producing allelochemical(s), and further demonstrated that a direct cell-to-cell contact was not necessary
for their mutual interactions. At last, a mathematical model was used to simulate growth and interactions of P. micans and S. costatum;
P. micans and K. mikimotoi in bi-algal cultures. It is proposed that allelopathy is an important process in marine ecology.
115
ISOLATION AND CHARACTERIZATION OF A
DOUBLE-STRANDED DNA VIRUS INFECTING A HARMFUL
DINOFLAGELLATE, HETEROCAPSA PYGMAEA
JinJoo Kim1, Yoshihito Takano2, Keizo Nagasaki3, Tae-Jin Choi1, Chang-Hoon Kim1
1
Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, Korea. E-mail: [email protected]
Institute for East China Sea Research, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
3
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Japan
2
Dinoflagellates are considered to be one of the most abundant and diverse groups of microphytoplankton, in which Heterocapsa pygmaea
is one of the dinoflagellate species newly identified in Korean coastal water. The virus screening was done by the microplate MPN method
using 0.22μm filtered seawater samples from each sampling site. When lysis occurred, the MPN method was repeated 3 times for clonal
virus lysate. Then its growth experiment was done, along with TEM observations for both negative staining and thin-sectioning for observing
the shape of the virus. Its intraspecies and interspecies test was also checked for viral specificity, and the genome type was checked
using 5 different kinds of enzymes. Here, we report the characteristics of a double-stranded DNA virus (HpDNAV) which infects the
harmful dinoflagellate Heterocapsa pygmaea. It is icosahedral and lacks a tail. The size is ca. 160-170 nm in diameter. So, HpDNAV is
a lytic phycodnavirus. The latent period of HpDNAV was estimated to be about 76 hours. This is the first isolated dinoflagellate infecting
virus from Korean coastal water, and the detailed characterizations of both Heterocapsa pygmaea and its virus are now under way.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 116
DOMOIC ACID-BOUND SUBSTANCE
IN SOME STRAINS OF BACTERIA
Masaaki Kodama1, Yoshinobu Takata2, Kenji Kobayashi3, Shigeru Sato4
1
Graduate School of Agricultural and Life Science, The University of Tokyo, Yayoi, Tokyo, Japan.
E-mail. [email protected]
2
Shin Nihon Kentei Kyokai, Kohoku-ku, Yokohama, Japan
3
Graduate School of Medicine, Gunma University, Maebashi City, Gunma, Japan
4
School of Marine Biosciences, Kitasato University, Ofunato City, Iwate, Japan
Domoic acid is a causative toxin for amnesic shellfish poisoning which was first found in Prince Edward Island, Canada in 1987 (Wright
et al., 1989). Since the causative plankton species in the Canadian incident was identified as Pseudo-nitzschia multiseries (Bates et al.,
1989), more than 20 species of Pseudo-nitzschia have been reported to produce domoic acid (Bates and Trainer 2006). It has been
pointed out that domoic acid production decreases much when the culture is made axenic, but it recovers or increases more than that of
the original culture when some strains of bacteria in the original culture are added to the axenic culture (Bates et al., 1995). Autonomous
domoic acid production of bacteria is not confirmed. From these, environmental bacteria are considered to be not involved in the production
of domoic acid directly but in the enhancement of the domoic acid production of P. multiseries. On the other hand, we found that some
genes of bacteria which enhance domoic acid production of P. multiseries are detected in the axenic culture of P. multiseries (Kobayashi
et al. 2003). We also reported that the direct contact of living bacteria is necessary for axenic strains of P. multiseries to produce high
amount of domoic acid (Kobayashi et al. 2009). We report here that some strains of bacteria possess substance(s) which seem to have
domoic acid as a part of the molecule.
116
MIXOTROPHY IN THE HARMFUL DINOFLAGELLATE
GYMNODINIUM AUREOLUM
Kyung Ha Lee, Hae Jin Jeong, Yeong Du Yoo, Nam Seon Kang, Eun Young Yoon,
Eric Potvin, Kyeong Ah Seong, Jae Seong Kim
School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University,
51-747 Seoul, Republic of Korea. E-mail: [email protected]
We investigated mixotrophy in the harmful dinoflagellate Gymnodinium aureolum. During an algae bloom in March 2008 in western
Korea, Gymnodinium aureolum was isolated and clonal cultures were established. G. aureolum identified based on morphological and
genetic analysis (GenBank accession no. FN392226). We firstly report here that the red-tide dinoflagellate G. aureolum, which has previously been thought to be autotrophic, is a mixotrophic species. We observed that G. aureolum fed on algal prey using a peduncle. We
found that G. aureolum ingested heterotrophic bacteria, the cyanobacterium Synechococcus sp., and small algal species that had equiv_ 11.5 μm. However, it did not feed on larger algal species (ESDς >
_ 12 μm) or the small diatom
alent spherical diameters (ESDs) of <
Skeletonema costatum. The specific growth rate for G. aureolum on the cryptophyte Teleaulax sp. increased continuously with increasing
mean prey concentration before saturating at prey concentration of ca. 190 ng C ml-1 (11.050 cells ml-1). At 20oC under a 14:10 h lightdark cycle of 20 μE m-2 s-1 condition, the maximum specific mixotrophic growth rate of G. aureolum on Teleaulax sp. was 0.169 d-1,
while its phototrophic growth (without adding prey) was 0.058 ng C grazer-1 d-1 (3.4 cells grazer-1 d-1) and 0.003 μl grazer-1 h-1, respectively.
The calculated in situ grazing coefficients for G. aureolum on co-occurring cryptophytes was up to 0.498 d-1, respectively. Bioassay
results showed that this G. aureolum is not toxic strain. This study shows that G. aureolum have a potential considerable grazing impact
on the algal populations.
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
ALLELOCHEMICALS PRODUCED BY THE
DINOFLAGELLATE ALEXANDRIUM TAMARENSE:
PARTIAL CHEMICAL CHARACTERIZATION
AND PUTATIVE MODE OF ACTION
Haiyan Ma, Bernd Krock, Urban Tillmann, Allan Cembella
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany.
E-mail: [email protected]
Allelopathic effects of Alexandrium spp. including growth inhibition and cell lysis of target species are believed to contribute to population
dynamics and bloom formation of the producing organisms. We isolated, purified, and partially characterized lytic compounds produced
by A. tamarense, based on detection and quantification of lytic activity by a Rhodomonas salina bioassay. A. tamarense produces two
types of lytic compounds with different polarities, with the less hydrophilic component showing much higher activity. Further characterization of this high activity fraction by MALDI-TOF mass spectrometry revealed the presence of large amphiphilic compounds with molecular masses ranging from 9kDa to 15kDa. Lytic activity appears unrelated to proteinaceous structures because no activity was lost
after tryptic digestion. Total sugar content was twice as high in the most purified fraction of a lytic Alexandrium strain compared to a
control non-lytic strain. Additionally, we investigated the putative mode of action of the membrane-disruptive lytic compounds. Based
on the Ca2+ influx behavior of a model mammalian PC12 cell line exposed to various lytic fractions, we conclude that these lytic compounds lyse target cells through direct disruption of the external membranes and that lytic activity is not related to ion-channel interaction.
Lysis of protistan targets is dose-dependently reduced by adding various sterols. Nevertheless, determination of sterol compositions of
A. tamarense as well as five other protist species sensitive to these lytic compounds indicates that the molecular targets of the lytic
compounds are not or not exclusively sterol components of the membranes.
117
ISOLATION AND CHARACTERIZATION OF AN ENZYME
INVOLVED IN POLYETHER-TOXIN BIOTRANSFORMATION
IN BIVALVES
Lincoln MacKenzie
Cawthron Institute, 98 Halifax Street, Nelson 7010, New Zealand. E-mail: [email protected]
Fractionation of polyethylene glycol extracts (anion exchange, hydrophobic interaction, hydroxyapatite chromatography, gel filtration)
of the hepatopancreas of the GreenshellTM mussel Perna canaliculus resulted in the recovery of a single band on protein SDSPAGE gels. This 66 kDa molecular weight protein corresponded to up to 116 times enrichment of hydrolytic activity against
pectenotoxin-2, okadaic acid diol-esters, and fatty acid esters of various chain lengths. The enzyme is a serine esterase with a
high specificity for PTX-2 and PTX-1 analogues but is not active against PTX-11 and PTX-6. The enzyme hydrolyzes palmitoylDTX-1 at a rate comparable with that of porcine pancreatic lipase, suggesting that under non-aqueous conditions this enzyme
may also be involved in the synthesis of okadaic acid esters in vivo. Progress on the elucidation of the structure and genetic determinants of this enzyme will be described.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 118
ALGICIDAL ACTION OF AN ASCOMYCETE
AND SOME CULTURABLE BACTERIA ON HARMFUL
PHILIPPINE DINOFLAGELLATE SPECIES
Kamille Joshua V. Manset, Rhodora V. Azanza, Deo Florence Onda
The Marine Science Institute, University of the Philippines, Velasquez Street, Diliman, Quezon City, Philippines.
E-mail: [email protected]
Harmful Algal Bloom (HAB) occurrences in recent years have created serious problems in the marine environment and the coastal populations. Several management methods have been tried or studied and one of which is the biological means, an approach being evaluated
in this continuing research. A total of 42 bacteria and an ascomycete were isolated from Sorsogon Bay, Sorsogon, Philippines, in August
and December 2009. Thirteen isolates, including the ascomycete Clavispora lusitaniae, exhibited a fast-killing action against Pyrodinium
bahamense var. compressum (strain PbcMZRVA042595) cells. They were further tested on four other harmful dinoflagellate strains (P.
bahamense var. compressum strain from Honda Bay, Alexandrium sp., Prorocentrum micans and Amphidinium carterae). Algicidal activity
was estimated based on motility, ecdysis, lysis and disintegration. The order of susceptibility to algicidal activity seems to be P. micans >
PbcHBRVA102505 > PbcMZRVA042595 > Alexandrium sp. > A. carterae. Pseudoalteromonas sp. demonstrated a fast-killing action
against all the tested dinoflagellate species. For the first time this study was able to demonstrate the possible role of an ascomycete (and
some culturable bacteria) in harmful algal bloom dynamics and their potential use in HAB mitigation.
118
ELUCIDATION OF SOME OF THE MYSTERIES
OF AZASPIRACIDS
J. K. Nzoughet1, L. M. Botana2, C. T. Elliott1
1
2
Institute of Agri-Food and Land Use, Queen’s University, Belfast, UK. E-mail: [email protected]
Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela,
Campus Universitario, 27002 Lugo, Spain
Azaspiracids (AZAs), in terms of phycotoxins, are a relatively new phenomenon. Still much needs to be uncovered about the reasons for
their sudden occurrence, their mode of action and reasons why they persist in contaminated shellfish for very long periods. Factors such
as pollution and climate change have been, as with an increasing prevalence of other phycotoxins globally been blamed for the emergence
of this toxin group. A large scale proteomic study of azaspiracid contaminated shellfish was undertaken to determine if specific toxinprotein molecular interactions could be detected and if so a determination as to if these may provide information which will help uncover
some of the mysteries surrounding AZAs. Three proteins were identified that strongly suggest mussels treat AZAs as harmful to their
health. These three up-regulated proteins are interlinked in relation to their biological significance as all are involved in self-defense system
mechanisms and associated with carcinogenesis. This further suggests that the contaminated mussel colonies have not been exposed
to AZAs from a historical perspective and thus they have not evolved sufficiently to nullify the toxic effects of the phycotoxins. Another
important finding in the study is that the presence of AZAs in mussels seems to coincide with the presence of a Methylobacterium
species. A surprising finding which came about from these studies was this bacterial species is capable of utilising AZA as an energy
source. The research to date points towards the high level of AZA-protein interaction as the main cause why depuration of contaminated
mussels has been unsuccessful. The interaction between the AZA producing algae and the bacterial species identified in the study, may
have an important role in understanding why AZA toxins appeared during the 1990’s and their reappearance is highly intermittent.
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
DEGRADATION OF PARALYTIC SHELLFISH POISONING
TOXIN IN THE CULTURE MEDIA INOCULATED WITH
THE ENVIRONMENTAL SAMPLE
Hiroshi Oikawa1, Youhei Fukui2, Masataka Satomi2, Yutaka Yano2
1
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi,
Hatsukaichi, Hiroshima, Japan. E-mail: [email protected]
2
National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Kanazawa,
Yokohama, Kanagawa, Japan
Paralytic shellfish poisoning toxin (PST) is a potent neurotoxin, and over ten species of marine dinoflagellates are identified as the toxin
producer. Many filter-feeding shellfishes have been known as the vector of the toxin, and several species of predatory shellfishes like
crabs and gastropods also accumulate the toxin from their prey shellfish. Thus the toxin was widely distributed to aquatic organism in the
food web, but the fate of the toxin in the natural environment remains to be fully elucidated. Since it is natural that the microbial process
is involved in the degradation of the toxin, we examined PST degradation activities of some environmental samples such as sea water,
sea mud and bivalves which include microbial consortia. These samples were inoculated into the one-tenth strength nutrient IFO 325
culture medium which consists of yeast extract and peptone with PST, and the change of PST content was analyzed by HPLC-FLD.
PST decreased to about 70% after 2 weeks in several cultures inoculated with bivalve samples, and the same reduction ability was preserved in the subculture. To examine the usage of PST by the bacterial consortia as sole carbon source, the degradation of PST was
also investigated in the culture medium consist of Difco Yeast Nitrogen Base (Becton, Dickinson and Company, YNB) with PST. But
the degradation activity of PST was not preserved in the YNB medium. These results suggest that the reduction of PST was caused by
the cometabolic process in IFO 325 medium.
119
PHAGOTROPHIC BEHAVIOR OF PROROCENTRUM
DONGHAIENSE DURING THE BLOOM PERIOD
IN THE EAST CHINA SEA
Linjian Ou, Shuwen Zhang, Yuzao Qi, Songhui Lu
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510632, China.
E-mail: [email protected]
Ever since 1998, large scale blooms of Prorocentrum donghaiense have been reported in the Changjiang (Yangtze) River estuary and
the adjacent coastal waters of the East China Sea (ECS) during spring (late April - May) annually. The mechanism of the bloom formation
of P. donghaiense is still not very clear. By far, more and more phototrophic dinoflagellates have been proved to be mixotrophic. Jeong
et al. (2009) found that P. donghaiense could feed on Skeletonema costatum and the ingestion rate was 0.012ng C dinoflagellates-1
d-1 in lab. A field cruise was designed to follow the bloom process of P. donghaiense from 7th ~ 24th May, 2010 in the Changjiang River
estuary and the adjacent coastal ESC. In this study, the fluorescent labelled Isochrysis galbana (FLI) and S. costatum (FLS) were used
to study the phagotrophic behavior of P. donghaiense in situ during the bloom period. Results showed that cells of P. donghaiense could
feed on both FLI and FLS although the ingestion frequencies were scarce among the cells tested. Most cells of P. donghaiense which
were found to feed on FLI and FLS were in the southern area (from 28.0 to 30.0oN and 121.7 to 123.5oE) where bloom occurred. The
ingested cells of FLI and FLS by P. donghaiense ranged from one to several. Natural cells were also found in some P. donghaiense.
The abundant S. costatum which co-occurred with P. donghaiense during this bloom in this area might be one nutrient source forP.
donghaiense through the mode of phagotrophy; however the contribution of this nutritional mode and its importance for the bloom
formation and persistence of P. donghaiense is not sure.
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PHYTOPLANKTON COMMUNITY COMPOSITION
AND DIVERSITY EFFECTS ON THE GROWTH
OF POTENTIALLY PATHOGENIC MARINE VIBRIO BACTERIA
Olof Peterson1, Ann-Sofi Rehnstam-Holm2, Maria Asplund2, Indrani Karunasagar3, Anna Godhe1
1
Dept. of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden. E-mail: [email protected]
Section for Aquatic Biology and Chemistry, Kristianstad University, SE-291 88 Kristianstad, Sweden
3
Dept. of Fishery Microbiology, College of Fisheries, Karnataka Veterinary Animal and Fisheries Sciences University,
Mangalore 575 002, India
2
The marine autochthonous bacteria of the genus Vibrio contain several species of outmost importance to human health and economic
interests, and the ecology is closely linked to the epidemiology of the bacteria. This study explores which phytoplankton species or phytoplankton community factors (e.g. diversity) affect the growth of Vibrio spp. in an oligotrophic tropical marine area. A follow up lab experiment tested the effect of increased phytoplankton species richness on the growth of a Vibrio species. The environmental study
indicated that there is a similarity in phytoplankton community composition between the samples with high Vibrio abundance. Three
diatom taxa, Pseudo-nitzschia spp., Dactyliosolen phuketensis and Leptocylindrus danicus were found to be most favorable to the bacteria
growth. However, several diversity factors, such as species richness, were also high contributors. The lab experiment did not find any difference between the included phytoplankton species. However there was a significant positive effect of phytoplankton compared to
growth in f/2 media only. Also, there was a significant effect of increasing phytoplankton species richness from one to two or three
species. Thus, this study shows that healthy phytoplankton cells can serve as an important carbon source for Vibrio spp. bacteria and
that increased species richness can positively affect the abundance of vibrios. This is new knowledge, which should be considered when
assessing algal blooms and their affect on pathogenic bacteria.
120
PIGMENT COMPOSITION IN THE DINOFLAGELLATE
GENUS DINOPHYSIS AND THE ASSOCIATED CULTURES
OF MYRIONECTA RUBRA AND CRYPTOPHYTE SPECIES
Pilar Rial1, Manuel Zapata2, Jose Luis Garrido2, Francisco Rodríguez1
Centro Oceanográfico de Vigo (Instituto Español de Oceanografía, IEO), 36200 Vigo, Spain.
E-mail: [email protected]
2
Instituto de Investigaciones Marinas (Consejo Superior de Investigaciones Científicas, CSIC),
C/Eduardo Cabello, 6, 36208 Vigo, Spain
1
Since the milestone of bringing into culture the first Dinophysis species (D. acuminata) in recent years, several other species have
been successfully maintained in the laboratory. This achievement has opened the possibility of new studies on the ecology and physiology of this former “elusive” dinoflagellate genus. In particular, there exists still an active debate about the nature of the plastids in
Dinophysis, whether they are only “kleptoplastids” or if it contains plastids of its own. This discussion has been only addressed from
the morphological and molecular perspectives. However, none of these studies have included the characterization of the pigment
composition of Dinophysis species despite these results could throw new light about the nature of its plastids. In the present study
we analyzed the pigment composition (both lipophylic and hydrosoluble pigments) in several Dinophysis species and their associated
cultures (their own prey, the photosynthetic ciliate Myrionecta rubra, and the cryptophytes preyed by the ciliate itself). The obtained
results allowed us to check any differences that could confirm that Dinophysis species contain any distinct pigments (i.e. plastids)
from those of cryptophytes and the ciliate M. rubra.
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BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
RAPID REDUCTION OF THE PST CONTENT
IN THE HETEROTROPHIC DINOFLAGELLATE NOCTILUCA
SCINTILLANS FED ON A TOXIC DINOFLAGELLATE
Evangelos Spyrakos1, Máximo Frangópulos2, Cástor Guisande1
1
2
Faculty of Marine Sciences, Universidade de Vigo, Lagoas-Marcosende, E-36200 Vigo, Spain. E-mail: [email protected]
Centro de Estudios del Cuaternario de Fuego-Patagonia (Fundación CEQUA), Av. Bulnes 01890, P.O. Box 6213029,
Punta Arenas, Chile
In order to investigate the ecological role of the heterotrophic dinoflagellate Noctiluca scintillans (Macartney) in the planktonic community
and the fate of Paralytic Shellfish Toxin (PST) in the planktonic food webs, ingestion rates of N. scintillans fed on the PSP-producer
dinoflagellate Alexandrium catenella were examined in laboratory conditions. The toxin content retained in N. scintillans prayed upon
A. catenella for 4 days in 12:12 h light:dark cycle conditions and then starved for 0, 3, 6, 9 and 12 hours was qualified through
HPLC. In parallel, the ingestion rates and the toxicity of N. scintillans fed on the dinoflagellate Prorocentrum micans were measured
as a control. Results showed that N. scintillans can actively feed on A. catenella and P. micans. The mortality (35%) and ingestion
rates (0.07 μg C ind-1 day-1) of N. scintillans fed on A. catenella were higher than these observed in the Noctiluca fed on P. micans.
The mean cellular toxin content of A. catenella in the stock culture ranged from 17 up to 22 fmol cell-1. The mean toxicity detected
in N. scintillans at Hour: 0 was 157 fmol ind-1. No retained toxicity was detected in Noctiluca specimens fed on P. micans. The
retained toxin in N. scintillans was gradually decreased with increasing the starvation time till a concentration of 12 fmol ind-1 at Hour:
12. The rapid reduction of the accumulated toxin shows that N. scintillans may be important as a regulator of PST in the planktonic
community by preventing its transfer or accumulation in the food web.
121
ALLELOPATHIC EFFECTS OF SPRING BLOOM
DINOFLAGELLATES ON CO-OCCURRING
PHYTOPLANKTON IN THE NORTHERN BALTIC SEA
Sanna Suikkanen, Paivi Sillman, Kristian Spilling, Anke Kremp
Finnish Environment Institute, Marine Research Centre, P.O. Box 140, FI-00251 Helsinki, Finland.
E-mail: [email protected]
Globally, dinoflagellate-dominated spring blooms are uncommon, but they regularly occur in the Baltic Sea, varying in importance with
diatom-dominated blooms. The reason for the significant interannual variability in the dominant taxa is unclear, and recently, the relative
contribution of dinoflagellates to spring bloom biomass has even increased in the area. We investigated if allelopathy can provide a
mechanism for the Baltic cold-water dinoflagellates to support their dominance, despite their lower rates of nutrient uptake and growth
compared with the co-occurring diatoms. Using batch cultures, we studied the allelopathic effects of three dominant vernal dinoflagellates;
Biecheleria baltica, Gymnodinium corollarium and Scrippsiella hangoei, on five typical spring bloom diatoms and one cryptophyte. We
also tested the effects of the dinoflagellates on each other. Both cell-free filtrates and live cells of the donor taxa were applied. Two of
the five diatoms, Melosira arctica and Skeletonema marinoi, were inhibited by filtrates and live cells of all three dinoflagellates, and also
Thalassiosira baltica was suppressed when co-cultured with the dinoflagellates. Only G. corollarium negatively affected Chaetoceros
cf. wighamii and Diatoma tenuis. In contrast, the cryptophyte Rhodomonas sp. was stimulated by all dinoflagellate treatments. Live S.
hangoei and B. baltica cells tended to inhibit the other dinoflagellates, whereas G. corollarium generally stimulated them. As some of
the major players of the diatom spring bloom can be suppressed by co-occurring dinoflagellates in culture, we conclude that allelopathy
may be one mechanism by which vernal dinoflagellates frequently outcompete diatoms and form intense spring blooms.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 122
ECOLOGICAL RELATIONSHIPS BETWEEN
DIATOM BLOOMS AND VIRUSES
Yuji Tomaru1, Kensuke Toyoda1,2, Keizo Nagasaki1
1
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi,
Hatsukaichi-shi, Hiroshima 739-0452, Japan. E-mail: [email protected]
2
Present address: Department of Botany, Keio University, Hiyoshi, Kohoku-ku, Yokohama, Kangawa 223-8521, Japan
Diatoms are one of the most abundant primary producers and play an important role in carbon cycles on our planet. They are also
important from the viewpoint of fisheries. Cultivation of the seaweed laver (Porphyra yezoensis) is of significant economic importance in
Japan; diatom blooms cause bleaching (discoloration) of the thalli due to depletion of nutrients. An example, from the end of 2000
through 2001, a large diatom bloom occurred in Ariake Sound in western Japan and led to an extremely poor harvest of P. yezoensis;
the damage was estimated at $108,000,000 compared to the year before (1999-2000). Therefore, it is essential to reveal the dynamics
of diatom blooms and the related factors. So far, a number of biological, chemical and physiological factors influencing diatom dynamics
have been investigated. Although no direct evidence showing viral influence on diatoms had been reported in 20th century, we succeeded
in isolating the first diatom virus in 2004. Since then, we have reported isolation and characterization of seven different diatom viruses.
All of them are icosahedral nonenveloped virus <40 nm in diameter, lacking a tail, harboring a single-stranded RNA or a single-stranded
DNA genome (5-11 kb in length), and cause lysis of their respective diatom host species. Diatom viruses in water columns rapidly
increased at the initiation of the host blooms and were detected at high frequency during the bloom periods. Hence, viruses may be potentially important in controlling diatom bloom dynamics in natural waters.
122
STUDY OF A NOVEL MARINE BACTERIUM ALGICIDAL TO
THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE
Wang Binxiang1,2, Zhou Yanyan1, Lu Jinling1, Su Jianqiang1, Tian Yuna Zheng Tianling1,2
1
Key Laboratory of MOE for Coast and Wetland Ecosystem, School of Life Sciences, Xiamen University,
Xiamen 361005, China. E-mail: [email protected]
2
State Key Laboratory of Marine Environmental Sciences, Xiamen University, Xiamen 361005, China
Interactions between bacteria and harmful algal bloom (HAB) species have been acknowledged as an important factor regulating both
the population dynamics and toxin production of these algae. A marine bacterium DHQ25 with algicidal activity to the toxic dinoflagellate
Alexandrium tamarense was isolated from the Donghai Sea area, China. Genetic identification was achieved by polymerase chain reaction
amplification and sequence analysis of 16S rDNA. Sequence analysis showed that the most probable affiliation of DHQ25 was to the
Î3-proteobacteria subclass and the genus Vibrio. Bacterial isolate DHQ25 showed algicidal activity through an indirect attack. Algicidal
activity assay indicated both axenic and nonaxenic Alexandrium tamarense were susceptible to DHQ25 when A. tamarense in latent or
stationary phase. The algicidal compounds produced by bacterium DHQ25 were heat tolerant under 60of, unstable in acidic condition
and could be easily synthesized regardless of variation in temperature, salinity or initial pH for bacterial growth. This is the first report of a
Vibrio sp. bacterium algicidal to the toxic dinoflagellate A. tamarense and the findings increase our knowledge of bacterial-algal interactions
and the role of bacteria during the population dynamics of HABs.
4. ABSTRACT BOOK (98-123)_Layout 1 10/22/10 2:29 PM Page 123
BIOLOGICAL INTERACTIONS: ALLELOPATHY,
MIXOTROPHY, PARASITISM, SYMBIOSIS, BACTERIA AND VIRUSES
POSTER PRESENTATIONS
ALLELOPATHIC EFFECTS OF COCHLODINIUM BLOOM IN
PEARL RIVER ESTUARY, CHINA, ON OTHER HAB SPECIES
Zhaohui Wang, Shan Jiang, Wei Kang, Langcong Lin, Yangguang Gu
Institute of Hydrobiology, Jinan University, Guangzhou 510632, China. E-mail: [email protected]
A bloom of Cochlodinium, covering over 300 km2, occurred in coastal waters of Pearl River Estuary, China, in October 2009. Water
samples in the bloom patches were collected during the bloom. The bloom causative species was identified as C. geminatum based on
morphological observation and 18 s rDNA LSU sequence. Phytoplankton was analyzed, and the effects of bloom sample on the growth
of the other three phytoplankton species Skeletonema costatum, Prorocentrum micans and Heterosigma akasiwo were studied. C. geminatum was the predominant species in phytoplankton community in bloom sample, with a cell density of 3.15x106 cells/L and percentage
proportion of 73.3%. This species displayed a high efficiency for producing cysts, but the shape of cyst was quite different from those
reported in both sediment and cultures of Cochlodinium species. The bloom water did not inhibit the growth of S. costatum, but accelerated
the growth to some extend. However, the growth of P. micans and H. akasiwo was significantly inhibited by the bloom water. The
inhibition substances were mainly in cell-free filtrates, and tended to be hot-resistive. The results suggest that allelopathy of C. geminatum
to other phytoplankton species play an important role in its bloom occurrence. Meanwhile, the feasibility in cyst formation of C. geminatum
and cyst accumulation in sediments might act as a benthic reservoir of the recurrent occurrence of its bloom in Pearl River estuary.
123
THE ROLE OF ALLELOPATHY IN THE SUCCESSION
OF HARMFUL ALGAL BLOOM IN THE EAST CHINA SEA:
THE CULTIVATED EXPERIMENTS OF HARMFUL ALGAL
SPECIES IN THE ALGAL BLOOM WATER
Weihong Zhao, Pengji Zhuo
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, China. E-mail: [email protected]
Except for the physicochemical environment, biotic factor play a significant role during the process of harmful algal bloom occurrence and
succession. Allelopathy is a significant biotic factor to influence the phytoplankton community structure. Allelopathy can affect the interspecies
competition of harmful algal bloom process, and then affect the community succession. This paper was using the typical algal bloom cellfree water collected during the succession process of the diatom bloom to the dinoflagellate bloom in the East China Sea in the April to
May of 2007 to cultivate the main algal bloom species, Prorocentrum donghaiense, Alexandrium tamarens and Skeletonema costatus in
the laboratory in order to observe if the allelopathy play some role in the algal bloom succession in the East China Sea. In the spring of
2007, there was a short term pico-flagellate bloom between the diatom bloom and dinoflagellate bloom. After the diatom bloom dispersion,
the water of pico-flagellates bloom in incubation time would inhibit the growth of Prorocentrum donghaiense, Alexandrium tamarens and
Skeletonema costatus. The water in pico-flagellates eruptional time would have very strong inhibit ional effect on the growth of Prorocentrum
donghaiense, Alexandrium tamarens, but weaker inhibit ional effect on the growth of Skeletonema costatus. The cultivated experiment
results essentially agreed with the on-site algal bloom succession process. The allelopathy plays a certain role in the algal bloom succession.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 124
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 125
NEW REGIONAL
HAB EVENTS
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 126
ORAL PRESENTATIONS
BLOOMS OF COCHLODINIUM POLYKRIKOIDES
ALONG THE COAST OF OMAN AND THEIR EFFECTS
Hamed Al Gheilani1, Adnan Al Azri2, Sergey Piontkovoski2, Sergey Debrotsov2, Al Issa Amri3,
Ishah Al Ambo Ali2, Sarah Al Jufaili4, Saoud Al Bousaidi1, Salem Al Hajri1, Ahmed Al Aisari1,
Hilal Al Shaqsi1, Nader Al Abri1, Khalid Al Hashmi2
1
Marine Science and Fisheries Center, Ministry of Fisheries Wealth, P.O. Box 427, Code 100, Muscat, Oman.
E-mail: [email protected]
2
Department of Marine Science, College of Agriculture and Fisheries, Sultan Qaboos University, Al Kwod, P.O. Box 36,
Code 123, Muscat, Oman
3
Quality Control Center, Ministry of Fisheries Wealth, P.O. Box 427, Code 100, Muscat, Oman
4
College of Medicine, Sultan Qaboos University, Al Kwod, P.O. Box 36, Code 123, Muscat, Oman
Intensive blooms of Cochlodinium polykrikoides caused massive fish kills in the wild and aquaculture farm and affected tourism along
coasts of Oman bordering the Arabian Gulf, Oman, and Arabian seas during 2008-2009. Water quality parameters were estimated for
microbes, heavy metals, toxicity and organic and inorganic compounds. Fish and shellfish samples were collected during the blooms
period and heavy metals, microbes, Parasite infection and toxicity were analyzed. The histopathological alterations in the normal and affected fish gill histology were studied using light and scanning electron microscopy. Phytoplankton species were identified using light and
scanning electron microscopy. The remote sensing images including temperatures and chlorophyll-a were analyzed prior, during and
after the blooms indicated. Physical-chemical parameters of the water in the region was monitored regularly to correlate the parameters
and to determine the mechanism of how C. polykrikoides could cause massive marine organisms kill.
126
FIRST RECORD OF A COCHLODINIUM POLYKRIKOIDES
BLOOM ALONG THE SOUTH COAST OF IRAN
Gilan Attaran-Fariman1, Christopher J. S. Bolch2
1
2
Chabahar Maritime University, Daneshgah Ave., Chabahar 99717-56499, Iran. E-mail: [email protected]
National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College,
University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia. E-mail: [email protected]
A bloom of the dinoflagellate Cochlodinium polykrikoides occurred in Iran’s southeast coast. An initial red-brown water discoloration was
observed in late April 2009 near the Straits of Hormuz between Persian Gulf and Oman Sea. Two weeks later the bloom had dispersed
to other coastal area along southeast coast of Iran and persisted during May and June. The bloom was observed in the 200-300 m
nearest the shoreline and extended to over 60 km of coastline. Maximum cell densities observed were 1.9 x 107 cells ml-1 and a chlorophyll-a maximum of (24.1 μg L-1) in late May 2009. Blooms were composed of Cochlodinium cells occurring in single cells and chains
of up to 8 cells in length. Cell density decreased during the SWM monsoon period and after end of the South West monsoon phytoplankton
composition changed. No major mortality of marine organisms was observed however the bloom was associated with a bad odor and
surface foaming that persisted for two months. In late September a bloom of Gymnodinium sp. reached a max density of 6.0 x 106 cells
ml-1. This bloom event turned the water color to red-brown without any mortality and lasted for only 3-4 days. Highest cell density occurred
at water temperatures of 29-39oC, and a salinity of 35-37 ppt.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 127
NEW REGIONAL HAB EVENTS
CIGUATERA IN MACARONESIA: AN EMERGING PROBLEM.
CONTRIBUTION TO THE IDENTIFICATION
OF GAMBIERDISCUS SPECIES
Santiago Fraga1, Francisco Rodríguez1, Amandine Caillaud2, Jorge Diogène2,
Nicolás Raho3, Irma Marín3, Manuel Zapata4
Centro Oceanográfico de Vigo, IEO (Instituto Español de Oceanografía), Subida a Radio Faro 50, 36390 Vigo, Spain.
E-mail: [email protected]
2
IRTA, Ctra. Poble Nou, Km 5,5, 43540 Sant Carles de la Ràpita, Spain
3
Centro de Biología Molecular “Severo Ochoa”, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
4
Instituto de Investigacións Mariñas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain
1
The first recorded case of ciguatera poisoning in NE Atlantic, and the first observation of Gambierdiscus in the Canary Islands were reported
in 2004. Since then, more cases of ciguatera were reported in the area and individuals of two different species of Gambierdiscus were
isolated and brought into culture. The larger species, about 100 μm in diameter, has a ventrally displaced Po plate and is proposed as a new
species. This species is a potent maitotoxin producer while ciguatoxin production is very low. The other species is smaller in size, has a wider
2’ plate and could be what Estela Sousa e Silva reported as Goniodoma sp. from Cabo Verde islands in the 50’s. In this communication,
details of the morphologies, phylogeny, toxins and pigments of these species will be given.
127
OSTREOPSIS CF. OVATA FUKUYO IN THE GULF
OF TRIESTE (NORTHERN ADRIATIC SEA):
TAXONOMY, TOXIN CONTENT AND ULTRASTRUCTURE
Giorgio Honsell1, Cecilia Battocchi2, Oriana Blasutto3, Marco De Bortoli4, Carmela Dell’Aversano5,
Sabrina Boscolo6, Roberto Marzari6, Antonella Penna2, Luciana Tartaglione5, Mark Poli7, Aurelia Tubaro4
1
Department of Biology and Plant Protection, University of Udine, via delle Scienze 91-93, 33100 Udine, Italy.
E-mail: [email protected]
2
Biomolecular Science Department, University of Urbino, viale Trieste 296, 61100 Pesaro, Italy
3
ARPA FVG, via Cairoli 14, 33057 Palmanova, Italy
4
Department of Materials and Natural Resources, University of Trieste, via A. Valerio 6, 34127 Trieste, Italy
5
Department of Chemistry of Natural Products, University of Naples, via Montesano 49, 80131 Naples, Italy
6
Department of Life Sciences, University of Trieste, via L. Giorgieri 10, 34127 Trieste, Italy
7
Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases,
Ft Detrick, Maryland 21701-5011, USA
Blooms and toxic events associated with Ostreopsis species have been increasing in the Mediterranean over the last few years, including the
Gulf of Trieste where Ostreopsis cf. ovata was first reported in 2006. The first Ostreopsis bloom (6,700,000 cell/L) in the Gulf of Trieste was
detected in very shallow waters on 28 September 2009. Cells were definitely identified as Ostreopsis cf. ovata Fukuyo by morphological
features (light microscopy and SEM) and molecular analysis. LC-MS analyses of both natural planktonic and cultured O. cf. ovata were carried
out using either a triple quadrupole MS operating at unit resolution in multiple reaction monitoring mode or a hybrid linear ion trap-Fourier Transform MS instrument operating in full MS mode at high resolution settings. The results revealed the presence of palytoxin and palytoxin-like
compounds with ovatoxin-a being the major component. The presence of native palytoxin was confirmed for the first time also by the immunocytochemistry using polyclonal and monoclonal anti-PLTX antibodies. SEM and TEM observations of cultures and natural populations showed
interesting ultrastructural features with new details on the formation of the mucilaginous network around the cells.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 128
SPATIAL AND TEMPORAL VARIATIONS OF PLANKTONIC
AND EPIBENTHIC CELLS OF OSTREOPSIS CF. OVATA
IN NW MEDITERRANEAN (LIGURIAN SEA)
Rodolphe Lemée1, Thierry Thibaut2, Stéphanie Cohu1,2, Aurélie Blanfuné1,2, Sophie Marro1, Luisa Mangialajo2
1
Laboratoire d’Océanographie de Villefranche, CNRS UMR 7093, Université Pierre et Marie Curie,
06234 Villefranche-sur-mer, France. E-mail: [email protected]
2
Laboratoire Ecosystèmes Côtiers Marins et Réponse aux Stress, EA 4228, Université de Nice-Sophia Antipolis,
Parc Valrose, 06108 Nice, Cedex 2, France
This work was done within the framework of MediOs 2 (Mediteranean Ostreopsis). The goal of this national French project was to acquire
and analyze pertinent scientific knowledge on Ostreopsis occurrence in areas as diverse as ecology, biology, chemistry, epidemiology or
socio-economic issues in the Mediterranean Sea, in order to provide a decision-making support for administrative action. Variability in
concentrations of planktonic and epibenthic cells of the toxic dinoflagellates Ostreopsis cf. ovata was studied in 2008 and 2009 in NW
Mediterrean Sea (from Cassis, France to Genoa, Italy). Different spatial and temporal scales were analyzed, from m to hundreds of km
and from hours to years. Spatial patterns indicated that temperature, nutrients and local hydrodynamics could be linked to the severity
and the length of a bloom in a specific area, even if great variances were observed, not always clearly relatable to biological substrate
characteristics or depth. Temporal patterns showed the occurrence of a large first bloom in June, July or August, according the area and
a second less important bloom can occur in autumn. A high-frequency sampling indicated a daily rhythm, with higher quantities of benthic
cells in the morning and higher concentrations of planktonic cells before night.
128
KARLODINIUM VENEFICUM APPEARED
AS CO-OCCURRING BLOOM SPECIES OF KARENIA
MIKIMOTOI AND PROROCENTRUM DOGHAIENSE
IN THE EAST CHINA SEA
Douding Lu, Hongxia Wang, Haiyan Huang,Ping Xia, Xingfeng Dai
Lab of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, SOA, 36 Baochu Bei road,
Hangzhou 310012, China. E-mail: [email protected]
A small unarmored dinoflagellete was recorded as co-occurring bloom species of Karenia mikimotoi and Prorocentrum doghaiense in the
East China Sea (ECS), in the late spring of 2006. The highest cell concentration of this species was 5x106 cells/L during the bloom.
The species is confirmed as Karlodinium veneficum (D. Ballantine) J. Larsen both morphologically and genetically. The mean cell length
of 70 measured cells was 15.3 μm with a standard deviation of ± 1.4μm and the mean width of cells was 11.7 μm with a standard
deviation of ±1.3 μm. The ventral pore is located at the left side of the apical groove. Nucleus is large and positioned centrally at hypocone.
There are two or four large irregular chloroplasts inside of the cell. The sequence length of ECS strain LAMB090611 and German strain
LAMB010601 is 640bp and 646bp, respectively. The GC content is 49%. The nucleotide similarity is 98%. The nucleotide divergence
is 0.003. The Phylogenetic tree based on rDNA ITS sequence shows that strain LAMB090611 and LAMB010601 of K. veneficum is
clustered together with 3 other strains of K. veneficum from Genbank. They are the identical species.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 129
NEW REGIONAL HAB EVENTS
ORAL PRESENTATIONS
TRENDS IN OSTREOPSIS CF. OVATA
PROLIFERATION ALONG THE NORTHERN
MEDITERRANEAN SEA
(NW MEDITERRANEAN AND ADRIATIC SEAS)
COASTAL WATERS
Luisa Mangialajo1, Nicolas Ganzin2, Stefano Accoroni3, Valentina Asnaghi4, Aurélie Blanfuné5,1,
Marina Cabrini6, Riccardo Cattaneo-Vietti4, Fabienne Chavanon2, Mariachiara Chiantore4,
Stéphanie Cohu1,5, Eleonora Costa4, Daniela Fornasaro6, Hubert Grossel2,
Françoise Marco-Miralles2, Mercedes Masó7, Albert Reñé7, Anna Maria Rossi4,
M. Montserrat Sala7, Thierry Thibaut1, Cecilia Totti3, Magda Vila7, Rodolphe Lemée5
1
Université de Nice-Sophia Antipolis, EA ECOMERS, Parc Valrose, BP 71, 06108 Nice, Cedex 2, France.
E-mail: [email protected]
2
Ifremer - Institut Français de Recherche Pour l’Exploitation de la Mer - Centre de Méditerranée,
Laboratoire Environnement / Ressources, ZP de Brégaillon, BP 330, 83507 La Seyne sur Mer, France
3
Dipartimento di Scienze del Mare, Università di Scienze del Mare, via Brecce Bianche, 60131 Ancona, Italy
4
Università degli Studi di Genova, Dip.Te.Ris., C.so Europa 26, 16132 Genova, Italy
5
Université Pierre et Marie Curie-Paris 6, Marine Microbial Ecology Group, Laboratoire d’Océanographie de Villefranche,
06230 Villefranche-sur-Mer, France
6
Istituto Nazionale di Oceanografia e Geofisica Sperimentale, Dipartimento di Oceanografia Biologica,
Via Auguste Piccard, 54, 34014 Trieste, Italy
7
Institut de Ciències del Mar (ICM-CSIC), Pg. Marítim de la Barceloneta 37-42, 08003 Barcelona, Spain
Harmful benthic microalgae blooms represent an emergent phenomenon in temperate areas, causing health and economic concern, especially in touristic areas. This is the case for the Mediterranean Sea, where Ostreopsis blooms occur in summer, with increasing regularity. Although a relationship with sea water temperature has been highlighted in some areas, other factors such as
wave exposure, nutrients and salinity, are potentially involved; at the present state of knowledge we are not able to forecast the
risk of Ostreopsis development. In this study we present the occurrence and proliferation of Ostreopsis in 15 Spanish, French
and Italian sites along the northern limit of the Mediterranean Sea. Benthic and planktonic cell abundances from 2007, 2008 and
2009 were pooled in order to search for possible general trends in Ostreopsis proliferation. The results highlight that the sea
water temperature does not seem to be a primary driver and that maximal abundance periods are Site-Year specific. Nevertheless
general trends are observed in the two considered basins: in north-western Mediterranean Sea higher cell abundances are mostly
recorded in mid-summer (end of July), while in the Adriatic Sea in early fall (end of September). Such results represent an important
step in the understanding of harmful benthic microalgae blooms in temperate areas, and represent a good base for managers in
the attempt of forecasting toxic phenomena.
129
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GROWTH, TOXICITY AND MORPHOLOGICAL VARIATION
OF OSTREOPSIS CF. OVATA UNDER DIFFERENT LIGHT
AND TEMPERATURE CONDITIONS
Marina Montresor1, Eleonora Scalco1, Giovanna Benvenuto1, Francesca Marino1, Rachele Rossi2,
Vittorio Soprano2, Adriana Zingone1
1
2
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected]
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute 2, 80055 Portici, Italy
In the last years, blooms of Ostreopsis cf. ovata repeatedly occurred along the Mediterranean coasts during summer and/or autumn,
causing problems to human health and economic activities. In order to gain insights on the factors regulating bloom dynamics of this
species, we tested growth responses of 3 strains over a combination of temperature (14, 18, 22, 26, 30 and 34oC), photoperiod (9L:15D,
12L:12D, 15L:9D), and irradiance (50 and 200 μmol photons m-2 s-1) conditions simulating those recorded in the natural environment
over the year. Ostreopsis cf. ovata can grow at temperatures between 18 and 30oC. At 14oC the species can survive but does not grow;
at 34oC cultures die. The maximum growth rates, from 0.56 to 0.82 divisions·day-1, were recorded at the two longer day length conditions
and at temperatures between 18 and 26oC. At low irradiance, no significant differences were recorded between growth rates at 12L:12D
and 15L:9D, whereas at higher irradiance, longer day length conditions often caused a decrease in growth. At the lowest and highest
temperatures morphologically aberrant cells were produced after a few days, while at the optimal growth conditions small cells became
rapidly abundant. Toxin analyses of strains grown under different temperature conditions are under way. As compared to its occurrence
in the natural environment, Ostreopsis cf. ovata is able to grow over a broader range of temperature and day-length conditions. The
species seems to perform better at longer day lengths and lower irradiances, which match their life conditions among macroalgal thalli in
the summer period.
130
FIRST REPORT OF RECURRENT TOXIC ALEXANDRIUM
BLOOMS
IN THE EAST CHINA SEA
Yunfeng Wang , Rencheng Yu , Douding Lu , Songhui Lü , Chuansong Zhang , Dedi Zhu ,
1
1
1
1
2
3
1
4
2
Qingchun Zhang , Tian Yan , Mingjiang Zhou
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, People’s Republic of China. E-mail: [email protected]
2
Second Institute of Oceanography, State Ocean Administration, Hangzhou 310012, People’s Republic of China
3
Research Center of Harmful Algal Blooms & Aquatic Environmental Science, Jinan University, Guangzhou 510632,
People’s Republic of China
4
Ocean University of China, Qingdao 266003, People’s Republic of China
A rapid increase of global distribution of toxic Alexandrium species and their associated paralytic shellfish poisoning (PSP) incidents, which posed
potential threats on the human health, mariculture industry and natural ecosystems, was observed in the last century. In China, toxic species in genus
Alexandrium, as well as PSP contamination in shellfish products, have been detected, but little information is available on the Alexandrium blooms.
From 2002 to 2007, during the field surveys organized to investigate the large-scale dinoflagellate blooms in sea areas adjacent to the estuary of
the Changjiang River in the East China Sea, recurrent Alexandrium blooms were found besides the large-scale blooms of Prorocentrum donghaiense.
Cells of Alexandrium tamarense and A. catenella were identified from the phytoplankton samples. During the investigation, Alexandrium blooms
were observed in April and May every year except for 2003. The area affected by the Alexandrium blooms could reach 5,000 km2, with the maximum
cell density at 7×105 cells l-1 in the surface water. Patches of Alexandrium blooms were mainly found in sea area near the Zhoushan Island (29.031.0 oN, 122.0-123.0 oE) along the 30m isobath, with salinity between 28 and 31, and temperature between 14 and 22oC. The blooms were insulated from the inshore area by the high-turbid water with turbidity over 15 NTU. The initiation date of the Alexandrium blooms observed every year
related well with the seawater temperature at bottom in early April. Cyst germination promoted by the warm water should be the most important
trigger for the Alexandrium blooms. Through analysis of the concentrated phytoplankton samples with the mouse bioassay method or high performance
liquid chromatography (HPLC), it was found that the toxicity were at the range of 0.46-2.68 μg STX eq l-1 seawater, and the cellular toxin content
was at the range of 17-88 fmol cell-1. PSP toxins detected with HPLC include C1, C2, GTX1, GTX2, GTX3, GTX4 and neoSTX, and C is the exclusively dominant toxin. The mechanism and potential impacts of Alexandrium blooms in this region require more intensive studies in the future.
1
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NEW REGIONAL HAB EVENTS
ORAL PRESENTATIONS
SUCCESSION AND EVOLUTION
OF HABS ALONG CHINESE COAST
Mingjiang Zhou
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China. E-mail: [email protected]
In 2008, a huge green tide caused by Entermorpha prolifera (Ulva prolifera) broken out along the Chinese coast of the southern Yellow
Sea which covered an area of >10,000 km2, brought critical threat to the 2008 Olympic Sailing Game and caused a loss of about 200
M USD directly. Previous data indicated that a clear succession of the dominant micro-algae HAB species from diatom to dinoflagellate
was observed particularly in the Changjiang River estuary and its adjacent sea areas in the East China Sea. The macro-algae Entermorpha
bloom added more question to how and why the HABs evolved along Chinese coast. Therefore, a new Chinese national HAB project,
CEOHAB-II (Chinese Ecology and Oceanography of Harmful Algal Blooms-II) was set up focusing on the HAB’s succession and evolution
along Chinese coast. Three key scientific questions related to CEOHAB-II are: “Key factors to control the distribution pattern and succession of HABs”, “The function of eutrophication process in driving HABs succession”, and “Pathways and mechanics of the degradation
of coastal ecosystem caused by HABs and its succession”. Based on previous data and 3 recent cruises in the spring of 2010, some
preliminary results will be delivered.
131
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 132
POSTER PRESENTATIONS
REVISITING THE HAB-MAP PROJECT
FOR SOUTH AMERICA
Rut Akselman1, Martha E. Ferrario2, Silvia M. Méndez3, Clarisse Odebrecht4, Gemita M. Pizarro5,
Sonia Sánchez Ramírez6, Gladys Torres7
1
Instituto Nacional de Investigación y Desarrollo Pesquero, V. Ocampo 1, B7602HSA Mar del Plata, Argentina.
E-mail: [email protected]
2
Facultad de Ciencias Naturales y Museo, Paseo del Bosque s/n, 1900 La Plata, Argentina
3
Dirección Nacional de Recursos Acuáticos, Constituyente 1497, 11200 Montevideo, Uruguay
4
Fundaçao Universidad Federal do Rio Grande, Departamento de Oceanografia, 96201-900 Rio Grande, Brasil
5
Instituto de Fomento Pesquero, Enrique Abello 552, Punta Arenas, Chile
6
Instituto del Mar del Perú, Esq. Gamarra y Gral. Valle s/n, Callao, Perú
7
Instituto Oceanográfico de la Armada, Vía Puerto Marítimo, Guayaquil, Ecuador
Spatial and temporal distribution of harmful microalgal species is essential in understanding harmful events and useful for risk assessment.
The ISSHA project HAB-MAP was created to establish a global referenced map of the known distribution of toxic marine microalgae species.
This study constitutes a second stage of the first HAB-MAP database of toxic marine microalgae species from coastal and shelf waters of
Brazil, Uruguay, Argentina, Chile, Perú and Ecuador developed up to 2005 on the basis of a literature review that included location, date, cell
abundance, toxin content, harmful effects and relevant environmental information. This updated database adds new references based on literature review for the 2005-2010 period from those species included at the IOC Taxonomic Reference List of Toxic Plankton Algae (except
cyanobacteria) and is complemented with georreferenced maps of distribution that will be published on-line. Taking into account the whole
region, seven new species were detected (Pseudo-nitzschia calliantha, P. cuspidata, Alexandrium minutum, Azadinium spinosum, Ostreopsis
ovata, Prorocentrum emarginatum, P. rathymum), eight species were associated to toxicity in new areas (Pseudo-nitzschia australis, P. calliantha, P. fraudulenta, P. multiseries, P. pungens, Alexandrium tamarense, Dinophysis acuminata, D. caudata), and an extension of its geographical distribution was registered for twenty three species. This updated database includes 47 taxa for the South American region here
considered. The new records of potentially toxic algal species registered during this period as well as the apparent geographical expansion of
other taxa, marks the importance of continuing this kind of regional cooperative research as is here presented.
132
AZADINIUM SPINOSUM ELBRÄCHTER ET TILLMANN
(DINOPHYCEAE) IS PRESENT AND ALSO CAUSED
BLOOMS AT THE SOUTHWESTERN ATLANTIC
Rut Akselman, Rubén M. Negri
Instituto Nacional de Investigación y Desarrollo Pesquero, V. Ocampo 1, B7602HSA - Mar del Plata, Argentina.
E-mail: [email protected]
Azadinium spinosum has been identified as a species producer of azaspiracids (AZAs), marine toxins reported to cause human poisoning
aetiologically similar to diarrhetic shellfish poisoning. AZAs are accumulated via food web by molluscs which in turn could cause human
poisoning after consumption of contaminated shellfish. In this study we report the presence of A. spinosum for the first time in the
Southwest Atlantic and analyse two successive blooms that this species has caused in northern shelf waters of Argentine. These
blooms occurred in a restricted location of the open shelf (~38o30΄-38o50΄S; 55o41΄-56o23΄W) during the spring in 1990 and 1991. In
November 1990 a first bloom (up to 9.03 106 cells liter-1) produced heavy discolorations, and morphological and taxonomical studies
showed that the causal organism was a new dinoflagellate genus. During September 1991 plankton composition and abundance were
analysed along a transect from the coast to the Malvinas Current. Results showed that the same dinoflagellate was the most important
species in middle shelf waters, with high biomass and abundance values (up to 3 106 cells liter-1) surpassing those of various Thalassiosira
typical of the spring bloom. The description of this new genus, according to the thecal analysis performed at that time using light and
scanning electron microscopy corresponds to the thecal tabulation, cell morphology and dimensions of the recently described Azadinium
spinosum. Presence of A. spinosum in this area opens several questions, the first related to toxin production given the regional importance
of bivalve commercial fisheries.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 133
NEW REGIONAL HAB EVENTS
RED TIDE AND HARMFUL ALGAL BLOOMS
IN OMANI WATERS
Hamed Al Gheilani
Marine Science and Fisheries Center, Ministry of Fisheries Wealth. P.O. Box 427, PC 100, Muscat, Sultanate of Oman.
E-mail: [email protected]
Red tide is a natural ecological phenomenon and often this event is accompanied by severe impacts on coastal resources, local
economies, and public health. The occurrence of red tides is more frequent in Omani waters in the recent years; many of them caused
fish kill, damaged fishery resources and mariculture, threatened marine environment and osmosis membranes of desalination plan.
However, a number of them have been harmless. The most common dinoflagellate Noctiluca scintillans is associated with the red tide
events in Omani waters. However, the presence of some toxic species, like Karenia selliformis, Prorocentrum arabianum, and Trichodesmium erythraeum, has also been reported. Although red tides in Oman have been considered a consequence of upwelling, red
tide outbreaks in Oman are not restricted to the summer upwelling season (May to September). Frequent algal blooms have been reported during December to March. HABs may have contributed to hypoxia and/or other negative ecological impacts, but health problems
associated with fish products have never been reported in Oman.
133
FIRST DETECTION OF OSTREOPSIS CF. SIAMENSIS
IN PORTUGUESE COASTAL WATERS
A. Amorim1, V. Veloso1, C. Battocchi2, A. Penna2
1
Instituto de Oceanografia, Faculdade de Ciencias, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
E-mail: [email protected]
2
Dept. of Biomolecular Sciences, University of Urbino, Vle Trieste 296, 61100 Pesaro, Italy
The coast of Portugal is located in the warm temperate/sub-tropical transition of the North Atlantic. It is part of the major discontinuity
in the eastern boundary of the NE Atlantic, being affected both by seasonal upwelling and water mass exchange with the Mediterranean
basin. In October 2007, following reports of Ostreopsis spp. nuisance blooms in the Mediterranean Sea, a sampling program aiming
at the early detection of Ostreopsis species along the Portuguese coast was initiated. Here, we report the first record of Ostreopsis cf.
siamensis outside the Mediterranean Sea. O. cf. siamensis was detected and isolated from the Atlantic coast of Portugal (37o57΄2N;
8o51΄53W). Three cultures were established and are kept in the culture collection of the Oceanography Centre, University of Lisbon.
Cells were studied by LM and SEM and one strain was analyzed for species-specific genotyping [internal transcribed spacer regions
(ITS) and 5.8S rDNA]. The morphology and arrangement of the thecal plates agrees with the description of O. siamensis. The final
alignment of the ribosomal sequences of the analyzed strain with all ribosomal sequences of Ostreopsis available in the database used
in this study, revealed total identity (100%) with O. cf. siamensis sequences belonging to the Mediterranean clade. These results extend
the geographical distribution of O. cf. siamensis outside the Mediterranean basin to 38o N, the highest latitude at which species of Ostreopsis have been reported from the Atlantic. Further studies will be needed to clarify the phylogenetic and phylogeographic position
of O. cf. siamensis in the Atlantic waters.
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GULF OF TRIESTE, NORTHERN ADRIATIC SEA:
FIRST RECORD OF OSTREOPSIS OVATA BLOOM
Oriana Blasutto, Massimo Celio, Chiara Suraci, Maria Venuti, Bruno Zanolin,
Alessandro Acquavita, Fulvio Stel, Giorgio Mattassi
Regional Environmental Protection Agency ARPA-FVG, Via Cairoli 14, 33057 Palmanova, Udine, Italy.
E-mail: [email protected]
Ostreopsis ovata is a tropical benthic dinoflagellate. During the last decade blooms of this potentially toxic species have been reported
along the Italian coasts. In Tyrrhenian and Ligurian Seas the occurrence of Ostreopsis ovata blooms has been associated to the death
of benthic organisms and/or to human health problems. In the Gulf of Trieste Ostreopsis ovata was observed for the first time in
2006. At the end of September 2009 a bloom of Ostreopsis ovata developed in a tidal pool of the gulf. The tidal pool is confined from
the rest of the beach and in contact with the open sea by means a natural reef. The pool is sheltered from wind blowing and from
wave action; it is characterized by a pebbly bottom where macroalgae are almost absent. During the bloom a mucilaginous brown
pellet coated the whole bottom. In the water in contact with the pebbles the dinoflagellate abundance reached 3x106 cells L-1 and
2.5x106 cells L-1 were found in the surface layer. From 1991 to 2003, in the Gulf of Trieste, an increase of sea water temperature
(0.1-0.2 oC/years) has been observed and the positive trend is still evident. This temperature trend together with a reduced trophic
level of the whole northern Adriatic have probably supported the dinoflagellate settling in the gulf of Trieste. In 2009 a spell of fine
weather with high sea water temperature and nutrient availability supported the development of the bloom of Ostreopsis ovata that
found the ideal conditions for its proliferation in the tidal pool.
134
THE FIRST BLOOM OF PSEUDO-NITZSCHIA AUSTRALIS
IN SOUTH ATLANTIC COAST OF MOROCCO
Karima Chaira1, Btissam Ennaffah2, Amina Dellal1, Abdelghani Chafik3
1
Laboratoire des Efflorescences Nuisibles, Institut National de Recherche Halieutique, Dakhla, Maroc.
E-mail: [email protected]
2
Laboratoire des Efflorescences Nuisibles, Institut National de Recherche Halieutique, Casablanca, Maroc
3
Département Qualité et Salubrité du Milieu Marin, Institut National de Recherche Halieutique, Casablanca, Maroc
During a routine analysis of monitoring program, a bloom of pennate diatom Pseudo-nitzschia was detected in Morrocan south coast
in February 2010. The microscopy observation showed the presence of one species of Pseudo-nitzschia, and the highest density
was more than 105 c/l. Then the bloom was reported monospecific, and affected 1 shellfish area, called Puertito (Atlantic coast). The
surface water temperature was 22oC. The analysis of electron microscopy define that the species blooming in this area is P. australis,
producer agent of domoic acid. The phycotoxins test revealed the trace of ASP in mussels (Mytilus galloprovincialis). The frequency
of toxic events increases in the region, and become a serious threat for aquaculture sector.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 135
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
OSTREOPSIS OVATA (DINOPHYCEAE) BIOFILMS
IN MICROCOSM EXPERIMENTS
Roberta Congestri1, Francesca Di Pippo1, Neil Thomas William Ellwood2, Patrizia Albertano1
1
LBA - Laboratory for Biology of Algae, Department of Biology, University of Rome, Via della Ricerca Scientifica 1,
00133 Rome, Italy. E-mail: [email protected]
2
Department of Geological Sciences, University of Rome Roma Tre, Largo San Leonardo Murialdo, 1, 00146 Rome, Italy
Ostreopsis HABs are recurrent summer events along the Middle Tyrrhenian coast (Italy, Mediterranean Sea) since the first record in
1999. Bloom usually occurs as thick cell aggregates, biofilms, embedded in an exopolymeric matrix on biotic and abiotic substrata. Other
potentially toxic dinoflagellates, cyanobacteria, diatoms and heterotrophs coexist in the biofilms. Two “hot sites” were identified based on
decadal monitoring of outbreak periodicity and intensity along Latium coast. Past molecular and toxin analyses revealed the presence of
Ostreopsis ovata in the assemblages which produced palytoxin and ovatoxin. In order to expand on Ostreopsis ovata bloom development
and dispersion within mucous flocs, a laboratory microcosm approach was used to grow biofilms sampled during bloom events in 2009
at the two sites. An incubator prototype was used to perform experiments at 25oC, 110 μmol photons m-2 s-1 and 50 L h-1 flow velocity
of K/2 medium. 3 runs were carried out to test the effect of sampling time, site and substrata on biofilm development and species succession. Growth curves indicated similar trends in biomass accrual. Common to all experiments was the reduction of species diversity
with shifts in biofilm composition and domination by cyanobacteria in the late growth stages. Ostreopsis ovata was healthy and proliferated
at initial and active stages, with CPC light absorption systems evidenced by CLSM spectral analysis. CLSM also showed patchy coverage
of the artificial substrata and voids and channels in the film. Substratum colonisation appeared a stochastic process. HPLC on matrix
sugars revealed variations in total amounts and composition.
135
MAPPING THE BIOGEOGRAPHY
OF HARMFUL ALGAL SPECIES
Henrik O. Enevoldsen1, Adriana Zingone2, Patricia A. Tester3, Edward Vanden Berghe4, Rut Akselman5
1
IOC Science and Communication Centre on Harmful Algae, University of Copenhagen, Farimagsgade 2D,
1353 Copenhagen K, Denmark. E-mail: [email protected]
2
Stazione Zoologica A. Dohrn, 1 Villa Comunale, 80121 Naples, Italy
3
National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, NC 28516, USA
4
OBIS Secretariat, 71 Dudley Road, New Brunswick, NJ 08901, USA
5
INIDEP, Paseo Victoria Ocampo N. 1, Esc. Norte B7602HSA, Mar del Plata, Buenos Aires, Argentina
Data on the known geographical occurrence of individual HAB species is systematically derived from published material and checked by
regional experts for consistency and synonymies. This long term project named “HABMAP” is a joint initiative of ISSHA and the IOC of UNESCO. For each of the toxic species in the IOC Taxonomic Reference List findings in a given area are summarized with information on
whether the species has been found with no apparent toxicity / harmful effect; the species has been found and revealed to be toxic in
laboratory analyses; or the species has been associated with an impact. HABMAP can be used to trace the dynamic biogeography of the
harmful species and the risk associated with them. Example distribution maps will be presented. Distribution records will be stored with the
Ocean Biogeographic Information System (OBIS) which is the largest repository of marine biogeographic information. This will facilitate interpretation of HABMAP data in the context of distribution of other species, as recorded in OBIS. OBIS includes tools for modeling species distributions and for querying on physical oceanography parameters. Also, data published through OBIS is made available to the Global Biodiversity
Information Facility (GBIF). GBIF and OBIS data on species involved in HABs will be incorporated within HABMAP after careful data validation.
This will allow incorporation of distribution records from many more sources than the HABMAP Project would be able to handle, and substantially
increase the data holdings available to HABMAP. The scientific community is invited to become involved in HABMAP.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 136
TOXIC PHYTOPLANKTON OCCURRENCE
IN THE PORTUGUESE MONDEGO RIVER ESTUARY
B. D. D. Fragoso1,3, J. M. Neto1, A. Newton2,3
1
IMAR - Institute of Marine Research, Department of Life Sciences, University of Coimbra, Largo Marques de Pombal,
3004-517 Coimbra, Portugal. E-mail: [email protected]
2
NILU-CEE, P.O. Box 100, 2027 Kjeller, Norway
3
CIMA - Centro de Investigao Marinha e Ambiental, FCT, Edificio 7, Universidade do Algarve, Campus de Gambelas,
8005-139 Faro, Portugal
The Mondego estuary is located in the central western Atlantic coast of Portugal with a Mediterranean temperate climate. Human activities
in the estuary include recreational fisheries, aquaculture and shellfish harvesting. The ReConnect project is a monitoring project funded
by Fundao para a Ciencia e Tecnologia, including for the first time, a regular monitoring of the Phytoplankton in the Mondego Estuary,
following the Water Framework Directive (WFD, 2000/60/EC). This study shows the phytoplankton abundances between March and
September of 2009. Water samples were collected every month, in six stations along the estuary using a Niskin bottle, species (>20 micron) were identified by inverted microscopy. Total phytoplankton and toxic species abundances were determined. Some of toxic species
found within the estuary include, Pseudo-nitzschia sp., Dinophysis sp., Gymnodinium catenatum and Prorocentrum micans. In the most
upstream estuary station (E23), no toxic species were found. Stations near to estuary river-mouth show higher abundances of toxic
species. For April and May, Pseudo-nitzschia sp. abundances represent almost half of total phytoplankton abundances at stations E02
and E05. In September occurrence of G. catenatum was recorded with a maximum of abundance at station E12 with 148 cells/ml with
respect to a total phytoplankton abundance of 306 cells/ml. Dinophysis sp. was present in samples from May to September with a maximum abundance in May of 12 cells/ml at station E02. Even if HAB species in the estuary show low abundances there is the potential
for toxic blooms, therefore monitoring of HAB in the Mondego estuary should be implemented in the future.
136
HABS IN SE SPAIN REGION,
SOUTH WEST MEDITERRANEAN SEA
Javier Gilabert, Nuria Alcaraz, Laura Lopez, Andres Gimenez
Department of Chemical & Environmental Engineering, Technical University of Cartagena (UPCT), Alfonso XIII, 52,
E-30203 Cartagena, Spain. E-mail: [email protected]
We show here the evolution of potential toxic algae in the South Eastern coast of Spain since an epidemic event occurred in the summer
of 2006 bringing more than a hundred people to receive some medical assistance. The event was produced in a very touristic located
area with three beaches in close embayment. The monitoring program has been expanding in time until monitor more than 80 beaches
in the SE coast of Spain some of them monitored weekly from June to September. Although no other epidemic event occurred since
then, it has been reported the presence of several species including Ostreopsis ovata. During the monitoring for 2009, environmental
parameters were also measured in some selected beaches to understand the possible effect of environmental conditions in blooming
algae. We also show maps of the spatial distribution along time of different taxa as well as its correlation to environmental parameters
trough the multivariate analysis.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 137
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
PSEUDO-NITZSCHIA CACIANTHA, A POSSIBLE
CAUSATIVE ORGANISM OF DOMOIC ACID ACCUMULATED
IN SPONDYLUS VESICOLOR IN NHA PHU BAY,
KHANH HOA PROVINCE, VIETNAM
Dao Viet Ha1, Takuo Omura2, Yoshinobu Takata2, Pham Xuan Ky1, Nguyen Thu Hong1,
Yasuwo Fukuyo2, Masaaki Kodama2
1
2
Institute of Oceanography, 01 Cau Da “Nha Trang”, Vietnam. E-mail: [email protected], [email protected]
The University of Tokyo, Japan
Domoic acid is a toxin responsible for amnesic shellfish poisoning (ASP). Pseudo-nitzschia multiseries was identified as a causative
plankton species in the first case of ASP occurred in Canada. Since then, 22 species of Pseudo-nitzschia have been found to produce
domoic acid. Domoic acid accumulation in bivalves has been monitored in association with environmental plankton species. However,
there is so far no clear data on the relationship between the level of domoic acid in bivalve and the abundance of plankton species which
is known to produce domoic acid. In the study on domoic acid in South East Asian countries, we found that bivalves belonging to a genus
Spondylus accumulate domoic acid much more than other bivalve species. In the monitoring on domoic acid level of S. vesicolor in association with that of plankton net samples in Nha Phu Bay, Khanh Hoa Province, Vietnam, we observed that domoic acid level in S.
vesicolor increased during when domoic acid was detected in plankton net samples. As Pseudo-nitzschia cells were dominantly observed
in domoic acid-rich fraction of plankton samples, these cells were isolated for clonal cultures. Among 11 established cultures, 8 cultures
were found to produce domoic acid, though the productivity was much lower than that of P. multiseries. All these toxic clones were
identified as Pseudo-nitzschia caciantha. These results show that P. caciantha is at least one of the causative species of domoic acid
which is accumulated in S. vesicolor in Nha Phu Bay.
137
TWO NEW RECORDS OF POTENTIALLY TOXIC
PHYTOPLANKTON SPECIES FROM THE MEXICAN PACIFIC
David U. Hernández-Becerril1, Sergio Escobar-Morales2, Sara P. Moreno-Gutiérrez1, Sofía A. Barón-Campis3
Instituto de Ciencias del Mar y Limnología Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán,
México, D.F. 04510 México. E-mail: [email protected]
2
Posgrado en Ciencias del Mar y Limnología, ICMyL-UNAM, Ciudad Universitaria, Coyoacán, México, D.F. 04510 México
3
Instituto Nacional de Pesca, Pitágoras 1320, Sta. Cruz Atoyac, México, D.F. 03310 México
1
Phytoplankton material obtained by net and bottle from locations in coasts of the Mexican Pacific (2008-2010), during different oceanographic cruises, has yielded a rich planktonic flora where diatoms and dinoflagellates constitute the main taxonomic groups, in terms of
both diversity and biomass or abundance. In this paper we report the finding of two potentially toxic species from the Mexican Pacific: the
minute, thecate dinoflagellate Azadinium spinosum Elbrächter et Tillmann, recently and originally described from the North Sea and considered a species producing azaspiracid toxins, and the chain-forming diatom species Pseudo-nitzschia multistriata Takano, which is considered a producer of the toxin domoic acid. The morphology of Azadinium spinosum was almost identical to the original description:
solitary, small cells (12.5-13 μm length, 7-7.6 μm width) with a conical epicone having a slightly protruded apical pore, and more rounded
hyposome having a posterior short spine, a very wide and excavated cingulum, first apical plate with a ventral pore, three intercalary
plates, 2a tiny and square. Pseudo-nitzschia multistriata was found solitary or forming short chains (generally 2 cells), in girdle view with
the typical sigmoid shape, and linear-shaped in valve view (38-45 μm length, 3-4 μm width), central interspace lacking, and two rows of
poroids per stria. At the moment there are no records of production of any of these two toxins in the Mexican Pacific directly associated
to the presence of these two species, although domoic acid has been detected, but related to other Pseudo-nitzschia species.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 138
FISH MORTALITY ASSOCIATED WITH BENTHIC
OSCILLATORIA SPP. BLOOMS IN ALEXANDRIA - EGYPT
Amany A. Ismael, Yusef Halim
Oceanography Dept., Faculty of Science, Alexandria University, Alexandria 21511, Egypt. E-mail: [email protected]
During March 2005 mass mortality of bottom feeding fish, Siganus rivulatus, occurred for the first time in the Eastern Harbour of Alexandria. This fish feeds on macroalgae specially Ulva sp. As a result, a survey of the potentially harmful epiphytic microalgae in the harbor
and along the coast of Alexandria was initiated. A total number of 126 samples were collected monthly along Alexandria Coast, from
June 2005 to December 2007. Surface temperature and salinity ranged from 16.9 to 30oC and 20.8 to 39.5 psu respectively. Twenty
one potentially harmful species are recorded. Among them four Cyanobacteria are considered as new records for Mediterranean waters;
Oscillatoria nigro-viridis, O. acutissma, O. formosum and Planktothrix planctonica. The causative organism responsible for the massive
fish mortality occurred was Oscillatoria acutissma; with a standing crop reached 107x102 filament fwg-1. The qualitative observations
showed that Oscillatoria spp. seems to prefer green macroalgae as host to red and brown algae. The correlation between Oscillatoria
spp. and the algal host showed a significant correlation between O. acutissima, O. nigro-viridis and Ulva spp. (p=0.002). No significant
correlation was found between the other two Oscillatoria spp. and the macroalgal species tested. The two species Oscillatoria acutissima
and O. nigro-viridis as a result, were perennial and widespread during the present study and appear to be favoured by moderate temperatures. In the Egyptian coastal waters earlier dramatic events of harmful algae occurred on several occasions due to planktonic blooms
of dinoflagellates. It is the first time that fish mortality occurred due to benthic cyanobacteria, particularly Oscillatoria acutissma. Our
current work will focus on the possible impacts of cyanobacterial species on coastal food webs.
138
OBSERVATIONS OF DINOPHYSIS TRIPOS
IN NORWEGIAN COASTAL WATERS
Torbjørn M. Johnsen, Evy R. Lømsland
Norwegian Institute for Water Research, Thormøhlensgate 53D, N-5006 Bergen, Norway.
E-mail: [email protected], [email protected]
Norwegian Food Safety Authority has since 1992 monitored the Norwegian coast for human toxic HAB species. About 50 localities,
covering the whole Norwegian coast from the eastern part of the Skagerrak to the Barents Sea, are now sampled weekly, most of them
having a sampling period from the middle of January to the middle of December. Several institutions are involved in the monitoring. Norwegian Institute for Water Research (NIVA) is monitoring the Norwegian West Coast and the northern coastal areas. During the monitoring
in 2009, Dinophysis tripos was observed for the first time in Norway. It first appeared in the middle of August at the west coast where
D. tripos was detected at four different localities. Just a week later it also appeared in the northern part of Norway, in the Norwegian Sea
area. During the following weeks it was detected weekly in these areas, and at the end of September it also appeared in the Barents
Sea region. Pared cells were frequently detected indicating that Dinophysis tripos actually was growing in Norwegian waters.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 139
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
FIRST REPORT ON THE PRESENCE OF SPIROLIDES
IN GREEK SHELLFISH ASSOCIATED WITH THE DETECTION
OF THE CAUSATIVE ALEXANDRIUM SPECIES
Panagiota Katikou1, Katerina Aligizaki2, Thetis Zacharaki1, Dimitrios Iossifidis3, Georgios Nikolaidis*
1
National Reference Laboratory on Marine Biotoxins, Institute of Food Hygiene, Ministry of Rural Development and Food,
3A Limnou street, GR 54627 Thessaloniki, Greece. E-mail: [email protected]
2
Department of Botany, School of Biology, Faculty of Sciences, Aristotle University, 54124 Thessaloniki, Greece
3
MALVA S.A., 13 Ilission street, 14564 Athens, Greece
* deceased
During the spring periods of 2008 and 2009, highly acute toxicity reactions upon intraperitoneal injections of mussel lipophilic extracts derived
from the area of Thermaikos Gulf, Greece, were observed during mouse bioassay (MBA). Symptomatology consisted of neurological signs,
convulsions, cramps within a few minutes after the injection and death within 30 minutes. The presence of spirolides was suspected due to
the precedent and simultaneous (winter and spring periods) detection of Alexandrium ostenfeldii/peruvianum, the potent producer of spirolide
toxins, at the same area, though in low abundances (<160 cells/lt). Samples from mussels (Mytilus galloprovincialis) which provided a positive
MBA test for lipophilic toxins with the above acute symptomatology were extracted with methanol and subjected to further analysis by ultra
performance liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS) using a Waters Acquity UPLC® system. The
method applied was a multi-toxin method able to determine and/or quantify the following lipophilic toxin groups: okadaic acid and dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides, gymnodimine and palytoxin and ostreocins, whereas certified standards from the
National Research Council, Canada, where available, were used for quantification purposes. Results of the analyses revealed the presence
of 13-desmethyl spirolide C (SPX1) in all analyzed mussel samples at concentrations up to 10 μg/kg mussel tissue, confirming the MBA
result, for which the limit of detection is estimated to be around 5.6 μg/kg shellfish. These findings constitute the first report on the presence
of both toxins of the cyclic imines group in Greek shellfish and A. ostenfeldii/peruvianum in Greek coastal waters.
139
POTENTIAL TOXIC PHYTOPLANKTON IN LEVANTINE
BASIN (EAST MEDITERRANEAN). CASE STUDY:
LEBANESE COASTAL WATERS
Sami Lakkis1, Vanda Novel-Lakkis2
1
2
Biology Department, Lebanese University, Beirut, Lebanon. E-mail: [email protected]
Laboratory of Plankton, National Centre for Marine Sciences, LNCSR, Batroun, Lebanon
The Levantine Basin, including the Lebanese sector (Eastern Mediterranean) situated in a sub-tropical region, is the more oligotrophic
water body of the Mediterranean, showing low standing crop and small primary productivity rate. Surface offshore seawater temperature
and salinity averages are the highest in the entire Mediterranean. The opening of Suez canal has enhanced biological invasion leading to
introduction of tropical phytoplankton species of Indo-Pacific into the Mediterranean. Most of those have established permanent populations
in the Levantine Basin, including the Lebanese coastal waters. They may have negative impact on the biodiversity of local pelagic microalgae
populations. Among the 400 phytoplankton taxa recorded in the Lebanese seawaters, 235 belong to the dinoflagellates and 160 are
diatoms. The class Dinophyceaea comprises the main toxic species, namely within Dinophysales, Gymnodiniales, Peridiniales and Prorocentrales. 18 potentially toxic species were reported, the majority belong to genera Pseudo-nitzschia, Dinophysis and Prorocentrum. However, the low density of these toxic species does not allow to creating red tide or eutrophication and thus no production of toxic substances.
Since there is no marine aquaculture activity along the Lebanese coast and the consumption of bivalves is uncommon in the country, no
intoxication accidents are reported.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 140
EVIDENCE FOR SAXITOXINS PRODUCTION
BY THE CYANOBACTERIUM APHANIZOMENON GRACILE
IN A FRENCH RECREATIONAL WATER BODY
Aurélie Ledreux1, Solène Thomazeau1, Arnaud Catherine1, Charlotte Duval1,
Claude Yéprémian1, Arul Marie2, Cécile Bernard1
1
FRE 3206 CNRS / MNHN Molécules de Communication et Adaptation des Micro-organismes, USM 505 Cyanobactéries,
Cyanotoxines et Environnement, Muséum National d’Histoire Naturelle, 57 rue Cuvier, CP 39, 75231 Paris, Cedex 05, France.
E-mail: [email protected]
2
Plateforme de Spectrométrie de Masse, FRE 3206 CNRS / MNHN Molécules de Communication et Adaptation des Micro
organismes, Muséum National d’Histoire Naturelle, France, 57 rue Cuvier, CP 54, 75231 Paris, Cedex 05, France
An increasing frequency and severity of cyanobacterial blooms in freshwater environments has been observed in the last few years. Consequently,
recreational and drinking water bodies are now monitored by local authorities to prevent animal and human poisoning related to cyanobacteria
and their toxins. In this context, a water body used for recreational activities (at Champs-sur-Marne, Paris suburb) was monitored from 2005
to 2008 for the chlorophyll α concentrations, occurrence of cyanobacteria and of microcystins, anatoxin-a and saxitoxins. Microcystis was the
dominant genus during the first two years (2005 to 2007), whereas Aphanizomenon became the dominant genus towards the end of the
survey (2007-2008). As a consequence, microcystins were initially detected at high concentrations, and were later superceded by saxitoxins
(STXs) in 2008. Using LC-MS/MS and the Neuro-2a cell-based assay, we confirmed that the STX and neoSTX variants were present in the
freshwater natural samples at concentrations higher than 3 μg eq.STX L-1 in raw water. Monoclonal cultures were obtained after an isolation
procedure then screened for the presence of the sxtA gene in order to attribute STXs production to the appropriate cyanobacterial taxa. SxtApositive strains were screened for their STX content using the Neuro-2a cell-based assay and LC-MS/MS. The results led to consider Aphanizomenon gracile as the most likely producer of STXs. The first description of STXs in a French recreational water body leads to discuss the
health risks associated with the presence of STXs alone or with its co-occurrence with other known cyanotoxins in recreational water bodies.
140
MONITORING PROGRAM FOR HARMFUL ALGAE BLOOMS
IN SALVADORAN WATERS: REPORT OF PYRODINIUM
BAHAMENSE FROM NOVEMBER 2009 TO JUNE 2010
Sergio Licea1, Armando Navarrete2, Vianney Castañeda3, José Bustillos-Guzmán4, Enrique Barraza5
1
Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apartado postal 70-305,
México City 04510, México. E-mail: licea@[email protected]
2
Instituto de Ciencias del Mar de El Salvador, Final 25 Ave. Norte, Ciudad Universitaria, San Salvador, El Salvador
3
CENSALUD, Universidad del Salavador, Final 25 Ave. Norte, Ciudad Universitaria, San Salvador, El Salvador
4
Northwest Biological Research Center, P.O. Box 128, La Paz, B.C.S. 23000, México
5
Ministerio del Ambiente y Recursos Naturales, Alameda Roosevelt y 55 Av. Norte, Edificio Torre, San Salvador, El Salvador
At the end of November 2009, several red patches were observed seven miles from the coast. This bloom was caused by Pyrodinium bahamense var. bahamense and P. bahamense var. compressum. The surface discoloration has remained since detection and began to fade
during the middle of May 2010. Observations since 2002 indicate that these events occur at a meso-scale in wide areas near Central
America every four to five years. Samples were collected at 72 sites over the continental shelf at the beginning of December 2009. In
addition, four sites along the coast were selected for monitoring throughout the event. Tissue samples of bivalves and tissues of died turtles
were analyzed for saxitoxin by mouse bioassay; analyses by HPLC is still pending. Satellite displays of concentrations of chlorophyll-a
provided a positive anomaly (1-4 mg m-3) for this period. Cell concentrations of Pyrodinium were determined by the Utermöhl method. Maximum value was 15.3 x 106 cells L-1 60 km from the coast. In contrast, only 22,000 cells L-1 were found at the coastal sites. The concentrations of saxitoxin ranged from 150.5 to 1426.62 STX eq/100 g in shellfish from samples collected from Salvadoran beaches. This is
the first time that the local population have not been reported intoxicated during this event, thanks to the monitoring program and corresponding the administrative closures. However, 61 dead marine turtles (Lepidochelys and Chelonia mydas) were found.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 141
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
OBSERVATIONS OF CHATTONELLA GLOBOSA
IN NORWEGIAN COASTAL WATERS
Evy R. Lømsland1, Torbjørn M. Johnsen1, Wenche Eikrem2
Norwegian Institute for Water Research, Thormøhlensgate 53D, NO-5006 Bergen, Norway.
E-mail: [email protected], [email protected]
2
Norwegian Institute for Water Research, Gaustadalléen 21, NO-0349 Oslo, Norway. E-mail: [email protected]
1
Chattonella globosa was first observed in Norway in September 2007 where it appeared in the eastern Skagerrak and later on the West
Coast of Norway presumably transported there by the Norwegian Coastal Current. The highest density of 15,000 cells/L was observed
in the light limited Inner Oslofjorden in November 2007. It reoccurred in September 2008 in the Skagerrak and became the dominant
autumn bloom species at our monitoring station in southern Norway (Arendal st.2, E08.8167, N58.3833 (EUREF89-WGS84)) where
it constituted 54% of the total algal carbon. As in the previous year, C. globosa spread to the West Coast reaching densities of 53,000
cells/L in early November. It co-occurred with Pseudochattonella sp. and Heterosigma akashiwo where the bloom resulted in the death
of 100 tons of caged fish. During the 2008 bloom, cells of C. globosa were observed containing Dictyocha fibula-like skeletons in
different stages of development. This observation was confirmed for 11 different localities along the southern part of the West Norwegian
Coast. These observations suggest that C. globosa and D. fibula are related and may be stages in a common life cycle.
141
BLOOM OF ALEXANDRIUM CF. TAMARENSE
IN OAXACA’S COAST, MEXICO, DURING MAY 2009
Ma. Esther Meave1, Ma. Eugenia Zamudio Resendiz1, Eduardo Herrera Galindo2
1
Universidad Autonoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, PC 09349,
Mexico, D.F. Mexico. E-mail: mem @xanum.uam.mx
2
Universidad del Mar, CU Puerto Angel, PC 70902, San Pedro Pochutla, Oax., Mexico
A bloom of Alexandrium, identified as A. tamarense (Lebour) Balech, was detected in 2009 on the coast of Oaxaca, Mexico. The discoloration event (reddish-brown) started on May 1st in Salina Cruz (Gulf of Thehuantepec) and due to the effect of wind it moved on a W
direction. The material was collected on the surface with a Van Dorn bottle, fixed with acetate Lugol΄s and counted in Sedgwick-Rafter
chambers. Densities were: 1.4-56.6 cells l-1. For identification, specimens were stained with trypan blue for at least 48 hours while in a
dried preparation, then wet again and squashed. Cells were examined with a Leica optical microscope DMLB. The organisms were identified as A. tamarense due to its morphology and main taxonomic characters. Cells were solitary or in pairs, L = 25-31 μm, TR = 24.522 μm, W = 29.6-32.3 μm and thickness 28.7-29 μm. Organisms presented a ventral pore on the upper left edge of the plate 1, a
connecting pore was in the middle of the pentagonal posterior sulcal plate. Sixth precingular plate was wide. The apical pore had comma
shape (length 5.6 μm and width 3.6 μm), and a small Po plaque was to one side. Mouse bioassay did not showed saxitoxin toxicity, although some times Alexandrium tamarense is reported as saxitoxin producer. Due to the fact that A. tamarense is reported for temperate
areas, and Oaxaca is on the Tropical Mexican Pacific, this could be a case of cryptic species.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 142
KARENIA SPECIES IN THE MEXICAN PACIFIC
Ma. Esther Meave, Ma. Eugenia Zamudio Resendiz
Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, PC 09349,
México, D.F. México. E-mail: mem @xanum.uam.mx
From a review of bottle phytoplankton samples collected in various localities of Mexican Pacific and especially in the Acapulco Bay, fixed
with Lugol and examined under an inverted microscope, nine species of Karenia were identified. The taxonomic characteristics assessed
were: shape, dimensions (including length / width ratio), nucleus position and chloroplasts’ shape; with these we recognize nine Karenia
taxa: K. asteriochroma, K. bicuneiformis, K. cf. breve, K. cf. brevisulcata, K. mikimotoi, K. papillonacea, K. selliformis and two unidentified
taxa. The only one who has been a flourishing is K. mikimotoi in Manzanillo Bay (March-31 to May-22, 2007) with a density of up to
8.7x106 cells/L in April. In Acapulco, the most common are K. mikimotoi and K. selliformis with densities of up to 1750 cells/L in surface
water (1-3m). We also found some organisms like K. breve, but smaller, that could be consistent with K. papillonacea stages of life
cycle, which is rare but widely distributed in the Mexican Pacific (upper Gulf of California, Acapulco and Gulf of Tehuantepec). The rarest
species and restricted to a certain region are: K. bicuneiformis in the Gulf of Tehuantepec and Acapulco, and K. asteriochroma in Acapulco.
Considering that for K. brevis the density basis for fishing bands is 5x103 cells/L, and that two of the species reported here (K. mikimotoi
and K. selliformis) are ichthyotoxic (hemolysins and gymnodiminas), we consider that these species could represented a risk in the
Mexican Pacific, also because they have been found associated with Gymnodinium catenatum.
142
HAB OF PYRODINIUM BAHAMENSE VAR. COMPRESSUM
AT THE SOUTHERN REGION OF THE MEXICAN PACIFIC
Esther Meave1, Ma. Eugenia Zamudio1, Adriana Hernández1, Francisco Gutiérrez1, Francisco Varona-Cordero1,
Ivanhoe Herrera-Moro1, Beatriz Pérez2, Mario Alarcón Tacuba2, Jesús Díaz Ortiz2, René Valdovinos Sánchez2,
Mario A. Alarcón Romero2, Saul Lopez2
1
Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, PC 09349,
México, D.F. México. E-mail: mem @xanum.uam.mx
2
Laboratorio Estatal de Salud Pública del Estado de Guerrero, Boulevard Vicente Guerrero, esq. Juan R. Escudero,
Cd. Renacimiento, Acapulco, Gro. C.P. 39715, México
A HAB of Pyrodinium bahamense var. compressum was registered in Acapulco Bay (Guerrero, México) from July 7th of 2010. This
bloom started in the state of Oaxaca (Huatulco Bay), but in Acapulco Bay it didn’t cause water discoloration, possibly due to the location
of the highest densities was from 3 to 5 m below the surface. The mean temperature was 28.9oC, the salinity was 30 psu and the phosphates, ammonium and nitrates concentration were normal (4.3, 0.3 and 1.6 μM respectively); phytoplankton biomass (Chl a) fluctuated
between 29.9 - 46.2 mg m-3. Strong rains were present before the event. Sedgwick Rafter counts were made at several depth samples.
The longest chains were 16 cells, with a mean of 8 cells. Occasionally bigger and less compressed single cells were found. Between the
dinoflagellates Pyrodinium was dominant (88%), although few Prorocentrum (P. gracile, P. triestinum) were found, and also some naked
dinoflagellates (Karenia sp., Gymnodinium catenatum and Gyrodinium fusus) were present. The highest Pyrodinium cell densities were
from 1.4 x 105 cells L-1 outside the Bay; inside the Bay the cell densities were 300x 103 cells L-1 at 3 m depth and 140 x 103 cells L-1
at 5 m. Diatoms were present but in small sizes. Pseudo-nitzchias were important reaching densities of 140 x 103 cells L-1. Saxytoxin
concentration reached values of 2092 μg STX eq. 100 g-1 in violet oyster (Chama Mexicana) in Punta Pilares, near Puerto Marques
Bay, therefore a closure was established.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 143
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
FIRST REPORT OF HETEROSIGMA AKASHIWO,
FIBROCAPSA JAPONICA AND CHATONELLA
MARINA VAR. ANTIQUA IN URUGUAY
Silvia M. Mendez1, Rut Akselman2, Carmelo R. Tomas3
1
National Direction of Aquatic Resources, Constituyente 1497, Montevideo, Uruguay. E-mail: [email protected]
National Institute for Fisheries Research and Development, Paseo V. Ocampo No 1, Mar del Plata, Argentine
3
Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Lane, Wilmington,
North Carolina, USA
2
The class Raphydophyceae had been reported worldwide associated to massive fish mortalities. The killing mechanism of raphidophyte
blooms includes physical clogging of fish gills by mucus excretion and gill damage by haemolytic toxins. There is evidence of the production
of superoxide and hydroxyl radicals as a potential mechanism of fish mortality by Chattonella and Heterosigma. Some species and strains
may contain brevetoxin-like neurotoxins and free polyunsaturated fatty acids. In South American coastal waters both Chattonella and
Heterosigma akashiwo were reported for Southern Brazil and H. akashiwo caused important cultured salmon mortalities in Chile. Massive
mortalities of fishes are frequent along the estuarine and marine coastal waters of Uruguay which are often ascribed to abrupt salinity
changes. Considering these episodes and the possible harmful effects of raphidophytes which are difficult to identify in preserved samples,
the National Harmful Phytoplankton Monitoring Programme included since 2007 the analysis of live samples. This allowed the first report
of H. akashiwo, Fibrocapsa japonica and Chatonella marina var. antiqua in Uruguayan waters. These species were observed in coastal
unpreserved samples in summer - fall (January to May), when water temperatures from 17 to 22oC and salinities of 24.7 to 33.4 occurred.
The presence should be taken into account as a risk factor of fish mortality in these waters. Further research efforts are needed to study
the accumulation of toxins produced by Raphydophyceae in fish and their potentially harmful or toxic effect in fish consumers.
143
SPECIES OF PSEUDO-NITZSCHIA POTENTIALLY
TOXIGENIC IN THE URUGUAYAN WATERS
Silvia M. Mendez1, Martha Ferrario2
1
2
National Direction of Aquatic Resources, Constituyente 1497, Montevideo, Uruguay E-mail: [email protected]
Faculty of Natural Sciences and Museum, National University of La Plata, Argentine
Plankton samples were collected weekly for harmful algae monitoring since 1980 along the Uruguayan coast. Toxic events caused by
Alexandrium tamarense, Gymnodinium catenatum, Dinophysis acuminata and D. caudata frequently affected the coastal mussels fisheries.
Pseudo-nitzschia toxic species are present in few concentrations all over the year; however blooms have been reported during summer.
The species of Pseudo-nitzschia potentially toxigenic identified in coastal marine waters of Uruguay up to now are Pseudo-nitzschia multiseries, P. fraudulenta, P. Pseudodelicatisima/calliantha, P. pungens, P. multiestriata and P. australis. The unique toxic event with detectable levels of Amnesic Shellfish Poisoning (ASP) by the standard HPLC method, was reported in December 2001 during a P.
multiseriata proliferation, reaching 1200 cells/l-1. This was the first toxic event of this species in the South Western Atlantic waters. P.
multistriata was reported for the first time in February 2009 in the coast of Uruguay reaching 2x106 cells l-1. Since this species was
detected previously in Brasilian waters and there is no report for higher latitudes, this is until now the southern limit of distribution for this
species in the Atlantic coast of South America. These Pseudo-nitzschia species are known to produce ASP around the world and
represent potential ASP toxicity producers in Uruguay.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 144
NEW RECORDS OF HABS SPECIES
AT MANZANILLO BAYS, COLIMA, MÉXICO
Alejandro Morales-Blake1, Héctor Ortiz-Lira1, Gabriela Carreón-López1, Edith Santos-Morales1,
Carlos Lezama-Cervantes1, Adrian Tintos-Gómez1, David Hernández-Becerril2
1
Facultad de Ciencias Marinas, Universidad de Colima, Km 20, Carr. Manzanillo-Barra de Navidad, Manzanillo, Colima, México.
E-mail: [email protected]
2
Instituto de Ciencias del Mar y Limnología, UNAM, A.P. 70-305, Cd. Universitaria, México D.F. 04510, México
Historical records of HABs at Manzanillo bays from 1986 to 2004 show the occurrence of 12 dinoflagellates taxa, 1 silicoflagellate and
1 ciliate as dominant species, with very high time variability and spatial distribution related with oceanographic conditions. HABs that resemble a monoculture have been recorded forming patches to mesoscale events. A monitoring program with weekly surveys was carried
out from March 2007 to June 2010, mainly during winter and spring when HABs appear. During this period we recorded new and dominant HABs species, additionally, we noted that these events changed from monospecific to multispecific HABs, developing as a dinoflagellates cocktail. 7 new species forming HABs were recorded, increasing our taxa list from 14 to 21 HABs species. The new microalgae
recorded from 2007 to 2010 are: Karenia mikimotoi, Gyrodinium instriatum, Karlodinium sp., Akashiwo sanguinea, Dinophysis caudata,
Pheopolykrikos hartmannii, Polykrikos schwartzii. We also found toxic or potentially toxic phytoplankton previously unrecorded: Alexandrium
spp., Karenia spp., Karlodinium spp. and Pseudo-nitzschia spp. Common HABs species in Manzanillo bays are: Cochlodinium
polykrikoides, Gymnodinium catenatum and Ceratium divaricatum. Furthermore, oceanographic data and ocean color satellite imagery
were used to describe the occurrence and variability of HABs in the region.
144
POTENTIALLY HARMFUL MICROALGAE
FROM SOUTH-CENTRAL CUBA
Angel Moreira1, Beatriz Reguera2, Carlos Alonso1, Augusto Comas1
1
2
Cienfuegos Environmental Research Centre, P.O. Box 5, PC 59350, Cienfuegos, Cuba. E-mail: [email protected]
Spanish Institute of Oceanography, P.O. Box 1552, PC 36200, Vigo, Spain
Benthic and planktonic toxic microalgae from south-central Cuba were surveyed intermittently between 2007 and 2009, mainly in the
estuarine complex Cienfuegos Bay and its adjacent coast. Thirty one taxa belonging to 18 genera were identified. The highest abundance
both benthic and planktonic species were recorded in dry season and the beginning of the rains which is directly correlated with a higher
incidence of ciguatera and other episodes associated to harmful algal blooms during this period. Ostreopsis siamensis and Gambierdiscus
toxicus were the dominant benthic species in the eastern coast. After strong rains, Ostreopsis ovata and Prorocentrum lima were the
dominants. Moderate abundance of the potentially ciguatotoxic Prorocentrum concavum, Prorocentrum rathymum and the ichthyotoxic
cyanophyte Lyngbya majuscula was registered on blooms of filamentous epiphytic brown macroalgae in beaches from Cienfuegos Bay.
Gymnodinium catenatum, Pyrodinium bahamense and Dinophysis ovum were first reported in the region. Blooms of toxic and non-toxic
dinoflagellates and of the diatom Pseudo-nitzschia multistriata have been observed in eutrophic zones of Cienfuegos Bay. A small episode
of dead fish was associated to blooms of Heterocapsa circularisquama and to the presence of Amphidinium carterae in the fishes’ gills.
Other toxic or noxious species have been seen accompanying blooms in other parts of the bay such as Cochlodinium polykrikoides,
Dinophysis caudata, Gonyaulax polygramma and Gonyaulax spinifera. High abundance of the toxic cyanobacteria Planktothrix isothrix,
Pseudoanabaena sp. and Microcystis aeruginosa was registered in the bay after tropical storms and hurricanes.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 145
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
FIRST OCCURRENCE OF THE FISH KILLING
RAPHIDOPHYTE CHATTONELLA SP. IN MALIAKOS GULF,
GREECE (EASTERN MEDITERRANEAN SEA)
K. Pagou1, I. Varkitzi1, A. Pavlidou1, I. Hatzianestis1, R. Psyllidou1, H. Kontoyannis1, X. Zeri1,
M. Yiagnisis1,2, G. Rigos1, A. Prapas3
1
Hellenic Centre for Marine Research, P.O. Box 712, PC 19013, Attica, Greece. E-mail: [email protected]
Laboratory of Ichthyology & Aquatic Animal Health, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece
3
Fish Pathology Department, Centre of Athens Veterinary Institutions, Ministry of Rural Development and Food,
Agia Paraskevi, Athens, Greece
2
Blooms of Raphidophytes have been responsible for massive fish-kills worldwide. The ichthyotoxic mechanism involves the production of a
brevetoxin-like neurotoxin, haemolytic fatty acids, gill damaging reactive oxygen species (ROS such as superoxide, hydrogen peroxide, hydroxyl
radicals) and gill irritating mucus. In spring 2009, the first massive kills of wild and cultured fish were recorded in Maliakos Gulf, which receives
high river runoffs, nitrogen and phosphorus inputs, domestic, industrial and agricultural wastes, thus creating a variable environment of
mesotrophic status with unbalanced nitrogen/phosphorus ratios (N/P). During the event, phytoplankton communities and a number of environmental parameters were investigated. The water column was found to be well oxygenated but stratified with surface masses of lower
salinity and temperature due to runoffs. Organic pollutants and heavy metals presented low values. Nitrates, nitrites, ammonium and phosphates
ranged at relatively low levels while silicates presented high values. N/P ratios indicated N limitation in most stations. The dominant species
Chattonella sp. (15-30 μm length) was dispersed throughout the Gulf and ranged from 0.05 to 1.3 106 cells L-1. Values greater than 0.5 106
cells L-1 were located at stations with N limitation mostly. The gills of dead cultured fish Sparus aurata, Dicentrarchus labrax and wild fish
Mugil cephalus, Pagellus erythrinus, Umbrina cirrosa and Dicentrarchus labrax were examined with microscopy and Chattonella sp. cells were
observed on them. Other potentially harmful species co-existed at low abundances in the water column, such as the dinoflagellates Karenia
brevis, Alexandrium minutum, Dinophysis cf. acuminata and the diatom Pseudo-nitzschia sp.
145
MASSIVE BLOOM OF COCHLODINIUM POLYKRIKOIDES
AND ITS IMPACTS IN THE UNITED ARAB EMIRATES WATERS
Anbiah Rajan1, Thabit Zahran Al Abdessalaam1, Rajasekhar Thankamony1, Yasser Othman1,
Shadab A. Khan2, Ebrahim A. Jamali3
1
Biodiversity Management Marine Sector, Environment Agency, P.O. Box 45553, Abu Dhabi, United Arab Emirates.
E-mail: [email protected]
2
Marine Environment and Wildlife Section, Dubai Municipality, P.O. Box 67, Dubai, United Arab Emirates
3
Marine Resources Research Centre, Ministry of Environment and Water, P.O. Box 21, Umm Al Quwain, United Arab Emirates
A prolonged bloom of Cochlodinium polykrikoides was observed from August 2008 to April 2009 in the United Arab Emirates waters. This
bloom was unique to the region in terms of its large spatial extent and impacts on the environment and economy. From its first detection in
Fujairah on the east coast of the country in the Gulf of Oman, the bloom moved anticlockwise around the coast eventually reaching the west
coast and the Iran coast to the north without touching coastal Abu Dhabi in the Arabian Gulf. The bloom resulted in extensive mortalities of
marine organisms including annelids, molluscs and fishes both farmed and wild. The phytoplankton counts from multiple sites, meteorological
data and water chemistry, as well as information on the physical characteristics of the sampling sites, provided an extensive spatial and
temporal data set. The bloom extended 1200 km of the coastline, lasted for more than eight months, and killed more than 600 tons of fishes.
The cell numbers were as high as 1.1 to 2.1 x 107 cells/L in the bloom area and the water was slimy, foul-smelling and dark brown in color.
The phytoplankton species composition and bloom durations significantly varied between sites. The hydrographic parameters and nutrient
concentrations also varied between bloom and non bloom areas. The dissolved oxygen concentration was very low (0.14 to 2.9 mg/L) and
hypoxic conditions prevailed in the bloom areas. We integrated monitoring data with remote sensing to analyze the development and progression
of the Cochlodinium polykrikoides bloom to further discuss its causes and impacts.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 146
BLOOMS OF COCHLODINIUM POLYKRIKOIDES
IN THE COASTAL WATER OF HORMOZGAN PROVINCE
(NORTH OF PERSIAN GULF - IRANIAN WATERS)
Fereshteh Saraji
Center for Marine and Coastal Studies (Cemacs) Faculty, University Sains Malaysia, 11800 USM, Penang, Malaysia.
E-mail: [email protected]
Phytoplankton bloom has been occurred regularly and normally in north of Persian Gulf, in south of Iran. Blooms and discoloration
water were occurred normally in Persian Gulf and duration of them was short and some time without harmful effect on aquatic system.
Navicula, Nitzschia, Noctiluca and Oscillatoria were species that caused these blooms. We didn’t see any mass mortality during these
blooms in Iranian water of Persian Gulf. In 28th September 2008 until April 2009 we observed extensive phytoplankton blooms and
brown discoloration water in coastal water of Hormozgan Province in south of Iran. During this time, we identified Cochlodinium
polykrikoides blooms. This bloom was observed in Persian Gulf for first time. An initial brownish big patch was observed in late of September in Strait of Hormoz and then extended to west along coastal water. This bloom coincided with mass mortality of invertebrate organisms, fish and macroalgae for eight months. The densities of Cochlodinium polykrikoides range were 1000 to 25 x 106 cells liter-1.
The maximum densities were recorded in November 2009 and with increasing temperature the density of this species was decreased.
After this event we began red tide monitoring in coastal water of our area. In this project, phytoplankton, zooplankton, benthos and
physic-chemical parameters are recorded monthly.
146
SPECIES OF DINOPHYSIS ASSOCIATED TO THE DETECTION
OF DIARRHEIC SHELLFISH TOXINS IN BIVALVES
AND TO HUMAN DIARRHEIC DISEASE EPISODE
(BUENOS AIRES PROVINCE, ARGENTINA)
Eugenia Sar1,2, Inés Sunesen1,2, Andrea Lavigne1,3, Alejandra Goya4, Carlos García5, Néstor Lagos5
1
División Ficología, FCNyM, UNLP, Paseo del Bosque s/n, 1900 La Plata, Argentina. E-mail: [email protected]
2
Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Argentina
3
Dirección de Pesca, Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires, Calle 51 esquina 12,
Torre 1, piso 7o, 1900 La Plata, Argentina
4
Depto. de Toxinas Marinas, Laboratorio Regional Mar del Plata, Centro Regional Buenos Aires Sur, SENASA,
Aviso Dorrego y Víctimas del 46, 7600 Mar del Plata, Argentina
5
Lab. de Bioquímica de Membrana, Depto. de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile,
Casilla 70005 - Correo 7, Código Postal 8389100, Santiago, Chile
Since January to April 2010, in Villa Gesell and Mar Azul coastal waters, diarrheic shellfish toxins were detected in three bivalve species by mouse
bioassay and the phytoplankton community consistently contained toxigenic species of Dinophysis with densities of the order of 103 to 104 cells
l-1. Additionally, three cases of human gastrointestinal illness associated with the consumption of cooked cockles were reported. The consumed
shellfish were positive for diarrheic toxins by mouse bioassay. The human disease was consistent with diarrhetic shellfish poisoning. The presence
of DST in the sampled shellfish and the cooked cockles was confirmed by HPLC. The toxins detected were okadaic acid (OA), dinophysis toxin
1 (DTX1) and their acylated derivatives, Acyl-OA and DTX3, respectively. This is the first record for Argentina of an outbreak of diarrhetic shellfish
toxins associated to the presence of Dinophysis species. The species determined were Dinophysis acuminata and D. caudata, previously
associated to the detection of DSP in bivalves from coastal waters of Uruguay. The present study was carried out in the framework of a monitoring
project of toxigenic harmful algae and toxins in coastal shellfish, developed by members of our Division and the Dirección de Pesca with the
support of SENASA. The DSP toxin analysis was done by HPLC with fluorescence on line detection in the Universidad de Chile.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 147
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
TEMPORAL AND SPATIAL VARIABILITY OF TOXIC
DINOFLAGELLATES SPECIES IN THE İZMIT BAY
Vildan Tüfekçi1, Dilek Ediger1, Çolpan Polat-Beken1, Mustafa Mantıkçı1, Hakan Atabay1,
Neslihan Balkıs2, Niyazi Deniz3
1
TUBITAK, Marmara Research Center, Environment Institute, 41470, Gebze-Kocaeli, Turkey,
E-mail: [email protected]
2
Department of Biology, Faculty of Science, Istanbul University, 34134 Vezneciler, Istanbul, Turkey
3
ARAS Ship & Marine Constructions Co., 34944 Tuzla, Istanbul, Turkey
The species composition and abundance of phytoplankton and related physico-chemical parameters were studied along The İzmit Bay
(Marmara Sea) at 5 stations monthly between November 2007 and December 2009. A total of 95 species were identified. Dinoflagellates
and diatoms were the most abundant groups in terms of species number; 45% of them being dinoflagellates. Moreover, 7 species were
identified for other groups (Euglenophyceae, Raphidophyceae, Dictyochophyceae). 13 different toxic species were detected during the
sampling period in the studied area. Ceratium furca and Ceratium fusus were the most common dinoflagellate species at all 5 sampling
locations throughout the sampling period. Their contribution to the total abundance was ranging from 1 to 27% and 1 to 17%, respectively.
The toxic species Prorocentrum scutellum (424124 cells L-1) and Prorocentrum micans (732000 cells L-1) contribution to total abundance
was 87% during June 2009 and 78% during October 2008, respectively. Gonyaulax sp. (especially G. fragilis) played an important role
in November 2007 comprising 52% of total phytoplankton density by reaching to 26650 cells l-1. Dinoflagellates contribution was highest
during summer and autumn. The mixture of several phytoplankton species in the studied area might have also played a key role in the
mucilage formation occurred during autumn 2007, 2008 and winter 2008, 2009. The mucilage producers Prorocentrum sp. and
Gonyaulax sp. have been identified as abundant species during autumn sampling period. Hydrography and nutrients dynamics of the
region played key role on temporal and spatial distribution of phytoplankton.
147
SPATIAL AND TEMPORAL DISTRIBUTION
OF OSTREOPSIS OVATA ALONG THE CAMPANIA COASTS
Adriana Zingone, Anna Ammendola, Maria Cristina Buia, Vincenzo Saggiomo, Giovanna Urciuolo
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected]
Since 1998 the toxic dinoflagellate Ostreopsis ovata has been causing problems to human health and to the benthic macrofauna along
the Mediterranean coasts. In summer 2007, 2008 and 2009, the distribution of O. ovata was investigated along the Campania region
coasts (Tyrrhenian Sea, Mediterranean Sea) with the aims of shedding light on the ecology of the species and improving the management
of the risks posed by its blooms. In August 2007, epiphytic microalgae were collected from benthic organisms at 40 stations scattered
along 450 km of coast. Ostreopsis ovata and other benthic dinoflagellates (Prorocentrum lima, Coolia monotis and Amphidinium carterae)
were found at the majority of the sampled sites, with the exception of the sandy shores of the Gulfs of Gaeta and Salerno. Peak abundances of O. ovata (up to 2 x 105 cells g-1 FWM) were recorded north of Naples city and at some stations of the Gulf of Salerno, whereas
minima (<103 cell g-1 FWM) were shown at the pristine stations of the southernmost areas. Weekly-fortnightly sampling at selected sites
of the Gulf of Naples over the summer 2007-2009 showed maximum seasonal abundance of O. ovata (up to 3 x 105 cells g-1 FWM)
always in mid July. In 2008, a second period of increase was detected at the end of the summer, possibly in relation with the persistence
of favourable meteorological conditions. No obvious relationships were found between O. ovata abundance and temperature, nutrients,
macroalgal substrates or hydrodynamic conditions at the different sites.
5. ABSTRACT BOOK (124-148)_Layout 1 10/22/10 2:31 PM Page 148
NEW REGIONAL HAB EVENTS
POSTER PRESENTATIONS
THE MEDITERRANEAN HABMAP: DISTRIBUTION
OF HARMFUL MICROALGAL SPECIES ALONG
THE MEDITERRANEAN COASTS
Adriana Zingone1, Marina Montresor1, Francesca Marino1, Katerina Aligizaki2,
Margarita Fernández-Tejedor3, Amany Ismael4
1
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected]
School of Biology, Faculty of Sciences, Aristotle University, 54124 Thessaloniki, Greece
3
IRTA, Carretera del Poblenou km 5.5, 43540 Sant Carles de la Ràpita, Spain
4
Oceanography Department, Faculty of Science, Alexandria University, Alexandria 21511, Egypt
2
Distribution maps of harmful species are a fundamental tool in order to identify risk areas as well as range expansion and contraction of
some species. In 2004, within the framework of the ISSHA project HABMAP, the first attempt to map Mediterranean harmful species
distribution (HABMAP-Med) was presented at the XI HAB Conference. In 2010, an update of the overview of harmful species distribution
along the Mediterranean coasts has been conducted by expanding the regional participation to the project and including new harmful
species that have been identified in several areas recently. The majority of records still concern the north Mediterranean coasts, although
a better coverage of the North African coasts is available as compared to the previous version of HAB-MAP-Med. The number of records
of toxic or potentially toxic species in the Mediterranean Sea has considerably increased over the years. This is particularly the case of
benthic dinoflagellates which were hardly addressed in studies of the last century. Problems related to DSP (diarrhetic shellfish poisoning)
and discolorations persist, while mucilage events associated with a number of different species, constitute another increasingly reported
phenomenon. However, a considerable amount of scientific interest has been redirected to Ostreopsis species due to their great impact
on human activities and public health over the last decade; among benthic dinoflagellates, the detection of Gambierdiscus in the eastern
Mediterranean Sea constitutes a notable record that points at a range expansion for species of this genus.
148
6. ABSTRACT BOOK (149-152)_Layout 1 10/22/10 2:32 PM Page 149
INLAND SEAS
AND HABS
6. ABSTRACT BOOK (149-152)_Layout 1 10/22/10 2:32 PM Page 150
POSTER PRESENTATIONS
FIRST RECORD OF FIBROCAPSA JAPONICA
IN THE MARMARA SEA: OCCURRENCE
AND GROWTH DURING MUCILAGE EVENTS
Yelda Aktan1, M. Karin de Boer2
1
2
Istanbul University, Fisheries Faculty, 200, 34470, Laleli, Istanbul, Turkey. E-mail: [email protected]
Marine Benthic Ecology and Evolution, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands
The harmful algal bloom (HAB) species Fibrocapsa japonica (Raphidophyceae) has a world wide distribution in temperate regions and
is occasionally responsible for mass mortality of fish. So far F. japonica was observed several times in the warm temperate region of
the western Mediterranean, but it has never been observed more east than the Adriatic Sea. This study shows the first record of F.
japonica in the Marmara Sea, Turkey. The Marmara Sea has a complex hydrological system, in a zone of transition between dense
(salinity 37-38.5‰) and warmer waters originating in the Mediterranean Sea, and cold, lower-salinity water (20-22‰) coming from
the Black Sea. The pycnocline lies at 10 to 30 m depth and varies seasonally. At October 2008 the first F. japonica cells were observed
near Prince Island in the Marmara Sea during a survey of a mucilage event. This event was associated with the massive presence of
diatoms and dinoflagellates and occurred on October and December 2008 along the north-eastern part of the Marmara Sea. The subsequent laboratory examination of the samples and the detailed morphological and molecular analysis showed conclusively that F.
japonica cells were present in this region but only at low concentrations, <12.103 cells l-1. The aims of this study are to give an overview
of the occurrence of a new HAB species, F. japonica, in the Marmara Sea during a mucilage event and to evaluate their growth and
relationship with environmental parameters.
150
GYMNODINIUM CATENATUM DOMINANCE
IN THE PHYTOPLANKTON OF MANILA BAY, PHILIPPINES,
DURING THE NORTHEAST MONSOON (NE)
Garry A. Benico, Rhodora V. Azanza, Gil Jacinto
The Marine Science Institute, University of the Philippines Diliman Campus, Quezon City, 1101, Philippines.
E-mail: [email protected]
An extensive baywide phytoplankton monitoring was conducted in Manila Bay during the Northeast Monsoon in 2010 in relation to the
continuing effort to understand extent of eutrophication in the bay and the possible occurrence of harmful species specially because it
has a long history of toxic blooms. Pyrodinium bahamense var. compressum blooms were associated with Paralytic Shellfish Poisonings
from shellfish collected in the bay from 1987 to about 2000. Phytoplankton samples were collected in 29 stations using Niskin sampler
for quantitative analysis and 20 μm plankton net for qualitative analysis. A total of 61 phytoplankton species were identified belonging to
four groups. Diatoms are generally the most dominant group with a mean density of 3094 cells/L. Dinoflagellates are the second most
abundant with a mean density of 2594 cells/L, followed by silicoflagellates with a mean density of 20 cells/L and blue-green algae with
a mean density of 2 cells/L. Diatom taxa with the highest mean densities are Chaetoceros spp. at 1657 cells/L, followed by Rhizosolenia
spp. at 595 cells/L and Bacteriastrum spp. at 215 cells/L. The major dinoflagellate species are Gymnodinium catenatum at 3186
cells/L, Ceratium spp. at 143 cells/L and Noctiluca scintillans at 75 cells/L. The genus Tintinopsis is the most abundant silicoflagellate
at 20 cells/L, while Trichodesmium sp. showed the highest density among blue-green algae at 2 cells/L. Stations 2, 3 and 5 which have
the highest phytoplankton count were dominated by Gymnodinium catenatum, a PSP -causative organism, attributing to 79.17%83.97% of the phytoplankton community.
6. ABSTRACT BOOK (149-152)_Layout 1 10/22/10 2:32 PM Page 151
INLAND SEAS AND HABS
SPATIAL AND TEMPORAL VARIABILITY OF ALEXANDRIUM
CATENELLA AND PSP IN SOUTHERN CHILE
(43O - 55O S) (MAY 2006 - MAY 2010)
Leonardo Guzmán1, Ximena Vivanco1, Gastón Vidal1, Gemita Pizarro2, Cristina Hernández3,
María Angélica Tocornal4, Maria Teresa Fauré5, Mauricio Palma1
1
Instituto de Fomento Pesquero, Balmaceda 252, Puerto Montt, Chile. E-mail: [email protected]
2
Instituto de Fomento Pesquero, Enrique Abello 0552, Punta Arenas, Chile
3
Secretaría Regional Ministerial de Salud, O’Higgins 276, Puerto Montt, Chile
4
Secretaría Regional Ministerial de Salud, Lord Cochrane 360, Aysén, Chile
5
Secretaría Regional Ministerial de Salud, Las Heras 825, Punta Arenas, Chile
In 1972 Alexandrium catenella and PSP were detected in the Strait of Magellan, southern Chile, presenting a sporadic presence and
restricted geographically until 1990. Later, recurrent annual blooms and PSP outbreaks and a northern expansion have occurred. The microalga was detected in 1992 in the region of Aysén, and in 1998 in the southern area of Chiloé Island. In October 2009 reached its
northern distribution at Calbuco (41o48’S; 73o10’W), being its southern border still at Seno Ponsonby (55o08’S; 68o43’W). The blooms PSP outbreaks show different annual patterns, due to its geographical and seasonal presence, but encompassing vast geographical areas
and showing, in general, low densities. In its northern area of distribution, are not present every year, reaching densities not higher than
1,000 cells l-1, but relative abundance has reached extremely abundant, and a PSP not surpassing 1,419 μg STX eq. 100 g-1 shellfish
meat. In its southern area of distribution, densities have been not higher than 53,800 cells l-1, but PSP concentrations have reached 27,159
μg STX eq. 100 g-1 shellfish meat, nevertheless the highest densities, 1,132,200 cells l-1, the highest relative abundances (mega abundant)
and the greatest PSP mean levels have been observed in the region of Aysén, in its central area of distribution. The interannual variability,
characterized by geographic sectors and periods of the year with higher probabilities to present the microalgae and paralytic poison in
shellfish, is suggesting that the bloom initiations and PSP outbreaks in the Chilean fjords are of climatic - oceanographic origin.
151
ALEXANDRIUM CATENELLA BLOOM AND
THE SUBSEQUENT DISTRIBUTION AND ABUNDANCE OF
CYST IN A FJORD ECOSYSTEM OF CHILEAN PATAGONIA
Paulina Montero1,2, Miriam Seguel3, Carmen G. Brito1, Paula Ortiz1, Giovanni Daneri1,2
1
Centro de Investigacion en Ecosistemas de la Patagonia (CIEP), Bilbao 449, Coyhaique, Chile. E-mail: [email protected]
Centro Basal COPAS Sur Austral, Universidad de Concepcion, Centro de Investigacion en Ecosistemas de la Patagonia, Chile
3
Centro Regional de Analisis de Recursos y Medio Ambiente (CERAM), Universidad Austral de Chile, Los Pinos s/n,
Puerto Montt, Chile
2
In the area of channels and fjords of southern Chile, blooms of Alexandrium catenella have been frequently detected (~2 years). The
last great bloom of this dinoflagellate in Northwest Patagonia was registered in March 2009. Within the study area (Puyuhuapi Fjord,
44oS) specific environmental conditions could be associated with the start of the bloom. Concentrations higher than 105 cells L-1 were
recorded in March, for three consecutive days. In the water column (2-20 m of depth), the bloom was associated with temperature
between 12 and 16oC, dissolved oxygen concentration of around 8 mg L-1 and salinities of ~15 psu (2m) and 31 psu (10-20 m). A
significant correlation was observed between A. catenella abundance with NO3 (r=-0.6, p<0.05) and PO4 (r=-0.7, p<0.05). After
the bloom, distribution and abundance of cyst (1-183 cyst cm-3 of wet sediment) was associated with coarse sand sediment (r=0.8,
p<0.05) and high organic matter content (r=0.9, p<0.05).
6. ABSTRACT BOOK (149-152)_Layout 1 10/22/10 2:32 PM Page 152
INLAND SEAS AND HABS
POSTER PRESENTATIONS
DEVELOPMENT OF POTENTIALLY TOXIC
PHYTOPLANKTON SPECIES IN COASTAL ZONE
OF THE NORTH-EASTERN PART OF THE BLACK SEA
Ol’ga Yasakova
Southern Scientific Centre of Russian Academy of Science, Chekhov’s street, 41, Rostov-on-Don, 344006, Russia.
E-mail: [email protected]
Monitoring of “red tides” in the North-Eastern part of the Black Sea was conducted since 2004. More than 300 samples were collected
monthly from sea surface within the network of stations in the daytime, fixed by the standard methods and analysed. 23 toxic and
potentially toxic species of phytoplankton, including invaders, were identified. Alexandrium species (A. ostenfeldii (Pauls.) Balech et
Tangen, Alexandrium sp.) were recorded among them. Prorocentrum (up to 25 mln.cell/m3), Dinophysis (up to 6 mln.cell/m3) and Ceratium species (2 mln.cell/m3) were the most abundant among potentially toxic Dinophyta within the Novorossiysk Bay. A number of
Pseudo-nitzschia species (up to 5.1 bln.cell/m3) were registered also. Mass propagation outbreaks of Chrysochromulina pontica Rouch.
(Chrysophyta) (up to 100 mln. cell/m3) (coastal area) and Dictyocha speculum Her. (0.035 mln. cell/m3) (off-lying part) were observed
in the Novorossiysk Bay. Extensive “red tide” caused by Scrippsiella trochoidea (Stein) Balech (3.14 g/m3) was recorded in wide coastal
zone between Anapa and Gelendzhik in March 2008. Chlorophyll concentration reached 7-10 mg/m3 in “water bloom” area. Operational
information concerning taxonomic, spatial and seasonal structure of toxic algae, dynamics of their distribution and development based on
satellite system can be used in the strategy of state local environmental monitoring.
152
7. ABSTRACT BOOK (153-157)_Layout 1 10/22/10 2:34 PM Page 153
INTRODUCTION
OF ALIEN SPECIES
AND HABS
7. ABSTRACT BOOK (153-157)_Layout 1 10/22/10 2:34 PM Page 154
ORAL PRESENTATIONS
IS THE EUROPEAN KARENIA MIKIMOTOI AN ALIEN?
Manal Al-Kandari1,2,3, Declan C. Schroeder2, Martin Edwards3,4, Marian Yallop5, Paul Hayes6
1
Mariculture and Fisheries Department, Kuwait Institute for Scientific Research, P.O. Box 1638, Salmiyah 22017, Kuwait.
E-mail: [email protected]
2
Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK
3
Faculty of Science and Technology, Biological Sciences, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, UK
4
Sir Alister Hardy Foundation for Ocean Science, Citadel Hill, Plymouth, PL1 2PB, UK
5
School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK
6
Faculty of Science, University of Portsmouth, St Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, UK
The genus Karenia G. Hansen and Moestrup 2000 consists of the well-known toxic species K. brevis (Davis) Hansen and Moestrup
2000 and K. mikimotoi (Miyake and Kominami ex Oda) G. Hansen and Moestrup 2000. K. mikimotoi was known under different names
in Pacific and European waters, and have been in a state of taxonomic turmoil for over 20 years. The seminal study by Hansen et al.
(2000) based on light and electron microscopy, nuclear-encoded LSU rDNA sequences, and pigment analysis confirmed that the European isolates formerly identified as Gyrodinium aureolum, Gyrodinium cf. aureolum, or Gymnodinium cf. nagasakiense, are conspecific
with the Japanese Gymnodinium mikimotoi. Consequently, the European and Japanese isolates were renamed with new genus name
Karenia. Here we will produce further evidence for the conspecific nature of European and Pacific isolates, however, we nonetheless
propose the separation of this species into distinct genotypes, thereby re-opening the debate of whether the European K. mikimotoi is
in fact a relatively new Pacific introduction.
154
DINOFLAGELLATE CYSTS AS TRACERS OF BALLAST
WATER EXCHANGE RISK IN PORTUGUESE PORTS
A. Amorim1, V. Veloso2, P. Chainho2, J. L. Costa2, A. Fernandes2, A. Costa3, P. M. Torres3,
J. Semedo4, M. J. Costa5
1
Centro de Oceanografia e Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa,
Campo Grande, 1749-016 Lisbon, Portugal. E-mail: [email protected]
2
Centro de Oceanografia, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal
3
Departamento de Biologia, Universidade dos Açores, Campus de Ponta Delgada - Apartado 1422,
9501-801 Ponta Delgada, Azores, Portugal
4
Instituto Portuário e dos Transportes Marítimos (IPTM), Rua General Gomes Araújo, Edifício Vasco da Gama,
1399-005 Lisbon, Portugal
5
Centro de Oceanografia e Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa,
Campo Grande, 1749-016 Lisbon, Portugal
The introduction of non-native species is currently considered as a major threat to biodiversity. In marine ecosystems ballast water exchange is a
major vector of unintentional introduction of alien flora and fauna. The coast of mainland Portugal is annually affected by HAB episodes ranging
from non-noxious red tide blooms to highly noxious toxic blooms, and may represent a high risk area for dispersal of harmful species. Portugal has
an international import-export trade balance which suggests a global high ballast water export and a low theoretical invasion pressure. On the other
hand, the domestic trade traffic between mainland Portugal and the Azores and Madeira islands suggests that ballast water exportation is mainly
from the islands to the continent. In order to evaluate the invasion pressure on mainland Portugal and its role as a donor area we analyzed the
maritime traffic in the Lisbon port between 2006 and 2008. We also examined ballast water and sediments for dinoflagellate cysts from 2 vessels
and conducted a dinoflagellate cyst survey in the ports of Lisboa and Ponta Delgada (Azores). Results indicate that dinoflagellate cyst assemblages
in ballast water tanks were dominated by Gymnodinium catenatum reflecting the recent bloom history of this species along the Iberian coast. Viable
cultures of G. catenatum and 4 other species could be established, including species for which no resting stage is known. The port surveys indicated
that while G. catenatum is the most abundant species in mainland Portugal there is no evidence of its present day occurrence in the Azores.
7. ABSTRACT BOOK (153-157)_Layout 1 10/22/10 2:34 PM Page 155
INTRODUCTION OF ALIEN SPECIES AND HABS
IS BALLAST WATER A SOURCE OF HARMFUL
DINOFLAGELLATES FOR CANADA’S COASTAL WATERS?
Suzanne Roy, Marie Parenteau, Andre Rochon
ISMER, Universite du Quebec a Rimouski, 310 Allee des Ursulines, Rimouski, Quebec, G5L 3A1, Canada.
E-mail: [email protected]
As part of the Canadian Aquatic Invasive Species Network (CAISN), we examined the presence of harmful dinoflagellates in ballast
waters of ships visiting ports on the East and West coasts of Canada. Three categories of ships (mostly bulk carriers) were considered:
(1) trans-oceanic, (2) coastal vessels that underwent ballast exchange and (3) coastal vessels with no ballast exchange. For the East
Coast, 46 ships were examined and harmful dinoflagellates were present in more than 90% of the ships, with a maximum abundance
close to 4000 cells per litre. A total of 16 harmful dinoflagellate species were found, including 4 non-indigenous species (NIS). Nine of
these (including the 4 NIS) are potentially diarrhetic shellfish poisoning (DSP) species, including several Dinophysis species. Ballast water
exchange (BWE) is mandatory for ships entering coastal waters of Canada and this measure is thought to greatly reduce the number of
foreign aquatic organisms entering coastal waters. Here, BWE reduced the abundance of harmful dinoflagellates, but increased species
richness, suggesting the upload of species during ballast exchange. For the West Coast, 45 ships were examined and 18 harmful dinoflagellate species were found (including 3 PSP and 8 DSP species), with a frequency of occurrence of 63%. Here BWE had no significant effect on the abundance or species richness of harmful dinoflagellates, but coastal vessels generally had a greater abundance
and species richness compared to trans-oceanics. Clearly, BWE is not efficient to eliminate harmful dinoflagellates from ships’ ballast
water for ships arriving to Canada.
155
THE POTENTIAL ALIEN SPECIES AND RELATED BLOOM
IN THE EAST CHINA SEA
Jinhui Wang1,2,3, Yanqing Wu1, Ren Xu2,3
1
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai, 200240, PR China.
E-mail: [email protected]
2
East China Sea Environmental Monitoring Center, SOA, Shanghai, 200137, PR China
3
Key Laboratory of Integrated Monitoring and Applied Technology for Marine Harmful Algal Blooms, SOA, Shanghai,
200137, PR China
The number of harmful algal blooms (HAB) in the East China Sea has doubled since the 1990s. Recently, changes in the biogeography
of harmful dinoflagellates (Heterocapsa circularisquama, Prorocentrum dentatum) and raphidophytes (Heterosigma akashiwo, Chattonella
marina) have been detected along the Chinese coast. Strong northward shifts in the spatial distribution of Phaeocystis globosa and
Karenia mikimotoi blooms have been documented. During 2007 and 2008 ballast water samples from international vessels in Shanghai
harbor were analyzed. Non-indigenous algae such as Heterocapsa circularisquama, Pseudo-nitzschia multiseries and Pseudo-nitzschia
multistriata were discovered. While P. multiseries and P. multistriata produce domoic acid, there have not been any reports of HABs with
this species. However, Heterocapsa circularisquama blooms have been observed with subsequent detrimental effects on marine ecology.
Some new species formed bloom in different part of China which never been found before; species introductions including issues of anthropogenic sources (e.g. ballast water) or natural systems (e.g. species range extension) will be analyzed based on geographical distribution and long series monitoring data.
7. ABSTRACT BOOK (153-157)_Layout 1 10/22/10 2:34 PM Page 156
POSTER PRESENTATIONS
HA AND HAB ALONG ABRUZZO COAST FROM 2007
TO 2010: CORRELATIONS WITH ENVIRONMENTAL
FACTORS AND NEW REPORTS
Cristina Ingarao, Tommaso Pagliani
Consorzio Mario Negri Sud - Environmental Science Centre, Via Nazionale 8/a, 66030, Santa Maria Imbaro, Italy.
E-mail: [email protected]
This work underlines the presence of endemical and allocthonous toxic species of the Abruzzo coast (Western Adriatic Sea). The presence
of new tropical harmful species belongs to 2007 when it was revealed the first appearance of Prorocentrum lima. After that summer,
other tropical toxic species such as Ostreopsis ovata, Coolia monotis and Prorocentrum levis were observed. Besides, the ichthyotoxic
species from Japan sea (Fibrocapsa japonica) has been detected before 2007. These species are known to produce toxins that could
be dangerous for human health. In this study six coastal sampling stations, a shellfish farm and four coastal rocky points have been monitored from 2007 to 2010. The study included the use of a phytoplankton net for sample collection. Chemical - physical and nutritional
parameters of sea water have been correlated with the abundances of Harmful Algal (HA) taxa. Temperature trend has been positively
correlated with harmful species (p<0.001) while salinity trend was negatively correlated itself (p<0.001), meaning that there was a highly
positive correlation between the occurrences of HA taxa and low values of salinity. Nutrient factors have been correlated with harmful
species such as DIN (Dissolved Inorganic Nitrogen), phosphate and silicate content. The statistical analysis suggested that harmful
species are sensitive to phosphate content (p<0.001). Besides, phosphorus could be considered the limiting factor for HA species. The
silicate content is quite negatively correlated (p<0.01). The counting of potential HA taxa in net samples represented a more accurate
estimation of potential HA abundances in the water column.
156
RESOLVING THE HISTORICAL RECORD
OF GYMNODINIUM CATENATUM AND OTHER
MICRORETICULATE CYSTS IN THE NE ATLANTIC
Sofia Ribeiro1,2,3, Ana Amorim2, Fátima Abrantes3, Marianne Ellegaard1
Department of Biology, University of Copenhagen, Øster Farimagsgade 2D, 1353 CPH-K, Denmark. E-mail: [email protected]
Centro de Oceanografia, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal
3
Unidade de Geologia Marinha, LNEG, Estrada da Portela, Zambujal, Apartado 7586, 2720-866 Amadora, Portugal
1
2
Gymnodinium catenatum has been responsible for the main PSP events in the west Iberian coast (Portugal and NW Spain) over the past
three decades. This chain-forming unarmoured dinoflagellate produces fossilizable microreticulate resting cysts. Sedimentary records
where these cysts have been naturally preserved are important temporal archives for investigating the palaeobiogeography of the species
and thus better understand its present day ecology and geographical range in relation to HAB events. However, the correct interpretation
of Gymnodinium catenatum’s cyst record has often been hampered by misidentification with co-occurring non-toxic Gymnodinium species
also producing microreticulate cysts, such as G. microreticulatum and G. nolleri. Species resolution within the microreticulate cyst morphotypes is challenging as their size ranges overlap to a certain extent, and only minute details in cyst reticulation allow for correct identification. In the west Iberian coast, G. microreticulatum has been confirmed to co-occur with G. catenatum by germination experiments,
but uncertainties remain regarding the presence of a Gymnodinium nollerii-like and/or other species. We examined the microreticulate
cyst record of several sediment cores along a latitudinal gradient in the Portuguese coast, and covering ca. the past two centuries. This
work was complemented by detailed morphological analysis aimed at resolving the record of G. catenatum with that of other microreticulate
species. By reconstructing the presence and abundance of G. catenatum cysts along W Iberia, we provide new evidence relevant to
further discuss the hypothesis of its recent introduction in the NE Atlantic.
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INTRODUCTION OF ALIEN SPECIES AND HABS
BIOFOULING TUNICATES ON AQUACULTURE GEAR
AS POTENTIAL VECTORS OF HARMFUL ALGAL
INTRODUCTIONS
Sandra E. Shumway1, Maria Rosa1, Gary H. Wikfors2, Bridget A. Holohan1
1
Department of Marine Sciences, University of Connecticut, 1080 Shennecosset Road, Groton, CT, USA.
E-mail: [email protected]
2
NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT, USA
Biofouling tunicates are ubiquitous in coastal systems and among the main overgrowers of aquaculture gear. Our study tests the hypothesis
that the proliferation of abundant tunicate fouling species and their subsequent transport, removal and transfer by aquaculturists provide
mechanisms for concentration and distribution of harmful algal cells to new areas. Wild-caught species of common, biofouling ascidians
(Styela clava, Ciona intestinalis, Molgula manhattensis, Bottryloides violaceus, Didemnum sp., and Botryllus schlosseri) were exposed individually to cultured strains of harmful algae (Prorocentrum minimum, Alexandrium fundyense, Alexandrium monilatum, Karenia mikimotoi,
Aureococcus anophagefferens, or Heterosigma akashiwo) at simulated bloom densities of each HAB. The ability of algal cells to pass
intact through the digestive system and remain viable was assessed. After feeding, ascidians were transferred and kept for 48 h in ultrafiltered seawater. After 24 and 48 h, biodeposits were collected and observed microscopically for the presence of intact and possibly
viable cells. Subsamples of biodeposits were transferred into algal culture medium and monitored for algal growth. Results and potential
mitigation strategies will be discussed regarding the risks of transporting harmful algal species through movement of shellfish aquaculture
gear and disposal of biofouling material.
157
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8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 159
CYANOBACTERIAL
ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
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ORAL PRESENTATIONS
HOW DOES ANABAENA SP. RESPOND TO ELEVATED
TEMPERATURE AND OCEAN ACIDIFICATION?
Andreas Brutemark, Jonna Engström-Öst, Anu Vehmaa
ARONIA Coastal Zone Research Team, Novia University of Applied Sciences & Åbo Akademi University,
Raseborgsvägen 9, 10600 Ekenäs, Finland
Due to ocean acidification, aquatic organisms experience substantial changes in pH and the ocean carbonate system. However, the organisms are not only subjected to an increased acidification since other changes occur simultaneously. Global warming will increase the
surface temperatures 1-7oC depending on region and predicting model. In the Baltic Sea, mass-occurrences of cyanobacteria are a
common phenomenon during summer. The impact of climate change on cyanobacteria is largely unknown and most research has focused
on single factors. Here we determine how elevated temperature and reduced pH, individually as well as combined, affect the cyanobacterium Anabaena sp. growth, oxidative stress and toxin production. Anabaena sp. was grown as non-axenic batch monocultures with
normal temperature and normal CO2, high temperature and normal CO2, normal temperature and high CO2, and finally as “greenhouse
effect” i.e. high temperature and high CO2. During the 11-day long experiment there was a positive response in chlorophyll a in the high
temperature treatments whereas no effect of CO2 could be observed. The results suggest that Anabaena sp. will be favoured by the
future conditions in the Baltic Sea, especially by increased temperatures.
160
TOXIN PRODUCTION AND BIOTIC FACTORS
-CASE STUDIES WITH ANABAENA AND NODULARIA
Jonna Engström-Öst1, Hedvig Hogfors2, Elena Gorokhova2, Sari Repka3
1
Novia University of Applied Sciences, Finland. E-mail: [email protected]
Stockholm University, Sweden
3
University of Turku, Esthonia
2
We studied biotic effects on growth and toxin production of two important bloom-forming cyanobacteria in the Baltic Sea. Anabaena sp.
and Nodularia spumigena were incubated with eukaryote phytoplankton and copepods. Presence of the foreign haptophyte Tetraselmis
negatively affected growth rate of Nodularia, whereas presence of the cyanobacterium strongly impeded growth of co-occurring cryptophyte Rhodomonas, but not of Tetraselmis. The experiments with Anabaena were performed at two sites, of which one with lower salinity
(Bothnian Bay). The results showed that both growth and intracellular microcystin concentrations of Anabaena were higher at the Bothnian
Sea site, in comparison to the site in the Gulf of Finland. Anabaena produced more toxin in the presence of foreign chlorophyte Brachiomonas, and less toxin when incubated with Rhodomonas. Nodularia, on the other hand, did not produce different amounts of toxin
when incubated with co-occurring Rhodomonas or foreign Tetraselmis. At the less saline site, toxin levels of Anabaena were higher. For
Nodularia, we showed that in the presence of algae Rhodomonas and Tetraselmis, nodularin levels increased in a concentration-dependent
manner, however, when copepods were present in the mixtures of algae and cyanobacterium, this increase was significantly less. Our
results demonstrate that responses of different cyanobacteria to other plankton are highly species-specific and they are essential to be
aware when managing areas suffering from toxic algal blooms.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
ZOOPLANKTON IMPACTS ON CYANOBACTERIA BLOOM
DEVELOPMENT IN AN OLIGOTROPHIC RESERVOIR
Hong Ying1, Burford Michele2, Matthew Whittle2, Doblin Martina1
1
2
University of Technology, P.O. Box 123, Broadway, 2007 Sydney, Australia. E-mail: [email protected]
Griffith University, 170 Kessels Rd, 4111 Nathan, Australia
While it has been well documented that freshwater eutrophication leads to cyanobacterial blooms, such blooms can also occur in waters
with relatively low nutrient concentrations e.g., subtropical reservoirs in south-east Queensland. This study tested the hypothesis that
cyanobacteria accumulations are facilitated through low mesozooplankton grazing pressure. Mesocosm experiments were performed in
Lake Wivenhoe, the largest subtropical reservoir in south-east Queensland. There were four treatments (comprising triplicate 800 L
plastic bags suspended in the epilimnion) including: no zooplankton (control), low zooplankton (natural abundance), high zooplankton (5X
natural abundance), and P-enriched high zooplankton (to achieve N: P ratios of 16:1). Bags were monitored every 1-2 days for physical
parameters, chl-a, dissolved nutrients, particulate carbon, and phytoplankton species composition. Instantaneous zooplankton grazing
and nutrient regeneration rates were also measured in parallel laboratory incubations on days 4 and 7. Phytoplankton bio-volume was
the same among the treatments after 7 days, while the proportion of potentially toxic cyanobacterium Cylindrospermopsis declined in low
zooplankton but not in high zooplankton treatments. The zooplankton clearance rates were extremely low (0.002-0.022 ml ind-1 h-1)
compared to literature values (0.300-5.100 ml ind-1 h-1) while P regeneration rates (0.012-0.015 μg ind-1 h-1) were relatively high
compared to values of various marine copepods (0.005-0.010 μg ind-1 h-1). Together, these results suggest that Cylindrospermopsis
abundance may be facilitated through a planktonic foodweb subsidy, whereby mesozooplankton consume other algae in preference to
Cylindrospermopsis, then regenerate nutrients that are then rapidly taken up by low P-adapted Cylindrospermopsis.
161
ABUNDANT EXISTENCE OF MICROCYSTIS-KILLER
AND INHIBITOR BACTERIA ON THE SURFACE OF WATER
PLANTS IN LAKE BIWA: POSSIBILITY FOR CONTROL
OF MICROCYSTIS BLOOMS
Ichiro Imai1, Tasuku Kido2, Ikuo Yoshinaga2
1
Plankton Laboratory, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
E-mail: [email protected]
2
Division of Applied Biosciences, Kyoto University, Kyoto 606-8502, Japan
Toxic blooms of cyanobacteria such as Microcystis have increasingly given negative impacts to freshwater ecosystems and water resources
for human activity. Hence there is an urgent need for the development of useful tools for preventing occurrences and/or reducing negative
impacts of cyanobacterial blooms. Microorganisms such as bacteria appear to be promising control agents against cyanobacterial blooms.
We investigated cyanobacteria-killer and -inhibitor bacteria on the surface of water plants such as Egeria densa and Ceratophyllum demersum
in Lake Biwa, Japan, from May to October in 2008. Bacteria were isolated using nutrient agar plates, and the killing ability was examined
for Microcystis aeruginosa by co-culture experiments. We discovered a huge number of Microcystis-killer and growth-inhibiting bacteria for
the first time from the surface of these water plants (3x105~3x106 CFU/g from E. densa, and 7x104~9x105 CFU/g from C. demersum).
Microcystis-inhibitor bacteria were also confirmed in collected water (200 CFU/mL on July 14). Most powerful bacteria killed entire Microcystis cells in culture within two days after the addition with the bacterial density of 105 cells/mL. We previously discovered abundant
existence of Microcystis-killer bacteria in biofilms on submerged reed stems of reed community and in water there. Based on these new
findings, we here propose a new possible prevention strategy for cyanobacterial blooms, i.e. large-scale combined developments of water
plant zones and reed communities in the coast of lakes and/or in the inner lakes that connect to the main lake with Microcystis blooms.
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MICROCYSTINS IN IRISH LAKES: INTERACTIONS WITH
ENVIRONMENTAL FACTORS AND THE INVASIVE ZEBRA
MUSSEL DREISSENA POLYMORPHA
Karen M. Mooney1, John T. G. Hamilton2, Stewart D. Floyd2, Robert H. Foy2, Christopher T. Elliott1
1
2
Institute of Agri-Food and Land Use, Queens University Belfast, Belfast BT9 5AY, UK. E-mail: [email protected]
Agri-Food and Biosciences Institute, Newforge Lane, Belfast BT9 5PX, UK
With the introduction of the Nitrates Directive in Northern Ireland attempts are being made to reduce nutrient loadings into water bodies.
Combined with increasing temperatures and the ongoing invasion of zebra mussel Dreissena polymorpha this is changing the phytoplankton balance of the lakes. D. polymorpha has been hypothesised and found to alter the phytoplankton composition of lakes due to
prolific filter feeding, especially increasing cyanobacterial populations. The main cyanobacteria species present in Irish lakes are all cyanotoxin producers, forming blooms in spring (minor) and late summer (major). We report the results of three seasonal surveys at 47 sites
across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins.
Seven toxins were tested for, five of which were found, with MC-LR, MC-RR and MC-YR being the most prevalent. Aphanizomenon,
Anabaena, Gomphosphaeria, Microcystis and Woronichinia were the most prevalent cyanobacterial genera encountered and associated
with the highest microcystin concentrations. The occurrence of several microcystin variants indicates that there may potentially be more
than one cyanobacteria species producing microcystins at many sites, which varies seasonally. Total microcystin concentrations varied
over three orders of magnitude; many sites were over 1 μgMC/L, with two sites being over the 10 μgMC/L WHO recreational exposure
limit. Multivariate statistical analysis indicates D. polymorpha are associated with environmental modification and higher microcystin production in invaded lakes and Polymerase Chain Reaction analysis indicates that monitoring for microcystin mcyB and mcyD gene presence is not a suitable indicator of lake toxicity.
162
BIOGEOGRAPHY OF PLANKTONIC CYANOBACTERIA
IN RELATION TO HUMAN ACTIVITY AND CLIMATE CHANGE
Judit Padisak
Department of Limnology, University of Pannonia, H8200 Veszprem, Egyetem u. 10, Hungary.
E-mail: [email protected]
As a consequence of lack of sexual reproduction, cyanobacteria undergo a very fast evolution which can be viewed rather as strain
selection than speciation. They adapt fast to unique sets of habitat properties. As group, they have wide tolerance levels; however, as
species they occur under well specified environmental conditions and that makes them ideal indicators. Nutrients that are most likely to
limit algal growth are phosphorus and nitrogen. Biogeochemisty of these elements is strikingly different. Phosphorus has a sedimentary
cycle and its transfer between different media (air, water, solid phases) is very slow since atmospheric transport is negligible. In aquatic
environments, N undergoes substantial oxidation number changes. In both nitrification and denitrification process it passes the oxidation
state 0, when it occurs in volatile gaseous from. As a consequence, aquatic ecosystems loose substantial amounts of N that need to be
resupplied through N-fixation. Without N-fixation the Earth’s ecosystems would run fast into serious, even lethal, N-shortage. Presence
and N-fixing cyanobacteria in aquatic environments suffices an important ecosystemic need and cannot be considered as a harmful phenomenon. Terrestrial soils absorb P quite efficiently; therefore natural input of N to rivers and lakes is much higher than that of P. Consequently in lakes typically P limitation of plant growth develops. Human P input (eutrophication) increases the ecosystemic need for N
that leads to development of potentially toxic blue-green algal blooms. The presentation will include examples of proliferation of blue
green algae in aquatic environments due to evolutionary-, human imposed- and climatic reasons.
8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 163
CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
ORAL PRESENTATIONS
GENOMIC ANALYSIS OF THE TOXIGENIC FILAMENTOUS
CYLINDROSPERMOPSIS RACIBORSKII / RAPHIDIOPSIS
SPP. CLADE: FASCINATING THINGS IN SMALL PACKAGES
Karina Stucken1,2, Uwe John2, Gernot Gloeckner3, Alejandro Murillo1, Katia Soto-Liebe1, Juan J. Fuentes1,
Alvaro M. Plominsky1, Allan Cembella2, Mónica Vásquez1
1
Department of Molecular Genetics and Microbiology, P. Universidad Católica de Chile, Santiago, Chile.
E-mail: [email protected]
2
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany
3
Berlin Center for Genomics in Biodiversity Research and IGB, Königin-Luise-Str. 1-3, D-14195 Berlin, Germany
Cyanobacteria are unique prokaryotes capable of oxygenic photosynthesis and with broad ability for alternative nitrogen utilization, including
N2 fixation. Cyanobacterial morphology is diverse, including unicellular and filamentous forms, and many species are invasive and cosmopolitan,
and some associated with harmful algal blooms (HABs). For example, the widely distributed invasive filamentous Cylindrospermopsis raciborskii
may produce either the hepatotoxin cylindrospermopsin (CYN) or neurotoxins such as saxitoxin (STX) and analogues. Blooms of C. raciborskii
are often associated with Raphidiopsis spp., including toxigenic strains of R. brookii. We sequenced the complete genomes of C. raciborskii
CS-505 and R. brookii D9, a CYN- and STX- producing strain, respectively. Surprisingly, genome sizes (3.89 Mbp for CS-505 and 3.2 Mbp
for D9) are the smallest known for multicellular cyanobacteria. Furthermore, these genera cannot be discriminated at the molecular level with
reference to 16S rRNA genes or complete genomes, requiring re-classification of the C. raciborskii/Raphidiopsis spp. clade. Remarkable
features of the strains include high synteny in both genomes, and the organization of nitrogen fixation and toxin synthesis gene clusters, as
an example of non-canonical genomic islands. Major differences are evident, however, in the degree of genomic plasticity, with higher numbers
of regulatory elements, repeated sequences, transposons and phage integrases found in strain CS-505 than in strain D9. In summary, both
strains appear to provide examples of allopatric speciation among cyanobacteria. Insights into genome regulation under different growth
condition point to the plausible role of new regulatory elements rather different from typical transcriptional regulators.
163
CLIMATE CHANGE AND THE IMPACT OF C:N
STOICHIOMETRY ON TOXIN PRODUCTION
BY HARMFUL CYANOBACTERIA
Dedmer B. van de Waal1,2,3, Linda Tonk1,4, Ellen van Donk2, Hans C.P. Matthijs1, Petra M. Visser1, Jef Huisman1
1
Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Nieuwe Achtergracht 127,
1018 WS Amsterdam, The Netherlands
2
Aquatic Ecology, Netherlands Institute of Ecology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
3
Marine Biogeosciences, Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570,
Bremerhaven, Germany. E-mail: [email protected]
4
Centre for Marine Studies, University of Queensland, St Lucia, Qld 4072, Australia
It is commonly argued that climate change is likely to promote harmful cyanobacterial blooms in freshwater lakes worldwide. Microcystis
and Planktothrix are common bloom-forming cyanobacteria that can produce a family of hepatotoxins known as microcystins. The microcystin variants are composed of different amino acids, and thereby differ in their carbon:nitrogen (C:N) ratio. We studied how the
availability of dissolved inorganic carbon (DIC) and dissolved inorganic nitrogen (DIN) affects the cellular C:N stoichiometry and microcystin
production of Microcystis and Planktothrix. Chemostat experiments showed that high CO2 concentrations under nitrogen-poor conditions
yield high cellular C:N ratios, although the microcystin composition is not affected. However, a high CO2 availability under nitrogen-rich
conditions did not affect the cellular C:N stoichiometry, but it strongly enhanced the cellular microcystin content. Particularly, the nitrogen-richest microcystin variant, microcystin-RR, showed a strong negative correlation with the cellular C:N ratio, both in laboratory and
in the field. Furthermore, our results indicate that the impact of cellular C:N stoichiometry on microcystin composition is mediated by the
amino acids produced by the cells. Climate change is likely to alter the carbon and nitrogen availability in many aquatic ecosystems, and
may thereby affect the elemental stoichiometry of harmful cyanobacteria as well as the nature of the toxins that they produce.
8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 164
POSTER PRESENTATIONS
ANABAENA SP. BLOOM AND THE OCCURRENCE
OF MICROCYSTINS FROM A EUTROPHIC POND
IN BANGLADESH
Sagir Ahmed1, Thomas Krueger2, Bernd Luckas2
1
Aquatic Resource Management Laboratory, Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh.
E-mail: [email protected]
2
Department of Food Chemistry, Institute of Nutrition, University of Jena, Dornburger Street 25, 07743 Jena, Germany
Eutrophication of freshwaters and appearance of cyanobacteria blooms have become a serious problem in Bangladesh. A bloom of Anabaena sp. occurred in a freshwater pond in Brahmanbaria. Bloom sample was collected and filtered through a glass fiber filter. Methanolwater extract of filtered cells was analyzed by high performance liquid chromatography (HPLC) with UV, MS and MS-MS detection. We
detected three types of microcystins viz, [D-Asp3, Dha7] Microcystin-LR, [Dha7] Microcystin-LR and Microcystin-LR. The total concentration of microcystins was 4.0 μgl-1, well above the WHO provisional guideline value for drinking water. Cyanotoxin risk assessment is
important to protect public health in Bangladesh where surface water is used as drinking-water source.
164
A POTENTIAL PROTECTIVE ROLE OF HESPERIDIN
AGAINST MICROCYSTIN-INDUCED HEPATIC
AND RENAL DAMAGE
S. Al-Jassabi
Institute of Biological Sciences, University of Malaya, Malaysia. E-mail: [email protected]
The presence of cyanobacterial toxin in drinking and recreational waters represents a potential risk to public health. Microcystin-LR (MCLR) is a potent cyclic hepatapeptide hepatotoxin produced by the blue-green algae Microcystis aeruginosa (M. Aeruginosa). Chemoprotectant studies suggest that membrane-active antioxidants may offer a protection against microcystin toxicity. The aim of this study is to
investigate the potential protective role of hesperidin in MC-LR-induced hepatotoxicity and nephrotoxicity in Balb/c mice. Pretreatment
of Balb/c mice with hesperidin (300 mg/kg bwt) given orally once a day for 14 days before treatment with toxin i.p. (75 μg toxin/ kg
bwt) according to LD50 value. The potential benefits of hesperidin were evaluated based on alanine aminotransferase (ALT), glutamyloxaloacetic transaminase (SGOT), and glutamyl-pyruvic transaminase (SGPT), as well as levels of total bilirubin, creatinine and urea nitrogen in the serum. Also, the level of lipid peroxidation (LPO), protein carbonyl contents, DNA fragmentation, methylglyoxal (MG)
accumulation, and antioxidant enzyme activities were determined in liver and kidney tissues. Our results demonstrated that pretreatment
of balb/c mice with hesperidin prevented all these biochemical changes. Hesperidin pretreatment also restored MC-LR effect on PP1
activity in the liver to control level. Hesperidin also showed a considerable antioxidant activity in the FRAP assay system. The multiple antioxidant activity of hesperidin was evident as it showed strong reducing power. These results provide evidence that inhibition of ROS
overproduction and maintenance of intracellular antioxidant status may play a pivotal role in the protective effects of hesperidin against
MC-LR induced hepatic and renal damage.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
SUPPLEMENTATION OF POTATO PEEL EXTRACT
PREVENTS MICROCYSTIN-LR-INDUCED HEPATOTOXICITY
IN BALB/C MICE
S. Al-Jassabi
Institute of Biological Sciences, University of Malaya, Malaysia. E-mail: [email protected]
Contamination of microcystin-LR (MC-LR) in drinking water is associated with several human diseases including cancer. It has been reported that oxidative stress plays a vital role in MC-LR-induced biochemical and molecular alterations. The aim of this investigation was
to improve the understanding of MC-LR-induced oxidative damage to proteins and DNA, and the role of potato peel extract (PPE) (which
is considered as a food waste) in alleviating MC-LR-induced damages in Balb/c mice. A significant increase in the levels of protein oxidation, DNA strand breaks, and DNA-protein cross-links was observed in the serum and hepatocytes of Balb/c mice exposed to 75 μg
toxin/kg bwt of mouse for 24h. Co-administration of PPE to MC-LR-exposed mice showed a substantial reduction in the levels of toxininduced oxidative products of protein and DNA. The results of this study support free radical-mediated toxic manifestations of MC-LR
and also, suggest that PPE supplementation can improve the MC-LR-induced molecular alterations.
165
CAN WE PREDICT THE EUTROPHICATION STATUS
OF LAKES AND RESERVOIRS USING LOW COST
ENVIRONMENTAL VARIABLES?
Cécile Bernard1, Arnaud Catherine1, David Mouillot2, Nicolas Escoffier1, Claude Yéprémian1, Marc Troussellier2
1
FRE 3206 CNRS / MNHN Molécules de Communication et Adaptation des Micro-organismes,
USM 505 Cyanobactéries, Cyanotoxines et Environnement, Muséum National d’Histoire Naturelle, 57 rue Cuvier,
CP 39, 75231 Paris, Cedex 05, France. E-mail: [email protected]
2
Laboratoire Ecosystèmes lagunaires, UMR 5119 ECOLAG CNRS-UM2-IFREMER-IRD, Université Montpellier 2,
Case 93, Place E. Bataillon, F-34095 Montpellier, Cedex 05, France
Eutrophication is a serious threat in many parts of the world. Identifying the environmental factors that determine the spatial distribution of eutrophicated
water bodies and developing predictive tools to mitigate this issue remain a challenge. The Paris area (named region Ile de France: IDF) was selected
because it shows deep contrasts in terms of the nature and of its large diversity of anthropogenic pressure in terms of its nature and intensity, and
it includes a wide range of lakes and reservoirs. IDF provides a good mesoscale example for evaluating the risk associated to cyanobacterial development towards human populations, and its relationship to the nature and intensity of human pressures. The regional scale chosen in this study constitutes a good “model” for studying the impact of human pressures on water bodies, because the studied area lies within a single first-order
hydro-ecoregion and it can thus be considered as a homogeneous entity regarding climate and geology. Data from 50 water bodies of the IDF were
analysed to determine if catchment scale environmental variables could predict concentrations of chlorophyll α (used as a proxy for eutrophication
status) of artificial lakes and reservoirs. The better predictive power and robustness of Random Forest models vs GAM was attributed to its better
ability for handling complex interactions between predictors and accounting for threshold effects. Our results showed that (i) simple (e.g. linear and/or
monotonic) relationships between catchment land use and water quality was only found for sub-regional datasets, and (ii) land use needs to be considered in association with complementary environmental variables (hydromorphological variables) to best assess its impact on water quality.
8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 166
CYANOTOXIN PROFILING
IN THE SUBALPINE DISTRICT LAKES
Leonardo Cerasino, Nico Salmaso
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Environment and Natural Resources Area,
Via E. Mach, 1, I-38010 S. Michele all’Adige (TN), Italy. E-mail: [email protected]
Lakes in the subalpine district have been experiencing an increasing of cyanobacteria blooms in the last 20-30 years. We have performed
a comparative survey over nine lakes located in the region (Garda, Maggiore, Como, Iseo, Lugano, Idro, Pusiano, Ledro and Levico) for
assessing the cyanotoxin diversity and distribution. The considered lakes cover a wide range of trophic and physiographic characteristics.
We employed LC-MS techniques for identification and quantitation of cyanotoxins. The study revealed the presence of cyanotoxins in
the whole group of lakes. In particular, microcystins were present in all nine lakes. Although nine different microcystins variants have
been identified overall, four on them were the most abundant, accounting for at least 94% of the total amount. The desmethyl-RR variant
was the dominant form in all lakes with the exception of Levico Lake (where the YR form was dominant). In addition to desmethyl-RR
and YR, LR and desmethyl-LR forms were the other most represented microcystins, although with much lower abundances. Moreover,
it was found that the total microcystin concentration was positively correlated to the trophic status of the lakes. Among alkaloids, anatoxin-a was found in few cases (with the highest concentration of 2.3 μg/L registered in Lake Garda). The anatoxin-a concentration appeared to be independent from trophic status and strongly linked, instead, to the water temperature and to the size of the water body.
Therefore anthropic and climatic factors seem to play different roles in the selection of different classes of cyanotoxins.
166
INFLUENCE OF CELL-FREE MEDIUM AND MICROCYSTIN
CRUDE EXTRACT OF MICROCYSTIS AERUGINOSA
ON THE GROWTH OF ZEA MAYS AND CHROMOSOMAL
CHANGES IN ALLIUM CEPA ROOT TIPS
Mostafa M. El-Sheekh1, Hanan M. Khiry2, Rania El-Shenody1
1
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt.
E-mail: [email protected]
2
National Institute of Oceanography and Fisheries, Alexandria, Egypt
This work aims to investigate the effect of exposure to cell-free medium and crude extract containing microcystin-LR of Microcystis aeruginosa
on the germination stages and cytology of Zea mays. The results of this work confirm the toxic effect of both cell-free medium and crude microcystins of M. aeruginosa of log and death phase on Zea mays germination and chromosomal changes of Allium cepa. Soaking of Z. mays
seeds for 24 hours in the cell-free medium during death phase of M. aeruginosa induced a significant reduction in root, shoot lengths, number
of lateral roots, fresh and dry weights, leaf area and pigment contents after 8 days more than log phase. Cell-free medium showed high
significant inhibition of mitotic index (P<0.001) and inhibition was more pronounced in death phase than in log phase. In addition, chromosomal
abnormalities were also detected in the root tips of Allium cepa treated with cell-free medium of M. aeruginosa. Soaking of Z. mays seeds for
24 hours in different concentrations of crude extracts of M. aeruginosa (100, 200, 300, 500 and 800 μg dry cells ml-1) from log phase showed
inhibitory effect of growth parameters, and no germination was detected with 800 μg dry cells ml-1. The data obtained support the idea that the
use of surface water containing cyanobacteria for crop irrigation can affect both plant yield and quality, and the accumulation of cyanobacterial
toxins in edible plants might pose a potential risk for human and animal health, if the MC intake exceeded the recommended tolerable limits.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
ALGAE INDICATORS OF SOME LAKES
IN SONGNEN PLAIN, CHINA
Yawen Fan, Fengyang Sui
Department of Biology, Harbin Normal University, Harbin 150025, China. E-mail: [email protected]
Songnen plain is one of the highest density of lakes areas in China, there are plenty of lake and pool group as low plain lake group in the
temperate semihumid area. Algae assemblages were investigated at 82 sampling sites of 32 lakes in Songnen plain from June to August
in 2008, and 164 samples were obtained. Sum of 237 taxa were found, including Bacillariophyta 28 genera 113 species 16 varieties
and 3 forms, Chlorophyta 19 genera 52 species 12 varieties and 1 form, Cyanophyta 9 genera 24 species, Euglenophyta 3 genera 13
species, Dinophyta 2 genera 2 species, Xanthophyta 1 genera 1 species. Bacillariophyta was the dominant in this place, followed by
Chlorophyta. The community composition of algae might be classified as Bacillariophyta- Chlorophyta type in Songnen plain. All the dominant species in different lakes were counted, and Melosira granulata, Melosira ambigua and Cyclotella meneghiniana can be used as indicator species in all studied area. Other dominant species such as Cymbella cistula and Scenedesmus bijuga were found in a small
number of lakes. The majority of taxa from 32 lakes belong to β-mesosaprobic zone indicators, and the dominant species existed in most
of 32 lakes were also β-mesosaprobic zone indicators; this showed that the water quality belong to β-mesosaprobic zone in Songnen
plain. In addition, few of industrial toxicology indicators of algae were found in some of the lakes.
167
THE DIVERSITY AND DYNAMICS OF CYANOBACTERIAL
COMMUNITY IN TEN MAJOR RESERVOIRS
OF SINGAPORE AS REVEALED BY rRNA-ITS DGGE
Pingping Gao, Caiping Feng, Yueat Tin Wong, Yuk Chun Chiam-Tai
Technology and Water Quality Office, Public Utilities Board, C4-03, 82, Toh Guan Road East, WaterHub, 608576, Singapore.
E-mail: [email protected]
Proper management of water supplies relies on prior knowledge of cyanobacterial diversity and cause of toxin production. As such, the
cyanobacterial composition in 10 major reservoirs of Singapore was assessed by rRNA ITS-DGGE on a monthly basis from April 2008March 2010 aimed to better understand the occurrence of cyanobacteria in a tropical climate. The result showed high diversity of cyanobacteria
in 9 out of 10 reservoirs and less cyanobacteria population for MacRitchie reservoir. Some species/strains were commonly found in some
reservoirs. In 3 reservoirs where inter-reservoir transfers and exchanges take place, comparable DGGE patterns were observed. The correlation
of the cyanobacterial population to environmental variables by canonical correspondence analysis (CCA) suggested that the physicochemical
variables of the water body are the major determinants of the cyanobacterial community. No obvious seasonal variation in the cyanobacterial
community was detected. However, it was observed that some reservoirs had increased diversity during the later stage of the study. Potential
toxic Microcystis aeruginosa was present in 80%-100% of the samples collected from 9 reservoirs, and Cylindrospermopsis raciborskii was
identified from 9 reservoirs and constituted 7.8%-100% of the total samples assayed. The sequence information of 30 ITS fragments (231
to 851bp), retrieved from 42 bands in DGGE profiles, revealed that the major population identified were uncultured cyanobacteria/microcystis
or uncultured algae from unknown taxa. The DGGE patterns also showed noticeable variation in the phytoplankton community in the reservoirs
during the study period. The correlation analysis between the DGGE data and water parameters is in progress.
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ALGICIDAL ACTIVITY OF THREE BACTERIA AGAINST
HARMFUL ALGAL BLOOMING SPECIES MICROCYSTIS
AERUGINOSA AND ITS RELATED GENES
Myung-Soo Han, Yoon-Ho Kang, Chong Sung Park, Seong-Hyeon Jo
Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791,
Republic of Korea. E-mail: [email protected]
We isolated three bacteria, Aeromonas bestiarum HYD0802-MK36, Pseudomonas syringae KACC10292T and Ps. aeruginosa PA14,
that are active against the harmful algal bloom-forming Microcystis aeruginosa. Following their introduction into axenic M. aeruginosa
cultures at final concentrations of 3 x 103, 3 x 104, 3 x 105 and 3 x 106 bacterial cells ml-1, Ps. aeruginosa initiated algicidal activity at 3
x 103 cells ml-1, while A. bestiarum and Ps. syringae initiated algicidal activity at 3 x 107 and 3 x 106 cells ml-1, respectively. Biochemical
assays revealed that A. bestiarum directly attacked M. aeruginosa by secreting extracellular algicidal substances that were localized in the
bacterial cytoplasmic membrane. Ps. syringae and Ps. aeruginosa indirectly attacked M. aeruginosa through algicidal substances released
from the bacteria. Host range tests of three species showed that Ps. aeruginosa possessed the broadest range against Scenedesmus
actus NIES 94, Stephanodiscus hantzschii UTCC 267 and Coelastrium cambricum HYS0706-C3. Ps. syringae also significantly inhibited
the growth of S. actus NIES 94. In the case of A. bestiarum, however, it appeared to attack M. aeruginosa NIES 298 in a speciesspecific manner. Strain Ps. aeruginosa secreted a heat stable chemical substance of < 3 kDa which may not be a protein compound.
Among eight Ps. aeruginosa TnphoA mutants, three corresponded to the genes pilA, gacA, and lasR exhibited significantly reduced algicidal activity on M. aeruginosa vs. that of wild type Ps. aeruginosa. In genetic complementation analysis of mutants pilA, gacA, and
lasR, moreover, two mutants (gacA and lasR) were restored completely algicidal function of wild type. These results suggest that two
genes (gacA and lasR) of Ps. aeruginosa are related to inhibit growth of M. aeruginosa.
168
DEVELOPMENT OF CELL-BASED PHYSIOLOGICAL
MEASUREMENTS ON THE TOXIC DIATOMS
PSEUDO-NITZSCHIA SPP.
Hélène Hégaret, Aurélie Lelong, Philippe Soudant
Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR6539, Institut Universitaire Européen de la Mer (IUEM),
Place Nicolas Copernic, 29280 Plouzané, France. E-mail: [email protected]
Assessing physiology of microalgae is a key to understand phytoplankton bloom dynamics and could also potentially help understanding
toxin production of harmful algal blooms (HAB). The diatom Pseudo-nitzschia is one of those potentially toxic microalgae. Bloom dynamic
and toxin production are related to cell physiological status. Our study was performed using flow cytometry, to assess microalgal physiology
at the cell-level. Different probes were developed on or adapted to Pseudo-nitzschia species. Cellular lipid contents were assessed using
BODIPY, mortality using SYTOX-green and esterase enzymatic activity using fluorescein di-acetate (FDA). Bacterial counts, associated
to Pseudo-nitzschia cultures, were performed by staining bacteria DNA with SYBR green. Photosynthetic parameters, such as quantum
yield (QY) and light curve were measured with a fluorometer, and red fluorescence (proportional to chlorophyll content) measured by flow
cytometry. Domoic acid production was measured using ELISA kits. All these measurements were then applied to follow physiological
variations over cell cycle (21 days) of Pseudo-nitzschia multiseries. All physiological parameters exhibited variations according to growth
phase, with decrease of chlorophyll fluorescence, lipid content during exponential phase and increase of cell toxicity during stationary
phase. Other physiological parameters, such as FDA and percentage of dead cells were not directly linked to growth phase.
8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 169
CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
CHARACTERIZATION OF A NEW CYANOBACTERIA-LYSING
BACTERIUM FROM LAKE HIROSAWA, JAPAN
Shingo Hiroishi1, Yoshitake Takao1, Yukari Takashima2, Naohiko Hosoda1, Takashi Yoshida3
1
Department of Marine Bioscience, Fukui Prefectural University, 1-1 Gakuencho, Obama, Fukui 917-0003, Japan.
E-mail: [email protected].
2
Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology (JAMSTEC),
2-15 Natsushima-cho, Yokosuka 237-0061, Japan
3
Department of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 6068502, Japan
Noxious cyanobacterial blooms were observed in many eutrophic lakes in the world, and sometimes cause the deaths of livestock and
wild life and create serious problems for freshwater management throughout the world. However, the precise mechanism of the blooms
dynamics is not clarified yet. The blooms sometimes decreased in cell number when cyanobacteria-lysing bacteria increased. These bacteria may play an important role at the occurrence and ending of the blooms. In this study, we report a novel bacterium with high cyanobacteria-lysing activity from Lake Hirosawa, Kyoto, Japan, using Planktothrix agardhii in a screening system. It lysed wide range host strains
including P. agardhii, Anabaena flos-aquae, Aphanizomenon flos-aquae and Microcystis aeruginosa. Phylogenetic analyses based on
16S rRNA gene sequence revealed a clear affiliation with the family Cytophagaceae and showed the closest phylogenetic relationships
with the species Sporocytophaga myxococcoides, Cytophaga hutchinsonii and C. aurantiaca. In addition, further physiological study indicated that it should belong to a novel genus within family Cytophagaceae.
169
DISTRIBUTION AND ENVIRONMENTAL CONTEXT
OF CYLINDROSPERMOPSIS RACIBORSKII OCCURRENCE
IN WESTERN POLAND
Mikołaj Kokociński1,2, Karolina Stefaniak2
1
2
Collegium Polonicum, Adam Mickiewicz University, Kościuszki Street 1, Słubice, Poland. E-mail: [email protected]
Faculty of Biology, Adam Mickiewicz University, Umultowska Street 89, 61-614 Poznań, Poland
In Western Poland, most toxin outbreaks come from the microcystin (MC) producer Planktothrix agardhii. However, a recent study
revealed a new toxin, cylindrospermopsin (CYN), also present in this region. Based on the same work, Cylindrospermopsis raciborskii is
considered a potential producer of this toxin, therefore a study on the distribution and ecology of this taxa has been conducted. Samples
were collected from 46 lakes during the summer seasons (from July to September) in 2008 and 2009. Integrated phytoplankton samples
from whole water column (polymictic lakes) or epilimnion (stratified lakes) were collected from one sampling station located in the middle
of the lake. At the same time, samples for chemical analysis of the water were collected. Temperature, pH levels, and conductivity of
were measured by a multimeter probe. The study showed that C. raciborskii was present in over 40% of the investigated lakes, and its
contribution to total biomass ranged from 0.07 to 13.88%. According to General Linear Model (GLM) analysis, the biomass of C. raciborskii was most significantly related to temperature, ammonium nitrogen, orthophosphate, total nitrogen and total phosphorus. According
to Detrended Correspondence Analysis (DCA), C. raciborskii was mostly related to Aphanizomenon ovalisporum, Aphanocapsa holsatica,
Scenedesmus communis and Tetrastrum glabrum. P. agardhii, Limnothix redekei, and other species from Oscillatoriales belonged to a
separate community. The study demonstrated that C. raciborskii occurred mainly in shallow and highly eutrophic lakes and possesses
the capability to occur in a wide range of temperatures.
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INFLUENCE OF CULTIVATION PARAMETERS ON GROWTH
AND MICROCYSTIN PRODUCTION OF MICROCYSTIS
AERUGINOSA (CYANOPHYCEAE) ISOLATED
FROM LAKE CHAO (CHINA)
Thomas Krüger 1,2, Nadine Hölzel1, Ralf Oelmüller2, Bernd Luckas1
1
2
Department of Food Chemistry, Institute of Nutrition, University of Jena, Dornburger Str. 25, 07743 Jena, Germany
Department of Plant Physiology, Institute of General Botany, University of Jena, Dornburger Str. 159, 07743 Jena, Germany.
E-mail: [email protected]
Lake Chao, a shallow eutrophic Chinese lake, experiences periodic occurrences of massive cyanobacterial blooms, especially of Microcystis
aeruginosa. It is extensively used as water source for drinking, agricultural irrigation, fishery, and recreation. Hence, the potential hepatotoxicity of those cyanobacteria can cause serious health hazards through exposure to microcystins (MCs). The correlation between
high nutrient concentrations of the water body and cyanobacterial mass proliferations was of interest. M. aeruginosa isolated from Lake
Chao (2005) was investigated in terms of growth parameters and microcystin production under varying nutrient concentrations (P, N)
and pH values (abiotic factors) as well as under the influence of spent medium of a non-toxic cyanobacterium (biotic factors). Significant
stimulating effects on growth were observed at the alkaline pH value (10.5), whereas toxin production was increased under phosphateP limitation (0.6 mg L-1 medium). Nitrate-N limitation indicated a decrease of growth and toxin production, whereas no significant influence
was observed within a broad concentration range (41.2-247.2 mgL-1 medium). In addition, cryopreservation of M. aeruginosa demonstrated a significant decrease of toxin production by time-dependent exposure with the cryoprotectant dimethyl sulfoxide (DMSO) under
thawing conditions, since no decline in biomass production was observed based on cell dry weight and chlorophyll α content.
170
TRANSFER OF FRESHWATER CYANOHAB TOXINS FROM
LAND TO SEA: EVIDENCE OF AN EMERGING THREAT
Raphael M. Kudela1, Melissa Miller2, Abdu Mekebri3, David Crane3, Stori Oates2, David Jessup2
1
Ocean Sciences Department, 1156 High Street, University of California Santa Cruz, CA 95064, USA. E-mail: [email protected]
Marine Wildlife Veterinary Care and Research Center, California Department of Fish and Game,
1451 Shaffer Road, Santa Cruz, CA 95616, USA
3
Department of Fish and Game, Water Pollution Control Laboratory, 2005 Nimbus Rd, Rancho Cordova, CA 95670, USA
2
“Super-blooms” of freshwater cyanoHABs with production of potent and environmentally persistent cyanotoxins, such as microcystins,
are an emerging global health issue of impaired freshwater habitat. Despite increases of these toxic bloom events in lakes, reservoirs and
rivers worldwide, surveillance of the marine environment for secondary impacts has been minimal, although marine outflows of microcystin-contaminated freshwater have been documented in several countries. Here we report deaths of sea otters due to microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of
exposure. Sea otters dying due to microcystin intoxication were often clustered near river mouths, embayments and harbors; areas with
potential to receive and retain concentrated plumes of contaminated fresh water. We developed a modification of the Solid Phase Adsorption Toxin Tracking (SPATT) passive sampling method to track microcystins in both source (freshwater) and impacted (estuarine and
coastal seawater) sites. Microcystin was detected in freshwater outflows of nutrient-impaired watersheds flowing into Monterey Bay after
major storm events, with low levels of microcystins present year-round in impaired water bodies. In laboratory studies, marine bivalves
consumed by sea otters and humans exhibited significant uptake and slow depuration of microcystins, suggesting a potentially serious
environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to top marine
predators and humans at the land-sea interface.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
ANALYSIS ON CRISPR SEQUENCES OF TOXIC BLOOMFORMING CYANOBACTERIUM MICROCYSTIS AERUGINOSA
Sotaro Kuno1, Takashi Yoshida1, Takakazu Kaneko2, Yoshihiko Sako1
1
2
Faculty of Agriculture, Kyoto University, 606-8502 Kyoto, Japan. E-mail: [email protected]
Faculty of Life Sciences, Kyoto Sangyo University, 603-8555 Kyoto, Japan
Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently recognized genomic locus conferring prokaryotic defense
system, where partial sequences derived from invading genetic elements such as bacteriophages and plasmids are incorporated as
spacers separating direct repeats. To gain insights into the diversity of the elements contributing to heterogeneity of the toxic bloomforming cyanobacterium Microcystis aeruginosa, we characterized CRISPR loci for several strains of M. aeruginosa and analyzed “infection
memories” recorded as spacers in the CRISPRs. As expected, a spacer obtained from a strain matched sequence of the cyanophage
Ma-LMM01 infecting M. aeruginosa, confirming the capability of the M. aeruginosa CRISPR as an infection memory. It is notable that
most of the spacers obtained in this study exhibited strain-specific distributions. Two spacers associated with known phages were
identified, indicating the signature of related phages having infected M. aeruginosa. Additionally, 19 spacers exhibited significantly high
similarities to plasmids originally found in particular M. aeruginosa strains. In conclusion this study revealed that different strains retain
greatly different “infection memories”.
171
TOXICITY AND MORPHOLOGY ASSESSMENT OF SEVERAL
TOXIN-PRODUCING CYANOBACTERIA STRAINS FOUND IN
BORNEO, MALAYSIA
Jasmina Majit, Ruhana Hassan, Samsur Mohamad
Faculty of Resource Science and Technology, University Malaysia Sarawak, 94300 Kota Samarahan Sarawak, Malaysia.
E-mail: [email protected]
This study was conducted to determine the potential toxigenic species of cyanobacteria from selected aquaculture ponds in Borneo
Island. Few previous studies had reported the occurrence of cyanobacterial species in algae-blooming pond. However, there is not much
information regarding the taxonomic study and toxicity analysis of those reported occurrence especially in Borneo Island. Thus, a preliminary
study of cyanobacteria toxicity and morphology was done where two states of Borneo were selected as study areas. Aquaculture ponds
in Sabah and Sarawak which are known to experience algae-bloom were chosen as sampling sites. Cyanobacterial strains belonging to
Microcystis sp., Nostoc sp., Anabaena sp., Cylindrospermopsis sp. and Lyngbya sp. were isolated and maintained in laboratory cultures.
Mass cultures of isolated cells were performed and the cells were harvested in their late exponential phase of growth. Each strain was
lyophilized and extracted using methanol to get the crude extract. Five different concentrations were tested in brine shrimp assay and
mouse bioassay was done via intraperitoneal injection of 1.0 ml of a lysate of toxin extract into white mice. The preliminary screening of
toxin extracts using brine shrimp and mouse bioassay indicate that the strains are potentially toxin producer species and subjected to
further examine using High Performance Liquid Chromatography (HPLC) method. For taxonomic studies, further analysis using Scanning
Electron Microscope will be done to identify the strains up to species level.
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THE EFFECT OF CLIMATE CHANGE ON THE BIOMASS
AND PRODUCTIVITY OF HETEROTROPHIC BACTERIA
GROWING TOGETHER WITH CYANOBACTERIAL
BLOOMS IN THE BALTIC SEA
Malin Olofsson1, Anders Torstensson2, Maria Söderberg3, Nina Sunesson4, Malin Mohlin5,
Melissa Chierici6, Lars Riemann7, Angela Wulff8
1
Department of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden. E-mail: [email protected]
2
Department of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden. E-mail: [email protected]
3
Department of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden.
E-mail: [email protected]
4
Department of Marine Chemistry, University of Gothenburg, Sweden. E-mail: [email protected]
5
Department of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden.
E-mail: [email protected]
6
Department of Marine Chemistry, University of Gothenburg, Sweden. E-mail: [email protected]
7
Department of Science, University of Linné, 39182 Kalmar, Sweden. E-mail: [email protected]
8
Department of Marine Ecology, University of Gothenburg, Box 461, SE 405 30 Göteborg, Sweden.
E-mail: [email protected]
A hot topic the last decades has been climate changes. And the Baltic Sea is not an exception with a predicted temperature increase of 3-5oC
and lowering of salinity due to increased precipitation. The concentration of CO2 is believed to rise from current values of ca 380 up to more
than 970 ppm by 2100. This could of course affect life of all trophic levels. During summer, cyanobacterial blooms (toxic and non-toxic species)
cover large parts of the Baltic proper. In our study, we wanted to test how these bloom-forming cyanobacteria and heterotrophic bacteria are
affected by lower salinity in combination with increased CO2. In particular, heterotrophic bacteria have been neglected in multifactorial studies
focusing on the proposed predicted changes. They may be affected by both salinity and CO2, but also indirectly by the phytoplankton or
cyanobacterial species present. Our study was performed during summer 2010 and here we will present how bacterial biomass and productivity
are affected by increased CO2 levels and lowered salinity in combination with possible changes in the cyanobacterial species composition.
172
BLOOM DYNAMICS AND CONTROLLING FACTORS OF
PERENNIAL TOXIN-PRODUCING MICROCYSTIS BLOOMS
IN CHINA’S LAKE TAIHU
Timothy G. Otten
Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell St, Morehead City, NC 28557, USA
Cyanobacterial harmful algal blooms (CHABs) are occurring at increasing frequencies worldwide due to warming temperatures and human induced eutrophication. Common surface bloom-forming genera include Anabaena, Aphanizomenon, and Microcystis, with the latter being one of the most prevalent
freshwater CHABs. Some Microcystis species and strains are capable of producing microcystin, a potent hepatotoxin responsible for many human and
animal poisonings documented over the past two centuries. The potential for toxin production cannot be determined by microscopic observation alone
due to the high degree of phenotypic plasticity inherent to Microcystis. Blooms of toxin-producing Microcystis are an increasingly prevalent environmental
health hazard worldwide, with serious consequences for human populations dependent on surface water for drinking and irrigation. A case study can
be made of this organism’s potency by observing the on-going crisis affecting China’s Lake Taihu. Lake Taihu is the third largest lake in China and
provides drinking water to over 10 million residents. The lake itself is very shallow (mean depth 2 m), well mixed and highly eutrophic due to a variety of
point (industrial and municipal wastewater discharges) and non-point (agricultural runoff and atmospheric deposition) nutrient inputs. For several months
each year the water is rendered undrinkable due to a massive, toxic bloom of Microcystis. The bloom was so severe in 2007 that a major drinking water
crisis impacted the region. Aside from being a health risk, the bloom also threatens the livelihood of the region’s residents dependent on the lake’s
fishing and tourism industries. Over the course of two summers water quality data and biological samples were collected at 8 sites from across the lake
and from all blooms encountered while on sampling transects. These samples, combined with physical, chemical and molecular data, have enabled us
to characterize these blooms both qualitatively and quantitatively. The primary objective of this research was to elucidate the environmental factors most
likely to promote toxin-producing bloom-forming strains. The microbial community from each sample was examined both microscopically and molecularly
by whole cell PCR allowing for 16S rRNA fingerprinting and verification of mcy toxin genes. ELISA was used for direct measurements of microcystin
(MC-LR) as well. The results of this work showed that blooms in Lake Taihu are dynamic, varying over time and space and composed of a variety of
Microcystis morphologies possessing distinct genotypes and varying degrees of toxicity ranging from non-toxic to highly toxic.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
MICROALGAL PIGMENTS TRACKING -POTENTIAL
AND LIMITATIONS FOR INLAND SYSTEM MONITORING
Monika Sobiechowska1,2, Katherine Alben2, Alicja Kosakowska1, Joanna Stoń-Egiert1,
Mirosława Ostrowska1, Bogdan Woźniak1
1
Institute of Oceanology, Polish Academy of Sciences, Powstańców Warszawy 55, 81712 Sopot, Poland.
E-mail: [email protected]
2
Department of Environmental Health Sciences, University at Albany and NY State Dept of Health, Empire State Plaza,
NY 12201 - 0509, USA
Pigment analysis allow for classification of plankton distribution in ocean systems, estuarine and freshwater ecosystems and sediments.
For inland basins, algal blooms are still of environmental health concern, affecting especially the near shore areas. For detection and
mapping of algal events variety of methods is being applied. Phycobiliproteins routine analyses e.g. fluorimetry and their accuracy are still
not as far advanced as chromatography for carotenoids or chlorophylls. For this work we gained insight into methods applicable for
cyanobacteria monitoring. We used phycobilins and carotenoids as taxonomic biomarkers to trace different groups of algae composition
in the Baltic Sea and the Great Lakes. We verified the utility and sensitivity of spectroscopic and chromatographic analyses, and their application for ongoing monitoring. In our study, monocultures of the blooms-causing cyanobacteria were grown and along with carotenoids
(HPLC), levels of phycoerythrin, phycocyanin and allophycocyanin were determined spectroscopically. Employing rapid extraction method
required for monitoring, phycobiliprotein concentrations were determined. Values for monocultures were compared with pigment standard
solution extracts, as verification of method efficiency and sensitivity for environmental samples.
173
REASSESSMENT OF THE TOXIN PROFILE OF CYLINDROSPERMOPSIS RACIBORSKII T3 AND FUNCTION OF
PUTATIVE SULFOTRANSFERASE IN SYNTHESIS
OF SULFATED AND SULFONATED PSP TOXINS
Katia Soto-Liebe1, Alejandro A. Murillo1, Bernd Krock2, Karina Stucken2, Juan J. Fuentes-Valdés1,
Nicole Trefault1, Allan Cembella2, Mónica Vásquez1
1
Laboratorio de Ecología Microbiana y Toxicología Ambiental, Pontificia Universidad Católica de Chile, Alameda 340,
6513492 Santiago, Chile. E-mail: [email protected]
2
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany
The toxigenic freshwater cyanobacterium, Cylindrospermopsis raciborskii T3, has been used as a model to study and elucidate the biosynthetic
pathway of paralytic shellfish poisoning (PSP) toxins. There are nevertheless several inconsistencies and contradictions in the published toxin
profiles of this strain. An initial report claimed that this strain produced the carbamoyl analogues saxitoxin (STX) and gonyautoxins 2/3 (GTX2/3).
A subsequent study by pre-column derivatization and HPLC analysis with fluorescence detection gave the profile: STX and the N-sulfocarbamoyl
derivatives C1/2. When strain T3 was later utilized to identify the sequence of genes putatively involved in PSP toxin biosynthesis (sxt gene
cluster), the toxin profile was considered to include STX, neoSTX (NEO), decarbamoyl STX (dcSTX) and the N-sulfocarbamoyl analogue B1.
Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C.
raciborskii T3, as well as the profile of PSP toxins in both strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS) has resolved many issues regarding the correlation between biosynthetic pathways, putative genes and the T3 toxin profile. Our reassessment of the
PSP toxin profile of T3 by LC-MS/MS confirmed production of only STX, NEO and dcNEO. Significant insights were gained via correlation
between specific sxt genes and their role in PSP-toxin production in both D9 and T3 strains. In particular, analysis of sulfotransferase functions
for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes yielded a revised view of the genetic mechanisms underlying the synthesis of sulfated and sulfonated analogues of STX in toxigenic cyanobacteria.
8. ABSTRACT BOOK (158-176)_Layout 1 10/22/10 2:35 PM Page 174
THE MICROCYSTIS AERUGINOSA CYANOPHAGE
ENCODING THE PHYCOBILISOME DEGRADATION GENE
Takashi Yoshida1, Yukari Takashima2, Shingo Hiroishi3, Keizo Nagasaki4, Yoshihiko Sako1
1
Department of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
E-mail: [email protected], [email protected]
2
Biochemistry and Food Technology Division, National Research Institute of Fisheries Science, Fisheries Research Agency,
2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan
3
Department of Marine Bioscience, Fukui Prefectural University, 1-1 Gakuen-cho, Obama, Fukui 917-0003, Japan
4
Harmful Algal Bloom Division, National Research Institute of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi,
Hatsukaichi, Hiroshima 739-0452, Japan
The host-like genes are often found in viral genomes. The best example of host-like gene of cyanophage is psbA gene encoding the
photosystem II core reaction center protein D1. After the decline of host protein synthesis by phage infection, phage D1 protein maintains
photosynthesis to supply energy for phage replication. Ma-LMM01 is a cyanophage infecting Microcystis aeruginosa. The phage genome
composed of 162,109-bp double-stranded DNA. However, the Ma-LMM01 genome encodes no homologs for psbA. Alternatively, the
genome of Ma-LMM01 encodes NblA homolog, which plays a key role in phycolibisome degradation to maintain host PSII system. This
suggests that mechanism of maintenance of PSII give the advantage to cyanophages to spend a long latent period.
174
MESOSCALE SURVEY OF CYANOBACTERIA
AND MICROCYSTINS IN WEST AND IRISH LAKES
Nicolas Touzet1, Anne Feeney1, Eoin Flood1, Dave McCarthy2, Aileen Gill2, Gerard Fleming2
1
Martin Ryan Marine Science Institute, School of Natural Sciences, National University of Ireland, Galway, Ireland.
E-mail: [email protected]
2
Microbial Oceanography Research Unit, School of Natural Sciences, National University of Ireland, Galway, Ireland
Eutrophication is usually associated with the development of toxic cyanobacterial blooms in continental waters. Those have caused a
variety of ecological disruptions, including poisoning incidents of various animals and humans following exposure to or consumption of
water contaminated with cyanotoxins. The dynamics of natural cyanobacterial populations have been studied in many countries due to
the potential noxious effects they can elicit. However, only few studies have investigated their spatial and temporal distributions at
mesoscale levels over extensive geographic areas encompassing several water bodies. In Ireland, water quality in lakes is of great importance from a human health aspect but also in terms of region’s attractiveness (fishing, tourism, recreation…). Mortalities of wild animals
have been associated with blooms of cyanobacteria in some Irish lakes and previous investigations mostly focused on identifying cyanotoxins in a selection of affected water bodies. This study reports the results obtained from a mesoscale survey carried out in west and
northwest Irish lakes during summer 2009. A regionalisation sampling framework was applied, based on a stratified sampling strategy of
water bodies according to ArcGIS analysis of river catchment attributes. The cyanobacterial community structure in the lakes sampled
was assessed through DGGE analysis. The potential for contamination with microcystins was determined by PCR-RFLP of the Mcy-A
gene and protein phosphatase 2A analysis of the planktonic fraction. A significant relationship was observed between the land cover type
adjacent to the lakes sampled and their trophic status.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
CYTOTOXICITY AND PROTEIN PHOSPHATASE INHIBITION
BY DEMETHYLATED MICROCYSTINS AND NODULARIN
Helena Ufelmann1, Thomas Krüger2, Bernd Luckas2, Dieter Schrenk1
1
Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Straße 52, 67663 Kiserslautern, Germany.
E-mail: [email protected]
2
Institute of Nutrition, Department of Food Chemistry, Friedrich-Schiller-University, Dornburger Straße 25, 07743 Jena, Germany
Microcystins (MCs) and nodularin (NOD) are cyclic peptide hepatotoxins produced by cyanobacteria. They exert their toxic effects through inhibition of hepatic protein phosphatases via covalent binding to the serine/threonine phosphatases 1 and 2A (PP1 and PP2A). Aims of this
study were to show the cytotoxic effects and the inhibitory action on PP2A and PP1 activities, of some microcystins and NOD, especially of
their demethylated congeners, in primary rat hepatocytes in culture. To explore hepato-cytotoxic effects, primary hepatocytes from male
Wistar rats were treated with concentrations ranging from 0.1 to 200 nM. The decrease in vitality was examined 24 h post treatment with the
resazurin reduction assay and the lactate dehydrogenase leakage assay. Furthermore, the inhibitory effects on protein phosphatases were
examined fluorometrically with methylumbelliferyl phosphate (MUP) at concentrations ranging from 0.001 to 100 nM using isolated mammalian
PP1 and PP2A. Additionally, Western Blotting Assays with ERK1/2 were carried out to explore effects of microcystins on pathways relevant
for liver tumor promotion. It could be shown that the hepato-cytotoxic effects of mono-demethylated variants were either as high as or higher
than those of their fully methylated (MCs at Asp3, Dha7; NOD at Asp1, Dhb5) congeners. The di-demethylated variants, however, showed
no toxic effects at the examined concentrations. The IC50-values for PP1 inhibition varied between 0.04 and 25.2 nM and for PP2A between
0.03 and 16.9 nM. For all examined substances except MC-LR, inhibition data correlated very well with the results from the cytotoxicity
assays. In addition, MC-LR activated the mitogen-activated protein kinase (MAPK) ERK1/2, probably through inhibition of PP1 and PP2A.
175
PHYSICAL-CHEMICAL AND BIOLOGICAL FEATURES
IN THE SCHINIAS-MARATHON NATIONAL PARK
AND OLYMPIC ROWING CENTRE (GREECE)
Dimitris Vayenos, Theodoros Zoulias, Panayiotis Panayotidis, Aleka Pavlidou
Hellenic Centre for Marine Research, 46,7 km Athens-Sounio Avenue, P.O. Box 19013, Anavissos, Greece.
E-mail: [email protected]
Temperature, salinity, conductivity, pH, dissolved oxygen, nutrient and chlorophyll-a concentrations were studied monthly from February
2009 to January 2010 from 11 sampling sites in the National Park of Schinias-Marathon. In parallel water sampling was carried out in
order to describe the seasonal succession of phytoplankton communities. The results showed that the wetland presents a very strong
gradient of conductivity, from fresh water environments to hyper-saline environments with strong seasonal nutrient availability which
probably is the forcing factors for an important spring plankton bloom. The study of phytoplankton community gave a total of 50 phytoplankton taxa. The Bacillariophyceae contributed with the highest number of species (17), followed by Cyanobacteria (15), Chlorophyceae
(9), Dinophyceae (5), Charophyceae-desmids (4). No major anthropogenic impacts where observed, thus Scinias-Marathon National
Park could be characterized as a typical naturally mesotrophic Eastern Mediterranean wetland.
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CYANOBACTERIAL ECOLOGY, PHYSIOLOGY
AND BIOACTIVE COMPOUNDS
POSTER PRESENTATIONS
MANIPULATING LIGHT AND OXYGEN USING MODIFIED
LOCAL SOIL TECHNOLOGY AND ITS EFFECT
ON VARIATION AND METABOLISM OF PHYTOPLANKTON
Dan Wang, Gang Pan
Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085,
People’s Republic of China. E-mail: [email protected], [email protected]
Chitosan modified local soil can flocculate cyanobacterial blooms, improve transparency of the water and manipulate redox conditions in the
sediment. In order to understand the fate of algae after flocculation and the effect of different light and O2 on this process, the composition
of phytoplankton community was studied from March to December in 2009 in a controlled enclosure in Lake Tai (China). Microcystis was
the dominant species throughout the period except that diatom was dominant in March. The percentage of cyanobacteria in April was lower
than that in June and October, but the diversity of phytoplankton community in April was higher than that in June and October. In the
incubation experiment, algal water of April, June and October in situ was flocculated by chitosan modified local soil and cultured in different
irradiance and redox conditions. After 75 days, the cyanobacteria of April changed to green algae or diatom under light and aerobic conditions,
and the cyanobacteria of June changed to diatom under dark-aerobic condition, while the dominance of cyanobacteria in October did not
change under all conditions. For the flocculated algae in April, measurement on O2 production in photosynthesis and consumption in respiration
indicated that the flocculated algae could sustain normal metabolism under light and aerobic conditions and contribute for oxygen enrichment,
but it did not sustain the normal photosynthesis ability and even aggravate oxygen deficit under dark and anaerobic conditions. The results
suggested light and oxygen played an important role in variation and metabolism of phytoplankton. This work suggested it is possible to use
modified local soils to transform harmful cyanobacteria into contributors for light and redox conditions improvement in shallow waters.
176
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 177
GENOMICS AND GENETIC
DIVERSITY OF HABS
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 178
ORAL PRESENTATIONS
NEW APPROACH USING REAL-TIME PCR METHOD
FOR THE QUANTIFICATION OF THE TOXIC OSTREOPSIS
CF. OVATA IN ENVIRONMENTAL SAMPLES
Cecilia Battocchi1, Federico Perini1, Anna Casabianca2, Stefano Accoroni3, Cecilia Totti3, Antonella Penna1
1
Dept. of Biomolecular Science, University of Urbino, Viale Trieste 296, 61121 Pesaro, Italy.
E-mail: [email protected]
2
Dept. of Biomolecular Science, University of Urbino, Via Saffi 2, 61029 Urbino, Italy
3
DISMAR, University of Ancona, Via Brecce Bianche, 60100 Ancona, Italy
Ostreopsis spp. is a benthic dinoflagellate known to produce palytoxin-like compounds. In recent years, serious cases of human poisoning
by toxic aerosol associated with Ostreopsis spp. blooms have been recorded along the Italian and Spanish coastlines. Two species (O.
cf. ovata and O. cf. siamensis) are being found with increasing frequency in the Mediterranean Sea, but when occurring together in
mixed blooms, their correct identification and counting by microscopy is very difficult due to the high variability of their morphometric characters. To this purpose, we designed a real-time PCR assay for the quantification of O. cf. ovata in field samples. Macrophyte and
seawater samples were collected at the Conero Riviera, (Ancona, Italy) between March and November 2009. A standard curve was
constructed with scalar dilutions from 106 to 2 copies of a plasmid containing a LSU rRNA fragment gene of O. cf. ovata. A second calibration curve was generated by amplifying the LSU fragment of a DNA pool from field samples representative of the bloom. This
procedure allowed us to normalize the variability of ribosomal DNA copy number per cells between analyzed samples and calculate the
number of cells per each sample. The quantification obtained by this molecular method was compared to the classical method of quantification by microscopy. This innovative molecular system of quantitative rt-PCR, which gives rapid, specific and sensitive results, can be
a useful tool in the control and management of harmful coastal Ostreopsis spp. blooms.
178
COMPARATIVE GENOMIC ANALYSIS OF THE
BIOSYNTHETIC PATHWAY FOR SAXITOXIN
AND SULFATED ANALOGUES IN CYANOBACTERIA
AND THE DINOFLAGELLATE ALEXANDRIUM MINUTUM
Allan Cembella1, Karina Stucken1,2, Uwe John1, Ines Yang1, Bernd Krock1, Gernot Gloeckner3,
Nicole Trefault2, Alejandro Murillo2, Katia Soto-Liebe2, Juan J. Fuentes2, Mónica Vásquez2
1
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany.
E-mail: [email protected]
2
Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile
3
Fritz Lipmann Institute for Age Research, Beutenbergstraße 11, 07745 Jena, Germany
Certain cyanobacteria share capacity for biosynthesis of tetrahydropurine neurotoxins, saxitoxin (STX) and analogues, with members of three genera
of marine dinoflagellates. Although the skeleton biosynthetic pathway to STX was described decades ago in the cyanobacterium Anabaena flosaquae and dinoflagellate Alexandrium tamarense based upon stable isotope labeling, the corresponding sxt gene cluster was identified only recently
from cyanobacteria. We sequenced the genome of the filamentous cyanobacterium Raphidiopsis brookii D9, and compared the sxt cluster and toxin
profile with that published for Cylindrospermopsis raciborskii T3. Our reassessment of the PSP toxin profile of T3 by LC-MS/MS confirmed production
of only STX, NEO and dcNEO, whereas D9 produces STX, O-sulfated gonyautoxins GTX2/3, and decarbamoyl analogues dcSTX and dcGTX2/3.
Significant insights were gained via correlation between specific sxt genes and their role in PSP toxin production in both D9 and T3 strains. Analysis
of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes yielded a revised view of the genetic mechanisms
underlying the synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria, with biosynthetic implications for dinoflagellates. We
constructed a normalized expressed sequence tag (EST) library for the dinoflagellate Alexandrium minutum, from a strain which produces primarily
gonyautoxins, and found expressed candidate genes plausibly but not conclusively related to toxin biosynthesis. Microarray application to A. minutum
and the cyanobacterium D9 revealed little evidence for transcriptomic regulation of STX biosynthesis. Extreme genomic complexity in dinoflagellates
argues for continued use of model cyanobacteria for studying this biosynthetic pathway and possible lateral gene transfer from cyanobacteria.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 179
GENOMICS AND GENETIC DIVERSITY OF HABS
ONE, NO ONE AND ONE HUNDRED THOUSAND
PLANKTOTHRIX SPECIES: IS LATERAL GENE TRANSFER
A NEGLECTED SOURCE OF GENETIC DIVERSITY?
Domenico D’Alelio, Andrea Gandolfi, Nico Salmaso
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Environment and Natural Resources Area,
Via E. Mach 1, I-38010 S. Michele a/Adige (Trento), Italy. E-mail: [email protected]
The systematics of the genus Planktothrix (cyanobacteria) basically relies on phenotypic and ecological traits. In fact, attempts towards
the definition of species by means of morphological, genetic and biochemical analyses are scanty and have been carried out using few
sympatric strains which probably under-represent the actual diversity. On the other hand, the idea that the whole genus could be monospecific is still supported. We isolated about 300 strains of Planktothrix from eight lakes in Northern Italy and analyzed their morphology
and genetics (i.e. for rpoC1 and rbcLX coding-genes). The rpoC1 gene was highly polymorphic, with about 30 single nucleotide polymorphisms, resulting in an overall diversity of ~6%; differences in rbcLX were less abundant. The strains clustered into two main genotype-groups: one was associated to the “species” P. rubescens, whose red-water blooms are reported in meso/oligotrophic lakes; the
other included sequences recombinant between P. rubescens and P. agardhii, the latter being a green-water bloom “species” reported
in eutrophic lakes. Furthermore, even sequences published on GenBank by previous authors and referring to strains putatively identified
as either P. rubescens or P. agardhii clustered in the “recombinant clade”. Our study, still in progress, points out the fact that recombination,
probably as lateral gene transfer, could potentially limit the formation of stable species within the genus Planktothrix; a potential consequence of this phenomenon might be that different scientists could attain distinct ideas of a single biological entity, depending on the
nature of each observer’s observation (i.e. a sort of “Pirandellian syndrome”).
179
COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF ALEXANDRIUM TAMARENSE REVEALS INDEPENDENT ORIGIN OF SAXITOXIN SYNTHESIS IN CYANOBACTERIA AND DINOFLAGELLATES
Jeremiah D. Hackett1, Jennifer H. Wisecaver1, Dave M. Kulis2, Donald M. Anderson2, Debashish Bhattacharya3,
F. Gerald Plumley4, Deana L. Erdner5
1
Department of Ecology and Evolutionary Biology, University of Arizona, P.O. Box 210088, Tucson, AZ 85721, USA.
E-mail: [email protected].
2
Biology Department Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
3
Department of Ecology, Evolution, and Natural Resources, Institute of Marine and Coastal Sciences, Rutgers University,
New Brunswick, NJ 08901, USA
4
Bermuda Institute of Ocean Sciences, St. George's GE01, Bermuda
5
Department of Marine Science, University of Texas, 750 Channel View Dr., Port Aransas, TX 78373, USA
We assembled a comprehensive transcriptome dataset for the saxitoxin-producing dinoflagellate Alexandrium tamarense combining over 10 gigabases of
expressed sequence tag, 454 and Illumina pyrosequencing data. The resulting assembly comprises 112,383 contigs with an average coverage of 100X,
from which 56,578 proteins were predicted. We also generated 454 transcriptome data for three additional toxic dinoflagellates: A. tamarense SPE10-03,
Pyrodinium bahamense, and Gymnodinium catenatum. Searching these data, we identified 274 potential homologs of 14 cyanobacterial saxitoxin genes
including all of the genes directly involved in toxin synthesis. We used these candidate proteins in phylogenetic analyses to determine their evolutionary
history. Four dinoflagellate candidate proteins group closely with cyanobacterial saxitoxin genes in phylogenetic analyses and are likely the homologs of sxtA,
sxtB and sxtG in dinoflagellates. SxtA is split into two proteins in the dinoflagellates with sxtA1 corresponding to the N-terminal portion with methyltransferase
and acyl carrier protein domains and sxtA2 corresponding to the C-terminal portion with the aminotransferase domain. Phylogenetic analyses of sxtA, sxtB
and sxtG homologs suggest that they were acquired independently in cyanobacteria and dinoflagellates from similar bacterial sources. The A. tamarense
transcriptome does not contain phylogenetically related homologs for the remaining saxitoxin genes. However, we identified candidate functional homologs
for all of the remaining saxitoxin synthesis genes that were acquired from a variety of sources. These results indicate that the saxitoxin synthesis pathways
in cyanobacteria and dinoflagellates were assembled independently using genes with similar functions, but from phylogenetically unrelated sources.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 180
GLOBAL ANALYSIS OF GROWTH PHASE-ASSOCIATED
TRANSCRIPTOMES IN THE TOXIC DINOFLAGELLATE
KARENIA BREVIS
Jillian Johnson1,2, Jeanine Morey2, Marion Beal2, James Ryan2, Frances Van Dolah1,2
1
Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina,
221 Fort Johnson Road, PC 29412, Charleston, South Carolina, USA. E-mail: [email protected]
2
NOAA Center for Coastal Environmental Health and Biomolecular Research, 219 Fort Johnson Road, PC 29412,
Charleston, South Carolina, USA
Molecular mechanisms regulating senescence and cell death in phytoplankton have received increased attention within the last decade
due to their prospective roles in population structuring, species succession, biodiversity/selection, bloom termination, and biogeochemical
cycling. To gain insight into gene expression indicative of aging and/or cell death in Karenia brevis, the toxic dinoflagellate responsible for
the near annual harmful algal blooms in the Gulf of Mexico, oligonucleotide microarrays were employed to monitor transcriptomic changes
over a complete growth curve. Mid-logarithmic phase (day 6) was used as the point of comparison to the transition into stationary (day
10), mid-stationary (day 14), and late stationary phase (day 18). Of the 10,263 genes assessed, 29% (2,959) of the features on the
array were significantly changing, suggesting a drastic reorganization of the K. brevis transcriptome at the transition from logarithmic to
stationary phase growth. Analysis of the distribution of genes with increased and decreased expression with respect to functional classes
(GO Slim, Modified Fisher’s Exact, FDR<0.05) identified coordinated expression of transcripts involved in energy production, calcium ion
homeostasis, regulation of gene expression, and response to stress at the transition from logarithmic to stationary phase growth. While
further research is needed to discern the functions of the K. brevis transcripts responsive to the transition between growth and maintenance
phases, this work may serve as a diagnostic tool for determining the growth status of field populations.
180
CHANGES OF THE GENETIC DIVERSITY AND POPULATION
STRUCTURE ALONG A GONYOSTOMUM SEMEN BLOOM
Karen Lebret, Emma Kritzberg, Rosa Figueroa, Karin Rengefors
Limnology, Department of Biology, Lund University, Ecology Building, 22362 Lund, Sweden. E-mail: [email protected]
Gonyostomum semen has spread and become more abundant in Scandinavian lakes during the last 30 years and is now considered as
a noxious and invasive species. This raphidophyte produces dense blooms and can dominate the phytoplanktonic community to up to
95%, showing high competitive capacity despite their fragile cells and low growth rate. Species with high genetic diversity have a potentially
higher capacity to respond to variations of environmental conditions. Moreover, the development of population structure and the changes
in genetic diversity along a bloom of microalgae are rather unknown, particularly in limnic systems. After germination of the cysts, the
genotypes best adapted to the current environmental conditions may become dominant, with potential succession of populations following
changes in environmental conditions. Alternatively, the genetic diversity may be maintained, without dominant genotypes taking over,
with potentially coexistence of different populations. To follow the development of the population structure during the different phases of
a G. semen bloom, 200 strains were isolated from lake Bökesjön (Sweden) in October 2008 (encystement phase) and between April
(germination of cysts) and October 2009. Amplified fragment length polymorphism (AFLP) analyses were performed on all the strains
to determine the genetic structure of the population at the different phases. Our results so far suggest that all individuals isolated are genetically unique, and that there is more than one population during the growth season.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 181
GENOMICS AND GENETIC DIVERSITY OF HABS
ORAL PRESENTATIONS
METATRANSCRIPTOMIC PROFILING OF A LARGE
PHYTOPLANKTON BLOOM IN PUGET SOUND
Ahmed Moustafa1,*, Jonathan Badger1, Micaela S. Parker2, E. Virginia Armbrust2, Andrew Allen1
1
Microbial and Environmental Genomics, J. Craig Venter Institute, San Diego, CA 92121, USA
School of Oceanography, University of Washington, Seattle, WA 98195, USA
* Present address: Department of Biology, American University in Cairo, New Cairo 11835, Egypt.
E-mail: [email protected]
2
We conducted an exploratory metatranscriptome sequencing in Puget Sound, focusing on eukaryotic microbes, particularly eukaryotic
phytoplankton, in the early fall of 2007 when diatoms and dinoflagellates blooms are known to occur frequently. Using 454 sequencing
of cDNA populations sampled from five different sites representing the major ecological niches in Puget Sound, we generated an average
of 200,000 environmental cDNA reads from each site with about 1,000,000 sequences. Our data show a gradient in the composition
of the phytoplankton blooms from diatoms to dinoflagellates in the south-north direction. We observed a major shift in the transcript
profile from Great Bend, which was dominated by the diatoms, to Hein Bank, which was dominated by the dinoflagellates. During the
sampling time at Trip Junction, in eastern Puget Sound, we did not detect any phytoplankton blooms and the collected phytoplankton
cells appeared stressed. Our metatranscriptomic data indeed indicate limited traces of dinoflagellates and diatoms. However, we identified
a large population of marine copepods in this area, mostly encoding motor-related proteins such as myosin, tropomyosin, and actin. Interestingly, in the same region, we found an up-regulation of genes encoded by dsDNA viruses compared to the other sites. Likely, the
presence of viruses and a large population of grazers led to the termination of the phytoplankton blooms prior to our sampling. Here, we
will present our results highlighting the dynamics of the phytoplankton blooms and the impact of environments stressors and interactions
among the microbial populations on gene expression patterns of the marine microbial assemblages.
181
DETECTION AND QUANTIFICATION OF PST-FORMING
DINOFLAGELLATES AND THEIR TOXINS USING
REAL TIME QPCR
Shauna Murray1, Maria Wiese1, Julia Suurbach1, Rouna Yauwenas1, Bob Moore1, Troco Mihali1,
Gustaaf Hallegraeff2, Brett Neilan1
1
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.
E-mail: [email protected]
2
School of Plant Science and Institute of Antarctic and Southern Ocean Studies, University of Tasmania, Private Bag 77,
Hobart Tas 7001, Australia
The coast of New South Wales, in south eastern Australia, is considered a climate change ‘hotspot’, due to a disproportionately large
increase in water temperature, recorded over the past 15 years. The impact of this temperature increase on the distribution and abundance of HAB-forming taxa is not clear. However, in recent years, blooms of Alexandrium catenella and Gymnodinium catenatum,
producing paralytic shellfish toxins (PSTs), have been reported, coinciding with PST accumulation in Sydney rock oysters (Saccostrea
glomerata) at levels below regulatory limits. To address the need for more sensitive and rapid detection and quantification methods, we
assessed the use of quantitative real time PCR to detect local strains of Alexandrium spp. and Gymnodinium catenatum, based on
primer sets targeted to high copy number ribosomal genes and other nuclear marker genes. We determined the stability of gene copy
number amongst species in this genus, and its suitability for cell quantification throughout the dinoflagellate growth cycle in these strains.
Using information from EST datasets of Alexandrium spp., we have investigated the potential for the detection of genes that putatively
involved in PST biosynthesis.
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METAGENOME ANALYSIS OF MARINE PHYTOPLANKTON
USING NEXT GENERATION SEQUENCER
Satoshi Nagai1, Goh Nishitani1, Daiki Noguchi2, Kazuo Abe3
1
Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan.
E-mail: [email protected]
2
Nippon Total Science, Inc. 456-2 Minomi-cho, Fukuyama 720-0832, Japan
3
Seikai National Fisheries Research Institute, 1551-8 Taira, Nagasaki 851-2213, Japan
By recent revolution of the sequencing technology, we can obtain a huge number of sequences at one time by use of next generation
sequencer (one million reads per one tip). This enables us to identify thousands species of plankton from one seawater sample at one
time. To investigate and compare plankton diversities, seawater samples were taken five times from two locations in 2009 and the DNAs
were extracted with 5% Chelex buffer (10 samples in total). The target region (ca. 500 bp) of the nuclear 18S-rDNA was PCR-amplified
using universal primers tagged 6 bp-oligonucleotide for discrimination of samples. The PCR products were mixed together after purification
by column cartridge (SUPREC-PCR, TaKaRa, Japan). The PCR products were sequenced using a Roche 454 next generation sequencer,
and ca. 50,000 sequences were determined. Each sequence was blast-searched, resulting in >960 different sequences from each location (in total >1,500 sequences) containing almost all plant phylum (21 algal classes except for cyanobacteria and prochlorophytes)
and many phylum in protozoa, fungi and Animalia. Regarding HAB species, Alexandrium (10 species), Chattonella (3), Pseudo-Chattonella
(2), Dinophysis (7), Gymnodinium (12), Gyrodinium (6), Karenia (2), Peridinium (7), Prorocentrum (12), Chaetoceros (8), Pseudonitzschia (8), Skeletonema (7), Thalassiosira (18), Cochlodinum polykrikoides, Heterosigma akashiwo, Pfiesteria piscicida and so on,
were detected. Thus, this PCR-Sequencing system is still really new, however, it is highly possible to be one of the most powerful monitoring tools for marine biodiversity (not only microalgae but also protozoa) in the near future.
182
A PHYLOGEOGRAPHICAL STUDY OF THE TOXIC BENTHIC
DINOFLAGELLATE GENUS OSTREOPSIS SCHMIDT
Antonella Penna1, Santiago Fraga2, Cecilia Battocchi1, Silvia Casabianca1, Maria Grazia Giacobbe3,
Pilar Riobó4, Cristiano Vernesi5
1
Dip. Scienze Biomolecolari, University of Urbino, viale Trieste 296, 61100 Pesaro, Italy. E-mail: [email protected]
2
Centro Oceanografico de Vigo (IEO), Subida a Radio Faro 50, 36390 Vigo, Spain
3
CNR-IAMC, Institute for the Marine and Coastal Environment, 98122 Messina, Italy
4
Instituto de Investigacions Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain
5
Research and Innovation Centre, Fondazione Edmund Mach, 38040 Viote del Monte Bondone (TN), Italy
Ostreopsis is a benthic dinoflagellate that produces potent toxins and is widespread in tropical and warm temperate coastal areas worldwide. In this study, we tested the hypothesis that as it is benthic, it would show distinct biogeographical patterns in comparison with
planktonic species. Sequence variability in ribosomal DNA markers was analyzed to provide the first phylogeographical study of this toxic
benthic dinoflagellate. Ribosomal DNA sequence data from nuclear LSU-5.8S genes and ITS regions were obtained from 82 isolates
of the Ostreopsis species, representing 26 localities sampled throughout different regions of the world. Molecular sequence data were
analyzed using different phylogenetic methods for phylogenetic inference. A statistical parsimony network was obtained based on concatenated LSU and 5.8S rDNA-ITS region sequences of the Mediterranean/Atlantic O. cf. ovata isolates to infer haplotype distribution
over their geographical range. Microscopy and toxin analyses were performed on cultured and live Ostreopsis material for taxonomic
characterization. The rDNA phylogeny revealed different clades corresponding to different species within the genus Ostreopsis. It was
found that in the species O. cf. ovata, different genetic lineages were correlated with macrogeographical distribution. O. cf. ovata was
found to be widely dispersed throughout the coastal areas of tropical and some warm temperate seas. In the Atlantic/Mediterranean
region it may constitute a panmictic population that is highly distinct from Indo-Pacific populations. O. cf. siamensis was found only in the
Mediterranean Sea, and strains identified as O. lenticularis and O. labens were found only in the Indo-Pacific region.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 183
GENOMICS AND GENETIC DIVERSITY OF HABS
ORAL PRESENTATIONS
GENETIC DIVERSITY AND LOCAL ADAPTATION
IN BLOOM-FORMING ANTARCTIC DINOFLAGELLATES
Karin Rengefors1, Ramiro Logares2, Johanna Laybourn-Parry3, Marie Svensson1
1
Limnology, Dept. of Biology, Lund University, Ecology Building, 22362 Lund, Sweden. E-mail: [email protected]
Institut de Ciències del Mar, CSIC, Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain
3
Bristol Glaciology Centre, School of Geographical Sciences, University of Bristol, Bristol, BS8 1SS, UK
2
Genetic differences among clones and populations of HAB species may have a major effect on their phenotypes, such as toxicity,
growth rates, and salinity tolerance. Moreover, genetic differentiation and divergence among populations may lead to reduced gene
flow and eventually speciation. Here, we investigated possible genetic divergence due to either local adaptation and/or geographic isolation. As a model system, we used marine dinoflagellates in recently formed Antarctic saline lakes. One species, Scrippsiella hangoei
forms blooms in the Baltic Sea, while the other, Polarella glacialis is common in both polar seas. Clonal strains of the two different
species were isolated from lakes of different salinities. To study local adaptation, we tested salinity tolerance in marine and limnic strains.
The genetic difference among the strains was determined using a DNA fingerprinting technique (AFLP). The AFLP analyses indicated
that the lake strains were more closely related to each other than to the marine strains. The salinity tolerance experiments showed that
the limnic strains had a wider salinity tolerance than the marine strains, and that the limnic strains were adapted to the salinity ranges
of their lake of origin. We tentatively suggest that the limnic populations have undergone local adaptation and may be genetically isolated
from the marine populations.
183
A DINOFLAGELLATE PRODUCER OF PINNATOXINS,
ISOLATED FROM NORTHLAND, NEW ZEALAND
Lesley Rhodes1, Kirsty Smith1, Sam Molenaar1, Rex Munday2, Andy Selwood1, Paul McNabb1
1
2
Cawthron Institute, 98 Halifax St East, PB2, Nelson, New Zealand. E-mail: [email protected]
AgResearch, Private Bag 3123, Hamilton, New Zealand
A dinoflagellate producer of pinnatoxins E and F was isolated from surface sediments collected from Northland, New Zealand, in the
austral summer of 2008/2009. The dinoflagellate appears to be a new species and is morphologically similar to other scrippsilloid species,
but with a distinctive ornamentation of the thecal plates and a truncated horn. It is a cyst former, with the motile form occurring most
readily at 25oC with urea as the nitrogen source; it is hypothesised that the cysts undergo mitotic division and this will be tested. Phylogenetic analyses show that the pinnatoxin producer groups within the Peridiniales and is genetically most closely aligned to species in the
genera Ensiculifera and Pentapharsodinium. The effect of the toxic dinoflagellate on invertebrate larvae and the uptake and depuration
rates of pinnatoxin by several species of shellfish and paddle crabs (Ovalipes catharus) are currently being investigated. Initial in vitro experiments with Pacific oysters (Crassostrea gigas) and GreenshellTM mussels (Perna canaliculus) determined that the total pinnatoxin
uptake following consumption of dinoflagellate cells over 3 days was only 2.3% of the 340 μg and 3.0% of the 519 μg of pinnatoxins
offered respectively. Depuration by oysters was more rapid than for mussels, with 50% of the pinnatoxins being depurated by oysters
per week. Extracts of mass cultures of the dinoflagellate were tested for toxicity in mice by intraperitoneal injection, gavage and voluntary
consumption and LD50 values were determined as 1.33 mg/kg, 2.33 mg/kg and 5.95 mg/kg respectively.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 184
EXTENSIVE GENETIC DIVERSITY AND RAPID POPULATION
SUCCESSION DURING BLOOMS OF ALEXANDRIUM
FUNDYENSE (DINOPHYCEAE) IN AN ISOLATED SALT
POND ON CAPE COD, MA, USA
Mindy Richlen1, Deana Erdner2, Linda McCauley1, Katie Libera1, Donald Anderson1
1
Woods Hole Oceanographic Institution, 266 Woods Hole Rd., MS 32, Woods Hole, Massachusetts 02543, USA.
E-mail: [email protected]
2
Marine Science Institute, University of Texas at Austin, 750 Channel View Dr., Port Aransas, Texas 78373, USA
In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments
and salt ponds on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing
industry. This study examined NMS populations of Alexandrium fundyense in 2006-2007 to investigate the genetic characteristics of localized blooms and assess their connectivity with Gulf of Maine (GOM) populations. Comparisons of genetic differentiation showed that
A. fundyense blooms in the NMS are comprised of at least three populations, which exhibited extensive clonal diversity and were genetically
distinct from populations in the GOM. In both project years we also observed rapid population succession of NMS populations, on the
order of approximately seven days. The succession observed during the course of the bloom may be due to differences in cyst dormancy
periods, or possibly driven by selection from environmental conditions, parasitism, and/or grazing. Alternatively, population turnover may
reflect mixing of distinct genotypes from a separated population; an expanded study of the system is underway. Project results also
highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale
and limited connectivity of NMS salt ponds provide a ideal system for investigating regulators of bloom development and termination,
such as life cycle events, as well as the influence of selective forces on population dynamics, all of which are otherwise difficult or
impossible to study in the adjacent open coastal waters or larger estuaries.
184
THE GENUS GAMBIERDISCUS
IN THE RYUKYU ISLANDS, JAPAN
Mahfuzur Rhaman Shah1, Genta Nagamura1, Yosuke Taira2, Kenichi Onodera2, Kazuyo Tsuha2,
Takeshi Yasumoto2, Shoichiro Suda1
1
2
University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan. E-mail: [email protected]
Okinawa Science and Technology Promotion Center, Marine Bioindustry Division, 12-75, Suzaki, Uruma,
Okinawa 904-2234, Japan
Occurrence of ciguatera fish poisoning (CFP) is rather rare in Japan, and the subtropical Ryukyu Islands are the only area where sporadic
but regular occurrence of CFP has been reported. Benthic dinoflagellates of the genus Gambierdiscus are the cause of CFP. Since G.
toxicus was established by Adachi et Fukuyo, 10 species are currently distinguished by morphology and phylogenetic analyses. In the
Ryukyu Islands, however, species level classifications and relation with toxin production are uncertain. The aim of this study was to clarify
the classification of Gambierdiscus spp. in the Ryukyu Islands using molecular and morphological approaches. We isolated Gambierdiscus
strains from various localities of Ryukyu Islands and approximately 100 strains were established. Using these strains, Gambierdiscus
species diversity was reevaluated. Phylogenetic analyses of the preliminary results of the sequences of 18S rDNA and 28S rDNA D1D3
regions showed that at least different four types were present. One type was closely related with G. australes but the other three observed
types did not fit closely with any known species. These types were distantly related with G. toxicus and G. paficicus, with G. caribaeus
and G. carpenteri, and with G. yasumotoi and G. ruetzleri, respectively. These three different types are potentially undescribed species.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 185
GENOMICS AND GENETIC DIVERSITY OF HABS
ORAL PRESENTATIONS
POPULATION STRUCTURE AND SPECIES GENETIC
DIVERSITY OF THE TOXIC DIATOM PSEUDO-NITZSCHIA
MULTISTRIATA IN THE GULF OF NAPLES
Sylvie Tesson, Gabriele Procaccini, Marina Montresor, Wiebe Kooistra
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected]
The pennate diatom Pseudo-nitzschia multistriata occurs in the Gulf of Naples since 1995 where it is observed in early summer and in
autumn. Although the species is a minor bloom former (max. 6.9X105 cells L-1), it is economically and environmentally relevant because
it produces domoic acid, the agent causing Amnesic Shellfish Poisoning. To assess population genetic structure in P. multistriata at the
LTER MareChiara in the Gulf of Naples, 362 strains have been cultured from cells isolated over the short early-summer bloom in 2009
and the autumnal blooms in 2008 and 2009. The strains belonged to a single morpho-species, but showed two markedly distinct internal
transcribed spacer (ITS) sequence types, defined as ITS-A and ITS-B. The majority of strains possessed both ITS types, though the proportion of ITS-AB-type strains differed between years and between the two bloom events. In addition, we conducted a population genetic
survey of the strains using seven species-specific polymorphic microsatellite markers. Results revealed the occurrence of two genetically
distinct populations that did not correlate with the grouping of the strains according to their ITS types. Mating experiments between compatible strains were successful irrespective of their ITS-type or their population assignment as deduced from their micro-satellites. These
results suggest that widely distributed coastal phytoplankton species such as P. multistriata form meta-populations composed of genetically
distinct sub-populations that even occur in sympatry.
185
COMPARATIVE PROTEOMICS REVEALS DIFFERENT
PROTEIN EXPRESSION PATTERN AND MODIFICATIONS
IN TOXIC AND NON-TOXIC ALEXANDRIUM TAMARENCE
STRAINS
Da-Zhi Wang, Cheng Li, Zhang-Xian Xie, Hua-Sheng Hong
State Key Laboratory of Marine Environmental Science/Environmental Science Research Center, Xiamen University,
182 Daxue Road, 361005 Xiamen, People’s Republic of China. E-mail: [email protected]
Alexandrium is a dinoflagellate genus commonly found throughout many regions of the world. This study compared protein profiles of
toxic and non-toxic A. tamarense strains collected from different geographic regions using a combination of two dimensional fluorescence
difference gel electrophoresis (2-D DIGE) approach and MALDI-TOF-TOF mass spectrometer analysis. The results showed that the
toxic and non-toxic A. tamarense strains presented different 2-DE protein patterns, and almost no common protein spots were found in
2-D DIGE gels between toxic and non-toxic strains, while 2-DE protein profiles presented the same patterns within toxic or non-toxic A.
tamarense strains. Several abundant proteins identified by MALDI-TOF-TOF MS, i. e. methionine S-adenosyl transferase, peridininchlorophyll a-binding protein, plastid protein NAP50, glyceraldehyde-3-phosphate dehydrogenase and 1,5-bisphosphate carboxylase
oxygenase form II (RuBisCO) exhibited different shift patterns in pI or molecular weight between toxic and non-toxic A. tamarense strains,
indicating protein modifications might occur in toxic A. tamarense strains. The results from LTQ OrbitTrap indicated that phosphorylation
of RubisCO was occurred in toxic A. tamarense while methylation of RubisCO was occurred in non-toxic A. tamarense. The present
study suggests that differential protein expression pattern and protein modifications between toxic and non-toxic A. tamarense strains
might be an acclimatization of cells to intracellular toxin, which provides new insights into cell adaptation to intracellular environment.
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9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 187
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
FURTHER EVIDENCE ON THE IDENTITY OF THE GREEK
DINOPHYSIS MORPHOTYPE CAUSING TOXIC BLOOMS:
A D. CF. OVUM RATHER THAN A D. CF. ACUMINATA
Katerina Aligizaki1, Dimitra Papaefthimiou2, Panagiota Katikou3, Konstantinos Koukaras4, Georgios Nikolaidis*,1
1
Department of Botany, School of Biology, Faculty of Sciences, Aristotle University, 54124 Thessaloniki, Greece.
E-mail: [email protected]
2
Institute of Agrobiotechnology, CERTH, Thermi - Thessaloniki, GR-57001, Greece
3
National Reference Laboratory on Marine Biotoxins, Institute of Food Hygiene, Ministry of Rural Development and Food,
3A Limnou str., GR-54627, Thessaloniki, Greece
4
Nearhus G.P. Company, R&D Department, Anaximandrou str. 69, GR-54250, Thessaloniki, Greece
*deceased
The identity of the Dinophysis morphotype prevailing in Greek coastal waters since 2000 and causing recurrent DSP toxicity in shellfish was
reinvestigated. The morphotypes characterized until now as D. cf. acuminata in Thermaikos Gulf (NW Aegean Sea), were recently shown to
actually be more related to D. ovum, based on morphological and molecular analyses. The use of cox1 mitochondrial marker enabled distinction
between D. acuminata and D. ovum and re-definition of the common Greek morphotype as D. cf. ovum. Dinophysis morphotypes from Thermaikos Gulf studied in the 2009 bloom were morphologically compared to D. cf. acuminata morphotypes of the same area from precedent
blooms (2000-2008); furthermore, similar specimens, that have been identified in the framework of the Greek HAB monitoring programme,
from numerous areas along the Greek coastal waters (Kavala coasts, Saronikos and Euvoikos Gulfs) during the last decade were also re-examined. Within this period, the highest cell densities of D. cf. ovum populations were recorded in Thermaikos and Saronikos Gulfs and were
accompanied by shellfish toxicity mainly in winter and spring. At the same periods, the only toxin detected in shellfish by HPLC-FLD and LCMS/MS was okadaic acid. Results of the present study indicate that the toxic Dinophysis morphotype blooming in Greek waters and causing
DSP episodes is common among the studied areas and is now characterized as D. cf. ovum. These findings contribute towards clarification of
the taxonomy of D. acuminata complex in the Mediterranean Sea and indicate the underestimated potential of D. ovum to cause toxic episodes.
187
EXPLORING WHY DINOFLAGELLATES ARE SO SENSITIVE
TO SMALL-SCALE TURBULENCE. THE MICROTUBULE
ASSEMBLAGE ALTERATION HYPOTHESIS
Elisa Berdalet1, Gisela Llaveria1, Philippe Huitorel2
1
2
Institut de Ciències del Mar, CSIC, Passeig Marítim de la Barceloneta 37-49, E-08003 Barcelona, Spain
CNRS, UMR 7009 and UPMC Univ Paris 06, UMR 7009, Lab. de Biologie du Developpement,
Observatoire Oceanologique BP28, 06234 Villefranche-sur-mer Cedex, France
Small-scale turbulence has been shown to slow down the growth and/or some phases of the cell cycle of some dinoflagellate species,
and it seems that turbulence or shear stress could alter the cell division processes. Here, we report our studies to investigate the unknown
underlying mechanisms of the observed responses. We tested the hypothesis that turbulence could alter the correct assemblage of microtubules composing the mitotic spindle involved in the chromosome segregation during the dinomitosis. In shaking experiments performed
on Akashiwo sanguinea, Prorocentrum micans and Oxyrrhis marina, microtubules were visualized through a combination of inmunocytochemistry (using anti-tubulin monoclonal antibodies) Confocal Fluorescent Laser Microscopy. Each species showed particular organizations of the nuclear, microtubular and other mitotic elements under still conditions. These mitotic organizations appeared unmodified by
turbulence. Thus, our results did not provide evidences to support the microtubule hypothesis. Some alternative hypotheses also linked
to the microtubular organization in the cell are suggested.
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DIEL TRANSCRIPT VARIATION
IN KARLODINIUM VENEFICUM
Holly Bowers, Ernest Williams, Kristy Lidie, Rose Jagus, Allen R. Place
Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Sciences,
701 East Pratt Street, Baltimore, MD 21202, USA. E-mail: [email protected]
Like other dinoflagellate genomes, the spliced leader transcriptome of Karlodinium veneficum contains large gene families which apparently evolved by extensive tandem gene duplication events. When transcript abundance for actin was measured over a 24 hour
period for cells in a 14:10 photoperiod, there was a nearly 4-fold drop in transcript abundance at the end of the light period followed
by an increase in the dark period to near light period levels. This pattern was replicated in transcript levels for two eurkaryotic initiation
factors, eIF4E-1 and eIF4E-2. In eukaryotes, eIF4E is a central component in the initiation and regulation of translation. Through its
interaction with the 5'-cap structure, eIF4E functions to recruit mRNAs to the ribosome. Identical diel changes were also found for
two putative proton channels. The overall findings indicate a generalized decrease in the transcriptome prior to the dark period followed
by an increase during the dark period.
188
DETECTION OF ALEXANDRIUM TAMARENSE
IN SCOTTISH WATERS USING TAQMAN PCR(QPCR)
AND WHOLE CELL FISH ASSAYS AND EVIDENCE
OF POSSIBLE HYBRIDIZATION
Jennifer Graham, Catherine Collins, Lyndsay Brown, Jean-Pierre Lacaze, Eileen Bresnan,
Elizabeth Turrell, Matthew Gubbins
Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB, UK. E-mail: [email protected]
Monitoring for toxic algal events requires the reliable identification of the phytoplankton to species level. Molecular sequencing (LSU
and ITS rDNA), whole cell FISH assays and chemical analysis (LC-MS-MS) of Alexandrium isolated from sediments and water
samples has highlighted the presence of both benign (GpIII) and PSP toxin producing (GpI) Alexandrium tamarense strains in Scottish
coastal waters. A method to differentiate easily between these morphologically similar strains is required to provide a better picture of
their composition in Scottish waters and to help understand environmental and seasonal conditions influencing their occurrence. To
this aim real-time TaqMan PCR (QPCR) assays were developed. Assays targeting the LSU and ITS rDNA were tested using clonal
cultures of different Alexandrium species and strains and assessed using field samples from shellfish aquaculture and harvesting
areas. Preliminary results with clonal cultures from Scottish waters suggested some cross-reactivity of the GpIII assay against DNA
from GpI. Further investigation using traditional PCR and primers targeting different regions of the ITS rDNA showed correlation between traditional and QPCR results with respect to level of cross reaction between the strains and assays. Sequencing of the “nonspecific” products confirmed presence of A. tamarense GpIII DNA in GpI isolates, despite these isolates having been generated from
single cells and subcloned several times. This suggests that some form of hybridisation is occurring in Scottish coastal waters between
A. tamarense Gps I and III. If so, this may have implications for the life history and toxin production of these “hybrid” isolates.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 189
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
UNDERSTANDING THE COMPLEX AND CONTROVERSIAL
PHYLOGENETIC RELATIONSHIPS AMONG CLOSELY
RELATED AND POTENTIALLY HARMFUL
DINOPHYSIACEAN GENERA
Sara M. Handy1,2, Jonathan Deeds2, Charles F. Delwiche1
1
2
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA
U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD 20740, USA.
E-mail: [email protected]
Select members of the dinoflagellate family Dinophysiaceae produce okadaic acid (OA), and dinophysistoxins (DTXs), the causative toxins
of diarrhetic shellfish poisoning (DSP). FDA guidance recommends <0.2 ppm OA+DTX1 in shellfish for human consumption, and is giving
consideration to the EU standard of <0.16 ppm total OA+DTXs. The US National Shellfish Sanitation Program currently provides no specific
guidance on appropriate testing for these toxins or monitoring for causative organisms. In recent years, DSP toxins were confirmed in both
shellfish and phytoplankton in US waters and the first ever shellfish harvesting closures from confirmed OA occurred in 2008 in the Gulf of
Mexico. At present, the taxonomy of this group is in flux. The ability to reliably distinguish the various members of the genus Dinophysis and
the closely related (sometimes considered synonymous), toxin producing genus Phalachroma is the first step towards understanding more
complex topics such as the determination of causative organisms during toxicity events, bloom development, food-web dynamics, etc. The
fact that, until recently, most of the dinophysiaceans were unculturable has made this topic particularly challenging. Through single cell analysis,
we examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites based on ribosomal
DNA. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from
three sites. This will hopefully provide further insight into the complex regulatory issues surrounding this complicated group of organisms.
189
A TALE OF TWO MORPHOTYPES: A NEW SPECIES
OF PROROCENTRUM EXHIBITS TWO DISTINCT
MORPHOLOGIES
Darren W. Henrichs1, Paula S. Scott2, Karen A. Steidinger2, Reagan M. Errera3, Ann Abraham4,
Heidi M. Sosik5, Robert J. Olson5, Lisa Campbell1,3
1
Department of Biology, Texas A&M University, College Station, TX, USA. E-mail: [email protected]
Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, St. Petersburg, FL, USA
3
Department of Oceanography, Texas A&M University, College Station, TX, USA
4
Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, Dauphin Island, AL, USA
5
Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, USA
2
Assessments of biodiversity have become increasingly important in recent years due to changes in climate, habitat, and anthropogenic influences, making identification of unique species a crucial first step. This process however, can be complicated by many factors, including
cryptic speciation and morphological variants of single species. We report on a new species of Prorocentrum from the Gulf of Mexico that
exhibits two distinct morphotypes, round and pointed. Both morphotypes were recently brought into culture using cells isolated from Port
Aransas, Texas, USA. Genetic sequences from ribosomal (SSU, LSU, ITS) and mitochondrial (cob, cox1) genes in both morphs were unique
from all other sequenced Prorocentrum spp. but identical to each other. Phylogenetic analysis showed this new species to be closely related
to P. micans and P. rhathymum. Population dynamics of both morphotypes and P. micans will be analyzed using daily time series data, spanning three years (2008-2010), from the Port Aransas, Texas, USA study site. Preliminary analysis shows the round morph is dominant within
the phytoplankton community during the spring while the pointed morph is present throughout the year in low concentrations. Supporting
data showing growth rate, potential toxin production, and morphological differences between the two morphotypes will also be presented.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 190
CHIMODINIUM SHIWHAENSE N. GEN., N. SP.,
A NEW PLANKTONIC HETEROTOPHIC DINOFLAGELLATE
FROM THE KOREAN COASTAL WATERS
Seon Kang1, Hae Jin Jeong1, Yeong Du Yoo1, Eric Potvin1, Eun Young Yoon1, Jae Seong Kim2
1
School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University,
Gwanak-ro, Gwanak-gu, 151-747 Seoul, Republic of Korea. E-mail: [email protected]
2
Red Tide Research Center, Kunsan National University, Daehangno, 573-701 Gunsan, Republic of Korea
The heterotrophic dinoflagellate Chimodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light,
scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU)
rDNA are reported. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged
in 16 rows. The apical groove of the species has a loop-like shape. The cingulum is displaced by 0.3-0.5 x cell length. Cell length and
width of live cells fed on the dinoflagellate Amphidinium carterae were 9.1-21.6 μm and 6.6-15.7 μm, respectively. C. shiwhaense has
nematocysts, trichocysts, peduncle, and pusule systems, while it does not have chloroplasts. The sequence of the SSU rDNA of this dinoflagellate is > 3% different from that of Warnowia sp. (FJ947040), Lepidodinium viride, Gymnodinium aureolum, Gymnodinium catenatum, Nematodinium sp. (FJ947039), and Gymnodinium sp. AF022196, while the LSU rDNA was 11-12% different from that of
Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that this dinoflagellate belongs to the
Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous
connective. Unlike Polykrikos spp. which have a taeniocyst-nematocyst complex, C. shiwhaense has nematocysts without taeniocyst. In
addition, C. shiwhaense does not have ocelloids in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Therefore,
based on morphological and molecular analyses, we suggest that this taxon is a new species within a new genus.
190
CLONING AND EXPRESSION OF VIRAL CAPSID GENES
FROM HARMFUL ALGAL BLOOM CAUSING ALGAE
Wonduck Kim1, Jang Min Park2, Si Wouk Kim2
1
2
Pioneer Research Center for Controlling of Harmful Algal Bloom, Chosun University, Gwangju, Korea
Department of Environmental Engineering, Chosun University, Gwangju, Korea
We attempt to express viral capsid proteins from harmful algae that cause blooming as an initial process of controlling harmful algal
blooming. In our study, capsid proteins of RNA viruses (HcRNAV34 and HcRNAV109) that infect Heterocapsa circularisquama strains,
one of the blooming forming dinoflagellates, were determined for expression study. Each protein is encoded from a single gene and their
sizes are relatively small, thereby efficient syntheses of the coat proteins in E. coli were expected. However, the genes contain significant
number of rare codons that may reduce the expression levels in E. coli, thus the genes were artificially synthesized for codon optimization.
At first, the synthesized genes were cloned into pET vectors and expressions were induced by IPTG. However, SDS-PAGE analysis
showed that negligible amount of the capsid proteins were produced, possibly due to protein degradation. Thus, gene fusion expression
systems, pETSUMO protein expression system and intein- mediated protein expression system, were considered for the expressions.
When the capsid genes were expressed with the tag genes in the expression vectors, pETSUMO and pTYB21, significant levels of
capsid proteins were synthesized. However, the proteins were aggregated in case protein inductions with IPTG were carried out at 37oC
or room temperature. To enhance solubilities of the proteins, the expressions were induced lower than 20oC, and considerable amount
of the soluble proteins were produced, which was confirmed by SDS-PAGE analysis and western blot analysis.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 191
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
DIVERSITY OF BENTHIC DINOFLAGELLATES
FROM PAPUA NEW GUINEA
Gurjeet Singh Kohli, Shauna A. Murray, Brett A. Neilan
School of Biotechnology & Biomolecular Sciences, University of New South Wales, Kensington, NSW 2052, Australia.
E-mail: [email protected]
The objective of this study was to conduct the first ever diversity survey of benthic dinoflagellates in the coral reefs of Papua New Guinea,
using a molecular clone library approach. Samples were collected from three different locations i.e. 1) Lion Island, Port Moresby, 2)
Lolata Island, Port Moresby and 3) Tavali, Alotau, Milne Bay, Papua New Guinea. As dinoflagellates are known to live epibenthically with
numerous macroalgae, specimens of Amphiroa sp., Turbinaria sp., Sargassum sp., Halimeda sp. and Padina sp. were collected and
their associated dinoflagellate populations were investigated. Dinoflagellate identification was achieved through DNA barcoding of a 0.45
kb region of the mitochondrial cytochrome b (cob) gene. Clone libraries were constructed and DNA sequences obtained for phylogenetic
analysis. Phylogenetic analysis revealed the presence of Amphidinium spp., Prorocentrum spp., and other Gonyaulacalean spp., which
were confirmed by microscopic examination. Additionally, microscopic analysis revealed the presence of Ostreopsis spp. as well. This is
the first study to apply the cob barcoding technique to identify dinoflagellate lineages in benthic environmental samples, and shows that
this is a promising tool for rapid identification of potentially toxic dinoflagellates.
191
PHYLOGENETIC RELATIONSHIPS, MORPHOLOGICAL VARIATION AND TOXICITY PATTERNS IN THE ALEXANDRIUM OSTENFELDII/ALEXANDRIUM PERUVIANUM SPECIES COMPLEX
Anke Kremp1, Pia Mäenpää1, Sanna Suikkanen1, Bernd Krock2, Harri Kankaanpää1,
Päivi Sillman1, Satoshi Nagai3, Po Teen Lim4
1
Finnish Environment Institute, Marine Research Centre, P.O. Box 140, 00251 Helsinki, Finland. E-mail: [email protected]
2
Alfred-Wegener-Institut für Polar- und Meeresforschung, Ökologische Chemie, Am Handelshafen 12, 27570 Bremerhaven, Germany
3
National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan
4
Faculty of Resource Science and Technology, University of Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Alexandrium ostenfeldii and Alexandrium peruvianum are morphologically similar species, characterized by an oblique first apical plate and the presence
of an unusually large ventral pore. According to the valid species descriptions they are differentiated by the shapes of the S.a. plate and the right anterior
margin of their 1’ plate. Both species have been identified from different parts of the world and both have been reported to produce Paralytic Shellfish
Toxins (PSTs) and/or Spirolides. We compared rDNA sequence data, morphology and toxin characteristics of cultured isolates of A. ostenfeldii and A.
peruvianum from different locations around the world to better understand the relationships between the two species and different geographic populations.
Phylogenetic analyses based on 3 different markers (18S, 28S, 5.8S/ITS region) revealed clear separation of tropical A. peruvianum from all other investigated A. ostenfeldii and temperate A. peruvianum populations. Together, the latter form a highly structured clade, where a cluster containing a geographically heterogeneous A. peruvianum morphotype and a genetically distinct group of Baltic A. ostenfeldii strains is opposed by a group of clusters
separating North Atlantic, North Sea and South Pacific A. ostenfeldii isolates. Japanese isolates branch separately, being however genetically more
closely related to the temperate A. ostenfeldii/peruvianum clade than to tropical A. peruvianum. The phylogenetic relationships were not clearly reflected
by patterns of morphology and toxin composition. Plate shapes were highly variable and the distinctive 1’ and S.a. features of either species were found
in both species and most populations. Tropical A. peruvianum produces PSTs, whereas spirolides are common in the temperate A. peruvianum morphotype. PST profiles were highly consistent in Baltic A. ostenfeldii populations. North Atlantic and North Sea A. ostenfeldii exclusively produced spirolides.
Together, our results suggest that the species concept of the A. ostenfeldii/A. peruvianum group needs to be revisited.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 192
DINOFLAGELLATES OF THE GENERA COOLIA,
OSTREOPSIS AND PROROCENTRUM FROM
THE SOUTHEASTER BAY OF BISCAY
Aitor Laza-Martinez, Emma Orive
Department of Plant Biology and Ecology, Faculty of Science and Technology, University of the Basque Country,
48940 Leioa, Spain. E-mail: [email protected]
Epibenthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrum isolated from coastal waters of the Southeaster Bay of
Biscay were identified morphologically by means of light microscopy including epifluorescence microscopy, and scanning electron microscopy. To gain insight into the specific identity of the strains, molecular phylogenetic analyses using the nuclear large subunit rDNA
(LSU) were performed to selected strains of the three genera. The morphological and phylogenetic analyses revealed the presence of
the following species: Coolia canariensis Fraga; Coolia monotis Meunier; Ostreopsis siamensis Schmidt, P. emarginatum Fukuyo, P.
fukuyoi Murray et Nagahama, P. lima (Ehrenberg) Dodge and P. rhathymum Loeblich III, Sherley & Schmidt. The most common of them
were Coolia monotis, Ostreopsis siamensis and Prorocentrum lima, which appeared at most sampling sites. Only the strains corresponding
to Ostreopsis siamensis and Prorocentrum lima were toxic for Artemia franciscana.
192
PROTEOMIC ANALYSIS OF ALEXANDRIUM TAMARENSE,
A DINOFLAGELLATE WITH UNSEQUENCED GENOME BY
MALDI-TOF/TOF AND AUTOMATED DE NOVO SEQUENCING
Cheng Li, Zhang-Xian Xie, Hong-Po Dong, Hua-Sheng Hong, Da-Zhi Wang
State Key Laboratory of Marine Environmental Science/Environmental Science Research Center, Xiamen University,
182 Daxue Road, 361005, Xiamen, People’s Republic of China. E-mail: [email protected]
Alexandrium is a widely spread dinoflagellate genus throughout many regions of the world, which is not only able to produce potent neurotoxins
resulting in paralytic shellfish poisonings, but also cause HABs along the coastal waters. However, the world wide lack of available genetic information of dinoflagellates limits our understanding of HABs and subsequently monitoring, mitigation and prevention of HABs. This study reported a multilayered, gel-based and underivatized strategy for de novo protein sequence analysis using a MALDI-TOF/TOF mass spectrometer
with the assistance of DeNovo ExplorerTM software. MASCOT, a highly specific stringent search was applied as the first layer screen to identify
either known proteins or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed,
the reminders were searched against dinoflagellate EST database using BLASTX. In the last layer, those non-confident hits were further
subjected to de novo interpretation by DeNovo ExplorerTM software. The de novo sequences passing a reliability filter were subsequently submitted to non-reduntant BLAST searches, and MS-BLAST hits are validated by a sampling technique. Using this layer identification manner,
144 proteins out of 156 protein spots from A. tamarense were confidently identified. Among of them, 77 proteins against NCBInr database,
35 proteins against dinoflagellate EST database, and 32 proteins by de novo sequencing and MS-BLAST, these proteins were involved in
various intracellular physiological activities. This study is the first effort to establish a completely automated approach to identify proteins from
unsequenced database of dinoflagellates, which provides a potential tool for various proteomic studies of dinoflagellates in future.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 193
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
MOLECULAR IDENTIFICATION AND PHYLOGENETIC
STUDY BASED ON THE PARTIAL LSU GENE SEQUENCE
FOR FIVE PSEUDO-NITZSCHIA SPECIES FROM
SOUTHEAST CHINA SEA
Junrong Liang1, Xin Lin1, Peng Wang1, Changping Chen1, Yahui Gao1, Yuzao Qi2
1
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems and Sch. of Life Sciences,
Xiamen University, Xiamen 361005, China. E-mail: [email protected]
2
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510630, China
Based on the data of D1-D3 domains of LSU rDNA sequences, five Pseudo-nitzschia species including seven strains isolated from
Southeast China Sea were identified. Five species were classified to two clades: clade 1 includes P. multistriata, P. brasiliana, P. pungens,
and clade 2 includes P. cuspidata, P. delicatissima. The phylogenetic relationship between the Pseudo-nitzschia species shows that the
classification based on the LSU rDNA is similar to the classicification based on the morphological based taxonomy (P. delicatissima group
_ 3μm and P. seriata group with frustule width >
_ 3μm). The interspecific nucleotide polymorphisms between Chinese
with frustule width <
Pseudo-nitzschia strains and the strains from other sea areas in the world were also analyzed in this study. There is no distinct difference
between the P. pungens strains from different locations. As for the P. delicatissima, the Italian strain has distinct difference compared
with Chinese and Scotland strain. And the P. cuspidata of Chinese strain is distinctly different from Italian strain. The analyses of LSU
rDNA variety within the same Pseudo-nitzschia species are very essential. The data are indispensable references for the application of
DNA probe based on LSUrDNA sequence for the Pseudo-nitzschia. In addition, the toxin analyses showed that no toxin was detected
for all the strains in this study. This study was supported by the NSFC (Nos. 40831160519, 40676080) and the NSFF (No. 2008J0139).
193
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION
OF DIATOM PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE)
FROM MALAYSIA
Hong-Chang Lim1, Chui-Pin Leaw2, Po-Teen Lim1
1
Faculty of Resource Science and Technology, University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
E-mail: [email protected], [email protected]
2
Institute of Biodiversity and Environmental Conservation, University Malaysia Sarawak, 94300 Kota Samarahan,
Sarawak, Malaysia
The genus Pseudo-nitzschia is one of the most well studied marine diatoms as fifteen of the species are known to produce the neurotoxin,
domoic acid (DA) that responsible for Amsenic Shellfish Poisoning (ASP). Thus far, little attention is given to the distribution of Pseudo-nitzschia
spp. in Malaysia. In this study, three Pseudo-nitzschia spp. were identified as P. pungens, P. brasiliana and P. dolorosa based on detailed ultrastructure under light and electron microscopy. Species identification was supported by LSU rDNA phylogenetic analyses. The application of
sequence-structure alignment based on the second internal transcribed spacer (ITS2) RNA transcript was also inferred to improve the phylogeny
which allows comparisons at deeper taxonomic levels. The highly conserved ITS2 secondary structures of Pseudo-nitzschia spp. were predicted
through free energy minimization and homologous modeling. Our results revealed two distinct types of ITS2 secondary structure models viz.
Type A and Type B. This region of ITS2, together with the structural information obtained was further incorporated into our phylogenetic
inference of Pseudo-nitzschia. The study revealed that the phylogenetic position of Pseudo-nitzschia spp. in the ITS2 sequence-structure tree
is better well resolved compared to the LSU rDNA tree topology. On the other hand, mapping of 14 morphological characters on Pseudonitzschia tree showed that the character of central interspace was of taxonomic diagnostic value. The other 13 characters and toxicity were
somehow homoplastic. Intraspecific genetic variation of P. pungen and P. brasiliana from Borneo was also investigated and our results showed
that Bornean populations of P. pungen were distinctly isolated which could be due to the surface current pattern in the South China Sea. This
study represents the first study of the Pseudo-nitzschia occurrence in the region with extensive morphological and genetic information.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 194
INTRAGENOMIC RDNA POLYMORPHISM (IRP) STATUS
AND IRP-GUIDED PHYLOGENY DELINEATE THE
A. TAMARENSE/FUNDYENSE/CATENELLA COMPLEX
TO TWO MAJOR CLADES
Senjie Lin1,2, Lilibeth Miranda1, Huan Zhang1
1
2
Department of Marine Sciences, University of Connecticut, Groton, CT 06340, USA. E-mail: [email protected]
South China Sea Institute of Oceanology, CAS, Guangzhou 510301, China
We analyzed 18S rRNA in monoclonal strains of A. fundyense by both direct and clone-based sequencing and detected extensive polymorphism. In five out of the six strains examined, the 18S rDNA amplicon revealed 42 polymorphic sites. Clone sequencing for polymorphic
amplicon libraries revealed sequences differing 0-2.5%. Phylogenetic analyses showed that the polymorphic sequences were not clustered
by strain but dispersed across strains, indicating the possibility of misidentifying a geographic population when analysis is based on a
single 18S rDNA clone sequence. Analyses based on all reported and new data indicated that the sequence polymorphism would not
impact the monophyly of the Alexandrium genus or that of the A. tamarense/fundyense/catenella species complex, but that sequences
from this complex clearly formed two major clades. One clade comprised A. tamarense and A. fundyense intermingled in various subclades,
with the polymorphic 18S sequences from each strain dispersed in all major subclades, suggesting these taxa to be one species. The
other clade contained A. catenella mixed with some A. tamarense and A. fundyense, with no evidence of 18S rDNA polymorphism. Applying the analysis to May 2008 toxic Alexandrium bloom in Long Island revealed high diversity of 18S rDNA but clearly one single Alexandrium species. This new phylogenetic approach may prove helpful in eventually resolving this three-species complex to practically useful
and simpler taxonomic units and classifying natural populations to a species.
194
SPATIAL AND TEMPORAL INTRAGENETIC VARIATION
OF ALEXANDRIUM OSTENFELDII IN THE BALTIC SEA
Pia Mäenpää1, 2, Rosa Figueroa3, Jaanika Blomster4, Anke Kremp1
1
Marine Research Centre, Finnish Environment Institute, P.O. Box 140, 00251 Helsinki, Finland
Tvärminne Zoological Station, University of Helsinki, J.A. Palmenintie, 10900 Hanko, Finland. E-mail: [email protected]
3
Limnology Department, University of Lund, SE-22362 Lund, Sweden
4
Department of Environmental Sciences, University of Helsinki, P.O. Box 65, 00014, Finland
2
Alexandrium ostenfeldii, (Paulsen) Balech & Tangen, is a toxic dinoflagellate which has been present as a background species in the
Baltic Sea for over a century. Recently, the species has started to form dense toxic, bioluminescent blooms which have successively
spread from the south into the central and northern parts of the Baltic Sea. We analyzed and compared the genetic structure of different
Baltic A. ostenfeldii populations to reveal possible spatial (different locations) and temporal (annual, life cycle succession) patterns that
could help explain the recent expansion. Material for the analyses was collected from 4 different bloom locations in the Baltic Sea and
from blooms of different years occurring in the Åland archipelago. Clonal strains were established from resting cysts and single cells and
studied for genetic variation using AFLP (Amplified Fragment Length Polymorphism) - markers. For each of the 180 A. ostenfeldii strains
5 to 6 primer pairs were applied and the resulting fragments were processed and analyzed with GeneMapper, FAMD, and STRUCTURE.
The AFLP data revealed high genetic variability within and among the examined populations, which clearly structures A. ostenfeldii in the
Baltic, despite their identical rDNA genes. We discuss whether the observed patterns of the species are linked to populations’ geographical
localities or ecophysiological adaptations (cold water-warm water populations). Special emphasis will be given to differences among
annual blooms as well as between blooming versus resting cysts populations.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 195
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
MORPHOLOGY AND MOLECULAR CHARACTERIZATION
OF OSTREOPSIS (DINOPHYCEAE) ISOLATES
FROM NORTHEASTERN BRAZIL
Mariângela Menezes1, Daniel Varela2, Luis A.O. Proença3, Javier Paredes2, Mathias Schramm3
Museu Nacional, Quinta da Boa Vista s/n, PC 20.940-040, São Cristóvão,
Rio de Janeiro, Brasil. E-mail: [email protected]
2
Centro de Investigación I-Mar, Universidad de Los Lagos, Casilla 557, Puerto Montt, Chile
3
Universidade do Vale de Itajai, P.O. Box 360, PC 88.3022-02, Itajai, Santa Catarina, Brasil
1
Analysis of the morphology and sequence of internal transcribed spacer (ITS) regions were carried out on two strains of Ostreopsis
isolated from northeastern Brazilian coastal waters (16o27´S and 39o05´W) collected in October 2004 from Coroa Alta Beach (L3)
and in January 2008 from Coroa Vermelha Beach, Santa Cruz de Cabrália (L2). Living material was collected from macroalgae and
clonal cultures were established and maintained in f/2 media, without silica at 20oC, 80 μmol·m-2•s-1, 12:12 h light-dark cycle and 32
PSU. Morphological analysis based on light and epifluorescente microscope of L3 strain showed characters overlapped with O. lenticularis
and O. siamensis while L2 was similar to O. ovata. The phylogenetic analyses supported the existence of two distinct populations in the
studied area. Brazilian isolate L2 grouped with isolates of O. cf. ovata from Mediterranean/Atlantic region whereas L3 segregated with
isolates of O. cf. ovata from Indo-Pacific populations. The sequence information argued that the differentiation degree of L3 strain with
the isolates from Indonesia seems to indicate a long-term separation and isolation of locally established population, rather than for recent
introductions of the species in the region. Financial support: CNPq.
195
GENETIC AND MORPHOLOGICAL DIVERSITY OF THE
GENUS OSTREOPSIS IN OKINAWAJIMA ISLAND, JAPAN
Atsushi Nakashima1, Md. Mahfuzur Rhaman Shah1, Yosuke Taira2, Takeshi Yasumoto2, Shoichiro Suda1
1
2
University of the Ryukyus, Senbaru, Nishihara, Okinawa 903-0213, Japan. E-mail: [email protected]
Okinawa Science and Technology Promotion Center, Marine Bioindustry Division, 12-75, Suzaki, Uruma,
Okinawa 904-2234, Japan
Okinawajima is located in the middle of the Ryukyu Islands. The Ryukyu Islands are a chain of islands approximately 1,000 km long in
the western Pacific and eastern border of the East China Sea, stretching between southern Kyushu, Japan to east of Taiwan. The islands
are strong influenced by the warm Kuroshio Current and were surrounded by well developing coral reefs before recent deterioration.
Benthic dinoflagellates of the genus Ostreopsis are putative producers of toxic substances such as palytoxin, ostreocin and relatives. Recently Ostreopsis spp., and especially O. ovata, O. siamensis and close relatives are problematic. Their blooms have been reported worldwide from several Mediterranean countries, as well as Brazil and New Zealand, and they are likely responsible for the mortality of benthic
invertebrates and cause serious problems for humans. Since O. siamensis established by Schmidt, 10 species are currently distinguished
based on morphology. In the Ryukyu Islands, Fukuyo described O. siamensis and O. ovata in 1981 and Hoiriguchi added O. labens (in
Murray 2009). Our recent observations have revealed that O. cf. lenticularis is also present. The aim of this study was to clarify the classification of Ostreopsis spp. in the Ryukyu Islands using molecular and morphological approaches. In particular, we will make reference
of newly acquired genetic sequences of O. ovata Fukuyo, for which the type locality is the Ryukyu Islands. Additionally, species’ relations
with toxin production will be discussed.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 196
THE DIATOM GENUS SKELETONEMA
IN KOREAN COASTAL WATERS
Jong-Gyu Park, Hyung-Joon Park
Kunsan National University, san 68 Miryong-dong, 573-701 Kunsan, Korea. E-mail: [email protected]
Skeletonema is a well known diatom genus distributed all over the world. Since 2005 several new species have been introduced into the
science, attaining up to more than 13 species. However, most Skeletonema species observed in Korean coastal waters have been
routinely classified as S. costatum without any other verifying processes taxonomically. To clarify the exact name of Korean species, molecular and morphological works were conducted for 38 strains isolated from Korean coastal regions from September 2007 to August
2009. Skeletonema was comprised mainly of S. marinoi and S. japonicum. S. costatum was not observed. Interstitial cell traits of S.
marinoi were similar with those in previous studies, but end cells had additional traits distinguishable from them. Unlike the reports that
terminal cells carry only one rimoportula process in the center of valve, three types of terminal process were observed in the Korean
strains; two rimoportula processes, one rimoportula and one siliceous process, and no rimoportula process. In case of S. japonicum, the
shapes of two strains were different from each other. While fultoportula processes of terminal cells in KSU DS01 were claw-like like
those of S. costatum, KSU TY02 had dentate processes like S. marinoi. Although the Korean strains displayed different morphologies
mutually, neighbor-joining and maximum parsimony analysis using SSU rDNA sequences showed that the strains of each species formed
an independent clade supported by bootstrap values of 54/83 in S. marinoi and 66/74 in S. japonicum.
196
POLYKETIDE SYNTHASES (PKS) IN DINOFLAGELLATES:
NEW INSIGHTS INTO THEIR CELLULAR LOCALIZATION
AND FUNCTIONALITY
Laura Pezzolesi1, Mackenzie L. Zippay2,3, Emily Monroe2,4, Rossella Pistocchi1, Frances M. Van Dolah2,3
1
Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Università di Bologna, Via S’Alberto 163,
48123 Ravenna, Italy. E-mail: [email protected]
2
NOAA Marine Biotoxins Program, Charleston, SC, USA
3
Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston, SC, USA
4
Current address: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA
Polyketides are a large family of secondary metabolites that are synthesized from acyl- CoA precursors by polyketide synthase enzymes (PKSs).
These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). To date, approximately 25
species of dinoflagellates have been found to produce polyketides. Recently, several putative PKS genes encoding ketosynthase (KS), ketoreductase
(KR), and both acyl carrier protein (ACP) and KS domains were identified from K. brevis. Their structure is unique in that their sequence is most
similar to Type 1 PKS, but separate catalytic domains reside on separate polypeptides, like Type II. Their altered expression in a non-toxic isolate
of K. brevis suggested their involvement in brevetoxin biosynthesis; however, their chloroplast localization resembles FAS. Since no information
exists on PKS proteins of other toxic dinoflagellates, we used antibodies developed against K. brevis PKS proteins to probe for the expression and
intracellular localization PKS domains in three harmful dinoflagellates (Karenia brevis, Ostreopsis ovata, Coolia monotis), one non-toxic species
(Karenia mikimotoi) and a raphidophyte (Fibrocapsa japonica) which is known to produce high concentrations of free fatty acids (FFA). All species,
including the raphidophyte expressed proteins cross-reactive with one or more K. brevis antibodies, however different protein sizes were reported
likely due to differences in species. These results lead us hypothesize that either (1) these proteins are FAS or (2) single PKS units could be
cobbled together to form complexes that synthesize different polyketide compounds and/or fatty acids in different species.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 197
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
5-HYDROXYMETHYLURACIL IS SYNAPOMORPHIC
TO THE DINOKARYOTES ONLY
Allen R. Place, Ernest Williams
Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Sciences,
Baltimore, MD, USA
Since the original description of 5-hydroxymethyluracil in the genomic DNA of dinoflagellates by Rae in 1973 there has been little further
research in extending these findings to other species. Accordingly, we re-examined the genomic DNA from members of various dinoflagellate genera and found that between 10 and 60% of the thymine bases where substituted by 5-hydroxymethyluracil. We also observed
elevated levels of 5 methyl cytosine. When we examined sister taxa to the dinoflagellates we did not find this modified base. This included
DNA from Perkinsus marinus, Oxyrrhis marina, and three species of Amoebophrya (Amoebophyra ex sanguinem, Amoebophyra ex instriatum, Amoebophyra ex K. veneficum). The replacement of the normal thymine base by 5-hydroxymethyluracil appears to be a prerequisite for the dinokaryon character to be manifested.
197
GENETIC VARIABILITY AND MOLECULAR PHYLOGENY
OF DINOPHYSIS SPECIES (DINOPHYCEAE)
OF MITOCONDRIAL COX1 GENE
Nicolás Raho1, Francisco Rodríguez Hernandez2, Beatriz Reguera2, Irma Marín1
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
E-mail: [email protected]
2
Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Aptdo. 1552, E-36280 Vigo, Spain
1
The genus Dinophysis comprises about 200 species widely distributed in oceanic, neritic and coastal waters. A few of these species
contain potent lipophilic toxins (i.e. okadaic acid, dinophysistoxins and/or pectenotoxins) responsible for diarrhetic shellfish poisoning and
other undesirable effects. The taxonomic identification of Dinophysis spp. is mainly based on their cell contour -size and shape of their large
hypothecal plates- and the shape of their left sulcal lists and ribs. Nevertheless, biogeographical intra-specific differences in morphology
and large differences in toxin profile and content have been reported, and the same strains of Dinophysis on a given locality exhibit a continuum of shapes from large vegetative specimens to small gamete-like cells as a result of their polymorphic life cycle. Morphological
variability adds uncertainty in the routine identification and quantification of phytoplankton samples in HAB monitoring programmes, especially
when close species, one weakly- and the other very toxic co-occur. Molecular biology may provide powerful tools to improve identification
of microalgae and their different life cycle stages in field samples. Mitochondrial DNA is a useful candidate as a molecular marker in closer
phylogenetic reconstructions because its genes are generally conserved, but they are subject to more variations than nuclear coding genes.
Here we analyze the functionality of mitochondrial gene cox1 as molecular marker for Dinophysis and its relation to other dinoflagellates
genera using as a model mitochondrial cox1 sequences from different species of Dinophysis from Galician coastal waters.
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MOLECULAR ANALYSIS OF THE DINOPHYSIS ACUTA
FROM HUELVA (SPAIN) CHLOROPLASTS FED
WITH DIFFERENT CRYPTOPHYTES
Nicolás Raho1, David Jaén2, Luz Mamán2, Pilar Rial3, Irma Marín1
1
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
E-mail: [email protected], [email protected]
2
Laboratorio de Control de Calidad de los Recursos Pesqueros, Consejería de Agricultura y Pesca,
Junta de Andalucía, DAP S.A., Spain
3
Instituto Español de Oceanografia, Centro Oceanográfico de Vigo, Aptdo 1552, 36200 Vigo, Spain
Dinophysis Ehrenberg is a genus of marine dinoflagellate, which is polyether toxins producer. For many years, the inability to maintain these organisms in laboratory cultures was the main obstacle in their study. Recently, a predator-prey line has been established in which D. acuminata,
mixotrophic nature, was fed with the ciliate Myrionecta rubra. In turn, M. rubra, which has an obligate endosymbiont, need the supplement of
cryptophyte algae Teleaulax genus to get good division rates. While it is known that photosynthetic species of Dinophysis have chloroplasts of
cryptophyte origin, during the last decade has been controversy about whether chloroplasts are permanent, or otherwise, are acquired periodically
by a process called kleptoplastidic, where Dinophysis steal the chloroplasts from other organisms. Different ultrastructural studies on the chloroplasts indicates that Dinophysis has permanent chloroplasts, based on the presence of only two membranes, the absence of endoplasmic reticulum and the absence of cryptophyte plastidial nucleomorph. On the contrary, it has been found a great similarity between the sequences of
plastidial genes in Dinophysis and cryptophytes, suggesting that the first gets the chloroplasts by kleptoplastidic process. However, these results
can be explained by a recent incorporation of plastids. To try to understand the origin of the chloroplasts of Dinophysis, in this work various
cultures of ciliated Myrionecta sp. from Huelva have been fed with different cryptophytes. In turn, these ciliates were used to feed different
cultures of Dinophysis acuta from the same region. In order to identify whether the chloroplasts of Dinophysis come from their preys or, on the
contrary, have been acquired throughout their evolutionary history, we examined the chloroplast psbA gene in these laboratory cultures.
198
ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM
THE ICHTYOTOXIC DICTYOCHOPHYTE
PSEUDOCHATTONELLA FARCIMEN
Ingvild Riisberg1, Simon M. Dittami1, Uwe John2, Russel Orr1, Kamran Shalchian-Tabrizi1,
Kjetill S. Jakobsen1, Bente Edvardsen1
1
2
Department of Biology, University of Oslo, P.O. Box 1066 Blindern, N-0316 Oslo, Norway. E-mail: [email protected]
Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany
Pseudochattonella farcimen (Eikrem, Edvardsen, et Throndsen) is an ichthyotoxic alga within the Dictyochophyceae (Heterokonta), which
has been shown to form blooms in Scandinavian waters every year since 1998. To improve our understanding of the biology of this alga
and to facilitate future genomic studies, we report the sequencing and analysis of >10,000 expressed sequence tags (ESTs) corresponding
to 8149 unique gene models from this species. A direct comparison with EST libraries from other heterokonta and haptophytes revealed
several functional categories to be significantly overrepresented among the P. facimen ESTs, such as genes involved in cell communication,
transporters, or genes targeted to cell organelles. Interestingly, P. farcimen possesses three putative polyketide synthases (PKSs),
probably belonging to two different families. Polyketides are structurally highly diverse and can exhibit a vast variety of functions such as
antibiotics and toxins. We also found a high proportion (1.4%) of proteins related to fatty acid metabolism, and counted eight fatty acid
desaturases and two phospholipase A2 genes. Three of the desaturases belong to a family of delta-4 desaturases, known so far only
from haptophytes, where they catalyze the conversion of n3-docosapentaenoic (n3-DPA) acid to docosahexaenoic acid (DHA). These
findings may partially explain the unusual fatty acid profiles observed in P. farcimen and are discussed both from an evolutionary point of
view and in relation the ichthyotoxic effects of this alga.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 199
GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
MORPHOLOGICAL VARIABILITY, TOXINOLOGY
AND GENETICS OF THE POTENCIAL LIPOPHILIC
TOXIN-PRODUCER DINOPHYSIS TRIPOS
Francisco Rodríguez, Laura Escalera, Beatriz Reguera, Pilar Rial, Pilar Riobó
Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Cabo Estai, Canido, 36200 Vigo, Spain.
E-mail: [email protected]
Dinophysis tripos (Gourret, 1883) exhibits a marked morphological variability and his small and intermediate forms may get confused with
those from Dinophysis caudata when their blooms co-occur. Sequence of its ribosomal DNA (LSU) place it, with D. caudata, in a clade
separated from the other larger clade that includes all mixotrophic species of Dinophysis. D. tripos has been included in the list of toxinproducing Dinophysis spp. on the basis of one single HPLC analyses with fluorescence detection. Here we examined morphological,
toxinological and genetic characteristics of D. tripos from: i) Field populations; ii) Single cell isolates; iii) Laboratory cultures of D. tripos
fed on Myrionecta rubra. During late autumn 2009, D. tripos was the dominant Dinophysis species in the Galician Rías Baixas (NW
Iberia) and there were no harvesting closures associated with its occurrence. OA derivatives and pectenotoxins were under detection
levels in LC-MS analyses of single cell isolates, net hauls rich on this species and laboratory cultures. Our results suggest that the Galician
strains of D. tripos are not toxin-producers and emphasize the need to re-examine, with last generation analytical techniques, the checklist
of toxin-producing Dinophysis spp. in different parts of the world.
199
RESTING STAGES IN THE GENUS BYSMATRUM
Cecilia Teodora Satta1, Silvia Anglès2, Esther Garcés2, Antonella Lugliè1, Nicola Sechi1, Jordi Camp2
1
Dipartimento di Scienze Botaniche, Ecologiche e Geologiche, Università di Sassari, Via Piandanna 4, 07100 Sassari, Italy.
E-mail: [email protected]
2
Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, Barcelona, Spain
The genus Bysmatrum Faust & Steidinger was erected in 1998 for three peridinoid red tide forming dinoflagellates previously placed in
the genus Scrippsiella and Peridinium. Other two species were later described, B. teres and B. granulosum. This genus includes typical
benthic sand-dwelling species that occur in tide pools and in sandy sediments. They are also epibiotic on macroalgae or corals. Bysmatrum
species are reported from warm temperate, tropical and subtropical areas. Species belonging to this genus have not been reported so
far in the Mediterranean basin. Nowadays, the life cycle of these species is not described and reproduction type is not well known, as
well as any data on genetic aspect are available. Cultures established from two different cyst morphotypes isolated from the sediment of
Fangar Bay (Ebre Delta, NW Mediterranean Sea) were attributed to a species of the genus Bysmatrum. The vegetative cells are 32.239.7 μm long and 32.2-39.7 μm wide. They show the typical plate pattern of the genus (Po, X, 4', 3a, 7'', 6c, 4s, 5''', 2'''') with the
second and third anterior intercalary plates not in contact with one another. First phylogenetic data for this genus were obtained. The different morphology of the two progenitor morphotypes suggests the possibility of different sexual routes in the life cycle of this species.
9. ABSTRACT BOOK (177-203)_Layout 1 10/22/10 2:37 PM Page 200
THE DISCOVERY OF PUTATIVE GENES FOR SAXITOXIN
SYNTHESIS IN DINOFLAGELLATES
Anke Stüken1, Russell J. S. Orr1, Ralf Kellmann2, Troco K. Mihali3, Brett A. Neilan3, Kjetill S. Jakobsen4
1
Microbial Evolution Research Group (MERG), Department of Biology, University of Oslo, P.O. Box 1066 Blindern,
316 Oslo, Norway. E-mail: [email protected]
2
Department of Molecular Biology, University of Bergen, P.O. Box 7803, 5020 Bergen, Norway
3
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Kensington,
Sydney, NSW 2052, Australia
4
Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo,
P.O. Box 1066 Blindern, 0316 Oslo, Norway
Saxitoxin (STX) is a potent neurotoxin, which occurs in fresh and marine waters worldwide. The toxin blocks sodium channels of nerve
cells, and may lead to paralysis and death. In freshwaters, STX is produced by several species of cyanobacteria; in marine waters, it is
thought to be produced by certain species of dinoflagellates. The genetic basis for STX synthesis has been characterised in several
species of cyanobacteria, but remains unknown in dinoflagellates. Alexandrium fundyense and A. minutum are two prominent STX producing dinoflagellate species. We have generated one cDNA library of each species using 454 sequencing technology. Each library contains >500,000 reads, an average read length of 350 bp and GC content of 57%. From these datasets, we have identified STX candidate
genes. Using PCR and Sanger sequencing, we have amplified these genes from a range of dinoflagellate strains, using genomic DNA
as well as cDNA as template. Here we will present our results on the identity and diversity of STX genes in dinoflagellates and compare
them to the STX genes in cyanobacteria.
200
THE DINOFLAGELLATE GENUS AZADINIUM - AN UPDATE
Urban Tillmann1, Malte Elbrächter1, Uwe John1, Bernd Krock1, Philip Messtorff1, Rafael Salas2, Michael Schweikert3
1
Alfred Wegener Institute, Bremerhaven, Germany
Marine Institute, Galway, Ireland
3
Biological Institute, Uni Stuttgart, Germany
2
Azaspiracids (AZAs) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin associated with human incidents of shellfish poisoning. Although knowledge of the toxins involved has been continually increasing, the biological source remained
elusive until the culprit for AZA production was recently unambiguously identified and described as a novel species within a newly created
genus Azadinium Elbrächter et Tillmann The toxigenic type species, a small photosynthetic dinoflagellate, was described as Azadinium
spinosum Elbrächter & Tillmann. Stimulated by this finding, further isolation of Azadinium-like species was performed so that currently
seven strains of Azadinium are available, belonging to three different species: A. spinosum (3 strains), A. obesum Tillmann & Elbrächter
(1 strain), and a third species not yet officially described (3 strains). Among these strains and species, only A. spinosum, available as
isolates from Scotland, Denmark and Ireland, turned out to produce azaspiracids, whereas all isolates of the other two species are nontoxic. We will present an update on the current status of knowledge on the genus Azadinium including various aspects of toxin profile,
morphology, phylogeny and ultrastructure.
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GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
DETECTION OF AND DISCRIMINATION WITHIN
THE NOVEL DINOFLAGELLATE GENUS AZADINIUM,
THE SOURCE OF AZASPIRACID TOXINS
Kerstin Töbe, Aboli Joshi, Urban Tillmann, Allan D. Cembella, Uwe John
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany.
E-mail: [email protected]
The dinoflagellate genus Azadinium Elbrächter et Tillmann comprises toxigenic and non-toxic members. Thus far, seven Azadinium
strains have been identified, and affiliated to three newly described species. These Azadinium representatives are difficult to differentiate
by traditional morphological analysis and the location of the genus with the dinoflagellate phylogeny also remains uncertain. We
therefore developed genus- and species-specific primers and TaqMAN MGB probes for a quantitative polymerase chain-reaction
(qPCR) assay. These primer and probes were successfully tested on DNA from cultured Azadinium species and are being applied to
natural plankton samples from the North Sea coast of Scotland and from along the west coast of Denmark. Additionally, we developed
fluorescence in situ hybridisation (FISH) probes to visualize Azadinium cells in field experiments and biogeographical distribution studies.
Here we present the first data generated from these field samples analysed with the Azadinium-specific primers and probes for qPCR
and FISH, respectively.
201
GENETIC DIVERSITY OF THE LUCIFERASE GENE
IN DINOFLAGELLATES
Martha Valiadi1, Ana Amorim2, M. Debora Iglesias-Rodriguez1
1
National Oceanography Centre, School of Ocean and Earth Science, University of Southampton, European Way,
Southampton SO14 3ZH, UK. E-mail: [email protected]
2
Centro de Oceanografia, Faculdade de Ciencias, Universidade de Lisboa, 1749-016 Lisboa, Portugal
Luciferase represents a family of proteins that are responsible for the biological production of light, a phenomenon termed bioluminescence.
Bioluminescence has been reported in several dinoflagellate species including some that are responsible for harmful algal blooms. Bioluminescence is thought to have an ecological significance as an anti-predation strategy and could play a role in bloom dynamics. While
some dinoflagellates produce visible light, others produce low levels of light invisible to the naked eye. Additionally, there are intraspecific
variations in the magnitude of cellular bioluminescence. Therefore, elucidating which dinoflagellate species carry the luciferase gene can
be used as an indicator of bioluminescence potential, which cannot be assessed based solely on optical measurements of either cultures
or mixed field populations. We tested 42 dinoflagellate species belonging to 5 orders for bioluminescence and for the presence of the
luciferase gene using a novel set of PCR primers designed to target several loci of the gene. To assess intraspecific variation, we have
also tested multiple strains of selected representatives of both bioluminescent and non bioluminescent species. We report novel partial
sequences of luciferase from several species and discuss the diversity of the luciferase gene in an evolutionary context.
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INTRA REGIONAL VARIATION AMONG ALEXANDRIUM
CATENELLA STRAINS FROM SOUTHERN OF CHILE:
MORPHOLOGICAL, TOXICOLOGICAL AND MOLECULAR
DIFFERENCES
Daniel Varela1, Javier Paredes1, Catharina Alves de Souza1, Miriam Seguel2, Andrea Sfeir2, Máximo Frangopulos3
1
Centro “i~mar”, Universidad de Los Lagos, Casilla 557, Puerto Montt, Chile. E-mail: [email protected]
Centro CERAM, Universidad Austral de Chile, Casilla 1327, Puerto Montt, Chile
3
Fundación CEQUA, Casilla 737, Punta Arenas, Chile
2
In Chile, Alexandrium catenella have been recorded during the last three decades, affecting the human healthy and marine resources.
This specie was first reported in Magellan’s region in 1972 and since that has been associated to recurrent toxic blooms, spreading progressively northward into channels and fjords of Chilean’s austral region (from 52o S to 42o S), and showing geographic differences in
both seasonal dynamic and toxicity level along this range. In this context, we combined a morphologic, toxic and molecular analysis of A.
catenella strains collected along this southern distribution. Morphologic analysis illustrated traits variation inside every strain, observing diacritic characters according the species definition but also others conferred to another species from the same genera. Through toxic
analysis, the strains showed important difference among them, mainly at toxin concentration and toxicity. While rDNA (partial LSU and
ITS) sequences analysis confirmed the Chilean strains as part of the Clade I (North American) of Alexandrium tamarense species complex,
but also showed a significant diversity among the strains. Thus, this intra regional variability could be discussed as result of environmental
heterogeneity and as an explanation, at least in part, for geographic differences in the outbreak associated to this specie in southern of
Chile. Financial support: Fondecyt No 1080548.
202
GENETIC DIVERSITY OF PSEUDO-NITZSCHIA BRASILIANA
(BACILLARIOPHYCEAE): A NEW RECORD SPECIES IN CHINA
Peng Wang1, Junrong Liang1, Xin Lin1, Changping Chen1, Yahui Gao1, King-Chung Ho2
1
Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems and Sch. Life Sciences
Xiamen University, Xiamen 361005, China. E-mail: [email protected]
2
Sch. Science & Technology, Open University of Hong Kong, Hong Kong SAR, China
Diatom Pseudo-nitzschia brasiliana Lundholm, Hasle & Fryxell is documented in China for the first time. Strains isolated from China
southeast coastal waters were characterized on the observation of light and electronic microscope. All strains were also tested for domoic
acid (DA) and no toxin was detected. The genetic diversity and phylogenetic relationships of different strains of P. brasiliana were assessed.
The LSU phylogenetic analyses together with morphological comparison strongly supported the fact that P. brasiliana isolated from China
and other locations of the world constituted one monophyletic clade, with P. americana placed as its sister taxon. Phylogenetic analyses
of the ITS rDNA sequences showed a clear separation between the five Asian strains and the three European strains. The ITS rDNA
secondary structure analyses making an impressive indicator of geogenetic variation. We identified one hemi-CBC in the loop of the
Asian group changing to the basis of helix I in the European group, which made an obvious difference in topological structures. Between
ICMB-176 and the other strains, there were two hemi-CBCs in helix III. Only One hemi-CBC was found between the Hongkong strains
and other Asian strains. The findings from this work reveal the distribution of P. brasiliana in China coastal waters and further provide
clues to our understanding of this species in terms of genetic diversity. This study was supported by the NSFF (No.2008J0139) and the
NSFC (Nos. 40831160519, 40676080).
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GENOMICS AND GENETIC DIVERSITY OF HABS
POSTER PRESENTATIONS
COMPARATIVE PROTEOMIC STUDIES
IN ALEXANDRIUM CATENELLA DH01 GROWN
UNDER DIFFERENT NUTRIENT CONDITIONS
Wei-Jie Zhang, Cheng Li, Hua-Sheng Hong, Da-Zhi Wang
State Key Laboratory of Marine Environmental Science / Environmental Science Research Center, Xiamen University,
182 Daxue Road, 361005 Xiamen, People’s Republic of China. E-mail: [email protected]
Alexandrium catenella DH01 is a key harmful algal blooms (HABs) species, widely spread along the coastal waters of China. This study
investigated differential expressed proteins of A. catenella DH01 grown under various nutrient conditions using 2D-PAGE and MALDITOF-TOF mass spectrometer. The results showed that a higher expression level of 27 proteins, such as sigma-54-dependent transcriptional regulator, cytidylate kinase, nitrogenase molybdenum-iron cofactor biosynthesis protein, was found in nitrogen-starved growth, but
their abundances decreased rapidly when nitrate was added to the culture medium. While 16 proteins including plastid protein NAP50,
chloroplast light harvesting complex protein were found to have a higher level of expression in nitrogen-replete growth. In phosphatestarved growth, 16 proteins involved in phosphate uptake (i.e. phosphate ABC transporter, periplasmic phosphate-binding protein) and
other physiological functions presented a higher level of expression, but their abundances were reduced when phosphate was added to
the phosphate-starved growth. While 3 proteins, putative transposase, Acyl-coenzyme A synthetases and AMP-(fatty) acid ligases were
found to be present in a higher amount in phosphate-replete growth. These results indicate that proteomic response of A. catenella to
nutrient variations is fast, and specific proteins highly expressed under different nutrient conditions might be involved in nutrient stress
adaptation or nutrient metabolisms, which might be used as potential indicators for different nutrient status. This work is a first step in the
dissection of the behavior of the dinoflagellate proteome under various nutrient conditions, and may provide new insights into the relationship
between dinoflagellate physiology and ambient nutrient status.
203
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 204
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 205
TOXINS:
CHEMICAL STRUCTURE
AND SYNTHESIS,
DETECTION AND
ANALYTICAL METHODS
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 206
ORAL PRESENTATIONS
LC-MS/MS ANALYSIS OF SCOTTISH BIVALVES DURING
AN UNPRECEDENTED LIPOPHILIC TOXINS EVENT
AND CORRELATION WITH PHYTOPLANKTON MONITORING
Clothilde Brunet1, Rebecca Kelly1, Keith Davidson2, Steve Morris1
1
Centre for Environment, Fisheries and Aquaculture Science (Cefas), The Nothe, Barrack Road, Weymouth,
Dorset DT4 8UB, UK. E-mail: [email protected]
2
Scottish Association for Marine Science (SAMS), Scottish Marine Institute, Oban, Argyll PA37 1QA, UK
In May 2009, a sample of common mussels (Mytilus sp.) collected for the Scottish official control monitoring program in a sea loch to
the North of the Clyde sea, returned a positive result for DSP by the biological assay using the method prescribed by the UK national reference laboratory. This result led to the closure of the production area. Harvesting restrictions remained in place for 16 consecutive
weeks. Quantitative LC-MS/MS analysis revealed that lipophilic toxin concentrations in the sample greatly exceeded the regulatory limit.
For investigative purposes, instrumental analysis was extended to include 69 samples in total collected on a weekly basis between 5th
May and 8th September 2009 and incorporated common mussels and Pacific oysters (Crassostrea gigas) both produced at the same
site, allowing inter-species comparison. Mussels from 3 neighbouring production areas affected by their own harvesting closures were
included to assess the wider geographical impact of the toxic event. The results highlighted the prevalence of two toxin groups: the
classical DSP toxins (okadaic acid, dinophysistoxins and acylated esters) and the yessotoxins. The toxin concentrations recorded during
this study were unprecedented in Scotland and reached level greater than 37 times the European regulatory level. Drawing on the data
from the official control phytoplankton monitoring program for samples collected in the vicinity of the shellfish, correlations were sought
between dinoflagellate (Dinophysis spp., Protoceratium reticulatum) cell densities and toxin concentrations. The results from the chemical
analysis will be presented. The phytoplankton and lipophilic toxins situation in the area will be discussed.
206
SUITABILITY OF PASSIVE SAMPLERS FOR TRACKING
OF DISSOLVED CIGUATOXINS. APPLICATION
TO GAMBIERDISCUS PACIFICUS IN CULTURE
Amandine Caillaud1, Pablo de la Iglesia1, Helena Eixarch1, Esther Barber1,
Normawaty Mohammad-Noor2, Jorge Diogène1
1
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou km 5.5, Sant Carles de la Ràpita, Tarragona, Spain.
E-mail: [email protected]
2
Borneo Marine Research Institute, University Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia
The suitability of the solid-phase absorption toxin tracking (SPATT) devices filled with the HP20 Diaion® resin for tracking of dissolved
Pacific type 1 CTX (P-CTX-1B) standard was evaluated. The in vitro ouabain/veratridine dependent neuroblastoma (Neuro-2a) cell-based
assay (CBA) was used as a sensitive and selective detection method for CTX-like activity. Excellent recoveries ranging from 85 (± 13.2)
to 90 (± 6.4) % were found at different times of resin exposure tested (at 24, 72 and 120 hours). SPATT was further applied to a 10Lculture of Gambierdiscus pacificus in different phases of growth. The Neuro-2a CBA for CTXs allowed the detection of CTX-like activity
in both particulate (cell filters) and dissolved (resins) fractions of G. pacificus culture. Additionally, qualitative and quantitative determination
of the different CTXs congeners was performed by liquid chromatography coupled with tandem mass spectrometry. Both methods agreed
identifying the highest content of dissolved CTXs during the stationary and decay phases of the culture. Interestingly, the profile of dissolved
CTXs congeners found in the culture medium (hydroxyl derivates of the CTX-3C in high proportion) differed from that found in G. pacificus
cells (CTX-3C and CTX4A/4B only in filters). Further LC-fractionations followed by CBA are currently ongoing to confirm the toxicity of
each congener. These results demonstrate the suitability of SPATT devices for CTXs monitoring. This tool may be of great interest either
to perform physiological studies on Gambierdiscus spp. or to be applied as a warning method for ciguatera in a given area.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 207
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
COMPLEX PALITOXIN-LIKE PROFILE OF OSTREOPSIS
OVATA. IDENTIFICATION OF FOUR NEW OVATOXINS
BY HIGH RESOLUTION LIQUID CHROMATOGRAPHY
-MASS SPECTROMETRY
Patrizia Ciminiello1, Carmela Dell’Aversano1, Emma Dello Iacovo1, Ernesto Fattorusso1, Martino Forino1,
Laura Grauso1, Luciana Tartaglione1, Franca Guerrini2, Laura Pezzolesi2, Rossella Pistocchi2
1
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, Via D. Montesano 49,
80131 Naples, Italy. E-mail: [email protected]
2
Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Università di Bologna,Via S’Alberto 163, 48100 Ravenna, Italy
In the last decades, the Italian coastlines have been plagued by the recurring presence of the benthic dinoflagellate Ostreopsis ovata.
Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous
studies evidenced the presence of ovatoxin-a, as the major toxin of the algal toxin profile together with small amounts of putative palytoxin.
In our ongoing research on O. ovata toxins we report herein on high resolution LC-MS and MS2 investigation of an O. ovata culture,
which revealed an algal toxin profile more complex than previously found. Particularly, the presence of putative palytoxin and ovatoxin-a
was confirmed and the occurrence in the extract of four new palytoxin-like compounds, ovatoxin-b, -c, -d, and -e, was highlighted. Elemental formulae very close to palytoxin’s have been assigned to the new ovatoxins and information has been gained about their structural
features. A quantitative study of the O. ovata culture extract indicated that ovatoxin-a represented almost 54% of the total toxin content
with the remaining 46% being constituted by the whole of ovatoxin-b, -c, -d and -e. Thus, the presence of the new ovatoxins should be
taken into account when LC-MS based monitoring programs of either plankton or contaminated seafood are carried out.
207
LIPOPHILIC MARINE TOXIN ANALYSIS BY LIQUID
CHROMATOGRAPHY TANDEM MASS SPECTROMETRY
Arjen Gerssen, Patrick P. J. Mulder, Hester J. van den Top, Hans van Egmond
RIKILT - Institute of Food Safety Wageningen UR, 2 Akkermaalsbos, 6708 WB, Wageningen, The Netherlands.
E-mail: [email protected]
Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish. Consumption of contaminated
shellfish can lead to severe intoxications and cause symptoms such as diarrhea, abdominal cramps and vomiting. European Union (EU)
legislation prescribes the use of a mouse or rat bioassay to test for the presence of these toxins. These assays are unethical and have a
poor sensitivity and selectivity. For this reason there is an urgent need for alternative methods. Most promising alternatives are the methods
based on liquid chromatography - tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method has been developed using alkaline
chromatographic conditions by which the toxins stated in legislation can be analysed in a single run. A solid phase extraction (SPE) clean
up procedure was developed to reduce the amount of matrix effects (ion suppression and enhancement) that can occur during LCMS/MS analysis. Combination of SPE clean up and alkaline chromatographic conditions resulted in significantly reduced matrix effects
for all matrices tested (mussel, scallop and oyster). The developed analytical method was in-house validated at regulatory limits using EU
Commission Decision 2002/657/EC as guideline. The method performed very well with respect to accuracy, repeatability, reproducibility,
decision limit, specificity and ruggedness. The method also performed excellent with respect to the lower limits proposed by European
Food Safety Authority (EFSA) for okadaic acid, dinophysistoxins and azaspiracid toxins and in a pre-validation study. Currently, a collaborative study is ongoing for the eight most important toxins of the lipophilic marine toxin group.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 208
AN INVESTIGATION INTO SOLID PHASE ADSORPTION
TOXIN TRACKING (SPATT) FOR THE DETECTION
OF DOMOIC ACID, THE AMNESIC SHELLFISH POISON,
IN SEAWATER
Guillaume Hermann1, Jean-Pierre Lacaze1, Laura Morley1, Guido Drago2, Evelyn Keady3,
Ana Cabado4, Matt Gubbins1, Elizabeth Turrell1
1
Marine Scotland Science, 375 Victoria Road, Aberdeen, AB11 9DB, UK. E-mail: [email protected]
2
Applied Enzyme Technology Ltd., Monmouth House, Pontypool, NP4 0HZ, UK
3
The National Diagnostics Centre, National University of Ireland, Galway
4
ANFACO-CECOPESCA, Campus Univ 16, 36310 Vigo, Pontevedra, Spain
Previously proposed as a tool to facilitate monitoring of lipophilic shellfish toxins (LSTs) in shellfish harvesting areas, Solid-phase adsorption
toxin tracking (SPATT) was assessed for its capability to adsorb and recover Domoic Acid, an amnesic shellfish toxin (AST) responsible
for many site closures in Scottish waters. Lab trials were performed to assess different potential candidate adsorbents before field studies
were undertaken at aquacultures sites in Scottish Waters (Loch Ewe). SPATT bags containing SP700 resin beads were deployed on a
weekly basis in conjunction with sampling of phytoplankton and local bivalve shellfish. During four sampling sessions through the summer
of 2009, an automated water sampler was deployed in the loch, flushing SPATT cartridges containing the same SP700 resin daily and
collecting a water sample for phytoplankton identification. SPATT extracts were analysed by two different quantitative methods of analysis:
by LC-MS/MS and by ELISA. Although no direct correlation could be drawn between the concentration of the algal toxins in SPATT extracts and the phytoplankton community in water samples, significant variation of toxin concentration could be observed between sampling
periods. Moreover, the finding of DA in the SPATT cartridge shows that the resin can adsorb DA sufficiently to allow subsequent extraction
and analysis. Results suggest that the use of SPATT cartridges may be employed as an early warning technology to inform shellfish harvesting strategies and potentially as a tool to improve understanding of AST events in Scottish waters.
208
NOVEL TOXINS PRODUCED BY THE DINOFLAGELLATE
KARENIA BREVISULCATA
Patrick Holland1, Feng Shi1,2, Masayuki Satake3, Yuka Hamamoto3, Veronica Beuzenberg1,
Lesley Rhodes1, Paul McNabb1, Rex Munday4, Emiko Ito5, Patrick Edwards6
1
Cawthron Institute, Private Bag 2, Nelson 7010, New Zealand. E-mail: [email protected]
2
Lincoln University, Lincoln, Canterbury 7647, New Zealand
3
Department of Chemistry, the University of Tokyo, Tokyo 113-0033, Japan
4
AgResearch, Ruakura Research Centre, Hamilton 3240, New Zealand
5
Chiba University, Chiba 260-8673, Japan
6
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
Karenia brevisulcata (Chang), a new toxic dinoflagellate of the genus Karenia, was isolated from a harmful algal bloom that occurred in
Wellington Harbour, New Zealand in 1998. This bloom affected most marine biota and caused long-term ecological damage. It also
caused respiratory distress in harbour bystanders. Cultures of K. brevisulcata produced a range of novel toxins including 6-8 lipid-soluble
Karenia brevisulcata toxins (KBTs) and 6 more water-soluble brevisulcatic acids (BSXs). Brevetoxins have not been detected. Small
quantities of KBT-F, KBT-G, BSX-1 and BSX-2 were isolated and purified from bulk cultures. Preliminary chemical and toxicological investigations show that KBT-F and -G are complex polyethers with m.w. 2054 / 2084, UVmax at 232nm and LD50 (mouse i.p.) 0.032
/ 0.040 mg/kg b.w. respectively. Proton NMR indicated the presence of two doublet and singlet methyls, an aldehyde, and a conjugated
double bond. BSX-1 and BSX-2 gave m.w. 916 / 872 (C49H72O16 / C47H68O15) and LD50 (mouse i.p.) 3.7 / 1.8 mg/kg b.w. respectively.
The structures are dicarboxylates without conjugated double bonds (UVmax 196nm) and the proton NMR show polyether rings with
similarities to A-ring opened brevetoxin-1. They are accompanied by BSX-4 and BSX-5 which are the lactone ring closed analogues. A
range of chemical and bioassay data will be reported for experiments directed at elucidating the structures and mechanisms of action for
these potent novel compounds.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
ORAL PRESENTATIONS
CERTIFICATION OF A REFERENCE MATERIAL
FOR MULTIPLE GROUPS OF SHELLFISH TOXINS
Pearse McCarron1,2, Sabrina Giddings1, Elliott Wright1, Ruth Perez1, Håkan Emteborg3, Cíara Nulty2,
Katharina Teipel3, Thomas Rundberget4, Jared I. Loader5,*, Christopher O. Miles4,5, Philipp Hess2,*, Michael A. Quilliam1
1
National Research Council Canada, Institute for Marine Biosciences, 1411 Oxford St, Halifax, Nova Scotia B3H 3Z1, Canada.
E-mail: [email protected]
2
Marine Institute, Rinville, Oranmore, Galway, Ireland; *current address: Ifremer, Rue de l’Île d’Yeu, Nantes, Cedex 03, France
3
European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg 111,
2440 Geel, Belgium
4
National Veterinary Institute, P.O. Box 750 Sentrum, NO-0106 Oslo, Norway
5
AgResearch Ltd., Private Bag 3123, Hamilton 3240, New Zealand; *current address: Hill Laboratories Ltd,
Private Bag 3205, Hamilton 3240, New Zealand
Significant progress in LC-MS methodology has enabled rapid analysis of shellfish for a wide variety of toxins. This alternative to traditional
mouse bioassay testing has become even more important due to recent changes in EU legislation, which now considers LC-MS as the
reference method. Certified reference materials (CRMs) are necessary for the development, validation and quality control of analytical
methods. Up to this point, CRMs have been available only for single toxin groups. A recent collaboration between several international
organisations on the production, characterisation and certification of a multi-toxin-group CRM will be discussed in this presentation. A
freeze-dried mussel tissue (Mytilus edulis) reference material has been prepared containing domoic acid, okadaic acid, dinophysistoxins,
azaspiracids, pectenotoxins, yessotoxins and spirolides. The homogeneity of the material was satisfactory, and stability studies revealed
no degradation of any toxin at temperatures up to +20οC over a one year period. Techniques have been implemented for accurate quantitation of the various toxins groups present. Based on this extensive work, certified values and associated uncertainties have been
assigned for the regulated toxins present in the CRM. This material, CRM-FDMT-1, will be useful for laboratories to assess complete analytical methods from sample extraction through to data processing and for ultimately verifying trueness of analysis.
209
A MASS SPECTROMETRY STUDY OF THE FRAGMENTATION
OF MICROCYSTINS IN NEGATIVE ION ELECTROSPRAY
Ernani Pinto1, Diogo de Oliveira Silva2, Dietrich Volmer3, Felipe Augusto Dörr1
Universidade de São Paulo, Av Professor Lineu Prestes, 580 - 05508-900, São Paulo - SP, Brazil. E-mail: [email protected]
Universidade Federal de São Paulo, Rua Prof. Artur Riedel, 275 - 09972-270, Diadema, SP, Brazil. E-mail: [email protected]
3
Universität des Saarlandes, Institut D-66123 Saarbrücken, Germany. E-mail: [email protected]
1
2
Microcystins (MC) are natural hepatotoxins characterized by seven-amino acid membered peptide ring system that are found in toxic
cyanobacteria. Demethylated variants (dm-MC) are relatively common and the amino acids dehydroalanine (Dha7) or dehydrobutyrine
(Dhb7) might be presented, instead of N-methyldehydroalaline (Mdha); or D-aspartic acid (Asp3) instead of D-erythro-β-methylaspartic
acid (D-MeAsp). In a conventional water/organic solvent system, without any other additives, deprotonated molecules of MC ([M H]-) may appear in negative mode electrospray mass spectrometry (ESI-MS) because of the presence of acidic functional groups.
They are not well-suited for collision-induced decomposition (CID) experiments in negative mode electrospray since its product ion
scan gives few detectable fragments. However, the fragmentation pattern is more stable than in the positive mode and the mechanisms
can be more easily understood. In this study, the fragmentation pattern of both MC and dm-MC were tested using ESI-MS in negative
mode either by Iontrap or QTOF. The detailed fragmentation mechanisms are discussed and diagnostic product ions are proposed
for the most abundant MC variants (MC-LR and MC-RR) as well as their desmethylated analogues (dm-MC-LR and dm-MC-RR).
Our results suggested that ESI-MSn in negative mode can be an alternative and powerful strategy to correctly assign the absence of
methyl groups in MC analogues.
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KARLOTOXINS - THEIR STRUCTURE,
MODE OF ACTION AND ECOLOGICAL FUNCTION
Allen R. Place
Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Sciences, Baltimore, MD, USA
The karlotoxins, like the amphidinols produced by dinoflagellates of the genus Amphidinium, have a hairpin-like structure with three distinct
regions: a polyol arm that exhibits variable hydroxylation and methylation; a hinge region containing two ether rings; and a lipophilic arm
that often includes conjugated trienes in amphidinols but instead in karlotoxins contains a terminal diene that gives these compounds
their distinctive UV spectra. We have determined the structures for the toxins first described in 1957 by Abbott and Ballantine for Gymnodinium veneficum (Latin veneficum - poisonous, bearer of venom). Two toxins were isolated, KmTx 4 and 59-E-chloro KmTx 4, which
exhibited both a shorter polyol arm and a shorter lipophilic arm compared to structure we have described for KmTx 2. We also determined
the structures for the karlotoxins found in the Chesapeake Bay, KmTx 1 and KmTx 3. KmTx 3 differs from KmTx 1 in having one less
methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 and 10-O-sulfo-KmTx 3 were identified
as well as 65-E-chloro-KmTx 1 and 10-O-sulfo-KmTx 1. Comparison of the hemolytic activities and sterol binding affinities of the newly
isolated compounds shows that potency and sterol binding affinity correlate positively with the length of the lipophilic arm and is disrupted
by sulfonation of the polyol arm. Further electrophysiological studies find that both amphidinol and karlotoxin share the ability to form
non-specific pores in biological membranes. These toxins appear to be targeted to desmethylsterols, like brassicasterol found in their
prey, and interact poorly with 4α methyl sterols containing a 8(14) double bond.
210
FATE AND EFFECT OF THREE COMMON
BREVETOXIN METABOLITES IN MICE
John S. Ramsdell1, Tod A. Leighfield1, Marie-Yasmine Dechraoui Bottein1, Noah Muha1, Zhihong Wang1,
Jennifer Maucher Fuquay1, Rex Munday2, Andrew I. Selwood3, Roel van Ginkel3, Jared I. Loader2,
Alistair L. Wilkins4, Christopher O. Miles2,4
1
NOAA-National Ocean Service, Charleston, SC 29412 USA. E-mail: [email protected]
2
National Veterinary Institute, Postboks 750 Sentrum, 0106, Oslo, Norway
3
Cawthron Institute, Private Bag 2, Nelson, New Zealand
4
AgResearch Ltd., Private Bag 3123, Hamilton 3240, New Zealand;
*current address: Hill Laboratories Ltd, Private Bag 3205, Hamilton 3240, New Zealand
Brevetoxin B (BTX-B) is a highly reactive molecule, due in part to a α,β-unsaturated aldehyde group at the terminal side chain, leading to
metabolites by reduction, oxidation and conjugation. We have investigated the fate and effects in mice of three common brevetoxin metabolites
found in shellfish, semi-synthesized from BTX-B in both stable and radioactive forms. BTX-B was reduced at C42 to yield dihydro-BTX-B.
BTX-B was conjugated to cysteine at the C50 olefinic group with S-oxidation and C42 aldehyde reduction to yield BTX-B2, which was further
modified by fatty acid addition via the cysteine-amide linkage to give N-palmitoyl-BTX-B2. Analysis of each metabolite by mouse N2A neuroblastoma assay revealed N-palmitoyl-BTX-B2 as the most potent brevetoxin, 12-fold > dihydro-BTX-B, and this corresponded well to OECD
Guideline 425 mouse bioassay where it was 19-fold > dihydro-BTX-B. However, N-palmitoyl-BTX-B2 was not well recognized by immunological
methods: 23-53-fold < dihydro-BTX-B by three different immunoassays. Intravenous administration of radiolabeled metabolites best fit two
phase elimination in which N-palmitoyl-BTX-B2 eliminated slower than other metabolites from the peripherial compartment. Distribution over
48-hr indicated that N-palmitoyl-BTX-B2 accumulated in liver 100-fold > the other brevetoxin metabolites, whereas B2 accumulated in kidney
nearly 10-fold > the other metabolites. These results indicate that brevetoxin metabolism can increase toxic potency more than an order of
magnitude, increasing both toxic activity at the level of brain cells as well as retention and distribution of toxin in the animal. Discrepancies in
immunoassay vs. mouse bioassay for N-palmitoyl-BTX-B2 may have significant regulatory and surveillance implications.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
ORAL PRESENTATIONS
VALIDATION STUDY OF THE “EU-HARMONISED
STANDARD OPERATING PROCEDURE FOR
DETERMINATION OF OA-GROUP TOXINS BY LC-MS/MS”
María L. Rodríguez-Velasco1, Adriano Villar-González1, Ana Gago-Martínez1,2
European Union Reference Laboratory for Marine Biotoxins (EU-RL-MB), Agencia Española de Seguridad Alimentaria
y Nutrición (AESAN), Estación Marítima S/N, Muelle de Trasatlánticos, Vigo, Spain. E-mail: [email protected]
2
Departamento de Química Analítica y Alimentaria, Universidad de Vigo, Campus Universitario Lagoas, Marcosende,
36310 Vigo, Spain
1
Different studies have been conducted for the simultaneous determination of lipophilic toxins by LC-MS within the framework of activities of
the Working Group (WG) on Liquid Chromatography-Mass Spectrometry of the European network of National Reference Laboratories (NRL)
for Marine Biotoxins. These studies showed important drawbacks, mostly associated with the use of a single and fixed protocol for this analysis.
In order to overcome these drawbacks, the WG agreed on taking the approach of validating methods independently for single group of toxins,
using a protocol with a fixed sample extraction and leaving open some other conditions (clean-up, concentration, LC and MS) and fulfilling
the agreed quality criteria. According to this approach, an Interlaboratory Validation Study for the Okadaic acid-group was carried out, and parameters such as accuracy, repeatability and between-laboratory reproducibility were evaluated for the method described in the EU-Harmonised
Standard Operating Procedure for determination of Okadaic Acid group in molluscs, using liquid chromatography coupled with tandem mass
spectrometry (LC-MS/MS). Twelve laboratories among ten European countries participated in the study, which involved the quantitative determination of free and total OA-group toxins: okadaic acid (OA), dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2). The results obtained
for Interlaboratory reproducibility (RSDR %) were between 12.6% and 34.9% for OA determination, with HorRat values between 0.49 and
1.57. In the case of DTX1, RSDR varied from 17.8% (HorRat =0.67) and 20.6% (HorRat =0.84). For DTX2 determination, the variability
was 43.4% (HorRat = 1.98). A detailed discussion of the results obtained in this study will be presented in this work.
211
PINNATOXINS AND SPIROLIDES IN NORWEGIAN BLUE
MUSSELS AND SEA WATER
Thomas Rundberget1, John A. B. Aasen2, Andrew I. Selwood3, Christopher O. Miles1
1
National Veterinary Institute, Department of Feed and Food Safety, PB 750 Sentrum, 0106 Oslo, Norway.
E-mail: [email protected]
2
Norwegian School of Veterinary Science, Department of Food Safety and Infection Biology, P. O. Box 8146,
Dep. 0033 Oslo, Norway
3
Cawthron Institute, Private Bag 2, Nelson, New Zealand
Pinnatoxins and pteriatoxins, members of the fast-acting cyclic imine group of algal toxins, were first identified in Japanese Pinna and Pteria
spp. after food poisoning in China linked to Pinna spp. Recently, new and known pinnatoxins were identified in shellfish, sediment, and
seawater from Australia and New Zealand. Although the structurally related spirolide toxins are better known and have a worldwide distribution
including Europe, pinnatoxins have not been reported in European waters. We surveyed shellfish for pinnatoxins and spirolides, by LC-MS/MS
analysis of extracts from Norway’s routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides
and pinnatoxin G were widespread; pinnatoxin G, spirolide C, 13-desmethylspirolide C, and 20-methylspirolide G were detected in 69%,
56%, 22%, and 77%, respectively, of the 166 shellfish samples. Although levels were generally low, concentrations of up to 115 μg/kg of
pinnatoxin G and 226 μg/kg of 13-desmethylspirolide C were detected. Similarly, pinnatoxin G, spirolide C, 13-desmethylspirolide C, and
20-methylspirolide G were detected in 100%, 35%, 96%, and 100% of seawater extracts obtained from 57 passive adsorption disks from
Norway in 2007. This suggests that pinnatoxins and spirolides are more widespread than previously thought, and that they or related compounds
could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of the toxin levels is unclear, but preliminary data indicates pinnatoxins to be more toxic to mice than spirolides by the
oral route, despite being less toxic by i.p. administration.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 212
ORAL PRESENTATIONS
LIPOPHILIC TOXINS AND PSP-TOXIN PRODUCING SPECIES
IN PHYTOPLANKTON FROM THE SOUTH EASTERN
PACIFIC, CHILE, REVEAL AN INCREASE IN TOXICITY
Nicole Trefault1, Nathalie Delherbe1, Blanca Pérez1, Bernd Krock2, Allan Cembella2, Mónica Vásquez1
Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas,
Pontificia Universidad Católica de Chile, Santiago, Chile. E-mail: [email protected]
2
Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany
1
Harmful algal blooms associated with potent toxins have increased in the last decades. In the southernmost waters of Chile most toxin
outbreaks come from Paralytic Shellfish Poisoning (PSP) produced by the recurrent and persistant dinoflagellate Alexandrium catenella, however there is only partial information about lipophilic toxins throughout the coastal waters of this country. We analyze the lipophilic toxin profile
and determine the genetic diversity and toxin analogues variation among clones of A. catenella isolated since 2008 from the most austral
waters of Chile. Our results show a great variety of lipophilic toxins, including azaspiracid and gymnodimine-1, previously undetected toxins in
Chile and in the eastern South Pacific. Sequencing analysis of the D1-D2 hypervariable region of LSU rDNA from the new isolated clones
of A. catenella showed no substantial divergence. Analysis of the PSP toxin composition and content of the new austral clones, however, revealed large differences between the existing clones and those previously isolated from Chile. On a per cell basis, the new clones of A.
catenella are nearly seven times more toxic than the Aysén (1994) clones, and exhibit an unusual toxin profile that stands out for the presence
of the most toxic analogues, saxitoxin and neosaxitoxin. These results indicate that the presence of phytoplanktonic toxins is a widespread
and growing phenomenon, whose danger is increasing, considering both the appearance of lipophilic toxins and the emergence of clones
from toxic dinoflagellates with the most potent PSP toxin analogues.
212
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
RECOMBINANT IMMONUCONJUGUATES
FOR THE DETECTION OF MICROCYSTINS
Sihem Aboulkacem, Aurélie Ledreux, Philippe Billiald
FRE 3206 CNRS / MNHN Molécules de Communication et Adaptation des Micro-organismes, USM 505 Cyanobactéries,
Cyanotoxines et Environnement, Muséum National d’Histoire Naturelle, 57 rue Cuvier, CP 39, 75231 Paris, Cedex 05, France.
E-mail: [email protected]
Microcystins represent a large family of hepatotoxins frequently encountered in freshwater ecosystems. The worldwide occurrence of
these toxins have raised the development of a vast choice of methods for their detection, but most of them are time-consuming and
require extensive sample processing. In that context, molecular engeneering of antibodies represent a mean to develop specific probes
with enhanced properties compared to conventional antibodies. Here we synthesized a gene encoding a recombinant antibody fragment
(scFv) using nucleotidic sequences coding for antibody 3A8 variable regions which is directed against MC-LR and cross-react with
related MCs (McElhiney et al., 2002). This gene was then inserted into a prokaryotic expression vector so as to produce a scFv fused
to the bacterial alcaline phosphatase. The expression of the fusion protein was optimized. It was fully functional and preserved MCLR binding and alcaline phosphatase activities. Such a genetic approach offers many advantages over chemical cross-linking of antibodies to colorimetric enzymes and may be adaptable to the detection of MCs in water samples. In addition, this genetic construction
could be used as a building-block to generate novel scFvs for which reactivity with MCs may be altered in order to be adapted to the
analysis of MCs diversity.
213
CHARACTERIZATION OF BREVETOXIN METABOLITES
IN CLAM (MERCENARIA SP.)
Ann Abraham, Kathleen R. El Said, Yuesong Wang, Edward L. E. Jester, Jennifer Hooe-Rollman,
Hudson R. Granade, Steven M. Plakas
Gulf Coast Seafood Laboratory, FDA, Dauphin Island, AL 36528, USA. E-mail: [email protected]
Brevetoxins produced by the marine dinoflagellate Karenia brevis are accumulated and extensively metabolized in filter-feeding molluscan
shellfish. Consumption of brevetoxin-contaminated shellfish causes neurotoxic shellfish poisoning (NSP). Here we report characterization
of brevetoxin metabolism in the hard clam (Mercenaria sp.). Brevetoxins in K. brevis bloom-exposed clams were examined using LCMS/MS, ELISA, and N2a neuroblastoma cell (cytotoxicity) assay. The principal brevetoxin of K. brevis (BTX-2, MH+: m/z 895) was not
detected in clam. Several brevetoxin metabolites were identified using LC-MS/MS. Major metabolites included products of oxidation/reduction and amino acid/peptide conjugation. Cysteine conjugates of BTX-2 (MH+: m/z 1018) and BTX-1 (MH+: m/z 990) were present
in abundance. We also identified taurine conjugates of oxidized BTX-1 and BTX-2. BTX-B5 (MH+: m/z 911), the oxidation product of
BTX-B2, along with its open A-ring analog (MH+: m/z 929), were also found in abundance. Oxidation represents a significant metabolic
pathway for brevetoxins in clams. Glutathione and related peptide conjugates of BTX-1 and BTX-2, as previously identified in the Eastern
oyster, were present in clam. Several brevetoxin-fatty acid conjugates were characterized structurally and found to be significant contributors
to the composite sodium channel-specific cytotoxicity.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 214
JELLETT RAPID TESTS UTILIZED AS THE PRIMARY PSP
TOXIN DETECTION METHOD FOR HARMFUL ALGAL
BLOOMS (HABS) IN UAE COASTAL WATER DURING 2009
Rashid Al Shihi, Ebrahim Al Jamali, Mohammad Alzarouni, Ali Aldhanhany
Marine Environment Research Centre, Ministry of Environment and Water, P.O. Box 21, Umm Al Quwain, UAE.
E-mail: [email protected]
Harmful algal blooms (HABs) are uncommon events in UAE coastal waters. Recently, however, a harmful red tide of the dinoflagellate
Cochlodinium polykrikoides occurred in the Arabian Gulf and the Sea of Oman from August 2008 to May 2009. This bloom had ecological, social, economical and food safety impact and unforeseen consequence on public health, marine environmental resources,
fishery industries, tourism industries and desalination plants. In the latter instance, a number of plants had to cease operations during
the red tide because of the high algal biomass that clooged filters and otherwise altered the efficiency of the desalination process.
During the Cochlodinium bloom, several other toxic HAB species were identified, including Gymnodinium catenatum, Pyrodinium bahamense, Dinophysis spp., and Pseudo-nitzschia spp. Given these observations, it was clear that a monitoring program for PSP, DSP,
and ASP toxins was needed for the region. Here we describe a study to assess PSP toxins detection in shellfish samples collected
from UAE coastal waters subsequent to the 2008/2009 bloom. Jellett Rapid Tests were used as a primary assay for PSP toxicity.
This method provides reliable, consistent, rapid results at a reasonable cost. No PSP toxins were measured during the study interval.
Thus this work serves as example of the use of simple screening methods as early wearing of HAB toxicity in regions with limited
analytical expertise for direct toxin measurement. Once positive results are observed, samples will then be sent to certified analytical
laboratories for confirmation. In areas such as the UAE, with infrequent HABS and no history of toxicity in shellfish, these Rapid Tests
offer a good measure of protection at a reasonable cost.
214
STRATEGIES FOR NEW PALYTOXIN DETECTION METHODS
Amparo Alfonso1, Beatriz Caramés1, Mª José Pazos2, Mercedes R. Vieytes3, Luis M. Botana1
Departamento de Farmacología, Facultad de Veterinaria, USC, 27002 Lugo, Spain. E-mail: [email protected]
Unidad de Microscopía Electrónica y Confocal, USC, 27002 Lugo, Spain
3
Departamento de Fisiología, Facultad de Veterinaria, USC, 27002 Lugo, Spain
1
2
Palytoxin, produced by dinoflagellates from the genus Ostreopsis but discovered, isolated and purified from marine soft coral belonging
to the genus Palythoa, is one of the most potent non-protein animal toxins. Its mechanism of action is related with the binding to the
Na+,K+-ATPase. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In addition
calcium fluxes and intracellular pH are also modified. These effects are inhibited by the cardiac glycoside ouabain. In this work we
develop two detection methods for palytoxin based on its binding with the Na+,K+-ATPase. In these methods, changes in fluorescence
polarization (FP) units and in the signal from a surface plasmon resonance (SPR)-based biosensor are measured. In the FP method,
Na+,K+-ATPase is labeled with a reactive succinimidyl-esther of carboxy-fluorescein, and changes in polarization degree of emitted
light are measured in the presence and absence of toxin. The signal obtained (FP units) decreases when toxin concentration increases
following a linear regression. In the second method the interaction of palytoxin over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in a BiaCore X biosensor and checking the binding signal K(obs) (observed rate constant).
These two new and specific palytoxin detection methods are fast, repetitive, easy to use, and highly sensible since toxic samples in
the range 20-500 nM can be quantified.
Funded by VIIth FP CP2008-1-555612 (Atlantox), 2009-1/117 (Pharmatlantic).
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 215
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
ADVANCE TESTS FOR NEW AND EMERGING TOXINS
IN THE ATLANTIC AREA: THE INTERREG FUNDED
PROJECT ATLANTOX
Luis M Botana1, Chris Elliott2, Cowan Higgins3, Ambrose Furey4, Vitor Vasconcelos5,
Jordi Molgo6, Ana G. Cabado7, Jorge Lago7
1
University of Santiago de Compestela, Spain
2
IAFLU, Queen’s University, Belfast, Ireland
3
Agri-Food and Biosciences Institute, Belfast, Ireland. E-mail: [email protected]
4
Cork Institute of Technology, Ireland
5
CIIMAR, Portugal
6
CNRS, France
7
ANFACO, Spain
The effects of climate change and global temperature rises are imminent and marine ecosystems are under the effects produced by
fluctuation in water temperature which is responsible for the production of favourable conditions for the growth of toxin-producing
phytoplankton. There is in place legislation to cover a number of toxins already identified and for which detection methods are available.
However, within the Atlantic area, toxins new to the region have been detected and it is important that methods are available to detect
emerging toxins to protect the valuable aquaculture regions of Galicia, Ireland and France. Whilst the detection of ciguatera toxin, palytoxin, tetrodotoxin and cyclic immines in the Atlantic region may at present be isolated occurrences, it may reflect a changing profile
in marine toxins. It is important that the aquaculture industry and food safety authorities have available methods to detect these new
toxins. The ATLANTOX project will develop rapid, reliable, non-animal based methodologies to detect both currently legislated toxins
and new and emerging ones. The project will establish a collaborative network for the collection and analysis of samples of marine
toxins and will promote the exchange of knowledge through mobility of researchers in the Atlantic area. The main components of the
project are, the collection and analysis of samples, the development of rapid anti-body based methods and functional assays, the prevalidation of developed methods and exploitation of the tests by both the aquaculture industry and food safety authorities.
215
COMPARATIVE STUDY OF THE MAITOTOXIN-LIKE
AND CIGUATOXIN-LIKE TOXICITY IN GAMBIERDISCUS SPP.
FROM DISTINCT GEOGRAPHICAL ORIGIN
Amandine Caillaud1, Santiago Fraga2, Katerina Aligizaki3, Normawaty Mohammad-Noor4, Jorge Diogène1
1
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou km 5.5, Sant Carles de la Ràpita, Tarragona, Spain.
E-mail: [email protected]
2
Centro Oceanográfico de Vigo, IEO (Instituto Español de Oceanografía), Subida a Radio Faro 50, 36390 Vigo, Spain
3
School of Biology, Faculty of Sciences, Aristotle University, 54 124 Thessaloniki, Greece
4
Borneo Marine Research Institute, University Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia
Marine dinoflagellates of the genus Gambierdiscus are potent producers of various toxins i.e ciguatoxins (CTXs) and maitotoxins (MTXs);
the CTXs being responsible for the occurrence of the ciguatera fish poisoning (CFP). In the present study, various strains of Gambierdiscus
spp., i.e. G. pacificus from Malaysia (strains GDSA01, GPSi, G10DC), Gambierdiscus sp. from Indonesia (strains VGO917, VGO920),
from Canary Island (strains VGO790, VGO791 and VGO792) and from Crete (strain KC81) have been examined for their CTX and MTX
production. The Neuroblastoma (Neuro-2a) cell-based assays with ouabain and veratridine pretreatment and SK&F 96365 pretreatment
were respectively used for the detection of the CTX-like and MTX-like toxicity in the crude extracts of the different Gambierdiscus strains.
CTX-like toxicity was detected in all the strains of Gambierdiscus spp. studied with estimations of the CTX-like compounds ranging from
1.1 to 1.7x10-3 pg CTX-1 eq cell-1. The higher CTX-like toxicity was measured for Gambierdiscus sp. from Canary Island. MTX-like toxicity
was detected in all the strains of Gambierdiscus spp. from Canary Island, Malaysia and Indonesia. MTX-like content was ranging from 138
ng MTX eq cell-1 for Gambierdiscus sp. from Canary Island (strain VGO790), to 1.72 ng MTX eq cell-1. The exceptionally elevated MTXlike production by Gambierdiscus sp. from Canary Island and the lack of MTX-like production by Gambierdiscus sp. from Crete may be
used as chemo-taxonomical characteristic to differentiate these two species. The production of CTX-like compounds by Gambierdiscus sp.
from Canary Island may explain the occurrence of recent CFP evoked in that area.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 216
MULTIDISCIPLINARY APPROACH APPLIED TO THE STUDY
OF SUSPICIOUS HARMFUL ALGAE: FIRST DETECTION
OF OKADAIC ACID PRODUCTION IN PROROCENTRUM
RHATHYMUM
Amandine Caillaud1, Pablo de la Iglesia1, Mònica Campàs1, Laurence Elandaloussi1,
Margarita Fernández-Tejedor1, Normawaty Mohammad-Noor2, Karl Andree1, Jorge Diogène1
1
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou km 5.5, Sant Carles de la Ràpita, Tarragona, Spain.
E-mail: [email protected]
2
Borneo Marine Research Institute, University Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia
The application of a multidisciplinary approach combining toxicology, biochemical and chemical analysis may be of great interest for the study of
suspicious harmful algae species. As an example, we present the first report that evidenced okadaic acid (OA) production by the species Prorocentrum rhathymum. The neuroblastoma Neuro-2a cell-based assay (CBA) has the capability to measure quantitatively cytotoxic effects of marine
toxins such as OA and its derivatives using a simple cell viability measurement. Toxic fractions were identified in P. rhathymum extracts. The protein
phosphatase inhibition assay (PPIA) is based on the colorimetric detection of the enzymatic activity, which is known to decrease in the presence of
lipophilic diarrheic toxins. The assay allowed the discrimination of fractions able to inhibit protein phosphatase, indicating the presence of OA or a
derivative with inhibition potency. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method allowed unambiguous identification
of the OA based on the chromatographic retention time and selective analysis of the corresponding molecular and product ions. Okadaic acid
standard curves by PPIA and Neuro-2a CBA allowed a quantitative estimation of 8.3 and 4.7 ng OA equivalent in 1×106 P. rhathymum cells, respectively. LC-MS/MS estimation was 2.9 ng OA equivalent in 1×106 cells. In addition, the use of a multidisciplinary approach may be synergistic
to investigate the presence of new toxins or analogues, or even bioactive compounds produced by microalgae. In our case, polar fractions of P.
rhathymum showed activation of PP2A associated to a decrease in Neuro-2a cell viability, which is currently under study.
216
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 217
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
KNOWLEDGE TRANSFER NETWORK FOR PREVENTION
OF MENTAL DISEASES AND CANCER
IN THE ATLANTIC AREA (PHARMATLANTIC)
Maria Jose Chapela1, Luis M. Botana2, Begona Espina2, Martina Ferreira1, Jorge Lago1, Juan M. Vieites1,
Vitor Vasconcelos3, Chris Elliott4, Jordi Molgo5, Ambrose Furey6, Custodia Fonseca7, Richard Kennedy8,
Cowan Higgins9, Marcel Jaspars10, Ana G. Cabado1
1
Microbiology and Biotoxins Area, ANFACO-CECOPESCA, Campus Univ. Vigo, 36210 Vigo, Spain.
E-mail: [email protected]
2
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
3
LEGE/CIIMAR/CIMAR_LA, Laboratory of Ecotoxicology, Genomic and Evolution, Centre of Environmental
and Marine Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal
4
The Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5AG,
Northern Ireland, UK
5
CNRS, Institut Federatif de Neurobiologie Alfred Fessard FRC2118, Laboratoire de Neurobiologie Cellulaire
et Moleculaire, 91198 Gif-sur-Yvette Cedex, France
6
PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research, Department of Chemistry,
NCork Institute of Technology, Bishopstown, Cork, Ireland
7
Faculdade de Ciencias e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8000-117 Faro, Portugal
8
Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland
9
Agri-Food and Biosciences Institute, Belfast, Ireland. E-mail: [email protected]
10
Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Old Aberdeen AB24 3UE, Scotland, UK
217
Climate change on the Atlantic Area is responsible for the settlement of favourable ecological conditions for marine compounds reproduction because marine organisms develop unique metabolic and physiological capabilities to be able to survive in such new
habitats. These sometimes negative effects can be taken in a positive way because marine compounds can be studied by research
centres of the Atlantic Area as new chemicals that should be used by enterprises to be benefited from such research in the future
(chemistry, pharmacology, environmental, maritime, fishing, etc). PHARMATLANTIC is a 36 months EU project promoted by the
Pharmacology Department of University of Santiago de Compostela (Veterinary Faculty). It was created on the necessity to provide
involved industries situated in the Atlantic seaboard with research and innovation advances in prevention of mental diseases and
cancer already developed by the most specialized centres in this field at EU level. A partnership of 10 EU institutions from Spain
(University of Santiago de Compostela and ANFACO-CECOPESCA), Portugal (CIIMAR and University of Algarve), France (Centre
National de la Recherche Scientifique), United Kingdom (Queen’s University Belfast and Agri-Food & Biosciences Institute) and
Ireland (Cork Institute of Technology and Dublin City University) and the main research centres, universities and industries are represented in this project. The main objective of PHARMATLANTIC is to build a stable network of knowledge transfer between researchers and industries by developing a joint method for the use and exploitation of marine resources through previous research
in order to further transfer to the enterprise from involved sectors.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 218
VALIDATION OF A REVERSED-PHASE HPLC/FD
METHOD FOR Β-METHYLAMINO-L-ALANINE
QUANTITATIVE ANALYSIS USING METHANOL ELUTION
INSTEAD OF ACETONITRILE
R. C. C. Cianca1,2, M. S. Baptista1, V. R. Lopes1,3, V. M. Vasconcelos1,3
CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, R. dos Bragas 289,
4050-123 Porto, Portugal. E-mail: [email protected]
2
Departamento de Biología Funcional y Ciencias de la Salud, Facultad de Biología, Campus Universitario de Vigo,
Universidad de Vigo, 36200 Vigo, Spain
3
FCUP, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
1
Diverse species of cyanobacteria have recently been discovered to produce the neurotoxic non-protein amino acid β-methylamino-Lalanine (BMAA). BMAA has been suggested as an important environmental factor for neurodegenerative disease such as amyotrophic
lateral sclerosis- Parkinsonism dementia complex (ALS/PDC) in Guam. Different methods have been described for BMAA determination.
In this study we have developed and validated a method for detection of BMAA in a cyanobacteria sample. The method includes protein
hydrolysis, AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) derivatization, and phase reverse methanol elution in HPLC/FD
system. Acetonitrile is the most widely used organic HPLC solvent. Since October 2008, the worldwide supply of acetonitrile was low
and continued through early 2009. We switched of the mobile phase from acetonitrile to methanol. We consider that elution strength
differs with acetonitrile and methanol. The required validation parameters, i.e., linearity, repeatability, specificity, precision, limit of detection,
and limit of quantification, were studied for detection of BMAA in lyophilized estuarine specie of Nostoc. The method can be employed
as an absolute quantification method for BMAA determination.
218
RAPID RESOLUTION LC-MS/MS FOR THE ANALYSIS
OF ASP TOXINS IN SHELLFISH: SINGLE LABORATORY
VALIDATION AND COMPARISON WITH LC-UV
Pablo de la Iglesia1, Esther Barber1, Gemma Giménez1, María Luisa Rodríguez-Velasco2,
Adriano Villar-González2, Jorge Diogène1
1
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou km 5.5, Sant Carles de la Ràpita, Tarragona,
Spain. E-mail: [email protected]
2
European Union Reference Laboratory for Marine Biotoxins (EU-RL-MB), Agencia Española de Seguridad Alimentaria
y Nutrición (AESAN), Estación Marítima S/N, Muelle de Trasatlánticos, Vigo, Spain
We present the application of rapid resolution liquid chromatography on a 1.8-μm particle-size column coupled with tandem mass spectrometry
(RRLC-MS/MS) for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the
isomers was achieved in less than 3 min. The method was intra-laboratory validated for direct analysis of crude extracts without further clean-up.
With this aim, six different shellfish matrices with relevancy for the aquaculture industry were considered. The method showed limits of detection
ranging from 0.05-0.09 mg/kg and a working range including the current maximum permitted level of 20 mg/kg in European Union and also
the level recently recommended by the European Food Safety Authority (EFSA) of 4.5 mg/kg. The averages of RSD found within the same
sample matrix were: 11%, 9%, 8%, for mussel, pacific oyster and clam, respectively; and 7% for smooth clam, wedge shell and the muricid
gastropod. Additionally, confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results
obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intra-laboratory for the analysis of blind samples
(n = 22; R2 = 0.9751) and inter-laboratory through the participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and
0.177 for low and high contaminated samples, respectively). In conclusion, RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrices.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 219
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
MECHANICAL AND IMMUNOAFFINITY EXTRACTION
OF PARALYTIC SHELLFISH POISONING (PSP) TOXINS
FROM CULTURES OF THE DINOFLAGELLATE
ALEXANDRIUM TAMARENSE
Raymond Devlin, Katrina Campbell, Christopher T. Elliott
Institute of Agri-Food and Land Use, Queen’s University of Belfast, David Keir Building, Stranmillis Road, BT9 5AG,
Belfast, Northern Ireland. E-mail: [email protected]
Paralytic shellfish poisoning (PSP) is the longest known and most widespread on a worldwide basis of the shellfish poisoning illnesses.
The extraction of PSP toxins from a toxic strain of the marine dinoflagellate Alexandrium tamarense CCMP-1493 with PBS buffer as an
alternate extraction solvent using various mechanical and/or physical procedures was investigated. The methods used for cell lysis and
toxin extraction included freeze-thawing, freeze-boiling, steel ball-bearing beating, glass bead beating, and water bath sonication. The
steel ball-bearing beating was determined to release a similar amount of toxin when compared to a modified reported standard extraction
method which was reported to release 100% of toxins from the algal cells and was therefore used in the next phase of the study. This
was to determine the feasibility of utilising an antibody coupled to novel magnetic microspheres (Ferrospheres-N) as a simple extraction
procedure for PSP toxins from a toxic strain of algae. The affects of increasing mass of Ferrospheres-N on the immuno-capture of the
PSP toxins from the toxic algal strain extracts were investigated. Toxic recovery was found to increase when an increasing mass of Ferrospheres-N was used until close to 100% of the toxin extracted from the cells was captured and eluted. Toxin recovery was determined
by comparison to an appropriate PSP toxin standard curve following analysis by the AOAC HPLC method.
219
DETERMINATION OF OKADAIC ACID ACYL-DERIVATIVE
AND DINOPHYSISTOXIN-3 IN MUSSEL SAMPLES
COLLECTED IN CHILOE ISLAND, SOUTHERN CHILE
Carlos García, Cristóbal Contreras, Christian Costa, Cindy Costa, Eric Tapia, Néstor Lagos
Laboratorio Bioquímica de Membrana, Facultad de Medicina, Universidad de Chile, Santiago, Chile. Grant FONDECYT 1090058.
E-mail: [email protected]
This work shows the detection of Diarrhetic Shellfish Poison phycotoxins, using HPLC-FLD with pre-column derivatization procedure
and HPLC-MS methods, in the analysis of shellfish extracts tested positive with the official DSP mouse bioassay. The shellfish samples
were collected in Chiloe Island, Southern of Chile. The amount of Dinophysistoxin-3 measured in the shellfish extracts were in average
above the international safe limits for DSP content in the shellfish extracts analyzed. From all DSP toxins analyzed, the hydrolyzed extract
samples appeared mainly DTX- 3 in concentrations ranging from 99.40 ± 1.22 to 257.73 ± 12.46 ng/g digestive-glands. The acylOkadaic Acid (acyl-OA) was also detected in some samples, ranging from 1.32 ± 0.9 to 3.07. ± 1.6 ng of DSP toxin/g digestiveglands. This is the first report of acyl-OA ever found in Chilean shellfish samples. This data shows that shellfish samples were contaminated
with a complex DSP toxins profile, in which DTX-3 is the major DSP toxin component, followed by DTX-1 and the acyl-OA as the minor
one. The important findings showed in this study are the presence of both acyl-derivates (DTX-3 and Acyl-OA) which are the product of
a main metabolic biotransformation that occurred inside the shellfish, in order to chelate DTX- 1 and OA, transforming them into DTX3 and the acyl-OA respectively. This complex DSP toxins profile and the permanent presence of both acyl-derivates could explain the
permanent diarrhea symptoms that experience patients who have ingested cooked shellfish in the southern of Chile.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 220
SCREENING OF LIPOPHILIC MARINE TOXINS USING
LIQUID CHROMATOGRAPHY COUPLED TO ORBITRAP
MASS SPECTROMETRY
Arjen Gerssen1, Patrick P. J. Mulder1, Jacob de Boer2
1
RIKILT, Institute of Food Safety Wageningen UR, 2 Akkermaalsbos, 6708 WB, Wageningen, The Netherlands.
E-mail: [email protected]
2
Institute for Environmental Studies, VU University, 1087 de Boelelaan, 1081 HV, Amsterdam, The Netherlands
Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the
analysis of the toxins that are stated in legislation. However, to date over 200 lipophilic marine toxins have been described in the literature.
To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100 000) for lipophilic marine toxins
has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin
(PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were analysed with and without
fragmentation. Based on their mass and specific fragmentation pattern, a large set of toxins was identified. A major complication of full
scan HRMS is the huge amount of data generated, which restricts the possibility of a fast search. The software program metAlign was
used to reduce the orbitrap MS data files 200-fold. The reduced data files were screened using ‘Search_LCMS’ an additional software
tool for metAlign. A search library was constructed for the identified toxins. An important feature of the library is that it can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested
by analysing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. For each of
the contaminated samples a toxin profile was determined.
220
EVALUATION OF CURRENT EUROPEAN OFFICIAL
CONTROL METHODS FOR THE DETECTION OF MARINE
BIOTOXINS IN THE GASTROPOD LITTORINA LITTEREA
(EDIBLE PERIWINKLE)
Cowan Higgins, Brenda Morrow
UK National Reference Laboratory for Marine Biotoxins, Agri-Food and Biosciences Institute, Belfast, Northern Ireland.
E-mail: [email protected]
European regulation EC 854/2004 requires that bivalve molluscs, gastropods and echinoderms are monitoring for the presence of marine
biotoxins as part of the official controls on animal produce intended for human consumption. Whether monitoring is undertaken on classified
production areas or through end-product testing, the official control methods are likely to be the methods used. There is limited data on
the applicability of current official methods of control on the analysis of species other than bivalve molluscs. Within the UK, the edible periwinkle is the most significant species of gastropod harvested both commercially and by the public, for human consumption. The current
study evaluated the recovery of periwinkle tissue from the shell by mechanical extraction, freezing and boiling. The suitability of the tissue
extracts for analysis for the presence of Paralytic Shellfish Poison (PSP), Diarrhetic Shellfish Poison (DSP) and Amnesic Shellfish Poison
(ASP) by the appropriate official control methods was evaluated. Periwinkle extracts produced no adverse clinical signs in either the PSP
or DSP mouse bioassays. HPLC analysis with UV detection for ASP showed no interfering peaks at the retention time for domoic acid or
epi-domoic acid. HPLC analysis (AOAC 2005.06) showed the potential for interfering peaks at the retention times of C1,2 but no other
interfering peaks were observed. Preliminary investigations into recovery of the analytical methods indicate that the methods are fit for purpose, however further data is required to establish if enhancement or suppression of the signal is an issue in the quantitative methods.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 221
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
DEVELOPMENT OF A RAPID LATERAL FLOW
SCREENING METHOD FOR DETECTION OF DOMOIC
ACID IN SHELLFISH
Waqass Jawaid1, Andrew B. Allan2, Julie Meneely1, Katrina Campbell1, Christopher T. Elliott1
1
2
Institute of Agri-Food and Land Use, Queen’s University, Belfast, UK. E-mail: [email protected].
Neogen Europe Ltd, Ayr, Scotland, UK
Amnesic shellfish poisoning (ASP) causing toxins are produced mainly by the toxigenic diatom strain Pseudo-nitzchia multiseries.
The ASP toxins include domoic acid (DA), as well as associated compounds such as epi-domoic acid. The major goal of the current
study was to develop a qualitative lateral flow device (LFD) to detect the ASP marine biotoxin DA in shellfish extracts. A rapid single-step LFD was developed based on a competitive immunoassay format, involving the immobilisation of toxin-protein conjugates
onto the nitrocellulose membrane. Polyclonal antibodies specific for DA, conjugated to coloured particles, resulted in a visible signal.
A rapid and simple sample preparation procedure was also developed. The developed LFDs were optimized for visual detection at a
cut-off value of approximately 10 mg DA/kg, i.e. half the EU regulatory limit, in shellfish meat. Inter- and intra- laboratory evaluations
demonstrate that DA can be detected at the intended levels from a variety of spiked and naturally contaminated shellfish matrices.
Further data will be presented at the conference.
221
FUNCTION ASSAYS OF LIPOPHILIC MARINE TOXINS
ACTING ON VOLTAGE-SENSITIVE NA+ CHANNELS
Satoshi Kawatake1, Kazuya Murata2, Takeshi Yasumoto1
1
Okinawa Science and Technology Promotion Center, 12-75 Suzaki, Uruma-shi, Okinawa, Japan.
E-mail: [email protected]
2
School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan
The ciguatoxins (CTXs) and brevetoxins (BTXs) are the causative toxins for CFP and NSP respectively. Both toxins bind to the same site
of the voltage-sensitive Na+ channels, implying the possibly of competitive binding assay for each other. Previous method using [3H]BTXB is though efficient, inconvenient because of the radioactivity. Chemiluminescent acridinium-BTXB2 ensured high sensitivity but
errors due to non-specific binding were significant. To improve the performance of the assays, we tested differently labeled BTXB or
BTXB2 for their suitability and partially purified channel protein to increase sensitivity. The results obtained with biotinylated or fluorescence
labeled BTXBs will be presented.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:39 PM Page 222
BIOCHEMICAL CHARACTERISATION OF THE UNUSUAL
POLYKETIDE SYNTHASE SXTA THAT INITIATES
SAXITOXIN BIOSYNTHESIS
Ralf Kellmann1, Helene M. Svendsen1, K. S. Jakobsen2
1
2
Department of Molecular Biology, University of Bergen, P.O. Box 7803, 5020 Bergen, E-mail: [email protected]
Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo,
P.O. Box 1066 Blindern, 0316 Oslo, Norway
Saxitoxin and its analogues are produced from arginine, acetate, methionine, and carbamoyl phosphate by a complex and unique biosynthesis pathway. We have recently uncovered the molecular genetic basis for saxitoxin biosynthesis in cyanobacteria, and identified corresponding genes in dinoflagellates. The saxitoxin biosynthesis (sxt) gene cluster encoded a type I polyketide synthase, SxtA, which had
an unusual domain structure consisting of four domains related to methyltransferase, GCN-5 related acetyl transferase, acyl carrier
protein, and 8-amino-7-oxononanoate synthase. The molecular genetic data in conjunction with intermediate metabolite analysis suggested
that SxtA initiates saxitoxin biosynthesis in three consecutive steps, where the acyl carrier protein is loaded with acetate by the GCN-5
acetyltransferase domain. The acyl carrier protein-bound acetate is then methylated by the methyltransferase domain, and the resulting
propionate is Claisen-condensed to arginine by the 8-amino-7-oxononanoate synthase domain. To conceptually verify the function of
SxtA, we have cloned and expressed sxtA in Escherichia coli BAP1 using the pET expression system. Enzymatic activity of purified SxtA
was investigated by measuring the consumption of substrates with HPLC.
222
THE ISOLATION OF AZASPIRACIDS FROM HIGHLY
CONTAMINATED SHELLFISH (MYTILUS EDULIS)
Jane Kilcoyne1, Ger Clancy1, Adela Keogh1, Conor Duffy1, Patricia LeBlanc2, Ian Burton2,
Mike A. Quilliam2, Christopher O. Miles3, Philipp Hess4
1
Marine Institute, Renville, Oranmore, Co. Galway, Ireland. E-mail: [email protected]
National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, Canada
3
National Veterinary Institute, Postboks 750 Sentrum, 0106, Oslo, Norway
4
IFREMER, 21105 Rue de l’Ile d’Yeu, Nantes, France
2
Azaspiracids (AZAs) pose a serious problem to the Irish shellfish industry with outbreaks occurring almost every year since they were first
detected in 1995. The AZA group includes more than 32 analogues. However, only AZA1, -2 and -3 are regulated by the EU. The EU
reference method for the detection of AZAs in shellfish is the mouse bioassay. Efforts are now focusing on replacing the mouse bioassay
with LCMSMS, which gives more reliable results and has no ethical associations. Certified standards for AZA1, -2, -3 are currently available
(in limited supplies) for global distribution. However, with increasing occurrences of toxic episodes worldwide, in addition to the forthcoming
replacement of the mouse bioassay with LCMSMS methods, the demand for such standards will be high. Furthermore, as the mode of
action of AZAs is still unknown, the availability of purified materials for toxicological studies is imperative. To date only AZA1-5 have been
isolated, characterised and studied for their toxicological effects. Here we describe the isolation of AZAs using a 7 step procedure initially
developed in Japan which was modified to increase recovery and purity. The material used for these batches was collected from the North
of Ireland which experienced an AZA outbreak in 2005 with AZA levels as high as 46 times above the regulatory limit. Isolation batches,
using contaminated M. edulis tissue, were performed using a series of partitioning and column chromatography steps. Recoveries and
purities were assessed by LCMSMS and NMR. In addition to AZA1, -2 and -3 several other AZA analogues were isolated.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 223
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
APPLICATION OF SOLID PHASE ADSORPTION
TOXIN TRACKING (SPATT) FOR FIELD DETECTION
OF DOMOIC ACID
Jenny Q. Lane1, C. Meiling Roddam1, Gregg W. Langlois2, Raphael M. Kudela1
1
2
University of California Santa Cruz, 1156 High Street, 95064 Santa Cruz, USA. E-mail: [email protected]
California Department of Public Health, 850 Marina Bay Parkway G165, 94804 Richmond, USA
Recent publications have identified the analysis of phycotoxins in sentinel shellfish as a problematic tool for environmental monitoring purposes. Domoic acid (DA), a neurotoxin produced by some species of the diatom Pseudo-nitzschia, can remain undetected in sentinel
shellfish stocks during toxic blooms and subsequent marine bird and mammal mass mortality events. Solid Phase Adsorption Toxin
Tracking (SPATT) has previously been described as a new tool useful for monitoring of lipophilic toxins; our purpose here is to evaluate
the applicability of SPATT for monitoring a hydrophilic phycotoxin, domoic acid. We identify three adsorptive resins for this purpose,
describe a new SPATT manufacturing and processing design, and present results from 24 months of approximately weekly SPATT deployments in Monterey Bay, California (USA). We present the patterns of DA-signaling from SPATT deployments in conjunction with
data from concurrent, traditional monitoring practices (cell counting, sentinel shellfish monitoring, particulate and dissolved toxin analysis)
and with predictive output from Pseudo-nitzschia bloom models previously described for the Monterey Bay region. Toxic Pseudo-nitzschia
bloom events observed in autumn 2009 and spring 2010 are presented as case studies through which we consider the utility of SPATT
under the circumstances of an ‘unusual’ autumnal bloom event (which, as an unusual event, was unanticipated from a regulatory standpoint) and a more ‘classic’ spring bloom event. Under ambient (non-bloom) conditions, all resins detected domoic acid when its presence
was not apparent from traditional monitoring, highlighting the ubiquity of low level or transient toxin events in the environment.
223
DETERMINATION OF TETRODOTOXINS (TTXS)
AND PARALYTIC SHELLFISH POISONING (PSP) TOXINS
IN PUFFER FISH FROM MALAYSIA AND JAPAN
BY MASS SPECTROMETRIC DETECTION
Bernd Luckas, Tina Helbig
Department of Food Chemistry, Institute of Nutrition, Friedrich-Schiller University of Jena, D-07743 Jena, Germany.
E-mail: [email protected]
Paralytic shellfish poisoning (PSP) toxins are produced by marine dinoflagellates as well as by some cyanobacteria, whereas tetrodotoxins
(TTXs) are formed mainly by bacteria. Consequently, both groups of toxins are present in marine and freshwater organisms. Especially,
TTXs are widespread in marine and terrestrial animals, particularly in puffer fish. However, puffer fish can contain both toxin groups.
PSP toxins and TTXs act as potent blockers of sodium channels. Therefore, positive results obtained by application of the mouse bioassay, the official method for the determination of PSP toxins and TTXs, have to be confirmed with regard to the causative toxin group.
The newly developed method for the simultaneous determination of PSP toxins and TTXs is based on the application of hydrophilic interaction liquid chromatography (HILIC) followed by mass spectrometric detection. The chromatographic separation of the PSP toxins
and TTXs in one chromatographic run was achieved using a zwitterionic (ZIC-) HILIC column. Puffer fish of different provenience was
analyzed concerning contamination with TTXs and PSP toxins using this method. Only TTXs were present in tissues of marine puffer
fish Lagocephalus wheelerie from Malaysia, whereas puffer fish from Japan was contaminated with PSP toxins in lower concentrations
in addition to high TTX concentrations.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 224
STUDY ON THE MECHANISMS OF MICE DEATH IN INTRA
PERITONEAL INJECTION OF OKADAIC ACID
Kenji Machii, Hodaka Suzuki
National Institute of Health Sciences, 1-18-1, Kamiyouga Setagaya-ku, Tokyo 158-8501, Japan. E-mail: [email protected]
Diarrhetic shellfish poisoning (DSP) is one of the problems by marine biotoxin that affects human health and makes a large economical
loss. Okadaic acid (OA) is the main toxin that suffers diarrhea. DSP toxins are usually assayed using mouse bioassay (MBA). But recent
social movement to protect animal experimentation is opposing even the animal experiment for food safety. There are some alternative
techniques for detecting DSP toxins, but there exist few method completely alternate the MBA. MBA may be used in most country in
the world for detecting DSP toxin, but precise mechanism of the course of death seems not be revealed. In this study we present several
values of blood test in mice injected with 4 micrograms of OA intra peritoneally. The values such as hematocrit, red blood cell (RBC)
count, white blood cell (WBC) count showed remarkable increase in one hour after the injection. We will further present the values of
blood chemical test.
224
RHYTHMICITY IN PSP-TOXIN SYNTHESIS
IN THE FRESHWATER CYANOBACTERIUM
RAPHIDIOPSIS BROOKII D9
Dinka Mandakovic1, Katia Soto-Liebe1, Alejandra Serrano1, Karina Stucken1,2, Mónica Vásquez1
1
Laboratorio de Ecología Microbiana y Toxicología Ambiental, Departamento de Genética Molecular y Microbiología,
Pontificia Universidad Católica de Chile, 6513492 Santiago, Chile. E-mail: [email protected].
2
Alfred Wegener Institute for Polar and Marine Research, Biosciences Division, Bremerhaven, Germany
Raphidiopsis brookii D9 is a filamentous cyanobacterium producer of paralytic shellfish poisoning (PSP) toxins (STX and its analogues
GTX2/3, dcGTX2/3 and dcSTX). We have used this cyanobacterium as a model to analyze synthesis patterns of these toxins under different culture conditions. In a previous attempt we observed that sodium chloride in the culture media produced a toxin release in a cyclic
way. To establish if daily variations exist in the synthesis of these toxins, we quantified the intracellular concentration of GTX2/3 every 4
hours, grown under continuous light during 24 hours. We observed a variation of the levels of GTX2/3 in cycles of 8 hours. The effect
of a 4 hour darkness pulse, with the purpose of synchronize the cells, was also assayed. By microarray analysis, we measured to 24
hours growth cultures, the expression of sxtE, gene with unknown function that belongs to the toxin synthesis cluster. This gene exhibits
a 1.5 positive fold change expression when toxin synthesis increases (grown under urea 1 mM) and a 1.5 negative fold change expression
when toxin synthesis decreases (grown without fixed nitrogen). Therefore, sxtE evidences variation of its expression in the same way as
toxin synthesis. In this same experiment, we tested the expression of kaiC, gene part of the circadian rhythm master regulator operon.
We observed no changes in gene expression under the two culture conditions. Consequently, we conclude that probably there must be
another rhythm regulator of the toxin synthesis in R. brookii D9.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 225
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
QUANTITATIVE DETERMINATION OF GYMNODIMINE-A
BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
IN CONTAMINATED CLAMS FROM TUNISIA COASTLINE
R. Marrouchi, F. Dziri, N. Belayouni, A. Hamza, E. Benoit, J. Molgo, R. Kharrat
Laboratoire des Toxines Alimentaires, Institut Pasteur de Tunis, B.P. 74, 1002, Tunis-Belvedere, Tunisia.
E-mail: [email protected]
Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin
responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for
extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone,
subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were
performed using a gradient of acetonitrile-water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate
with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland,
respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation
was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated
clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed
that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the
first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantification of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.
225
TOXIN VARIABILITY IN CULTURED AND NATURAL
POPULATIONS OF ALEXANDRIUM FROM SOUTHERN
SOUTH AMERICA - EVIDENCES OF DIVERSITY
AND ENVIRONMENTAL REGULATION
Nora G. Montoya1, Vanesa K. Fulco2, Mario O. Carignan1, Jose I. Carreto1
1
Instituto Nacional de Investigacion y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo 1,
7602HSA-Mar del Plata, Argentina
2
Centro Nacional Patagonico, B. Brown s/n, 9120 Puerto Madryn, Argentina. E-mail: [email protected]
Cell content and composition of paralytic shellfish toxins of 10 cultured strains and 6 natural populations of Alexandrium from the Argentine
sea, were analyzed. These data were compiled with previously published data into a comprehensive view of the toxin composition of the
complex A. tamarense / A. catenella from southern South America. The N-sulfocarbamoyl derivatives C1,2 were predominant in almost
all the cultured strains. The second major derivatives were GTX1,4, although the GTX1,4/C1,2 ratio varied largely. Some strains contain
relatively high amounts of GTX2,3 and/or neoSTX. In all strains STX was a minor component whereas GTX5 was present only in A.
catenella isolates. Similarity analysis based upon toxin profiles showed that cultured strains from Argentine, Brazil, Chile and Uruguay
clustered together. However, whereas some strains from the same geographic area exhibited different toxin profiles, and consistently fell
out in separate subgroups, strains from Chile are grouped in a unique subgroup. In contrast to cultured strains, C1,2 were minor components among field populations. The highly toxic GTX1,4 were predominant in all spring field populations. Moreover, their toxin cell
content and toxicity were several times higher that showed by the cultured strains. Field populations are more closely related to one
another than to the cultured strains. Variability in toxin content and composition of A. tamarense field populations were well correlated
with in situ temperature and nitrate concentration.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 226
TOXIN PROFILES FROM PROROCENTRUM LIMA
AND PROROCENTRUM SP. ISOLATES
FROM BRAZILIAN COASTAL WATERS
Silvia M. Nascimento1, Monika Rapkova-Dhanji2, Steven Morris2
1
Laboratorio de Ecologia e Recursos Marinhos, Universidade Federal do Estado do Rio de Janeiro (UNIRIO),
Av. Pasteur, 458, 22.290-240, Brazil. E-mail: [email protected]
2
Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset, DT4 8UB, UK
Toxin profiles from two strains of Prorocentrum (P. lima and Prorocentrum sp.), isolated from Rio de Janeiro, Brazil, were described
using liquid chromatography mass spectrometry. Cultures were maintained in L-2 medium, at 24±2oC, 12:12 h light/dark cycles and
irradiance of 60 μmol photon flux.m-2 s-1. These were divided prior to harvesting by centrifugation. One replicate was immediately boiled
to inhibit enzymatic hydrolytic conversion of toxin analogues and to preserve original toxin profiles; the second replicate was not boiled.
Toxins were determined at points along the growth curve of P. lima, while Prorocentrum sp. cultures were harvested only at the exponential growth phase. Toxins were extracted from cells using 90% methanol. Extracts were qualitatively screened for a selection of OA
and DTX1 esterified derivatives such as DTX4, 5 and 6s, plus diol esters. Based on chromatographic peak area responses, boiled extracts generally exhibited higher proportions of esterified compounds compared to non-boiled samples. The DTX4 methylated diol ester
of the parent toxin, DTX1, was the most abundant in P. lima cells. Respectively, cellular concentrations of okadaic acid (OA) and dinophysistoxin-1 (DTX1) ranged from 1.7 to 62, and 0.008 to 49 pg cell-1. Prorocentrolide toxin was not detected in the P. lima cells. No
DTX1 was found although OA (1.3-22 pg cell-1) and the prorocentrolide toxin were produced by Prorocentrum sp., and a small number
of OA derivatives were also observed. This is the first study to report the toxin profiles of two Prorocentrum species isolated from the
coastal waters of Brazil.
226
MOLECULAR TAXONOMY AND TOXIN PROFILE
OF OSTREOPSIS CF. OVATA (DINOPHYTA)
FROM RIO DE JANEIRO, BRAZIL
Silvia M. Nascimento1,2, Eliliane V. Corra2, Mariangela Menezes3, Daniel Varela4,
Javier Paredes4, Denise S. Dagnino5, Monika Rapkova-Dhanji6, Steven Morris6
1
UNIRIO, Av. Pasteur, 458, 22.290-240, Brasil. E-mail: [email protected].
2
PP-ERN, UENF, Av. Alberto Lamego, 2000, 28.013-602, Brasil
3
Laboratorio de Ficologia, Museu Nacional, UFRJ 20940-040 Rio de Janeiro, Brasil
4
Centro de Investigacion I-mar, Universidad de los Lagos, Casilla 557, Chile
5
LBT, UENF, Av. Alberto Lamego, 2000, 28.013-602, Brasil
6
CEFAS, Barrack Road, Weymouth, Dorset, DT4 8UB, UK
Since 1998, blooms of the epi-benthic dinoflagellate Ostreopsis cf. ovata have been reported in Arraial do Cabo and Bazios, Rio de Janeiro,
Brazil. High O. cf. ovata abundances were reported in this region in June and December 2006 and April and September 2007. This dinoflagellate
can produce one of the most lethal marine toxins, palytoxin and its analogues. In this study, the morphology, molecular biology and toxin profile
of two strains of O. cf. ovata isolated from Bazios, Rio de Janeiro, were investigated. Cultures of strains E7 and B7 were grown at 24±2oC, 12
h light:12 h dark cycles and 60 μmol.m-2s-1, and further concentrated by centrifugation for molecular biology and palytoxin and ovatoxin determinations. Toxins were extracted using 80% methanol. The partial data of DNA genetic sequences obtained from the nuclear region (ITS) of strain
E7 confirmed that this is O. cf. ovata and grouped this isolate in a robust clade with isolates from Brazil and Italy. There were no morphometric
and morphological differences between the two strains and the natural population of the species, studied during a bloom event. The production
of palytoxin and ovatoxin was assessed using ouabain inhibited haemolytic assays with rabbit erythrocytes and LC-MS/MS. Extracts of the two
strains caused hemolysis that was positively related to O. ovata cell numbers. The hemolysis was partially inhibited by ouabain, a specific inhibitor
of palytoxin. This result may indicate the presence of other haemolytic compounds besides putative palytoxin. Toxin data will be reported.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 227
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
MALDI-TOF-MS DETECTION OF MARINE TOXINS
Ken-ichi Onodera, Kazuyo Tsuha, Yohsuke Taira, Takeshi Yasumoto
Okinawa Science and Technology Promotion Center, 12-75 Suzaki, Uruma, Okinawa, 904-2234, Japan.
E-mail: [email protected]
We studied the applicability of MALDI-TOF-MS method for quick and sensitive detection of marine toxins. The following toxins were
clearly detected at levels of 2 ng/well or below: azaspiracids, ciguatoxins, gambieric acids, maitotoxins, okadaic acids, ostreocins, palytoxin,
pectenotoxins and yessotoxins. Toxins belonging to a difficult group to analyze by LC-MS due to relatively high molecular weights and
presence of ionic charge groups could be detected after a simple sample preparation. We applied the method to a hemolytic and cytotoxic
compound found in Gambierdiscus sp. (GH8 strain) collected from coasts of Okinawa islands Japan and identified the compound to be
monodesulfomatitoxin based on the observed ion at m/z 3297 [M-Na]-. Absence of matitoxin, gambieric acid, and other ciguatera related
compounds in this strain was also confirmed. The simplicity of test samples preparation, instrument operation, and the high sensitivity of
the method suggested its suitability for detection of marine toxins.
227
IMPLEMENTATION OF ANALYTICAL METHODS
FOR DETECTION OF PALYTOXINS IN SHELLFISH
Roberta Orletti1, Alfiero Ceredi2, Gianluca De Grandis3, Tatiana Graziosi1, Cassandra Mengarelli3,
Silvia Pigozzi2, Fabio Principi3, Elena Riccardi2, Simone Bacchiocchi1
1
Istituto Zooprofilattico Umbria-Marche, 3 Via Cupa di Posatora, PC 60131, Ancona, Italy. E-mail: [email protected]
Fondazione Centro Ricerche Marine, 2 Viale Vespucci, PC 47042, Cesenatico, Italy
3
ARPA Marche Dipartimento di Ancona, 106 Via Colombo, PC 60127, Ancona, Italy
2
Blooms of Osteopsis ovata have been recently reported in several areas of Italian coastline and in Marche region too, specifically along
the coast of Monte Conero. Different Ostreopsis species are proven to produce analogues of palytoxins (PlTXs), which can represent a
health hazard through the food chain. Since 2006, with the co-occurrence of Ostreopsis ovata in the seawater, wild mussels collected
along the coast of Monte Conero have been found positive to the mouse bioassay for polar lipophilic toxins, with an unusual very rapid
death of mice, even if no cases of toxicity in humans have been reported. Actually in Europe there are neither regulatory limits for PlTXs
in seafood nor official methods of analysis. Three different analytical methods for detecting palytoxins in shellfish were implemented and
compared: a specific bioassay (MBA PlTXs), an hemolytic assay (HNA) and an instrumental one, using a QQQ-LC-MS. MBA PlTXs is
specific but shows a low sensitivity, HNA has a good sensitivity and a low susceptibility to interferences from other toxic compounds, LCMS is the most adequate for determining palitoxins, but requires a high economic investment by laboratories. At the moment a combination
of different methods is used for monitoring samples, above all if contaminated with complex toxic profiles.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 228
THE SEASONAL DISTRIBUTION OF MICROCYSTINS IN EIGHT
TISSUES OF COMMON CARP (CYPRINUS CARPIO): ARE THE
ANTIOXIDANT MARKERS AFFECTED?
T. Papadimitriou1, S. Tsikogias2, S. Georgiou2, I. Leonardos1, I. Kagalou3, K. Moutou2
1
Department of Biological Applications & Technologies, University of Ioannina, 45100 Ioannina, Greece.
E-mail: [email protected]
2
Department of Biochemistry & Biotechnology, University of Thessaly, 26 Ploutonos street, 41221 Larissa, Greece
3
Department of Ichthyology & Aquatic Environment, University of Thessaly, 38446 Volos, Greece
Cyanobacteria and their secondary toxic metabolites can pose a serious threat to animals and humans. Microcystins can accumulate in
edible and non edible tissues of fish. The aim of the present study was to investigate the response of antioxidant systems of common
carp inhabiting Lake Pamvotis, (Greece), to natural yearly fluctuations of total microcystin (MC) concentrations. Human health hazards
from fish consumption are also discussed. Common carp were collected in October, June and December around a year. Total MC concentration was determined in water, scum and various fish tissues by a commercial Microcystin ELISA kit. Catalase activity, TBARS,
GSH and GSSG concentrations were determined in the liver, kidney and brain and correlated to MC concentrations. MC concentrations
varied significantly in water and scum during the year (80.68-2239.25 and 116.75-4220.30 ng/l, respectively). No significant seasonal
fluctuations were observed in tissue MC concentration. Liver exhibited the highest MC concentrations followed by kidney. All antioxidant
parameters measured varied significantly with season. Contrary to results generated under controlled conditions of MC administration,
no significant correlations between antioxidant markers and tissue MC concentrations were observed in this study, stressing the importance
of studying biomarker systems under natural conditions of seasonal variation and exposure. Common carp is considered as a wide commercial fish in many regions of Greece. According to the MC concentration in muscle of the fish, it is most likely unsafe to consume
Common carp from Lake Pamvotis.
228
DEVELOPMENT OF A NEW HPLC-FLD METHOD FOR
THE DETERMINATION OF DSP TOXINS USING
THE FLUORESCENCE TAGGING REAGENT
3-BROMOMETHYL-7-METHOXY-1,4-BENZOXAZIN-2-ONE
Christina Pyrgaki1, Nikolaos S. Thomaidis2, Ioannis Hatzianestis1
1
2
Hellenic Centre for Marine Research, P.O. Box 712, Anavyssos, 19013 Athens, Greece. E-mail: [email protected]
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis Zografou,
157 84 Athens, Greece
A new sensitive HPLC method for the determination of the diarrheic shellfish toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) in
mussel tissues was developed and validated. The extraction of the analytes from the dried (lyophilized) mussel samples was achieved by
using acetonitrile, followed by solid phase extraction of the mussel extract on C18 cartridges. The eluant contained the toxin analytes
was then derivatized, using the sensitive reagent 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one, with the addition of N-ethyldi-isopropylamine as catalyst base. The derivatives were further cleaned up by normal phase SPE using alumina cartridges and the toxins
were determined by HPLC on a C18 analytical column using acetonitrile:water gradient elution and fluorescence detection (excitation at
345nm – emission at 440nm). The optimization of the derivatization reaction was based on experiments for: (1) the choice of the most
proper polar aprotic solvent for the specific SN2 derivatization reaction; (2) the determination of the optimum reagent concentrations; (3)
the selection of the optimum temperature-time combination for the successful completion of the reaction. Calibration was performed by
the standard addition method and was linear in a typical analytical range of 40-400 ng/g wet tissue.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 229
TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
CHARACTERIZATION OF AN EPOXIDE HYDROLASE
FROM KARENIA BREVIS
Kathleen S. Rein, Pengfei Sun
Florida International University, USA. E-mail: [email protected]
The polyether ladder motif of a number of marine algal toxins is exemplified by molecules such as brevetoxins, ciguatoxins and
prymnesisns. More than 25 years ago, Nakanishi hypotheized that an epoxide hydrolase catalyses a key reaction in the biosynthesis
of this class of compounds. This has been demonstrated to some extent in bacterial polyethers, but not in marine protists. We have
cloned and expressed an epoxide hydrolase from Karenia brevis, the Florida Red Tide dinoflagellate and producer of the brevetoxins.
The characterization of this enzyme will be described in particular with respect to substrate selectivity and regioselectivity of the
epoxide opening reactions.
229
OSTREOPSIS OVATA ISOLATED FROM RAROTONGA,
COOK ISLANDS
Lesley Rhodes1, Kirsty Smith1, Rex Munday2, Lyn Briggs2, Andy Selwood1
1
2
Cawthron Institute, 98 Halifax St East, Nelson, New Zealand. E-mail: [email protected]
AgResearch, Private Bag 3123, Hamilton, New Zealand
The genus Ostreopsis has been isolated from many geographically distant regions, such as temperate Australasia and the tropical
Caribbean. Species from this genus are known to produce ostreocins. One species, O. ovata, has formed blooms in the Mediterranean Sea in recent years, causing respiratory illnesses in this region. Epiphytic O. ovata cells have recently been isolated from
the south coast of Rarotonga, attached to the calcareous green macroalga, Halimeda sp., which grows in the lagoonal reef environment. Extracts of cultures of this isolate were negative in a haemolysis assay for palytoxin-like compounds, and are now being
tested for toxicity in mice. Extracts will also be analysed using a novel LC-MS method for detection of palytoxin and palytoxin analogues. Ostreopsis spp. isolated from sub-tropical Northland, New Zealand, and from South Australia (which has an etesian climate),
will be characterised to determine whether O. ovata is present, and these isolates will be compared with the Rarotongan isolate.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 230
COMPARISON OF LC-MS/MS AND HAEMOLYSIS ASSAY
FOR THE ANALYSIS OF PALYTOXINS IN SEAFOOD
Elena Riccardi, Monica Cangini, Alfiero Ceredi, Filippo Magnani, Anna Milandri, Roberto Poletti,
Marinella Pompei, Silvia Pigozzi
Fondazione Centro Ricerche Marine, V.le A. Vespucci 2, PC 47042, Cesenatico (FC), Italy.
E-mail: [email protected]
A liquid chromatography tandem mass spectrometry method for the analysis of palytoxins in seafood was optimized. It involves
extraction with aqueous methanol, SPE purification and an on-line pre-concentration step before injection into the LC-MS/MS system.
Purified extracts were tested also by a haemolysis assay (HNA), which includes the use of ouabain as palytoxin antagonist for enhanced
specificity. Here we compare results obtained for a set of field samples collected along Italian coasts in the period 2008-2009. Blue
mussel, clam and sea urchin samples were tested in parallel showing a good agreement between the two techniques. A comparison
of fully quantitative data (PlTXs concentration above the limit of quantification of both methods) was done using a two tailed paired ttest, which showed no difference in the results obtained from LC-MS/MS and HNA. HNA proved to be a highly sensitive method,
which doesn’t require expensive equipment. On the other hand LC-MS/MS allowed the determination of the specific toxin profile of
Italian seafood. The collected samples were mainly contaminated by ovatoxin-a, a recently described analogue of palytoxin for which
no toxicity data are available. Both techniques represent useful tools to produce representative occurrence data of PlTX-group toxins
in shellfish and seafood from Italy.
230
DETERMINATION OF PALYTOXINS IN SAMPLES
FROM OSTREOPSIS OUTBREAKS IN LLAVANERES
(NW MEDITERRANEAN COAST)
Pilar Riobó1, José M. Franco1, Isabel Bravo2, Santiago Fraga2, Isabel Ramilo2, Pilar Rial2,
Francisco Rodriguez2, M. Montserrat Sala3, Magda Vila3
1
Instituto Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, 36208 Vigo, Spain. E-mail: [email protected]
Instituto Español de Oceanografía (IEO), Subida a Radio Faro 50, 36390 Vigo, Spain
3
Institut de Ciències del Mar (ICM-CSIC), Pg. Marítim de la Barceloneta 37-42, 08003 Barcelona, Spain
2
Dinoflagellates of the genus Ostreopsis have been related to harmful episodes in many Mediterranean coastal areas since 1998. In
August 2004 one important event occurred in Llavaneres beach (Catalan coast, Spain) affecting 74 people with rhinitis and breathing
problems. Since then, many Mediterranean heavy blooms of Ostreopsis have coincided with respiratory problems in people staying near
the beach, suggesting a possible link between them. To test this hypothesis, samples of seawater, macroalgae, benthic marine invertebrates and aerosols were collected from this location during 2009 and 2010 in the framework of the EBITOX project to determination
of palytoxin. Different extraction procedures have been used depending of the sample type. The extracts were analysed by haemolytic
assay and LC–FLD and the presence of palytoxin was confirmed by LC-MS. Strains isolated from Llavaneres were cultured in the laboratory and analysed by the same chemical and biological methods. Toxins from field samples and cultures were compared. Presence
of palytoxin in macroalgae and cultures was confirmed but it has not yet been detected in aerosol filters or dissolved in seawater with the
methods used. Therefore, the hypothesis remains unresolved.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
APPLICATION OF IMMUNOAFFINITY CHROMATOGRAPHY
FOR THE SELECTIVE EXTRACTION OF DOMOIC ACID
PRESENT IN DIFFERENT SEAFOOD MATRICES
Socorro Rivera-Vila1, Jesús Pérez-Linares1, José Manuel Leão-Martins1, Pedro Reis-Costa2, Ana Gago-Martínez1
Laboratorio de Química Analítica y Alimentaria, Facultad de Ciencias, Universidad de Vigo,
As Lagoas-Marcosende 36310, Vigo, Pontevedra, Spain. E-mail: [email protected]
2
Instituto de Investigação das Pescas e do Mar (IPIMAR), Av. Brasilia 1449-006, Lisboa, Portugal
1
Increasing efforts were made in the development of analytical techniques for a reliable determination of marine toxins, which included the
use of Mass Spectrometry for confirmation. An alternative for the selective extraction of Domoic Acid present in different seafood matrices,
using immunoaffinity chromatography is presented in this work based on a previous study carried out by this research group, which
results, as well as its efficiency compared with conventional solid phase extraction procedures have been reported elsewhere. This work
presents the application of this approach for the solid phase extraction of domoic acid present in different contaminated seafood: mussels,
scallops, octopuses, cuttlefishes, cockles, Atlantic surf clams and oysters. The effect of the matrix on the efficiency of the immunoaffinity
extraction was evaluated by using HPLC-UV and further confirmation by HPLC-MS. The optimal load capacity of the SPE-IAC columns
was determined within 100-200ng DA to an efficient binding with the antibody. The results obtained showed a clear influence of the
matrix, affecting the recovery yield. Mussel and scallop samples exhibit the lower matrix influence with recoveries of 102.9%±3.8 and
103.2%±1.2, respectively while cockle and oyster samples showed the higher matrix influence with recoveries of 94.4%±0.94 and
93.5%±1.6, respectively. Comparison of SPE-IAC with C18 and SAX confirmed the overestimation of DA content in all samples using
conventional cleanup cartridges because of their low capability to eliminate natural interferences, especially in cuttlefish, octopus and
clam samples. This work shows the great potential of immunoaffinity chromatography for a reliable determination of domoic acid avoiding
further confirmation by Mass Spectrometry.
231
INFLUENCE OF AUTECOLOGICAL PARAMETERS ON
THE FORMATION OF YESSOTOXINS BY PROTOCERATIUM
RETICULATUM FROM THE GERMAN BIGHT (NORTH SEA)
Karin Röder1, Gunnar Gerdts2, Tina Helbig1, Bernd Krock2, Allan Cembella2, Mona Hoppenrath3, Bernd Luckas1
1
Department of Food Chemistry, Institute of Nutrition, Friedrich-Schiller University of Jena, Dornburger Str. 25,
07743 Jena, Germany. E-mail: [email protected]
2
Alfred-Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany
3
German Centre for Marine Biodiversity Research, Senckenberganlage 25, D-60325 Frankfurt, Germany
Yessotoxin and its analogues (YTXs) are a group of marine biotoxins provoking a huge scientific interest in the past. This interest based
upon the potency of YTXs to induce Diarrhetic Shellfish Poisoning-like symptoms in the classical Mouse Bioassay. A large scale culture
of the YTX-producing strain of Protoceratium reticulatum isolated from the German Bight (North Sea) was analysed concerning its
complex toxin profile. Toxin-composition analysis of this endemic P. reticulatum strain revealed that YTX was the dominant marine biotoxin
but also the putative 1a-homo YTX was prominent to some extent. In addition, several other YTXs analogues were detected: 32-arabinoside YTX, two putative keto YTXs, 44,45,46-trinor YTX, 45-OH YTX, 45-OH COOH YTX, 44,55-di OH YTX, and some other
minor-YTXs. Furthermore, this strain was investigated to elucidate the toxin formation influenced by several autecological parameters. In
particular the N/P ratio, temperature and salinity were modified. The influence of the growth phase on YTX formation was also under investigation. P. reticulatum showed the highest growth at 15oC and higher salinities. On the other hand, higher temperature led to a
reduced growth, whereas the total YTX concentrations were higher at lower temperature. Furthermore, P-limited cultures exhibited the
highest cell quota of YTX at the end of the stationary phase; and a dramatic effect occurred under P limitation at 15oC when the toxicity
caused by YTXs increased to ten fold higher values.
10. ABSTRACT BOOK (204-238)_Layout 1 10/22/10 2:40 PM Page 232
PREPARATION OF STABLE ISOTOPE-LABELED
CYLINDROSPERMOPSIN AND THE APPLICATION
TO LC-MS ANALYSIS
Tomoharu Sano1, Hiroo Takagi1, Kimiyo Nagano1, Masataka Nishikawa1, Kunimitsu Kaya2
1
National Institute for Environmental Studies, Laboratory of Intellectual Fundamentals for Environmental Studies,
Tsukuba, Ibaraki 305-8506, Japan. E-mail: [email protected]
2
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Cylindrospermopsin is an alkaloid hepato-toxin produced by water bloom-forming cyanobacteria. LC-MS is a powerful method for analysis
of cylindrospermopsin, but there is no commercially available stable isotope (SI)-labeled cylindrospermopsin as a surrogate for corrections
of recovery and ionization efficiency. We tried to prepare SI-labeled cylindrospermopsin as a surrogate for accurate LC-MS analysis. SIcontaining TS-15 medium was used for cultivation of Cylindrospermopsis raciborskii. After 6 months pre-cultivation, the toxic strain of
Cylindrospermopsis raciborskii was mass-cultured in 15N-NaNO3 containing TS-15 medium. After harvest, cells were lyophilized. The
15
N-labeled cylindrospermopsin was extracted according to our previous method. The SI-labeled cylindrospermopsin was purified using
HPLC with RP- and HILIC column. The structure of the SI-labeled cylindrospermopsin was confirmed with the MS and NMR spectra.
The SI-labeled cylindrospermopsin was very useful for accurate LC-MS analysis. RSD of the analysis (n=5) was less than 0.5% with SIlabeled cylindrospermopsin.
232
A SENSITIVE ASSAY FOR PALYTOXINS AND OSTREOCINS
USING LC-MS/MS ANALYSIS OF CLEAVAGE FRAGMENTS
FROM MICRO-SCALE OXIDATION
Andrew Selwood, Roel Van Ginkel, Patrick Holland, Michael Boundy, Lesley Rhodes, Paul McNabb
Cawthron Institute, Private Bag 2, Nelson 7010, New Zealand. E-mail: [email protected]
Direct analysis of palytoxin compounds by LC-MS with electrospray ionisation is difficult due to the complex mass spectra. They are large
molecules (FW 2500 - 2800 Da) containing three nitrogens which favours formation of multivalent parent ions with the charge spread
across multiple different cationised species, all with large 13C isotope contributions. This leads to ambiguities in assigning the true MW
and low and variable sensitivity and specificity for trace detection using SIR or the preferred MRM transition [M.H2]2+ > m/z 321, especially
in biological extracts. To simplify analysis and improve sensitivity for trace analysis, a method has been developed that uses LC-MS/MS
analysis of the substructures generated by oxidative cleavage of the macro-structure. Periodate oxidation of palytoxins or ostreocins generates two nitrogen-containing fragments. One fragment is common for all palytoxins and ostreocins and is analysed to quantify the sum
of all toxins present. The other fragment varies depending on the parent toxin and is analysed for confirmation of the particular palytoxin
or ostreocin present. The conditions for micro-scale oxidation of palytoxin were optimised and a rapid analysis was for the fragments was
established using LC-MS/MS (Acuity uPLC coupled to a Quattro Premier TSQ with Z-spray ion source, Waters-Micromass). Linear calibration ranges (MRM) for the quantitation and the confirmation fragments from oxidation of palytoxin and ostreocin-D were established.
LOQs with confirmation were ca 1 ng/mL. The method is applicable to both algal cultures and shellfish and has demonstrated the
potential to be used as a tool for research and regulatory testing.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
A FIRST APPROACH TO DISTINGUISH CYTOTOXIC
EFFECTS OF DIARRHEIC AND NON-DIARRHEIC MARINE
TOXINS USING TWO HUMAN INTESTINAL CELL LINES
Anne-Laure Serandour, Floriane Leurs, Rachelle Lanceleur, Valerie Fessard
French Agency for Food, Environmental and Occupational Health Safety, P.O. Box 90203, PC 35302, Fougeres, France.
E-mail: [email protected]
Human poisoning due to ingestion of seafood contaminated by algal toxins is a worldwide problem. The monitoring of phytoplankton
and phycotoxins is required in shellfish production sites to avoid food-safety problems. Among the alternative methods developed to
replace the mouse bioassay for the detection of lipophilic toxins, only few cell based assays have been investigated. In this study, cytotoxicity of diarrheic (OA and AZA1) and non-diarrheic (PTX2 and YTX) phycotoxins were investigated in two human intestinal cell
models (enterocyte-like Caco2 cells and mucus-secreting-like HT29-MTX cells) with the neutral red uptake assay. Undifferentiated
and differentiated cells were exposed for 48h before IC50 determination. Overall, the neutral red assay is more sensitive for detection
of cytotoxic effects of phycotoxins in undifferentiated cells than in differentiated cells. Moreover, IC50 obtained in differentiated HT29MTX cells are lower than in differentiated Caco2 cells. Interestingly, mucus-secreting HT29-MTX are more sensitive to diarrheic phycotoxins (OA+AZA1) than to non-diarrheic phycotoxins (PTX2+YTX), whereas no difference in cytotoxicity between both types of
toxins can be detected with enterocyte-like Caco2. Further investigations will be performed by RT-qPCR to study the cellular response
considering markers involved in diarrhoea such as mucins and interleukins.
233
SEARCH FOR TOXIC STRAINS OF OSTREOPSIS SPP.
IN OKINAWAN COAST AND THE CAUSATIVE AGENTS
Yosuke Taira, Zensai Teruya, Arisa Nakamura, Takeshi Yasumoto
Okinawa Science and Technology Promotion Center, Japan. E-mail: [email protected]
Marine dinoflagellates Ostreopsis spp. are known to produce Ostreosin-D (Ost-D), ovatoxin, and other analogs of palytoxin (PLTX), but
the frequency of the occurrence of toxic strains and the type of toxins produced remain to be elucidated. We isolated 23 clones of Ostreopsis spp. from Okinawan coast and detected toxicity in three clones by sheep blood hemolytic assays and Neuro-2a cell assays. Two
clones (OIK2, OA21) were used to study the causative agents (OIK2-Tx, OA21-Tx). Both the toxins caused slow hemolysis and were
antagonized by ouabain analogous with PLTX. A Sep-pack C18 column was used to remove salts and obtain toxins in acidic propanolwater (4:6). In subsequent HPLC on a C8 column, both OIK2-Tx and OA21-Tx showed retention times close to that of PLTX. However,
neither OIK2-Tx nor OA21-Tx produced ions corresponding to PLTX or Ost-D in LC-MS and MALDI-TOF analysis. Instead, occurrence
of toxins smaller than palytoxin was suggested in both fractions. Though further study is needed to characterize or identify the toxins, the
present results reveal the structural and genetic diversity of palytoxin-related toxins and their producers.
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PHYTOPLANKTON CAUSES AN INCREASE IN TOXICITY
BY PRODUCTION OF LIPOPHILIC TOXINS AND PSP-TOXINS
IN CHILEAN COASTAL WATERS
Nicole Trefault1, Nathalie Delherbe1, Blanca Pérez1, Bernd Krock2, Allan Cembella2, Mónica Vásquez1
1
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile,
Santiago, Chile. E-mail: [email protected], [email protected]
2
Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany
Harmful algal blooms associated with potent toxins have increased in the last decades. In the southernmost waters of Chile most toxin
outbreaks come from Paralytic Shellfish Poisoning (PSP) produced by the recurrent and persistent dinoflagellate Alexandrium catenella,
however there is only partial information about lipophilic toxins throughout the coastal waters of this country. We analyze the lipophilic
toxin profile and determine the genetic diversity and toxin analogues variation among clones of A. catenella isolated since 2008 from the
most austral waters of Chile. Our results show a great variety of lipophilic toxins, including azaspiracid-1 and gymnodimine A, previously
undetected toxins in Chile and in the eastern South Pacific. Sequencing analysis of the D1-D2 hypervariable region of LSU rDNA from
the new isolated clones of A. catenella showed no substantial divergence. Analysis of the PSP toxin composition and content of the new
austral clones, however, revealed large differences between the existing clones and those previously isolated from Chile. On a per cell
basis, the new clones of A. catenella are nearly seven times more toxic than the Aysén (1994) clones, and exhibit an unusual toxin profile
that stands out for the presence of the most toxic analogues, saxitoxin and neosaxitoxin. These results indicate that the presence of phytoplanktonic toxins is a widespread and growing phenomenon, whose danger is increasing, considering both the appearance of lipophilic
toxins and the emergence of toxic dinoflagellates clones producing the most potent PSP-toxins analogues.
234
EVALUATION OF CYTOTOXIC AND HEMOLYTIC ASSAYS
FOR DETECTION AND QUANTIFICATION
OF CIGUATERA-RELATED TOXINS
Kazuyo Tsuha, Takeshi Yasumoto
Okinawa Science and Technology Promotion Center, Japan. E-mail: [email protected]
Neuroblastoma 2a cells are widely used for detection/quantification of an important marine toxin group that acts on voltage sensitive
sodium channels (VSSC) and includes brevetoxins, ciguatoxins, saxitoxins, and tetrodotoxins. The assay is highly sensitive but produces
false positives when cytotoxic compounds, most notably free fatty acids, are present. Use of a small alumina column was effective to
eliminate fatty acids. Fatty acids also have to be removed from extracts before carrying out hemolytic assays on maitotoxins. Antagonistic
reagents, tetrodotoxin against ciguatoxins and S&KF96365 against maitotoxin, are also useful to specify toxins in question. Using the
neuro-2a and hemolytic assays, we screened 32 strains of Gambierdiscus toxicus for production of ciguatera related toxins. None of the
strains tested produced ciguatoxins but two strains showed positive reaction in hemolytic assays. The causative hemolysin was partially
purified and identified to be monodesulfo MTX by MALDI-TOF-MS analysis.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
HOMOGENEITY AND STABILITY OF PARALYTIC SHELLFISH
POISONING REFERENCE MATERIALS GENERATED
THROUGH SHELLFISH FEEDING OF MASS CULTURED
TOXIC ALGAE
Andrew Turner, Robert Hatfield, Andy Powell, Wendy Higman
Cefas, Barrack Road, Weymouth, Dorset, DT4 8UB, UK. E-mail: [email protected]
In order to validate and implement analytical methods for the quantitation of PSP toxins in shellfish, there is a clear requirement for the
availability of suitable contaminated shellfish reference materials. Over the past 2 years at Cefas, experiments have been conducted to
contaminate samples of a variety of shellfish species through feeding experiments with high concentrations of toxic algae. Feeding experiments using a variety of Alexandrium and Gymnodinium strains have been performed successfully to date, generating a variety of
shellfish materials containing a range of different PSP toxin profiles and overall levels of toxicity. Post-production work has concentrated
on determining the homogeneity and stability of these materials, initially focussing on the potential use of the materials as laboratory reference materials for application to the AOAC 2005.06 HPLC-FLD method. A variety of techniques have been investigated to prolong
the stability of the materials including the use of different storage conditions, use of artificial matrix stabilisers and the use of gamma irradiation. Homogeneity and stability data will be presented from a range of shellfish reference materials highlighting the potential for the
various techniques in the preparation and provision of PSP laboratory reference materials.
235
PSP TOXINS PROFILE ALONG THE CROATIAN
ADRIATIC COAST
Ivana Ujević1, Romana Roje2, Ivona Marasović1
1
2
Institute of Oceanography and Fisheries, Šetalište Ivana Meštrovića 63, 21000 Split, Croatia. E-mail: [email protected]
Student of Erasmus Mundus Master of Biodiversity and Conservation, Doverska 9, 21000 Split, Croatia
Paralytic Shellfish Poisoning (PSP) is caused by the saxitoxins, potent neurotoxins that cause paralysis and death in consumers of contaminated shellfish. This phenomenon may have serious health and economic consequences. Present study is the first attempt to determine PSP toxin types and establish a complete list of PSP toxins for Croatian coastal waters. 3408 samples of shellfish (Mytilus
galloprovincialis, Ostrea edulis, Pecten jacobaeus, Flexopecten proteus) and ascidians (Microcosmus sulcatus) from 25 sites inside breeding and harvesting areas along the Croatian coast of the Adriatic Sea were sampled during July 2000 until December 2009. Preliminary
PSP Mouse Bioassay test (AOAC Official Method 959.08.) showed the presence of PSP toxins for the period December 2008 through
May 2009. In the present study, analysis was conducted by the Lawrence HPLC method (First Action 2005.06 AOAC Official Method)
with pre-chromatographic oxidation of the PSP toxins and fluorescence detection for determination of saxitoxin, neosaxitoxin, gonyautoxins
2 and 3 (combined); gonyautoxins 1 and 4 (combined); decarbamoyl saxitoxin, gonyautoxins 5, C- 1 and C- 2 (combined); and C- 3 and
C- 4 (combined). The aim was to establish an instrumental method for continuous qualitative and quantitative monitoring of PSP toxins
accumulated in farmed animals to achieve required health standards according to Regulation (EC) No. 853/2004. Qualitative analysis
showed the presence of the five following PSP toxins STX, dcSTX, GTX 2,3, dcGTX 2, GTX 5 and C 1,2. Quantitative analysis revealed
STX and GTX 2,3 as dominant toxin types in these samples.
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DETERMINATION OF PARALYTIC SHELLFISH POISONING
TOXINS IN SHELLFISH BY RECEPTOR BINDING ASSAY:
A COLLABORATIVE STUDY
Frances M. Van Dolah, Spencer A. Fire, Tod A. Leighfield, Gregory J. Doucette
Marine Biotoxins Program, NOAA National Ocean Service, Center for Coastal Environmental Health & Biomolecular Research,
219 Fort Johnson Rd., Charleston, SC 29412, USA. E-mail: [email protected]
An AOAC collaborative study was conducted on the receptor binding assay (RBA) for paralytic shellfish poisoning toxins. Nine laboratories
from seven countries participated. In addition, two laboratories analyzed the same samples by the Lawrence HPLC method in order to
assess the STX congener composition and three laboratories performed the AOAC mouse bioassay. A total of 21 shellfish homogenates
were extracted in 0.1 N HCl and the extracts analyzed by RBA in three assays run on separate days. Samples included naturally contaminated shellfish of different species collected from several geographic regions, which contained varying saxitoxin congener profiles
due to their exposure to different PSP-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis)
from the U.S. east and west coasts, Mediterranean mussel (Mytilus galloprovincialis) from Chile, green mussel (Perna viridis) from New
Zealand, Atlantic surf clam (Spisula solidissima), scallop (Plactopecten magellanicus) from the U.S. east coast, and butter clam (Saxidomus
giganteus) from the west coast of the US. All samples were provided as whole animal homogenates, except scallop, for which hepatopancreas was homogenized. Samples were selected representing a range of concentrations below and above the regulatory limit.
Among the naturally contaminated samples, 5 were blind duplicates used for calculation of RSDr. At the time of this writing analyses are
still in progress. Results of the study will be presented, including sr, sR, RSDr, RSDR, recovery and Horrat values obtained.
236
MASS PRODUCTION OF DIARRHEIC SHELLFISH
POISONING (DSP) TOXINS IN LARGE SCALE CULTURES
OF THE DINOFLAGELLATE PROROCENTRUM LIMA
(EHRENBERG) DODGE
Ioanna Varkitzi1, Kalliopi Pagou1, Edna Graneli2, Christina Pyrgaki1, Ioannis Hatzianestis1
1
2
Hellenic Centre for Marine Research, P.O. Box 712, Anavyssos, 19013 Athens, Greece. E-mail: [email protected]
Marine Ecology Department, Linnaeus University, 39182 Kalmar, Sweden
Biotechnological importance of toxic marine dinoflagellates includes food safety, biomedical, pharmacological and research applications
of their toxins. Lack of sufficient dinoflagellate toxin quantities for investigational purposes remains a significant limitation. Some of these
toxins, that are available in small amounts, are quite expensive while their chemical synthesis is complex and costly. Therefore, producing
quantities of dinoflagellate toxins requires mass cultures of these microalgae, which however exhibit slow growth and increased sensitivity
to turbulence. Because of this, dinoflagellate mass culturing is a highly demanding and problematic task. Prorocentrum lima is a dinoflagellate that synthesizes DSP and ciguatera toxins, causing shellfish and fish poisonings in many coastal areas. In our study, P. lima was
grown in large scale semi-continuous cultures under controlled laboratory conditions, with f/2 medium and P deficiency, according to
previous findings of ours. Maximum culture volume was 300L and maximum biomass was 20690 cells mL-1. Doubling time of cell
biomass was 3.7 days. DSP toxins production started to increase on day 12 and maximum toxicity was obtained on day 21. Maximum
yield of toxins was 63.66 ng mL-1 for okadaic acid (OA) and 8.07 ng mL-1 for dinophysistoxin 1 (DTX1). Toxin quota in P. lima cells was
88.7% OA and 11.3% DTX1. Potential total yield of toxins with our culturing system was 19.1 g OA and 2.4 g DTX1, while reference
standards usually contain 100μg of OA or DTX1. The culturing system designed for our study has a volume capacity of 500L.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
UNRAVELING OF SAXITOXIN SYNTHESIS
IN DINOFLAGELLATES
Wiese Maria1, Murray Shauna1, Mihali Troco1, Hallegraeff Gustaaf2, Neilan Brett1
1
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.
E-mail: [email protected]
2
School of Plant Science and Institute of Antarctic and Southern Ocean Studies, University of Tasmania, Private Bag 77,
Hobart Tas 7001, Australia
Saxitoxin (SXT) and its analogs are nitrogen rich neurotoxins and causative agents of the paralytic shellfish poisoning (PSP). Bloomforming gonyaulacoid dinoflagellates of the genus Alexandrium, and Pyrodinium, as well as a single gymnodinoid species, Gymnodinium
catenatum, are marine producers of PSP toxins (PSTs). Certain freshwater species of cyanobacteria have been also identified to produce
PSTs. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production
in dinoflagellates remains elusive. Unknown is also the reason for the occurrence of toxic and non-toxic strains within one species of
cyanobacteria and dinoflagellates. In order to elucidate the capacity for SXT production in dinoflagellates several approaches have been
tested. The design of degenerate primers based on sequence information from cyanobacteria and available ESTs was performed to
amplify sxtA encoding for the S-adenosylmethionine synthase (SAM), a domain of the polyketide silimar enzyme responsible for saxitoxin
synthesis initiation. Expression patterns of sxtA were investigated in a qPCR assay in comparison to reference genes and genes relevant
to the overall metabolism of the dinoflagellate, such as a glutamin synthase and the chlorophyll a-c binding protein.
237
THE USE OF IMMUNOASSAY TECHNOLOGY IN
THE MONITORING OF ALGAL BIOTOXINS IN FARMED
SHELLFISH
Annette Wilson1, Evelyn Keady1, Joe Silke2, Robin Raine1
1
2
The Martin Ryan Institute, National University of Ireland Galway, Galway, Ireland. E-mail: [email protected]
Marine Institute, Rinville, Oranmore, Galway, Ireland
The use of immunoassay technology as an adjunct method for the monitoring of biotoxins in shellfish was investigated at aquaculture
production sites in Killary Harbour, Ireland, during the summer of 2009. Sub-samples of mussels (Mytilus edulis) collected at three
locations in Killary Harbour were taken from batches to be analysed by the National Monitoring Agency (Marine Institute) for Diarrhetic
Shellfish Poisoning (DSP) toxins, using chemical and biological methods. A commercially available immunoassay kit (DSP ELISA, Abraxis)
was used to detect and semi-quantify DSP toxins. The concentrations of toxins before and after hydrolysis were compared with those
derived from chemical methods (Liquid Chromatography-Mass Spectrometry, LC/MS) and the mouse bioassay to assess the reliability
and accuracy of the kit. A positive relationship was found between the toxin concentrations detected by the immunoassay and LC/MS.
Levels ranged from below limit of detection.
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TOXINS: CHEMICAL STRUCTURE AND SYNTHESIS,
DETECTION AND ANALYTICAL METHODS
POSTER PRESENTATIONS
SIMULTANEOUS ANALYSIS OF CIGUATOXINS
IN FISH FLESH BY LC/MS/MS
Kentaro Yogi1, Naomasa Oshiro2, Masahiro Hirama3, Takeshi Yasumoto1
1
Okinawa Science and Technology Promotion Center, 12-75 Suzaki, Uruma, 904-2234, Okinawa, Japan.
E-mail: [email protected]
2
Okinawa Prefectural Institute of Health and Environment, 2085 Aza-Ozato, Ozato, Nanjo, 901-1202, Okinawa, Japan
3
Department of Chemistry, Graduate School of Science, Tohoku University, 6-3 Aramaki, Aoba-ku, Sendai, 980-8578,
Miyagi, Japan
The ciguatera fish poisoning (CFP) occurs worldwide and thus needs proper means for toxin detection and quantification. The toxin
profiles, crucial for designing analysis, were studied in detail in French Polynesia but remains to be clarified in other Pacific regions. The
obstacles to the task are the paucity of reference toxins and the very low toxin levels to be quantified. Using synthetic and natural toxin
standards, we developed a sensitive simultaneous LC/MS/MS method for five ciguatoxin congeners of frequent occurrence: ciguatoxin
(CTX), 52-epi-54-deoxyCTX, CTX4A, CTX3C and 51-hydroxyCTX3C. In electrospray ionization the CTXs exhibited prominent sodium
adduct ions, which hardly yielded product ions even at high collision energy and enabled us to minimize the interference from matrices.
Solid-phase extraction sequentially using PSA, ODS and florisil cartridges efficiently removed interfering matrices in flesh extracts. Addition
of ammonium formate to a mobile phase also reduced matrix effects. Ion peaks were well separated and the limits of detection (S/N=3)
and quantitation (S/N=10) for standard CTXs were estimated to be 0.25 pg and 1 pg on-column, respectively. Detection of 50 pg/g
CTX in flesh judged as non-toxic (<175 pg CTX/g) by mouse bioassay confirmed the sensitivity of this method. In fish samples implicated
in CFP, CTX, 52-epi-54-deoxyCTX and 54-deoxyCTX were detected and the former two quantified. The method suits for practical use
and enabled us to clarify the toxin profiles in Okinawa.
238
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MECHANISMS
OF TOXICITY
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 240
ORAL PRESENTATIONS
EFFECTS OF TOXIC CYANOBACTERIA IN THE DIET
ON PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS
IN RATS EXPOSED VIA FOOD - SIMPLE SIMULATIONS
OF THE FOOD WEB TRANSFER
Ondr̀΄ej Adamovsky΄1, Radovan Kopp2, Andrea Zikova΄2, Jir̀΄ί Kohoutek1, Hana Paskerova΄1,
Stanislav Navra΄til3, Miroslava Palίkova΄3, Ludè΄k Bla΄ha2
1
Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 3,
62500 Brno, Czech Republic
2
Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic
3
University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic
Cyanobacteria produce numerous bioactive compounds including widely studied peptide hepatoxins - microcystins (MCs). Aims of the present study were to provide a model simulation of the simple food chain for evaluation of impacts cyanobacteria on the mammal (rat) physiology under different exposure scenario. Rats were fed with the diet enriched by fish meat and/or toxic Microcystis biomass and/or isolated
MCs for 28 days and then studied for number of parameters. Although considerable amounts of microcystins were administered to rats, only
minor trace levels of MCs were found in the rat tissues as determined with tandem LC/MS (maximum concentrations around 5 ng/g fresh
weight). Besides levels of MCs, we studied parameters of oxidative stress in the liver. Group with the highest concentration of MCs in the diet
had increased level of lipid peroxidation (LPO) and significantly elevated activities of glutathione S-transferase (GST) and glutathione reductase (GR). Other parameters of oxidative stress, i.e. levels of GSH, enzymatic activities of GPx, SOD and CAT were not modulated in the
present study. Series of hematological parameters were also investigated but most of the analyzed indices did not show any clear differences
among groups. With regard to immune parameters, statistically significant stimulation of the antibody response to model antigen KLH was
observed in the group with the highest MCs level. Our study demonstrates that oral exposure to high dose of microcystin in the diet may induce some detoxification responses, oxidative stress and immune modulations. However, effects observed in the present investigations were
relatively weaker in comparison with previously published studies. Supported by Czech projects NAZV QH71015 and MSM 6215712402.
240
THE MANILA CLAM RUDITAPES PHILIPPINARUM: A NEW
MODEL SPECIES TO INVESTIGATE THE GENETIC BASIS
OF RESISTANCE TO PARALYTIC SHELLFISH TOXINS?
Monica Bricelj1, Susan Ford2, Scott MacQuarrie3, Christophe Lambert4, A. Barbou4, V. Trainer5, C. Paillard4
1
Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901, USA
2
Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, New Jersey 08349, USA
3
MacQuarrie Research Consultants, 1142 Ketch Harbour Road, Ketch Harbour, Nova Scotia B3V 1K6, Canada
4
Institut Universitaire Européen de la Mer, LEMAR, Technopôle Brest Iroise, 29280 Plouzané, France
5
NOAANorthwest Fisheries Science Center, 2725 Montlake Blvd. Seattle, WA 98112, USA
The Manila clam, Ruditapes philippinarum, a native of the western Pacific, has been deliberately introduced to Pacific North America and Europe,
including both Atlantic and Mediterranean coasts of France, where established populations support valuable commercial fisheries. Manila clams
accumulate intermediate levels of paralytic shellfish toxins (PSTs) relative to other bivalves. This study determined a) the individual susceptibility of
Manila clams to toxic Alexandrium spp. as measured by effects on in vivo burrowing behavior and in vitro nerve response, and b) the effects of exposure to Alexandrium spp. on hemocyte concentrations and function, and the progression of brown ring disease (BRD), caused by Vibrio tapetis.
PSTs slowed the progression of BRD, rather than aggravating the disease, as initially hypothesized, but they produced no overall detrimental effects on hemocyte parameters. Individual variability in susceptibility to PSTs in a clam population from southern Brittany, France, characterized by
relatively low PSP levels, was confirmed from both burrowing and nerve responses. A burrowing assay indicated that 45% of the adult test population was resistant to PSP, while the remaining 55% was sensitive, i.e. was incapacitated following ~1d exposure to a high-toxicity Alexandrium
isolate. Clams from this region also exhibited 5-fold individual variability in nerve resistance to saxitoxin. Prior findings of phenotypic variability in sensitivity to PSTs led to the discovery of a Na+ channel mutation that confers resistance to PSTs in softshell clams, Mya arenaria. Thus T. philippinarum
may provide an alternate model species in which to investigate the molecular/genetic basis for resistance to PSTs.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 241
MECHANISMS OF TOXICITY
ADAPTATION OF FRESHWATER MUSSELS
TO CYANOBACTERIAL TOXINS: COMPARISON
OF BIOTRANSFORMATION AND PHYSIOLOGICAL COSTS
Vanessa Burmester1, Claudia Wiegand2
1
Institute of Freshwater Ecology and Inland Fisheries, Department of Ecophysiology and Aquaculture, Müggelseedamm 301,
12587, Berlin, Germany
2
Institute of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M. Denmark.
E-mail: [email protected]
Freshwater mussels as Dreissena polymorpha and Unio tumidus with high filtration activities may accumulate cyanobacteria and their toxins during cyanobacterial blooms and lysis. Physiological adaptations against the toxic effect enable organisms to live in cyanobacterial contaminated water bodies. Biotransformation and excretion capacities have been demonstrated for D. polymorpha. This study focused on
comparing the activities of biotransformation (glutathione S-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT) in the invasive D. polymorpha to the indigenous U. tumidus in response to cyanotoxin exposure (10 and 50 μgL-1 microcystinLR) for 24h and 7d. A further aim was to correlate the detoxification attempts to the physiological costs for the organisms (glycogen content).
Enzyme activities and glycogen content of D. polymorpha were measured in whole mussel tissue and of U. tumidus in digestive gland, gills,
mantle and food. The cGST activities were increased for the whole exposure period in D. polymorpha and didn’t change in the different tissues of U. tumidus. The catalase activity didn’t change during the whole period in both species. The glycogen content decreased after 24h
in both species indicating the energy requirements due to the stress caused by the MC-LR exposure. We conclude that D. polymorpha is
capable of detoxification of MC-LR but at the expense of energy. The results suggest that U. tumidus is less able to detoxify MC-LR via
biotransformation enzyme GST. Nevertheless, there was an enhanced requirement for energy, as indicated by reduced glycogen contents
in both mussel species. Compared to U. tumidus the invasive D. polymorpha seems to be better adapted to cyanotoxin exposure.
241
CYANOBACTERIA FROM TRI AN RESERVOIR, VIETNAM,
AND TOXICITY OF CYANOBACTERIAL COMPOUNDS
TO DAPHNIA MAGNA
Thanh-Son Dao1,2, Lan-Chi Do-Hong3, Claudia Wiegand4
1
Leibniz Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 301, 12587 Berlin, Germany.
E-mail: [email protected]
2
Institute for Environment and Resources, 142 To Hien Thanh Street, District 10, Ho Chi Minh City, Vietnam
3
Vietnam National University - Hochiminh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam
4
University of Southern Denmark, Institute of Biology, Campusvej 55, 5230 Odense M, Denmark
Tri An Reservoir provides drinking water for millions of people in Southern Vietnam. Due to it mesotrophic status, cyanobacteria were monitored,
isolated and cultivated for toxin analysis. Simultaneously, toxicity of cyanobacteria was tested with Daphnia magna at different aspects including enzymatic, behavioral responses and life traits of the animals. Daphnids were exposed to crude extracts of cyanobacterial isolates or cultures for enzyme activity or behavioral change studies. Animals were also exposed chronically for two generations to microcystin (MC) in 5 or 50 μg L-1 starting
at neonate stadium. Life history traits were observed for the first generation for two months and the second generation for one week. Twenty seven
species of cyanobacteria were recorded and Microcystis was common in the reservoir. Total MC concentration in scum samples was up to 0.64
mg g-1 dry weight. Cyanobacterial biomass temporally and spatially varied ranging from 0.009 - 0.834 mg L-1. Isolates caused sudden changes in
animal behavior and crude extracts induced an increase of antioxidant enzyme activities of daphnids. Survivorship decreased during chronic exposure with increasing MC concentration. Low concentration of MC slightly affected the growth and reproduction of parent daphnids. However, high
concentration caused a strong reproduction reduction. Age to maturity of the offspring increased and their survival decreased after parent generation was exposed to the toxin, even if the offspring were raised in control medium. Growth of offspring declined with increasing toxin concentration.
Moreover, cessation of the eggs/embryos was observed and malformation of neonates caused by cyanobacterial toxins was firstly recorded.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 242
FATE OF THE RELEASED TOXIN IN A XENIC BI-SPECIES
CULTURE OF DINOPHYSIS ACUMINATA AND ITS PREY’S
PREY, TELEAULAX AMPHIOXEIA
Na Ha, Geumog Myung, Jung-Rae Rho, Wonho Yih
Department of Oceanography, Kunsan National University, Kunsan 573-701, Republic of Korea.
E-mail: [email protected]
Among the 3 phototrophic protist species, Dinophysis acuminata is a peduncle feeder on a mixotrophic red tide ciliate Myrionecta rubra
which in turn is an engulfment grazer on its cryptophyte prey Teleaulax amphioxeia. Filtrates of the bi-species culture of D. acuminata
(strain KNU DA-MAL01) and its prey’s prey, T. amphioxeia (strain KNU CR-MAL01) exhibited strong cytotoxicity to Artemia nauplii
when cell concentration of the co-occurring T. amphioxeia is over 105 ml-1. Cytotoxicity of the fresh daily filtrates, however, was detectable only in subsamples on day 3 during the 6 days’ cultivation experiment, to imply that the released toxic material in the xenic coculture should lose its cytotoxicity within 24h. Interestingly, the toxic filtrate on day 3 could exert its toxicity to Artemia nauplii for longer
than 4 days after the daily subsampling. It seems that the particle fraction of the subsamples on day 3 somehow affected the cytotoxicity of the released toxin in the bi-species culture. Contribution of different size fractions of the particles in the xenic co-culture to the fate
of the released toxin will be discussed on.
242
PERSISTENT EFFECTS OF ACUTE DOMOIC ACID
EXPOSURE AFTER RECOVERY FROM NEUROBEHAVIORAL
EXCITOTOXICITY
Kathi Lefebvre1, Frederico Farin2, Theo Bammler2, Richard Beyer2, Emma Hiolski1, Mark Myers1, Elizabeth Frame1
1
NOAA Fisheries, Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle, Washington 98112, USA.
E-mail: [email protected]
2
Department of Environmental and Occupational Health Sciences, School of Public Health and Community Medicine,
University of Washington, Seattle, Washington 98105, USA
Domoic acid (DA) is a naturally produced phycotoxin that is responsible for a neurotoxic illness known as Amnesic Shellfish Poisoning
(ASP). Clinical signs of acute DA poisoning have been well defined for many species and include severe forms of neurobehavioral excitotoxicity such as seizures and erratic spiral-swimming in mammalian and fish models, respectively. Victims of toxicity can recover after
suffering symptoms and appear to return to “normal” behavior. However, little is known about the underlying impacts that may persist long
after recovery. To further understand the mechanisms of neurotoxic injury associated with DA exposure, we examined the transcriptome
of whole brains from zebrafish (Danio rerio) at multiple time points after receiving one symptomatic dose of DA via intracoelomic (IC) injection. Only fish that exhibited DA-induced spiral-swimming followed by complete recovery to “normal” behavior were examined in this
study. Whole brains were sampled at four time points from DA exposed fish and from time-matched control fish that were IC injected
with the vehicle (PBS). Microarray analysis comparing gene expression between exposed and control treatments yielded 917, 671, 159,
and 89 differentially expressed genes (>
_1.5-fold, p<
_0.05) at 24 hrs, 48 hrs, 1 wk, and 2 wks after exposure, respectively. A summary
of the functional and biological processes represented by these genes will be presented. These data provide a basis for identifying pathways of DA-induced injury that persist and may have long-term health consequences.
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MECHANISMS OF TOXICITY
ORAL PRESENTATIONS
CYTOTOXIC AND MORPHOLOGICAL EFFECTS
OF MICROCYSTIN-LR IN IN VITRO MODELS –
A COMPARISION BETWEEN HEPG2, VERO-E6,
MDCK AND CACO-2 CELL LINES
Carina Menezes, Elsa Alverca, Elsa Dias, Filomena Sam-Bento, Sérgio Paulino, Paulo Pereira
National Health Institute Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal.
E-mail: [email protected]
Microcystin-LR (MCLR), a potent hepatotoxin, is transported selectively into the cells through specific membrane polypeptides mostly present in the liver. These transporters are also expressed in the brain, kidneys and intestine, although the toxicity of MCLR in these cell types
is still poorly understood. In this study, morphological, ultrastructural and biochemical analyses were performed in hepatic, renal and intestinal cell lines in order to evaluate the toxicity of MCLR obtained from semi-purified cyanobacterial extracts. Our results show that after 24h
of exposure, MCLR induces the viability decrease of all cell lines, in a concentration-dependent manner. HepG2 cells are the most susceptible,
followed by Vero, MDCK and CaCo-2. Ultrastructural analyses show that subcytotoxic concentrations of MCLR induce the formation of large
cytoplasmic vacuoles, particularly in the HepG2 cell line. Immunofluorescence microscopy reveals that these vacuoles are enriched in LC3B
protein, suggesting autophagy as an early cellular response of HepG2 and Vero cells to MCLR. At cytotoxic MCLR concentrations, lysossomal dysfunction in both cell lines occurs prior to mitochondrial disruption, as demonstrated by the specific labeling with Acridine Orange
and Rhodamine-123. This suggests that besides mitochondria, lysossomes may also be an MCLR-early target. Immunolocalization and western blot analysis of the endoplasmic reticulum anti-apoptotic protein GRP94 show distinct MCLR-induced effects in Vero and HepG2 cells:
re-localization of GRP94 within Vero cells and decrease of GRP94 expression in the HepG2 cell line. These results demonstrate that all the
studied cell lines are susceptible to MCLR although with cell type specificity and differential organelle targeting.
243
AGE RELATED ACUTE EFFECTS OF MICROCYSTIN-LR
ON DAPHNIA MAGNA BIOTRANSFORMATION
AND OXIDATIVE STRESS
Rafael Ortiz-Rodrίguez1, Claudia Wiegand2
1
Institute of Freshwater Ecology and Inland Fisheries, Department of Ecophysiology and Aquaculture, 301 Müggelseedamm,
12587 Berlin, Germany. E-mail: [email protected]
2
University of Southern Denmark, Institute of Biology, 55 Campusvej, 5230, Odense M. Denmark.
E-mail: [email protected]
As a result of eutrophication and other factors, bloom-forming species of cyanobacteria dominate freshwater ecosystems with its consequent nuisance and hazards due to toxins. The most potent and studied cyanobacterial toxins are the microcystins, especially the variant microcystin-LR.
The zooplankter Daphnia magna plays a central role in freshwater pelagic food webs and its populations are negatively affected by cyanobacteria
due to nutritional inadequacy, feeding interference and toxic effects on growth, reproduction and enzyme activity. Particularly, biotransformation
and antioxidant enzymes play a key role on D. magna capacity to deal with MC-LR. Aim of our study was to investigate the effects of MC-LR on
biotransformation and antioxidant enzymes (glutathione S-transferase and catalase), lipid peroxidation and the enzymes lactate dehydrogenase and
pyruvate kinase on D. magna. F0 and F1 generations were tested due to the increasing evidence of transgenerational effects of cyanotoxin exposure. Acute effects were tested by comparing neonates (<3 days old) to young adults (7 days old), exposed to increasing concentrations of MCLR (0, 5, 10, 50 and 100 μg/L) for 24 and 48 hours. Age related, differential dose dependant changes on the biotransformation and antioxidant
enzymes and inhibition of lactate dehydrogenase occurred. Neonates’ antioxidant enzyme activities are increased or maintained at high MC-LR
concentrations. Transgenerational effects are investigated by exposing F0 generation to 10, 50 and 100 μg/L for 48 h and subject the subsequent brood (F1) to the same exposures. Results comparing effects of F1 to F0 and the neonates from the previous exposure will be presented.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 244
CHRONIC CIGUATERA FISH POISONING:
POTENTIAL MOLECULAR MECHANISMS
FOR CHARACTERISTIC INFLAMMATORY DISTURBANCES
James C. Ryan1, Ritchie C. Shoemaker2
1
Oceans and Human Health Initiative, Hollings Marine Lab, NOAA-National Ocean Service, Charleston, SC, USA.
E-mail: [email protected]
2
Center for Research on Biotoxin Associated Illnesses, Pocomoke, MD, USA
Ciguatoxins are extremely potent neurotoxins produced by tropical marine dinoflagellates. Over 100,000 people annually experience
acute ciguatera poisoning from consuming toxic fish with roughly 5% of these victims developing chronic ciguatera (CC), a widespread,
multisymptom, multisystem, chronic illness that can last tens of years. Patients with CC typically experience many persistent symptoms
such as fatigue, cognitive deficits and pain. Recently, we identified an underlying chronic inflammatory component to CC, giving traction
for both diagnosis and treatment of the disease. This work discusses some of these objective immune abnormalities identified by blood
testing, such as VIP, MSH, C4a and TGF and how they contribute to maintenance of the disease state. Now that we can identify the
presence of these serum abnormalities, we are beginning to explore why only a small percentage of patients transition from the acute
phase of exposure into the chronic disease state. We have just begun to examine the molecular mechanisms by which ciguatoxins could
hyper-activate immune cells, as well as how a pre-disposition of the host response, based on certain alleles of HLA gene loci, could lead
to a chronic illness triggered by ciguatoxin. Finally, we examine similarities to other chronic disease, driven by immune dysfunction, and
how shared mechanisms may speed understanding and treatment for CC.
244
TOXICOLOGICAL MECHANISMS OF AZASPIRACID:
AN EMERGING ALGAL TOXIN IN US WATERS
Michael J. Twiner1, Philipp Hess2, Racha El-Ladki1, Suzanne Butler1, Gregory J. Doucette3
1
Department of Natural Sciences, University of Michigan - Dearborn, 4901 Evergreen Rd, Dearborn, MI 48128, USA.
E-mail: [email protected]
2
IFREMER, Département Environnement, Microbiologie & Phycotoxines, Rue de l’Île d’Yeu, 44311 Nantes Cedex 03, France
3
Marine Biotoxins Program, NOAA/National Ocean Service, 219 Fort Johnson Rd, Charleston, SC 29412, USA
Azaspiracids (AZA) are an algal toxin group known to accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no regulatory requirements or standards in many of the other
countries where AZAs have recently been identified. In Puget Sound (WA, USA), the presence of AZAs in shellfish and in samples collected using solid phase adsorption toxin tracking (SPATT) disks provide compelling evidence of this emerging risk. Efforts are now underway to better define the effects and mechanism of action for the various AZA analogues. Our investigations have employed in vitro
cell models to characterize the potential toxicological impacts of AZA1, AZA2 and AZA3, and the use of DNA microarrays (i.e., gene chips)
has yielded valuable insights as to possible biochemical pathways targeted by AZA1. Studies with several mammalian cell types yielded
low nanomolar EC50 cytotoxicity values, making the AZAs one of the most cytotoxic algal toxin groups known. Our current focus is on
further defining the specific mechanism of action, which appears to involve inhibition of cholesterol biosynthesis. Future experiments will
continue to characterize analogue-specific cellular and biochemical responses as well as the toxicological effects of AZAs on various
model organisms such as bacteria, developing brine shrimp, and fish.
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MECHANISMS OF TOXICITY
ORAL PRESENTATIONS
UNRAVELING OF SAXITOXIN SYNTHESIS
IN DINOFLAGELLATES
Maria Wiese1, Shauna Murray1, Troco Mihali1, Gustaaf Hallegraeff2, Brett Neilan1
1
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.
E-mail: [email protected]
2
School of Plant Science and Institute of Antarctic and Southern Ocean Studies, University of Tasmania, Private Bag 77,
Hobart Tas 7001, Australia
Saxitoxin (SXT) and its analogs are nitrogen rich neurotoxins and causative agents of the paralytic shellfish poisoning (PSP). Bloom-forming gonyaulacoid dinoflagellates of the genus Alexandrium, and Pyrodinium, as well as a single gymnodinoid species, Gymnodinium catenatum, are marine producers of PSP toxins (PSTs). Certain freshwater species of cyanobacteria have been also identified to produce
PSTs. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. Unknown is also the reason for the occurance of toxic and non-toxic strains within one species of
cyanobacteria and dinoflagellates. In order to elucidate the capacity for SXT production in dinoflagellates several approaches have been
tested. The design of degenerate primers based on sequence informations from cyanobacteria and available ESTs was performed to amplify sxtA encoding for the S-adenosylmethionine synthase (SAM), a domain of the polyketide silimar enzyme responsible for saxitoxin
synthesis initiation. Expression patterns of sxtA were investigated in a qPCR assay in comparison to reference genes and genes relevant
to the overall metabolism of the dinoflagellate, such as a glutamin synthase and the chlorophyll a-c binding protein.
245
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POSTER PRESENTATIONS
IMMUNOFLUORESCENCE LOCALIZATION
OF MICROCYSTIN-LR IN HEPATIC (HEPG2)
AND RENAL (VERO-E6) CELL LINES
Elsa Alverca1, Carina Menezes1, Elsa Dias1, Filomena Sam-Bento1, Sergio Paulino1, Jose M. Leo-Martins2,
Ana Gago-Martinez2, Paulo Pereira1
1
National Health Institute Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal.
E-mail: [email protected]
2
Departamento de Quemica Analatica y Alimentaria, Facultad de Ciencias, Campus Universitario de Vigo, Universidad de Vigo,
36310 Vigo, Spain
The cellular mechanisms underlying microcystin-LR (MCLR) toxicity are still not fully understood, particularly in non-liver cells. In a previous study, we showed that in HepG2 and Vero cell lines, the MCLR cytotoxicity involves a crosstalk among several organelles. However, its subcellular localization was never analyzed. To address this issue, immunofluorescence labelling with a monoclonal antibody
against MCLR was performed in both cell lines following a 24h exposure to 25μg.ml-1 MCLR. Our results showed that exposed cells
presented positive anti-MCLR labeling, contrary to the control cells, confirming the specificity of the antibody. Furthermore, in HepG2
cell line, most of the cells were positively labelled, showing a green signal dispersed throughout the cytoplasm, together with numerous bright foci of various sizes. On Vero cell line a smaller proportion of positively labelled cells was observed and the anti-MCLR labeling appeared in a discrete punctuated pattern, with only few cytoplasmic foci. In both cell lines no nuclear labeling was ever observed.
These results demonstrate that independently of the cell type, MCLR mostly accumulates within discrete cytoplasmic foci. However,
the hepatic cell line appears to be more efficient in internalizing MCLR than Vero cells.
246
EFFECT OF RISING SEAWATER TEMPERATURES
ON THE GROWTH AND TOXIN PRODUCTION
OF PROROCENTRUM LIMA
Aldo Aquino-Cruz1, Duncan A. Purdie1, Steven Morris2
1
School of Ocean and Earth Science, National Oceanography Centre, University of Southampton, European Way, SO14 3ZH,
Southampton, UK. E-mail: [email protected]
2
Centre for Environment, Fisheries & Aquaculture Science (Cefas), Barrack Road, DT4 8UB, Weymouth, UK
Benthic toxic dinoflagellates are increasingly observed to occur in shallow productive coastal waters where elevated seawater temperatures
may cause more rapid growth and higher toxin levels. The aim of this research is to investigate the effect of rising seawater temperature
on the growth and toxin content of the benthic/epiphytic toxic dinoflagellate Prorocentrum lima. Clonal isolates of Prorocentrum lima from
the Fleet Lagoon, southern of England, were cultured under a range of temperatures (5-30°C) to determine the effect of seawater temperature on their growth rates and toxin production. Isolates were maintained in 1 L flask with f/2 media at irradiance of 35-65 μmol m-2
s-1 (12L:12D photoperiod) and changes in cell density, photosynthetic efficiency (Fv/Fm), nutrient uptake and toxin production measured
every few days. Growth rates increased from 0.5 d-1 at 5°C to a maximum of 0.18 d-1 at 15°C then decreased to 0.14 d-1 at 30°C. Highest Fv/Fm ratios were correlated with the exponential phase and showed similar pattern of response to growth rate at the different temperatures. Maximum cell abundance was apparently limited by dissolved inorganic carbon availability rather than nitrate or phosphate. Liquid
chromatography electrospray mass spectrometry (LC-MS/MS) analysis of P. lima cell extracts showed cellular concentrations of okadaic
acid (OA) and dinophysistoxin-1 (DTX-1) increased with increasing seawater temperature. Toxin content increased during the stationary
phase with maximum concentration of 15.1 pg[total OA]cell-1 and 6.48 pg[total DTX1]cell-1. The effect of increased sea water temperature particularly for shallow water regions will be discussed in relation to P. lima toxin production and growth.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 247
MECHANISMS OF TOXICITY
CHARACTERIZATION OF NOVEL TUMOR PROMOTING
METABOLITES IN CYANOBACTERIAL MIXTURES
(BIOMASSES AND EXUDATES) BY IN VITRO ASSAYS
Katerina Bartova1,2, Jiri Kohoutek2, Klara Hilscherova1,2, Ludek Blaha1,2
1
Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University,
Kamenice 3, 625 00 Brno, Czech Republic
2
Centre for Cyanobacteria and Their Toxins, Lidicka 25/27, 657 00 Brno, Czech Republic
Our study aimed to investigate in vitro toxicity of complex cyanobacterial samples including intracellular mixtures (extracts of biomass) and
extracellular mixtures (exudates / spent-media). Samples were collected from 11 different cultures of cyanobacteria including mostly species
known to form toxic cyanobacterial blooms (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii, Anabaena flos-aquae,
Cylindrospermopsis raciborskii etc.). Cyanobacterial extracts and exudates were tested for their potencies to inhibit gap-junctional intercellular communication (GJIC) in rat liver stem-like cells WB-F344, which is an established in vitro marker of tumor promotion (Toxicon 2009,
53:519–524). Intracellular as well as extracellular mixtures significantly downregulated GJIC, and the observed effects differed among different cyanobacteria. However, the effects were not related to the presence of known cyanotoxins such as microcystins (MCs) or cylindrospermopsin (CYN). Exudate of the laboratory culture of C. raciborskii (not producing MCs or CYN) was the most active, and it was
selected for further investigation to screen for causative agent. The sample was fractionated by semi-preparative HPLC on C-18 column
using gradient elution with water/methanol. Two fractions containing highly hydrophobic compounds significantly inhibited GJIC, and they
were further fractionated and analysed with tandem LC-MS/MS. Our study documents the presence of unknown metabolites produced extracellularly during the growth of cyanobacteria that may act as tumor-promoters, and currently running analyses indicate that phospholipid
(membrane) derivatives might be the causative agents of GJIC inhibition. The research was supported by the Czech National Science Foundation grant No. 524/08/0496, infrastructure is supported by the project CETOCOEN (CZ.1.05/2.1.00/01.0001).
247
THE ZEBRAFISH EMBRYO AS A “HIGH-OUTPUT”
MODEL FOR IDENTIFICATION, ISOLATION
AND CHARACTERIZATION OF CYANOBACTERIAL TOXINS
John Berry1, Asha Jaja1, Katherine Walton1, Miroslav Gantar2, Patrick Gibbs3, Michael Schmale3
1
Department of Chemistry and Biochemistry, Marine Science Program, Florida International University, 3000 NE 151st Street,
North Miami, FL, USA. E-mail: [email protected]
2
Department of Biological Sciences, Florida International University, 11200 SW 8th Street, Miami, FL, USA
3
Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker
Causeway, Miami, FL 33149, USA
The zebrafish (Danio rerio) embryo - specifically owing to a number of practical advantages - is emerging as an important vertebrate
model for a wide range of studies, spanning disciplines of genetics and molecular biology, biotechnology and toxicology. From a toxicological perspective, the zebrafish embryo presents a dynamic and diverse set of targets, and as such represents a “high output” model
system. We have utilized the zebrafish embryo to investigate freshwater cyanobacterial toxins, and particularly those that inhibit or impair vertebrate developmental pathways (“developmental toxins”). Specifically, this approach has been utilized to both characterize developmental toxicity of known cyanobacterial toxins, as well as to identify, isolate and characterize previously unknown metabolites,
including apparently novel compounds from otherwise toxigenic taxa. Examples of these applications will be presented.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 248
DIFFERENTIAL TOXIN RESPONSE OF PSEUDO-NITZSCHIA
SPECIES AS A FUNCTION OF NITROGEN SUFFICIENCY
AND SOURCE: A COMPARATIVE STUDY
OF P. MULTISERIES AND P. CUSPIDATA
William P. Cochlan1, Regina L. Radan1,2, Maureen E. Auro1,3, Julian Herndon1, Vera L. Trainer4
1
Romberg Tiburon Center for Environmental Studies, San Francisco State University, 3152 Paradise Drive, Tiburon, CA 94920,
USA. E-mail: [email protected]
2
Ocean Sciences Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA
3
Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
4
NOAA Northwest Fisheries Science Center, 2725 Montlake Blvd. E., Seattle, WA 98112, USA
Toxigenic diatoms on the west coast of North America experience a variety of nutrient conditions. Experiments designed to elucidate the effects
of nitrogen nutrition and sufficiency on cell growth and domoic acid (DA) production in large- and small-celled species of Pseudo-nitzschia demonstrate that there is no common response. Under nitrogen-replete, batch culture conditions, P. multiseries (ca. 95 x 6 μm) and P. cuspidata (ca.
50 x 2 μm) grew equally well on nitrate, ammonium and urea. Cellular DA concentrations (particulate DA normalized to cell abundance) also were
very similar for exponentially growing P. cuspidata regardless of the N substrate, whereas the cellular DA levels for urea- and ammonium-grown
cultures of P. multiseries were greater than nitrate-grown cells. Nitrogen-limited continuous cultures and N-starved batch cultures of P. multiseries
were utilized to determine if the degree of N-sufficiency alters substrate-mediated toxicity. At all tested levels of N-sufficiency, both inorganic and
organic N forms supported the growth of this diatom equally well, but urea-grown cells produced higher cellular DA concentrations compared to
those grown on either nitrate or ammonium. Shipboard experiments using natural phytoplankton assemblages collected off Washington State provided similar results; consistently greater toxicity for assemblages amended with ammonium or urea than those grown on nitrate alone. These laboratory and field results demonstrate that form of nitrogen available for growth is an important regulator of toxicity of developing coastal blooms,
and should be considered when evaluating the potential effects of cultural eutrophication on the growth of harmful diatoms.
248
LIVER EROD AND GST ACTIVITIES FOLLOWING
INTRAPERITONEAL EXPOSURE OF WHITE SEABREAM
(DIPLODUS SARGUS) TO PARALYTIC SHELLFISH TOXINS
PRODUCED BY GYMNODINIUM CATENATUM
Pedro R. Costa1, Patricia Pereira1, Marisa Barata2, Lydia Nicolau2, Pedro Pouso-Ferreira2
1
IPIMAR - National Institute of Biological Resources, INRB I.P., Av. Brasilia s/n, 1449-006 Lisbon, Portugal.
E-mail: [email protected]
2
IPIMAR - National Institute of Biological Resources, INRB I.P., Av. 5 de Outubro s/n, 8700-305 Olhao, Portugal
The re-emergence of Gymnodinium catenatum in 2005 after a ten years hiatus of absence in the Portuguese coast, initiated the present investigation
on effects of paralytic shellfish toxins (PST) in small planktivorous fishes. The aim of this study is to investigate changes in metabolism and activities of
detoxication enzymes of a combination of PSTs produced by Gymnodinium catenatum. The typical PST profile of G. catenatum includes the hydrophilic
sulfamate (C and B toxins) and decarbamoyl (dcSTX, dcGTX and dcNeo) toxins as well as the hydrophobic hydroxybenzoate analogues (GC1-3). Twenty
one juveniles of white seabream (Diplodus sargus) were intraperitoneally (i.p.) exposed to two nominal doses (2.5 and 1 μg STX eq kg-1) of hydrophilic
PSTs and two concentrations of hydrophobic PSTs, which corresponded to the respective counterpart of the hydrophilic PSTs produced by a 15 L G.
catenatum culture. The compounds were semi-purified via SPE and determined via HPLC-FLD and HILIC-MS. Fishes exposed to the highest dose of
hydrophilic PSTs, 2.5 μg STXeq kg-1, died within 1 h after the i.p. injection. Four specimens exposed to the highest dose of hydrophobic PSTs died within
24 h. The activities of ethoxyresorufin O-deethylase - EROD (phase I enzyme) and glutathione S-transferase -GST (phase II enzyme) were measured in
liver of fish specimens 48 h, 6 and 10 days after i.p. injection of the sublethal doses. Modulation of biotransformation enzymes were recorded in liver of
fish exposed to toxins, particularly hydrophilic PSTs. A significant (p<0.05) reduction of EROD activity was measured in fish injected with 1 μg STXeq of
hydrophilic PSTs kg-1 at 48h and 6 days after exposure. Although not significant, higher EROD activity was measured in fishes exposed to hydrophobic
toxins than fishes exposed to the saline solution (control). A significant induction of GST activity was measured 48 h after exposure to 1 μg STXeq of hydrophilic PSTs kg-1. Although not significant, reduction of GST activity measured 48 h after i.p. injection of the two concentrations of hydrophobic analogues was observed. These results suggest different roles for EROD and GST in the metabolism of the hydrophilic and hydrophobic PSTs.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:41 PM Page 249
MECHANISMS OF TOXICITY
POSTER PRESENTATIONS
THE ICHTHYOTOXICITY OF FIBROCAPSA
JAPONICA (RAPHIDOPHYCEAE) IS NOT CAUSED
BY ENDOTOXINS ALONE
M. Karin de Boer1,2, Claire Boerée2, Gemma Kulk2, Sacha Sjollema2, Tea de Vries2, Jan van der Heul3,
Adriaan Rijnsdorp3, Anita G. J. Buma2
1
Marine Benthic Ecology and Evolution, CEES, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands.
E-mail: [email protected]
2
Ocean Ecosystems, ESRIG, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands
3
Aquaculture and Fisheries Group, IMARES, Haringkade 1, 1976 CP IJmuiden, the Netherlands
Fibrocapsa japonica is a potential ichthyotoxic HAB species. Fish kills caused by F. japonica have so far been ascribed to both mechanical damage and toxin activity. In the presented study we tried to gain more insight in the mechanisms behind the ichthyotoxicity of F. japonica by performing direct and indirect fish tests. In different experiments living cells and cell extracts from different growth phases of a Dutch
F. japonica strain were directly and indirectly exposed to fish larvae of the Dutch flat fish Solea solea. During and after exposure samples
for toxin activity, cell density, fish mortality and behaviour were taken. In addition fish larvae were exposed to a range of brevetoxin and
haemolysin concentrations. The exposure of fish larvae to living F. japonica cells showed lethal effects after exposure times of 6-7 days.
In contrast, exposure to F. japonica cell extracts did not show measurable effects. Results of direct and indirect fish exposure to living
cells and haemolytic activity inside and outside F. japonica cells showed similar trends at the different cell concentrations. When fish larvae were present the concentrations of brevetoxins appeared to increase while at the same time algal growth was negatively affected.
Since differences were found in larval mortality and behaviour when exposed to living algae or pure toxins, we conclude that a combination of vectors, and not brevetoxin action alone, is responsible for the ichthyotoxic effect of F. japonica cells.
249
TRANSEPITHELIAL PERMEABILITY OF CACO-2 CELLS
MONOLAYERS TO 13 DESMESPX-C
Begon~a Espin~a1, M. Carmen Louzao1, Paz Otero1, Mercedes Alvarez2, Luis M. Botana1
1
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
E-mail: [email protected]
2
Laboratorios CIFGA, Plaza de Santo Domingo, 1, 27001 Lugo, Spain
Spirolides (SPXs) are lipophilic toxins included in the cyclic imines group of marine phycotoxins. These compounds are produced by the
dinoflagellates Alexandrium ostenfeldii and Alexandrium peruvianum and seafood from European and American countries were found to
accumulate them. SPXs induce coordination and movement decrease, abrupt locomotion movements, general trembling, lacrimation excess, respiratory distortion and even dead when injected intra-peritoneally to mice. In order to predict the intestinal absorption of the 13
desmethyl spirolide-C (13 desmeSPX-C) we developed transepithelial assays using differentiated CaCo-2 cells monolayers. We used
them when we obtained a transepithelial electric resistance (TEER) measurement of 400 Ω cm2 at least. 13 desmeSPX-C added to the
donor compartment only could trespass to the receptacle going through the monolayer. Then, we took samples each a short time period
that were analyzed and quantified by the liquid chromatography-tandem mass spectrometry technique (LC/MS-MS). Thus, we found that
13 desmeSPX-C is able to go through CaCo-2 monolayers without affecting their integrity. Furthermore, the linear relationship between
concentration of toxin added to the donor compartment and the quantity of 13-desmeSPX-C found in the receptacle points to a passive
diffusion of this toxin through the CaCo-2 monolayer. Finally, given the apparent permeability of 8x10-6 cm/s calculated, 13-desmeSPX-C human intestinal absorption prediction can be estimated to be almost 100%.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 250
CYLINDROSPERMOPSIN CAUSES A CYTOTOXIC
RESPONSE IN RAT HEPATOCYTES CLONE 9
~a1, M. Carmen Louzao1, Mercedes R. Vieytes2, Luis M. Botana1
Lorena Ferna΄ndez-Gonza΄lez1, Begon~a Espin
1
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
E-mail: [email protected]
2
Departamento de Fisiologίa Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain
Cylindrospermopsin (CYL) is a hepatotoxic alkaloid, reported as one of the most important freshwater algal toxins, mainly produced by
Cylindrospermopsis raciborskii. It is known that, at cellular level, CYL is a potent inhibitor of the protein and glutathione synthesis. As previously demonstrated in other studies, the hepatocytes are the most sensitive cellular model to study this toxin. So we tested the effect
of CYL on the viability of Clone 9, a cell line of rat hepatocytes, measuring their metabolic rate by using an indicator which becomes fluorescent when it is reduced as a consequence of the cell metabolism. Thus, we found that CYL decreased the metabolic rate of Clone
9 cells in a dose-dependent manner. Furthermore, we observed that 8 hours of incubation with micromolar concentrations of CYL were
enough to produce a significant fall. After 24 hours, also lower concentrations decreased the hepatocytes metabolic rate. Clone 9 cells
viability felt until 17% with 72 hours of incubation, for this incubation time the IC50 calculated was 1.52 μM. In conclusion, we can state
that Clone 9 hepatocytes are sensitive to a cytotoxic effect produced by CYL, dependent on concentration and time of exposure.
250
IN VITRO GENOTOXICITY INVESTIGATION
OF AZASPIRACID-1 AND PECTENOTOXIN-2
WITH THE MICRONUCLEUS ASSAY
Valérie Fessard, Annick Mourot
Anses, Toxicology of Contaminants, La Haute Marche, BP 90203, 35302 Fougères Cedex, France.
E-mail: [email protected]
Azaspiracid (AZA) and pectenotoxin (PTX) have been both associated with the lipophilic toxins complex until recently. Even though their
occurrence in shellfish has been reported for a wild now, their toxicological effects, including genotoxicity, have been only partly investigated. Moreover, their modes of action are still unknown and the toxicological data available are not sufficient to complete risk assessment. Due to the high risk for human health, genotoxicity is one of the first effects tested when a new compound is developed. It is also
a keypoint for toxin investigation. Among the in vitro assays for genotoxicity testing, the micronucleus test can detect both chromosomes
breakage and loss. To evaluate the genotoxicity of AZA1 and PTX2, the micronucleus assay was performed on the human intestinal cell
line Caco-2. Both undifferentiated (24h after seeding) and differentiated cells were treated with AZA 1 or PTX 2 for 24h. To obtain differentiated cells, cultures were maintained for 21 days after reaching confluence. Responses for micronucleus assay indicated that neither AZA nor PTX induced an obvious response of chromosome breakage or loss. Even though cytotoxicity may remain low, however,
experiments with higher concentrations are restricted due to the toxin disposal. For both AZA-1 and PTX-2, further investigation on
genotoxicity should be conducted in order to conclude on their genotoxic potential mainly by in vivo assays.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 251
MECHANISMS OF TOXICITY
POSTER PRESENTATIONS
IS DINOPHYSIS SPP. RESISTANT TO DIGESTION
BY COPEPODS?
Constantin Frangoulis1, Sandra Jansen2, Christian Wexels-Riser3, Isabelle Maneiro4, Kalliopi Pagou1, Edna Granéli5
1
Institute of Oceanography, Hellenic Centre for Marine Research, Former American Base of Gournes, P.O. Box 2214, 71003
Heraklion, Crete, Greece. E-mail: [email protected]
2
Alfred Wegener Institute for Polar and Marine Research, Bussestrasse 27, D-27570 Bremerhaven, Germany
3
Department of Marine and Arctic Biology, University of Tromsο/, N-9037 Tromsο/, Norway
4 ΄
Αrea Ecologίa, Universidad de A Corun~a, Campus A Zapateira s/n, 15071 A Corun~a, Spain
5
Marine Ecology Department, Linnaeus University, 39182 Kalmar, Sweden
Dinophysis cf. acuminata and Dinophysis norvegica cells inside faecal pellets produced by copepods (Calanus helgolandicus, Temora longicornis, Acartia clausi and mixed copepods population) were examined by light- and scanning electron microscopy in different regions: North
Sea, Atlantic Ocean and Eastern Mediterranean Sea. The Dinophysis spp. cells inside the pellets appeared intact in all these regions,
whereas in some cases all other material present in the pellets (e.g. diatoms) was strongly broken or amorphous. Experiments with micromanipulators could determine the strength of these cells to breakage by copepods. This resistance to digestion by copepods may have
essential impact on the transport and fate of these cells and their associated toxins in marine systems.
251
IN VITRO DIGESTION CHALLENGE TO INVESTIGATE
THE AVAILABILITY OF AZASPIRACID TOXINS FROM BLUE
MUSSEL (MYTILUS EDULIS) WHEN CONSUMED
Jennifer Geraghty1,2, Conor Duffy1, Barry Foley2
1
2
Marine Institute, Rinville, Oranmore, Co. Galway, Ireland. E-mail: [email protected]
Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland. E-mail: [email protected]
Azaspiracids (AZA) are regulated lipophilic marine phycotoxins that cause Azaspiracid Shellfish Poisoning (AZP). The availability of toxins when consumed can contribute to the overall toxic effect of the toxin and may have implications for established regulatory limits. Little is also know about the
pharmacokinetics of AZAs in mammalian tissue but in order to design pharmacokinetics studies, the effect of the digestion process needs to be
understood.
The bioaccessability of Azaspiracids in cooked (10 minutes at 90°C) and uncooked shellfish (Mytilus edulis) was investigated to determine the extent to which passage through mammalian gastrointestinal track could affect AZA toxins availability for subsequent absorption. The digestive simulation was designed to mimic the conditions found in the gastrointestinal tract using synthetic saliva, gastric and pancreatic juices. Samples of
homogenized blue mussel tissue were subjected to the various pH conditions and times normally encountered in the gastrointestinal tract, along
with agitation of the samples at 37°C. At incremental time intervals the reaction was stopped and LCMSMS analysis of AZA-1, AZA-2 and AZA3 was carried out on undigested tissue and digestive fluids. Initial findings show that cooking of the shellfish tissue had a significant effect on the
release of AZAs from the tissue during the digestive simulation. The concentration of AZAs in the uncooked mussel tissue reduced significantly
(AZA-1, 64%) over the course of the digestion simulation whereas for cooked mussel tissue the majority of AZAs toxins remained in the tissue.
This suggests that AZA toxins are less available for absorption in cooked as opposed to uncooked shellfish tissue.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 252
A COMPARISON OF NUTRIENT EFFECT ON HAEMOLYTIC
ACTIVITY PRODUCED BY TWO FISH-KILLING SPECIES:
GYMNODINIUM MIKIMOTOI AND HETEROSIGMA AKASHIWO
Victoria Hewlett1, Premlata Kshatriya1, Charles Trick1,2
1
2
Department of Biology, University of Western Ontario, N6A 5B7, London, Canada. E-mail: [email protected]
Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario,
N6A 5B7, London, Canada
A persistent HAB event occurred off the coast of United Arab Emirates (UAE) in September 2008 and caused massive fish kills and coastal
ecological devastation. A harmful algae species, Heterocapsa pygmaea, has been isolated from this red-tide event in the Persian Gulf.
Although H. circularisquama has been known to kill bivalves and produce haemolytic toxins, little has been known about the toxicity of
H. pygmaea. Also, environmental conditions that may induce toxicity are not well studied. The effect of relative nutrient abundance (N:P
ratio) and algal growth phase on haemolytic activity of H. pygmaea was determined and compared with a well-known fish killing species,
Heterosigma akashiwo. Each strain was grown in batch culture under different N:P amendments and haemolytic activity was determined
spectrophotometrically based on the lysis of rabbit erythrocytes. Haemolytic activity of H. pygmaea was consistent regardless of the nutrient abundance, whereas haemolytic activity of H. akashiwo was induced at nitrate- and phosphate-limited conditions. Additionally, the
level of haemolytic activity produced by H. pygmaea was higher at stationary growth phase. Since haemolytic activity of both harmful algal
species was induced under different nutrient amendments and growth phases, the results of this study suggest that toxic mechanisms
of each algal species are regulated differently.
252
THE EFFECTS OF LIGHT, TEMPERATURE AND SALINITY
ON GROWTH OF KARENIA BREVIS
M. Husain, Sh. Ibrahim, A. Al Sarawi, F. Al Bloushi, G. Al Otaibi
Environment Public Authority – Kuwait, AlSurrah P.O. Box 221, 45703, Kuwait. E-mail: [email protected]
Over the past 10 years, there has been an increase in the occurrence of harmful algal blooms such as those of the toxic dinoflagellate
Karenia brevis in Kuwaiti waters, further perpetuating threats to marine environments and to human health. K. brevis can cause neurotoxic shellfish poisoning (NSP) and respiratory irritation in humans. In this study we focused on culture isolates from Kuwait Bay and the
southern area. We examined the effects of light, temperature and salinity on the growth rate of K. brevis from Kuwaiti waters. Growth
rates of K. brevis were determined under various combinations of irradiance (15, 30, 50, 60, and 123 mmol m-2 s-1), salinity (25, 30,
35, 40 and 45), and temperature (20, 25, 30, 35 and 15°C). Maximum growth rates varied from 0.10 to 0.45 div day-1 with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 15 mmol photons m-2 s-1 for any experiment.
Maximum growth rates at 15°C were much lower than at other temperatures.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 253
MECHANISMS OF TOXICITY
POSTER PRESENTATIONS
PALYTOXIN-INDUCED CYTOTOXICITY IN HUMAN
INTESTINAL CELL LINE CACO-2
M. Carmen Louzao1, Lorena Ferna΄ndez-Gonza΄lez1, Begon~a Espin~a1, Eva Cagide1, Isabel R. Ares1,
Mercedes R. Vieytes2, Luis M. Botana1
1
2
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain
Departamento de Fisiologίa Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain
Palytoxin is a highly toxic compound associated with seafood intoxications leading symptoms like vasoconstriction, hemorrhage, pulmonary hypertension or death. The toxin binds to the plasma membrane Na+/K+ -ATPase, converting this pump into an open ion channel. Nevertheless, information relating palytoxin action and injuries in gastrointestinal cells is scarce. Our goal was to study the intestinal
cytotoxic effect of palytoxin. With this purpose we choose Caco-2, colon adenocarcinoma cells, and measured cellular viability through
a microplate metabolic activity assay. Even though at the beginning of the experiment the metabolic rate of Caco-2 cells increased, soon
palytoxin triggered a dose-dependent decrease in cell viability. This reduction was obvious after 24 hours incubation, reaching only
20% of viability. Pretreatment of cells with ouabain, a cardiac glycoside that specifically inhibits the Na+/K+ -ATPase, avoids the decrease on viability induced by the toxin. Therefore, those results indicate that action of palytoxin on the Na/K+ ATPase triggers the cytotoxic effect on Caco-2 cells.
253
REGULATION OF THE T CELL RECEPTOR COMPLEX
EXPRESSION AND APOPTOTIC EVENTS INDUCED BY
YESSOTOXIN IN MOUSE LYMPHOCYTE CELL LINE EL-4
A. Martίn-Lόpez, Juan J. Gallardo-Rodrίguez, Asterio Sa΄nchez-Mirόn, Francisco Garcίa-Camacho,
Antonio Contreras-Gόmez, Emilio Molina-Grima
University of Almerίa, 04120 Almerίa, Spain. E-mail: [email protected]
We have studied the effects of the marine phycotoxin yessotoxin (YTX) on the cell surface expression of the T cell receptor complex by
the mouse T lymphocyte cell line, EL-4. Immune system cell lines are relevant targets to study the immunotoxic potential since yessotoxin has been reported to suppress the immune functions. This study reports results from exposing cells to increasing concentrations of
YTX for 72 h. Concentrations of the same order have been previously found in mice bloodstream after oral administration of YTXs. Immunofluorescence technique showed YTX is able to regulate the expression of the component of the TCR complex, CD3. Biochemical
and morphological changes characteristic of apoptotic cell death were examined in this cell line. Annexin-V staining of phosphatidylserine exposed at the cell surface indicated many apoptotic cells after 24h culture with 50nM of YTX, with an occasional increase in necrotic
cell population (propidium iodide staining). Cells exposed to 25nM of YTX showed apoptosis after 48h culture. Moreover, YTX induced
morphological changes in the EL-4 cell line. The temporal sequence of morphological changes in cells was evaluated continuously using
light microscopy. The cell viability Trypan Blue assay showed that yessotoxin decreased the cell viability in a dose-dependent. Acknowledgements: This research was supported by the Consejerίa de Innovaciόn, Ciencia y Empresa de Andalucia (P07-CVI-03193 and P09TEP-5375) and the Ministerio de Ciencia e Innovaciόn de Espan~a (CTQ2008-06754-C04-02/PPQ).
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 254
TOXICITY OF CYANOBACTERIAL BIOMASS TO BIRDS –
EFFECTS IN TESTES INCLUDING DETOXIFICATION
PARAMETERS AND HISTOLOGY
Veronika Paskova1, Klara Hilscherova1, Hana Bandouchova2, Veronika Damkova2, Jiri Pikula2
1
Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University,
Kamenice 126/3, 625 00 Brno, Czech Republic
2
Department of Veterinary Ecology and Environmental Protection, Faculty of Veterinary Hygiene and Ecology,
University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic
Our previous studies demonstrated toxic effects of cyanobacteria in birds (Japanese quails) in various experimental set-up including acute,
sub-chronic and reproduction toxicity, co-exposures with toxic metals and vaccination. This study aimed to assess the testicular toxicity
of the complex cyanobacterial biomass administered to Japanese quails in the food during eight weeks (daily dose of 61.62 μg microcystins; 26.54 μg MC-RR, 7.62 μg MC-YR and 27.39 μg MC-LR). There was no mortality in both controls and biomass-exposed birds
and no alterations of other reproductive parameters such as egg viability or egg hatching. However, males exposed to cyanobacteria in
food had significantly atrophic testes compared to control birds. Interestingly, variable responses were observed in parameters related to
oxidative stress and detoxification. Biomass-exposed birds had elevated activities of catalase but decreased activities of glutathione peroxidase and surprisingly lower levels of lipid peroxides in testes. Other studied biochemical parameters (i.e. glutathione level and activities of glutathione reductase, glutathione-S-transferase and superoxide dismutase) were not affected by cyanobacteria. In conclusion,
the cell defensive system in bird testicular tissue seems to be insufficient to protect against toxic effects of complex cyanobacterial biomass. Long eight-week exposure could result in significant attenuation of oxidative stress but the overall outcome of exposure was significant damage to testes in birds. Acknowledgements: Research is supported by the Czech Ministry of Education projects No.
MSMT0021622412 and MSM6215712402. Infrastructure is supported by the project CETOCOEN (CZ.1.05/2.1.00/01.0001).
254
EFFECTS OF THE PHYCOTOXIN PALYTOXIN
ON THE HUMAN HACAT KERATINOCYTES IN VITRO
SKIN IRRITATION MODEL
Marco Pelin1, Alvaro Barreras Garcia1, Silvio Sosa1, Chiara Florio2, Roberto Della Loggia1, Aurelia Tubaro1
1
2
Materials and Natural Resources Department, University of Trieste, Via Valerio 6, 34100 Trieste, Italy. E-mail: [email protected]
Life Science Department, University of Trieste, Via Giorgieri 7/9, 34100 Trieste, Italy
Palytoxin (PLTX), isolated in tropical and subtropical areas from Palythoa zoanthid corals and from Ostreopsis dinoflagellates, is one of
the most dangerous and poisonous compounds known. In recent years, Ostreopsis ovata blooms were recorded also in the Mediterranean
Sea. From 1998 to 2006 episodes of respiratory distress with a 5% incidence of dermatitis stood out in people exposed to marine
aerosol during O. ovata blooms, especially along the Italian coasts of Puglia, Liguria and Tuscany. Analysis of microalgae samples revealed the presence of a putative PLTX as well as of ovatoxin-a, a PLTX analog. To clarify the entity of the skin distress induced by PLTX,
we characterized its effects on an in vitro irritation model, the HaCaT human keratinocytes. We found that PLTX induced a concentration-dependent cytotoxic effect. Indeed, cell mass (Sulforhodamine B assay) and mitochondrial activity reduction (MTT assay) occurred
with EC50 values of 0,47±0,09 and 0,06±0,01 nM, respectively, after 4h of PLTX exposure. The cytotoxic effect was inhibited by 10
μM ouabain (a blocker of the Na,K-ATPase), but not by the presence of the Ca2+ chelator EGTA (1mM). Moreover, in cells exposed to
PLTX for a time as short as 1h, a concentration-dependent and ouabain-sensitive ROS production (NBT assay) was observed, suggesting
that PLTX cytotoxic effect on human keratinocytes is a consequence of the osmotic imbalance and oxidative stress that follow the toxin
interaction with the Na,K-ATPase. In conclusion, these results raise concern about the effects of skin exposure to PLTX during both recreational and professional activities along seaside.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 255
MECHANISMS OF TOXICITY
POSTER PRESENTATIONS
PROTEOMIC RESPONSE OF A TOXIC DINOFLAGELLATE
ALEXANDRIUM TTAMARENSE CI01 TO ANTIBIOTICS,
NEOMYCINE
Meng-Zi Si, Cheng Li, Hua-Sheng Hong, Da-Zhi Wang
State Key Laboratory of Marine Environmental. Science/Environmental Science Research Center, Xiamen University,
182 Daxue Road, 361005, Xiamen, People’s Republic of China. E-mail: [email protected]
A. tamarense is a widely spread dinoflagellate species, which is not only able to produce potent neurotoxins causing paralytic shellfish
poisoning (PSP), but also results in harmful algal blooms (HABs) along the coastal waters. This study identified and characterized differential expressed proteins of A. tamarense CI01, a toxin-producing dinoflgellate species, grown with and without neomycin (Nm), an
aminoglycoside antibiotic, using 2D-PAGE and MALDI-TOF-TOF mass spectrometer. The toxin content, cell growth and morphology as
well as the extracellular bacteria abundance were also investigated. The results showed that the presence of Nm (1000μg/ml) remarkably inhibited toxin production of A. tamarense CI01 and decreased extracellular bacteria abundance. However, Nm produced no significant effects on cell growth and cell morphology. Comparison of 2-DE protein profiles of A. tamarense CI01 grown with Nm and without
Nm showed that 33 proteins were upregulated and 18 proteins were downregulated in A. tamarense CI01 with 1000μg/ml Nm. The
results from MALDI-TOF/TOF MS analysis showed that these proteins were involved in macromolecular metabolism, signal transduction, photosynthesis and some other particular functions, i.e. luminescence. Among of them, the expression of luciferin-binding protein
(LBP) was depressed significantly in Nm-treated A. tamarense CI01, suggesting antibiotics affected the illuminescence of A. tamarense
CI01. This study indicates that the presence of antibiotics might affect the synthesis of proteins involved in toxin biosynthesis or change
the signaling pathway related to the toxin biosynthesis, which declines the toxin production capacity of A. tamarense.
255
RAPID AND DRASTIC DECREASE OF BODY
TEMPERATURE IN MICE INTRAPERITONEALLY
INJECTED WITH OKADAIC ACID
Hodaka Suzuki
National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: [email protected]
The mouse bioassay for diarrheic shellfish poisoning (DSP) toxins has been used as the official method in Japan. This method has also
been widely used in many countries of the world. We have been studying about the pathological and pathophysiological changes of the
mice after intraperitoneal injection of DSP toxins. In this study, we found and report here, the rapid and drastic decrease of body temperature in the mice intraperitoneally injected with okadaic acid (OA). Lethal dose of OA (4μg/mouse) was injected intraperitoneally into
4-week-old, male ICR mice, weighting 18-20g. The rectal temperature of the mice was measured before and every 1 hour after injection by using an electronic thermometer. In some experiments, body surface temperature of the mice was monitored, instead of measuring the rectal temperature, by using a thermograpy camera. At 1 hour after injection, the rectal temperature decreased to below 35ºC
in all the mice injected with OA. And at 2 and/or 3 hours after injection, the rectal temperature decreased to below 30ºC in some mice.
Such rapid and drastic decrease of the body temperature in the mice was also detectable by using a thermography camera. The lethality of the mice was 40-80% in each experiment. The rapid and drastic decrease of the body temperature was observed within a few hours
in all the mice injected with lethal dose of OA. On the other hand, lethality of the animals was 40-80% at 24 hours after injection. These
results indicate the possibility that the decrease of the body temperature could be used as a new index for judging the results of the mouse
bioassay instead of death of the animals. This new index might make the mouse bioassay more sensitive and time-saving without big
changes in the conventional protocol.
11. ABSTRACT BOOK (239-256)_Layout 1 10/22/10 2:42 PM Page 256
MECHANISMS OF TOXICITY
POSTER PRESENTATIONS
CLONING AND EXPRESSION ANALYSIS
OF A TYPE PKS I GENE IN PROROCENTRUM LIMA
Nai-Sheng Zhang, Jing-Qian Tang, Wei-Dong Yang, Hong-Ye Li, Jie-Sheng Liu
Department of Biotechnology, Jinan University, Guangzhou 510632, People’s Republic of China. E-mail: [email protected]
To provide information for the HABs toxin biosynthesis mechanism and explore the functional role of polyketide synthases (PKSs) in the
synthesis of diarrheic shellfish poisoning (DSP) toxin, partial putative PKSI gene in Prorocentrum lima was amplified, and the 5- and 3adjacent unknown sequences of the isolated PKSs fragment were amplified and sequenced. The production of okadaic acid (OA), a main
component of DSP toxin, and expression of PKSI gene under different nutrient conditions in P. lima was observed by ELISA and RTPCR, respectively. Besides of the previous 675 bp sequence registered in Genbank (accession No: EF521601), 2984 bp continuous
sequence of PKSs gene, which corresponds with full ketosynthase (KS) domain and acyltransferase (AT) domain in type-I modular
polyketide synthases, were obtained from genomic DNA of P. lima and submitted to GenBank (accession: HM486498). In parallel with
OA production, PKSI gene expression conspicuously varied under different nutrient conditions in P. lima. A significantly positive correlation was observed between OA production and PKSI gene expression under different nutrient conditions with a R2 of 0.9831, suggesting that PKSs may play an important role in production of DSP toxin.
256
POSSIBLE INVOLVEMENT OF HEMOLYTIC ACTIVITY
IN THE CONTACT-DEPENDENT LETHAL EFFECTS
OF THE DINOFLAGELLATE KARENIA MIKIMOTOI
ON THE ROTIFER BRACHIONUS PLICATILIS
Yanan Zou1, Yasuhiro Yamasaki1, Yukihiko Matsuyama2, Kenichi Yamaguchi1, Tsuneo Honjo3, Tatsuya Oda1
1
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan.
E-mail: [email protected]
2
Seikai National Fisheries Research Institute, 1551-8, Taira-machi, Nagasaki 851-2213, Japan
3
Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki,
Fukuoka 812-8581, Japan
We investigated the effects of two strains (SUO-1 and FUK) of the dinoflagellate Karenia mikimotoi on the rotifer Brachionus plicatilis.
The SUO-1 strain was highly toxic to rotifers, whereas the FUK strain was less toxic. After 10-hour incubations, the survivorship of rotifers exposed to SUO-1 and FUK was 20% and 95%, respectively. Both the cell-free culture supernatant and the ruptured cell suspension prepared from these strains were not toxic to rotifers. Furthermore, when direct contact between K. mikimotoi and rotifers was
interrupted with a cell-impermeable membrane (3-μm pores), the toxicity of both the SUO-1 and FUK strains of K. mikimotoi to rotifers
were completely inhibited even after a 24-h exposure. Cell suspensions of SUO-1 showed hemolytic activity toward horse erythrocytes,
but the FUK strain did not. The cell-free supernatant and the ruptured cell suspension of SUO-1 showed no significant hemolytic activity. These results suggest that this highly toxic strain of K. mikimotoi causes lethality in rotifers by direct contact in which live cell-mediated hemolytic activity might be a contributing factor.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 257
NOVEL SENSOR
TECHNOLOGIES
FOR BIO-SENSING
APPLICATIONS
IN HAB RESEARCH
AND MONITORING
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 258
ORAL PRESENTATIONS
MIDTAL: WP 1: TOXIN DETECTION WITH A MICROARRAY
Katrina Campbell1, Jos Buijs2, Anne-Catherine Huet3, Natalia Vilarin~o4, Linda Medlin5,
Luis M. Botana4, Christopher T. Elliott1
1
Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University Belfast, David Keir Building,
Stranmillis Road, Belfast, BT9 5AG, UK. E-mail: [email protected]
2
GE Healthcare Bio-Sciences, Rapsgatan 23, 75184 Uppsala, Sweden
3
Centre d’Economie Rurale (CER Groupe), Département Santé, rue Point du Jour 8, 6900 Marloie, Belgium
4
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario,
27002 Lugo, Spain
5
Marine Biological Association of UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK
Regardless of ethical and technical concerns, for phycotoxin analysis in shellfish the mouse bioassay remains the reference method of
analysis for the global monitoring of diarrheic (DSP) and paralytic shellfish poisoning (PSP) toxins. Alternative methods of detection to
the biological methods have been described but to date each analytical method is specific for a particular toxin and chemical analogues
with each group of toxins requiring separate tests. An ideal scenario for the monitoring of marine biotoxins in shellfish and water samples
would be to incorporate multiple toxin detection into a single assay format. Surface plasmon resonance (SPR) technology has been displayed as a highly promising immunoassay based tool. This technology offers rapid real time detection requiring minimal toxin standards
which is crucial because of their limited availability. A micro-fluidic immobilization device and prototype multiplex SPR biosensor designed
for the detection of up to 16 bio-molecular binding interactions in a 4 line by 4 channel array on a single chip has been developed in the
EU project BioCop. This dual system was evaluated in its ability to be fit-for-purpose for the simultaneous detection of four key phycotoxins. Domoic acid, okadaic acid, saxitoxin and neosaxitoxin calibration curves were achieved in separate flow channels with IC50s of
2.6, 4.9, 1.9 and 2.6ng/ml. This detection system exhibits enormous potential for multiple phycotoxin screening on a single chip with
the additional benefit of being able to distinguish between toxin families using this format. Toxic shellfish and algal samples were evaluated following simple extraction protocols.
258
MIDTAL: WP1 & WP2: ENHANCEMENT OF MICROARRAY
HYBRIDIZATION SIGNAL FOR THE DETECTION
OF MULTI-SPECIES OF TOXIC ALGAE
Jixin Chen1, Linda K. Medlin1, Wiebe Kooistra2, Edna Graneli3, Beatriz Reguera4, Robin Raine5, Bente Edvardsen6,
Jane Lewis7, Chris Elliott8, Yolanda Pazos9, Lucie Maranda10
1
Marine Biological Association of UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK. E-mail: [email protected], [email protected]
2
Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy, 3 Marine Ecology
Department, Linnaeus University, SE-39182 Kalmar, Sweden, 4 Instituto Espan~ol de Oceanografίa, Centro Oceanogr΄afico de Vigo, Aptdo
1552, 36200 Vigo, Spain, 5 Martin Ryan Institute, National University of Ireland, University Road 1, Galway, Ireland, 6 Department of Biology,
University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway ,7 School of Biosciences, University of Westminster, 115 New Cavendish
Street, London W1W 6UW, UK, 8 Institute of Agri-Food and Land Use, Queen’s University of Belfast, Belfast BT7 1NN, UK, 9 Instituto Tecnoloxico Para o Control do Medio Marino de Galicia - Intecmar, Peirao de Villaxoan S/N, Villagarcia de Arousa, 36611 Spain, 10 Graduate
School of Oceanography, University of Rhode Island, Narragansett, RI 02882-1197, USA
MIDTAL is a new EU FP7 project that aims to produce a universal microarray for the detection of toxic algae and their toxins. Probes already
tested in a FISH format were adapted to a microarray format and their specificity retested. A microarray with more than 100 probes targeting
to over 30 species (strains) of toxic algae is developed in MIDTAL project (Microarrays for the Detection of Toxic Algae). An important consideration in application of MIDTAL microarray to field sample is the efficiency with which the target molecule is captured by, or hybridized to, surface-immobilized oligos. For RNA, secondary and tertiary structures of the target strand can significantly decrease capture efficiency. To
overcome this limitation, RNA is often fragmented to reduce structural effects. In this study, the hybridization conditions for the microarray for
detection of toxic algae are optimized by applying different lengths of probes and RNA / DNA fragmentation. 5 different-length probes derived
from 18nt toxic algae probes were tested, which shows length of probes significantly change the signal intensity, which refers to that longer
probes should be used. Experiments on metal based fragmentation metal ion catalyzed base hydrolysis of RNA are carried out to test the effect of fragmentation of RNA on the signal intensities of different probes located on microarray. It shows that slight fragmentation can increase
the signal/noise ratio for most probes, while excessive fragmentation of DNA will lead to lower signal intensity and less probes detected.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 259
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
NEW APPROACH FOR UNDERWATER DETECTION
OF DOMOIC ACID
Florent Colas1, Marie-Pierre Crassous1, Wayne Litaker2, Sébastien Laurent1, Emmanuel Rinnert1,
Chantal Compère1, Patrick Gentien1
1
2
Ifremer, Technopôle Brest-Iroise, BP70 29280 Plouzané, France. E-mail: [email protected]
National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, North Carolina, USA
Toxic species of the genus Pseudo-nitzschia are particularly dangerous because they produce domoic acid (DA), a neurotoxin that can
bioaccumulate and rapidly transvector throughout the food chain via phytoplankton feeder organisms. The production of toxins within and
among toxigenic phytoplankton species depends on environmental parameters, such as temperature, salinity, nutrients and trace elements
concentrations. Cell number cannot be easily related to toxicity. Systems able to detect the toxin at the early stage of the bloom would
be of great interest. An underwater transducer based on Surface Plasmon Resonance spectroscopy was then designed. This optical technique can detect refractive index changes as low as 10-6 corresponding to few picograms of molecules. Our system will be described.
Experiments for measuring its sensitivity and detection limit will be exposed. An SPR biosensor assay was then developed for underwater detection of domoic acid. At first the chip is functionnalized with DA through modified OEG chemistry. The sample is mixed with the
antibodies and the mixture is then injected in the sensor after 30 min of incubation time. Antibodies that did not react with the DA are
then quantified. Such an assay enabled us to detect DA at concentration of the order of 0.1ppb in calibration solutions during laboratory
and shipboard experiments. These first results will be discussed.
259
NOVEL FISH GILL CELL LINE ASSAY TO RESOLVE
THE PRECISE FISH-KILLING MECHANISM
BY CHATTONELLA MARINA (RAPHIDOPHYCEAE)
Juan José Dorantes-Aranda1, Gregory M. Woods2, César D. Tovar2, Aurélie Godrant3, Andrew L. Rose4,
T. David Waite3, Gustaaf M. Hallegraeff1
1
Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia. Email: [email protected]
2
Menzies Research Institute, University of Tasmania, Private Bag 23, Hobart, Tasmania 7001, Australia
3
Water Research Centre, School of Civil and Environmental Engineering, The University of New South Wales, Sydney, New
South Wales 2052, Australia
4
Southern Cross GeoScience, Southern Cross University, Lismore, New South Wales 2480, Australia
We developed a simple in vitro assay using the rainbow trout cell line RTgill-W1 to assess the mechanism of ichthyotoxicity of C. marina. Gill cells remained viable in seawater-based media for up to 3 hrs using an insert support with permeable membranes for microplates, which allowed us to perform acute toxicity assays on whole-cells, supernatant and sonicated Chattonella-cell suspensions.
Mexican strain CMCV-1 was the least toxic (up to 35% loss of viability), whereas Australian (CMPL01) and Japanese (N-118) strains
showed the highest toxicity associated with sonicated-cell preparations, with up to 71 and 65% loss of viability, respectively. Toxic
effects were sometimes equally observed in both whole-cell and sonicated-cell suspensions of N-118 and CMCV-1, with up to 57
and 35% loss of viability, respectively. In contrast, the supernatant always had the lowest toxicity for N-118 (up to 50% loss of viability). A modification of this assay was used to test toxicity of various fatty acid fractions, with pure EPA being more toxic (up to
98.5% loss of viability) than OTA (37% loss of viability when both were combined), and superoxide on its own showing negligible toxicity (14% loss of viability). These two simple, reproducible screening tests represent a more sensitive alternative to whole fish ichthyotoxic bioassays for research on fish-killing marine microalgae.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 260
FIELD-APPLICABLE COLORIMETRIC DETECTION
OF ALEXANDRIUM TAMARENSE SPECIES COMPLEX
USING PEPTIDE NUCLEIC ACID PROBES
AND A SYMMETRICAL CYANINE DYE
Janice Duy1, Rosemary L. Smith2, Scott D. Collins2, Laurie B. Connell3
1
Graduate School of Biomedical Sciences, University of Maine, Orono, ME 04469, USA. E-mail: [email protected]
Laboratory for Surface Science and Technology, University of Maine, Orono, ME 04469, USA
3
School of Marine Sciences, University of Maine, Orono, ME 04469, USA
2
The marine dinoflagellates comprising the Alexandrium tamarense species complex produce a suite of toxins responsible for paralytic shellfish
poisoning (PSP), which can result in serious illness or death of animals (including humans) who ingest shellfish that have accumulated these poisons. “Early warning” systems are employed to estimate the abundance of Alexandrium cells in the water, because even low cell densities (which
may not discolor the water) may be harmful. However, current detection methods rely on microscopic cell counting, which is time-consuming
and requires special training because of the almost-indistinguishable morphological similarities between toxic and non-toxic Alexandrium species.
The use of molecular probes targeted to these organisms permits specific identification of toxic Alexandrium species, but detection generally involves fluorescence or polymerase chain reaction (PCR), both of which require expensive reagents and equipment. We have developed a rapid,
specific colorimetric assay using peptide nucleic acid (PNA) probes and the symmetric cyanine dye 3,3′-diethylthiadicarbocyanine iodide
[DiSC2(5)]. Short (16-base) PNA probes are used to capture target Alexandrium sequences. PNA-DNA hybridization in the presence of DiSC2(5)
causes the dye to change color immediately from blue to purple, which can be quantitatively measured by an increase in the solution absorbance
at 540 nm. This assay has been tested using synthetic DNA and RNA oligonucleotide sequences and exhibits an unoptimized detection limit of
250 nM, with single-base mismatch resolution. The total sample volume is a 20 μL. This method is very simple and specific, and can be integrated with an electronic device for quantitative and high-throughput formats.
260
MIDTAL: WP2 CALIBRATION FOR THE VARIABILITY
IN CELLULAR RNA CONTENT OF SELECTED HAB SPECIES
Johannes A. Hagstrom1, Linda K. Medlin2, Wiebe Kooistra3, Edna Graneli1, Beatriz Reugera4, Robin Raine5,
Bente Edvardsen6, Jane Lewis7, Chris Elliott8, Yolanda Pazos9, Lucie Maranda10
1
Marine Ecology Department, Linnaeus University, SE-39182 Kalmar, Sweden. E-mail: [email protected]
2
Marine Biological Association of UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK
3
Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
4
Instituto Espanol de Oceanografia, Centro Oceanografico de Vigo, Aptdo 1552, 36200 Vigo, Spain
5
Martin Ryan Institute, National University of Ireland, University Road 1, Galway, Ireland
6
Department of Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway
7
School of Biosciences, University of Westminster, 115, New Cavendish Street, London W1W 6UW, UK
8
Institute of Agri-Food and Land Use, Queen’s University of Belfast, Belfast BT7 1NN, UK
9
Instituto Tecnoloxico Para o Control do Medio Marino de Galicia - INTECMAR, Peirao de Villaxoan S/N, Villagarcia de Arousa, 36611, Spain
10
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882-1197, USA
Ten partners representing 6 European countries and USA participate in the EU-funded project “Microarrays for the Detection of Toxic Algae (MIDTAL)”.
The aim is to develop a DNA chip (microarray) for rapid, simultaneous detection and quantification of most HAB species causing problems in European
marine coastal areas. MIDTAL will use hierarchical probes targeting ribosomal RNA (rRNA) molecules for species/strain identification. The fluorescent
signal intensity in the microarray is a function of the amount of RNA extracted from cells and used in the assay. It is therefore of paramount importance
for the quantitative aspect of the chip (i.e. its ability to estimate cell numbers) that variation in RNA content per cell under different environmental conditions is known for each species. To understand better the potential variability in RNA, three strains of each species represented in the microarray were
exposed to optimal conditions for nutrients (N and P for all species, Si for diatoms), irradiance, temperature, and salinity. When cultures had reached log
phase, stress condition of each variable (above and below optimum) was initiated. Optimum conditions were used as control. Cell counts and total RNA
quantification were conducted on subsamples collected at 24, 48 and 72 hours after stress conditions were established. We present the combined results of experiments run by all MIDTAL partners. These datasets will help MIDTAL to construct calibration factors to translate fluorescence signal intensity from the microarray into cell numbers. When calibrated, the microarray can be used to strengthen the EU’s ability to monitor for toxic algae.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 261
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
ORAL PRESENTATIONS
MIDTAL (MICROARRAYS FOR THE DETECTION OF TOXIC ALGAE)
Jane Lewis1, Linda Percy1, Marco Berzano1, Linda K. Medlin2, Wiebe Kooistra3, Edna Graneli4, Beatriz Reguera5,
Robin Raine6, Bente Edvardsen7, Chris Elliott8, Yolanda Pazos9, Lucie Maranda10
1
School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK. E-mail:
[email protected]
2
Marine Biological Association, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK
3
Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
4
Marine Ecology Department, Linnaeus University, SE-39182 Kalmar, Sweden
5
Instituto Espan~ol de Oceanografίa, Centro Oceanogra΄fico de Vigo, Aptdo 1552, 36200 Vigo, Spain
6
Martin Ryan Institute, National University of Ireland, University Road 1, Galway, Ireland
7
Department of Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway
8
Institute of Agri-Food and Land Use, Queen’s University of Belfast, Belfast BT7 1NN, UK
9
Instituto Tecnoloxico Para o Control do Medio Marino de Galicia - Intecmar, Peirao de Villaxoan S/N,
Villagarcia de Arousa, 36611 Spain
10
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882-1197, USA
Microalgae in marine and brackish waters of many parts of the world regularly cause harmful effects, considered from the human perspective, in that
they cause economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. For adequate management of these
phenomena, monitoring of microalgae is required. However, the effectiveness of monitoring, that it is time consuming, and cell morphology as determined by light microscopy, may be insufficient to give definitive species and toxin attribution. In the EU FP7 project MIDTAL (microarrays for the detection of toxic algae), our goal is to achieve rapid species identification using rRNA genes as the targets. Certain regions of rRNA genes with different
degrees of conservation can be targeted for probe design to recognize species or even strains. We are also developing a microarray including antibody reactions to specific toxins produced by these microalgae because even when cell numbers are low, toxins can be present and can be accumulated in the shellfish. Microarrays are the state of the art technology in molecular biology for the processing of bulk samples for detection of target
RNA/DNA sequences. Existing rRNA probes and antibodies for toxic algal species/strains and their toxins are being adapted and optimized for microarray use. The purpose of MIDTAL is to support the common fisheries policy, aid the national monitoring agencies by providing new tools for the
rapid identification of toxic algae and their toxins so they can comply with ECC directive 91/1491/CEE and reduce the need for the bioassays.
261
MIDTAL WP3: FIELD TESTING FOR TOXIC ALGAE
WITH A MICROARRAY
Gary McCoy1, Robin Raine1, Linda K. Medlin2, Wiebe Kooistra3, Edna Graneli4, Beatrix Reguera5, Bente Edvardsen6,
Jane Lewis7, Chris Elliott8, Yolanda Pazos9, Lucie Maranda10
1
Martin Ryan Institute, National University of Ireland, Galway, Ireland
Marine Biological Association of UK, The Laboratory, Citadel Hill, Plymouth, UK
3
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
4
Marine Ecology Department, Linnaeus University, SE-39182 Kalmar, Sweden
5
Instituto Espan~ol de Oceanografίa, Centro Oceanogra΄fico de Vigo, Spain
6
Department of Biology, University of Oslo, Blindern, 0316 Oslo, Norway
7
School of Biosciences, University of Westminster, London W1W 6UW, UK
8
Institute of Agri-Food and Land Use, Queen’s University of Belfast, N. Ireland
9
INTECMAR, Peirao de Vilaxoa΄n, Villagarcίa de Arosa 36611, Spain
10
Graduate School of Oceanography, University of Rhode Island, Narragansett, USA
2
One of the key tasks in the FP7 project MIDTAL (microarrays for the detection of toxic algae) is to demonstrate the applicability of microarrays to monitor harmful algae across a broad range of ecological niches and toxic species responsible for harmful algal events. Monitoring sites used in national biotoxin and toxic phytoplankton monitoring programmes have been selected in seven countries across
Europe for this purpose. At each sampling site samples are collected, filtered, and the rRNA has been extracted, labelled and applied to
the microarray chip which is then scanned and the signal intensity from <120 probes is measured and analysed. This ongoing process
will be carried out over two years. Cell counts and toxin analyses are also performed on all samples, counting as far as is possible each
species that is present on the microarray. Abundances are reconstructed from the signal intensity and compared with actual field data.
Preliminary results from year one sampling are presented.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 262
ORAL PRESENTATIONS
IN SITU CHARACTERIZATION OF PHYTOPLANKTON
COMMUNITIES USING A NOVEL SUBMERSIBLE IMAGING FLOW
CYTOMETER – FLOWCAM®
Harry Nelson1, Chris Sieracki1, Carlton D. Hunt2, Derek Michelin2, Patrick Curren2, Doug Pape3, Michael Neal3,
Caleb Chitwood3
1
Fluid Imaging Technologies, Inc. 65 Forest Falls Drive, Yarmouth, Maine 04096, USA. E-mail: [email protected]
Battelle, 397 Washington Street, Duxbury, Massachusetts 02332, USA
3
Battelle, 505 King Avenue, Columbus, Ohio 43201, USA
2
The study of plankton dynamics and particularly harmful algae is limited by a lack of data at the appropriate temporal and spatial scales.
This is in part caused by the lack of in situ tools that can sample continuously, forcing researchers and monitors to depend on limited data
measured by labor intensive laboratory methods or reliance on surrogate parameters. Research and monitoring in ocean and coastal regions would also benefit from an ability to quantify and characterize plankton on a continuous, real-time basis, and at higher spatial resolution. To address these measurement needs, we have adapted a well established digital imaging flow cytometer, Fluid Imaging
Technologies’ FlowCAM®, for in situ deployment. The new Submersible FlowCAM model retains the capabilities of the FlowCAM instrument and can operate autonomously at depths to 200 meters under user controlled operations. The instrument captures an image
of every analyzed particle for further analysis and cell identification by the user using image recognition algorithms. Whereas the submersible
FlowCAM is primarily designed for deployment on moorings/buoys, it can also be operated in a profiling mode and adapted for deployment on AUVs. This presentation describes this new instrument, presenting data from profiling and time-series deployments.
262
BIOSENSORS AS ROUTINE ANALYTICAL TOOLS
OF TOXIC ALGAE
Jahir Orozco, Linda K. Medlin
Observatoire Océanologique, Laboratoire Arago, Banyuls Sur Mer 66650, France. E-mail: [email protected]
Biosensors are powerful tools for species detection. The study of toxic algal biodiversity and distribution is greatly facilitated by the application of rRNA probes. In the EU project, ALGADEC, a biosensor was developed with 17 probe sets for toxic algae. This biosensor uses
electrochemical detection of the species in a sandwich hybridisation. A thiol (biotin) labeled capture probe was immobilised on gold (carbon) electrodes. rRNA extracted from both field material and cultures was applied to the sensor and allowed to hybridise to the capture
probe. A signal probe with a horseradish peroxidase label was then applied, followed by an antibody to the HRP and a substrate. The
electrical signal obtained from the redox reaction was proportional to the amount of rRNA applied to the biosensor, which in turn was proportional to the number of cells harvested. Optimization of the hybridization process was already achieved in a previous work. Elucidation
of the different steps of the fabrication process from the electrochemical point of view, orientation of the probes, proof of concept with
different algal species and evaluation of the influence of the transducer platform geometry and material in the biosensor analytical performance are the main achievements reported here.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 263
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
MICROARRAY DETECTION OF TOXIC SPECIES
IN THE PLANKTONIC DIATOM GENUS PSEUDO-NITZSCHIA
Lucia Barra1, MariaValeria Ruggiero1, Jixin Chen2, Pasquale De Luca1, Adriana Zingone1, Diana Sarno1,
Marina Montresor1, Wiebe Kooistra1
1
2
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy. E-mail: [email protected]
Marine Biology Association, Citadel Hill, Plymouth, UK
Several species of the diatom genus Pseudo-nitzschia produce domoic acid, a neurotoxin linked to amnesic shellfish poisoning. Morphological identification is error-prone, because of the presence of pseudo-cryptic species in the genus, making it difficult to correctly identify species in LM. Species-specific probes against the ribosomal RNA assist in the detection of toxic Pseudo-nitzschia species. Using
these probes in FISH can aid identification, but is still labour intensive. In the frame of the EU project MIDTAL we test the use of microarrays as tool for the detection of numerous toxic species simultaneously. We assessed specificity of 44 LSU rRNA probes of twenty
Pseudo-nitzschia species. Most of these probes were species-specific. Because RNA is used as a proxy for species occurrence, the concept of microarrays for detection purposes is jeopardized if rRNA content per cell varies radically with physiological state. Therefore, we
assessed the effects of temperature, and shortage of Si, P and N on RNA content per cell. Apart from these validation experiments we
also have assessed the feasibility of the method directly by hybridizing RNA harvested from plankton samples taken in the Gulf of Naples
against a series of species-specific probes and genus-specific backup-probes. Comparisons of the results with cell counts in LM and sequence tallies in rDNA clone-libraries from the same phytoplankton samples show similarities demonstrating the feasibility of the use of
microarrays for the detection of a range of toxic and non-toxic Pseudo-nitzschia species.
263
AN SPR BIOSENSOR FOR DETECTION
OF THE HARMFUL DINOFLAGELLETE ALEXANDRIUM
Amber R. Bratcher1, Laurie B. Connell1, Paul Millard2
1
2
School of Marine Sciences, University of Maine, 5735 Hitchner Hall, Orono ME, USA. E-mail: [email protected]
Laboratory for Surface Science and Technology, University of Maine, Orono ME, USA
Dinoflagelletes of the genus Alexandrium can produce dangerous amounts of toxins at such low cell densities that water discoloration
often associated with Harmful Algal Blooms (HABs) is not always evident. Species that produce toxins are difficult to distinguish morphologically from non-toxin producing species, and current identification methods are expensive, time-consuming, and require special training. Development of a rapid, low-cost and easy-to-use device to detect and monitor Alexandrium would be an important advancement,
as HABs vary interannually in location, intensity, and duration.
Our research focuses on RNA detection of multiple species of Alexandrium using peptide nucleic acid (PNA) probes and surface plasmon resonance (SPR). A portable SPR biosensing instrument has been developed with collaborators that will allow for rapid detection of
Alexandrium at suspected bloom sites. Using this method, we have achieved reliable detection of oligonucleotide sequences specific to
A. fundyense and A. ostenfeldii with good discrimination between PNA probes designed specific to each species. Preliminary results have
also shown detection of RNA extracted from cultured A. fundyense. These results indicate that this method of detection shows great promise as a tool for detecting and monitoring HABs.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 264
SPR METHODS FOR DETECTING EMERGING TOXINS
TO EUROPEAN WATERS
K. Campbell1, S. A. Haughey1, Amparo Alfonso Ranca2, Luis M. Botana2, C. T. Elliott1
1
Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University of Belfast, David Keir Building,
Stranmillis Road, Belfast, UK. E-mail: [email protected]
2
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario, 27002
Lugo, Spain
The toxins currently monitored in shellfish within the European Union are the amnesic, diarrheic (DSP) or lipophilic and paralytic shellfish poisoning (PSP) toxins. In recent years however contamination of shellfish has occurred with toxins not prevalent to European waters such as tetrodotoxin and palytoxin. Currently, these emerging toxins have been detected using the mouse bioassay (MBA). However,
with the imminent implementation of the AOAC HPLC method for PSP analysis and LC-MS for lipophilic/DSP analysis to replace the
MBA these toxins may go undetected posing a severe human health risk. Surface plasmon resonance (SPR) technology has been displayed as a highly promising immunoassay based tool for phycotoxin analysis. This technology offers rapid real time detection requiring
minimal toxin standards which is crucial because of their limited availability. Two individual SPR screening methods designed as inhibition assays were developed for the monitoring of tetrodotoxin and palytoxin. The individual immobilisation of each toxin onto separate
CM5 biosensor surfaces is described. Monoclonal antibodies raised to tetrodotoxin and palytoxin were employed and detection limits
of ng/ml were achieved for each assay. These SPR assays have sufficient sensitivity in that tetrodotoxin and palytoxin can be detected
in the range of the European regulatory limit for PSP toxins and the proposed European Food Safety Authority (EFSA) regulatory limits respectively. This biosensor technology could have an important role in providing an “easy-to-use” screening alternative to the mouse
bioassay procedure and current confirmatory techniques for these emerging toxins.
264
BIOCONCENTRATION OF PENTABROMODIPHENYL
ETHER BY SKELETONEMA COSTATUM UNDER
DIFFERENT NUTRIENT CONDITIONS
Chao Chai, Xundong Yin
College of Resources and Environment, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China.
E-mail: [email protected]
Eutrophication and persistent organic pollutants are two types of serious marine pollution problems in present worldwide. Using Skeletonema costatum as our research subject, the bioconcentration of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) by this alga under
conditions of nitrogen deficiency, phosphorus deficiency, silicon deficiency and eutrophic media was studied. The results showed that
the bioconcentration of BDE-99 by Skeletonema costatum decreased in the following order: phosphorus deficiency > silicon deficiency
> eutrophic media > nitrogen deficiency. Furthermore, the bioconcentration of BDE-99 when expressed as percentage uptake of available BDE-99, decreased in the following order: phosphorus deficiency > silicon deficiency > nitrogen deficiency > eutrophic media.
On the whole, the bioconcentration of BDE-99 by Skeletonema costatum in nutrient deficiency media was more than in eutrophic media
and the bioconcentration in phosphorus deficient media was significantly higher than in other media. The bioconcentration factors of
BDE-99 by Skeletonema costatum based on lipid weight were 80.39×104, 13.61×104, 12.77×104, 9.05×104 ng•g−1/ng•ml−1
under conditions of phosphorus deficiency, silicon deficiency, nitrogen deficiency and eutrophic media, respectively. Correlation analysis indicated that there was significant correlation between bioconcentration and total lipid content of Skeletonema costatum.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 265
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
NEW APPROACH FOR THE DETECTION
OF TOXIC ALGAE SPECIES
Florent Colas, Marie-Pierre Crassous, Sébastien Laurent, Catherine Dreanno, Chantal Compère
Ifremer, Technopôle Brest-Iroise, BP70 29280 Plouzané, France. E-mail: [email protected]
Up to now, toxic species are monitored by expensive and time-consuming laboratory analysis of seawater samples. After a 24h decantation, the samples are observed with an optical microscope and the cell concentration per species is determined. In this context,
systems able to detect HAB species directly underwater would be of great help. An underwater transducer based on Surface Plasmon Resonance spectroscopy was then designed. Shipboard experiments demonstrated that our system can detect refractive index
changes as low as 10-6 corresponding to few picograms of molecules down to 20m depth. An assay based on an SPR biosensor was
then developed for detecting Alexandrium minutum. First, a culture (AM89BM) was lysed following a CTAB DNA extraction protocol.
Then, part of the D1 D2 specific sequence coding for 28S ribosomal subunit was amplified by PCR. Then, double stranded PCR products were digested by λ-exonuclease to obtain single stranded DNA. Afterwards, purified single stranded DNA was diluted in running
buffer just before injection in the biosensor. PCR products of 200, 400 and 600-mer were injected at concentration ranging for 10 to
100nM. It was then demonstrated than the shorter the PCR product, the faster the detection but the greater the detection limit. A detection limit of few nM could be extrapolated. This first set of experiments was run in laboratory for assessing the whole assay. Its incorporation for underwater detection of toxic species will be discussed.
265
POTENTIAL OF DIATOMS AS NANOSTRUCTURES
FOR MARINE TOXIN SENSING
Diana Garibo, Margarita Ferna΄ndez-Tejedor, Jorge Diogène, Mònica Campàs
IRTA, Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain. E-mail: [email protected]
Diatoms are unicellular microalgae with cell walls composed of silica. They consist of two valves with ordered and symmetrical striae
and porous at the nano/micrometer scale. Diatoms are becoming highly interesting in the nanotechnology field due to their nanostructural characteristics, which suggest their potential in multiple applications such as drug delivery, nanofabrication, functional nanopatterning and biosensor development. Regarding this last example, diatom nanostructures offer several advantages such as the possibility
to increase the active surface area for biomolecule immobilisation and thus decrease the detection limits.
In this work, several species of centric diatoms with radial geometry have been cultured: Conscinodiscus wailesii, Cyclotella meneghiniana, Melosira nummuloides and Thalassiosira pseudonana. The organic matter has been removed by strong acid treatment and
afterwards clean frustules have been concentrated by centrifugation. Once frustules cleaned and concentrated, several methods
have been tested in order to immobilise them on supports of different material (gold and carbon) and fabrication process (screenprinting and photolithography). In a first approach, electrodeposition of diatoms has been attempted by adjusting the pH of the diatom solution to basic conditions in order to ionise the silanol groups and by applying a positive potential to the electrode. In another
strategy, frustules have been modified with (3-mercaptopropyl)triethoxysilane in order to provide diatoms with sulfhydryl groups for
their self-assembling on gold supports. After diatom immobilization, frustules will be biofunctionalised with molecules such as a protein phosphatase enzyme or an anti-okadaic acid monoclonal antibody, for the development of biosensors for the detection of
lipophilic diarrheic marine toxins.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 266
CONJUGATION OF PROTEIN PHOSPHATASE
TO MAGNETIC PARTICLES FOR THE DEVELOPMENT
OF BIOSENSORS FOR OKADAIC ACID IN THE FRAME
OF THE BIOKA PROJECT
Diana Garibo1, Jorge Diogène1, E. Devic2, Jean-Louis Marty3, Idoia Unzueta4, Marίa Bla΄zquez4, Mònica Campàs1
1
IRTA, Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain. E-mail: [email protected]
2
GTP Technology, Rue Pierre et Marie Curie, 31681 Labège Cedex, France
3
IMAGES EA4218, Université de Perpignan-Via Domitia (UPVD), Building S, 52 Av. Paul Alduy, 66860 Perpignan Cedex,
France
4
INKOA Sistemas S.L., Ribera de Axpe 11, Edificio D-1, Local 208, 48950 Erandio, Spain
The aim of the BIOKA project is the development of electrochemical biosensors for the detection of okadaic acid (OA). The consortium is formed by
INKOA Sistemas S.L., coordinator, GTP Technology, company responsible for enzyme production, IMAGES-UPVD, a biosensors’ group, and IRTA,
subcontracted institution expert on biosensors and marine monitoring. Electrochemical biosensors for OA based on the protein phosphatase (PP) inhibition have already been developed. Nevertheless, the limits of detection (LODs) attained may not be appropriate for their applicability in naturallycontaminated samples. The reason for these not low enough LODs is the diffusion barrier created by the photopolymer used in the enzyme
immobilisation on electrodes. In order to solve this limitation, we propose the development of a biosensor based on the use of magnetic particles (MPs)
as PP immobilisation carriers. To this purpose, a genetically-engineered PP has been obtained, which contains a His tail that allows its conjugation to
Ni-modified MPs via coordination chemistry. First, the enzyme activity has been characterised. Afterwards, PP has been conjugated to MPs. Several
parameters of the conjugation protocol have been optimised. Colorimetric experiments have shown that the conjugation is successful and that PP retains the activity. Moreover, PP-MP conjugate is inhibited by OA, although in a lower extent than free (non-conjugated) PP. Work is in progress to
obtain the corresponding electrochemical biosensor. It is expected that the high sensitivity inherent to the electrochemical detection together with the
immobilisation through MPs will decrease the LODs compared to those achieved by colorimetry or previous electrochemical biosensors.
266
NEW SIMPLE AND RAPID METHODS
FOR TOXIC ALGAE DETECTION
Fabienne Gas, Beatrice Baus, Laetitia Pinto, Valerie Tanchou, Eric Quamaneur, Veronique Le Berre,
Jean-Marie Fransois, Elisabeth Antoine, Catherine Dreanno, Marie-Pierre Crassous, Chantal Compere
CEA Marcoule, Plateforme Biopuces de la Genopole de Toulouse Midi Pyrenees, IFREMER, France.
E-mail: [email protected]
A whole cell enzyme-linked immunosorbent assay (ELISA) was designed for the detection of Alexandrium minutum. It displays no detectable cross-reactivity with most algae found in similar biotopes and sensitivity and specificity when used for the analysis of natural seawater samples.
- A one step immunochromatographic assay, which involves the use of two distinct monoclonal antibodies directed against surface antigens of A. minutum for a qualitative but highly specific assay suitable for rapid on-site testing (less than 15 min).
- Biochips for the detection of four species.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 267
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
A BIOSENSOR FOR PHYTOPLANKTON CULTURES
L. Giannoudi1, I. Varkitzi1, P. S. Petrou2, C. Pyrgaki1, S. E. Kakabakos2, K. Pagou1
1
Hellenic Centre for Marine Research, 46,7 km Athens-Sounio Ave., P.O. Box 19013, Anavissos, Greece.
E-mail: [email protected]
2
Immunoassay/Immunosensors Lab. I.-R./R.P., N.C.S.R. “Demokritos”, Aghia Paraskevi, 15310 Athens, Greece
The development of easy-to-use, sensitive, specific and cost-effective monitoring tools for the detection of Harmful Algal Bloom (HAB)
toxins is of great importance for the marine environment. Such monitoring tools could contribute to an efficient, early-warning system,
and thus improve public safety response mechanisms and management strategies for both the control of HABs and the protection of
the natural ecosystems. Such a biosensor is being developed for the rapid detection of toxins and its potential use as a multi-analyte
device. More specifically, it is an amperometric sensor, which uses screen-printed electrodes, coupled with antibodies that have previously been tested with an immunoassay procedure (ELISA), in order to detect HAB toxins, such as okadaic acid (OA) and its analogue DTX1. ELISA calibration experiments performed in the laboratory have shown a detection limit of OA standards to be 0.1 ng/ml
and 50 pg/ml in a microtitre plate format. These experiments have been transferred to the screen printed electrodes, for a simplistic
detection of the toxins, both in laboratory cultures of toxic species and in field samples. Prorocentrum lima diluted culture samples were
detected in the range of 10 ng/ml to 40 ng/ml in the ELISA system. The biosensor results were the same as the ELISA ones having an error of 3% to 4%, indicating a good compatibility of the sensor with the ELISA. In short, experiments have demonstrated that
the biosensor could be used for real sample analysis.
267
APPLICATION OF FLUORESCENCE IN SITU
HYBRIDIZATION (FISH) METHOD TO DETECT
HETEROSIGMA AKASHIWO
Guo Hao, Chen Bin, Liang Bin
National Marine Environmental Monitoring Center, People’s Republic of China. E-mail: [email protected]
In this paper, species-specific probes against cytoplasmic rRNA and nuclear rDNA of Heterosigma akashiwo were designed on the basis
of its LSU and ITS sequences, and the FISH protocols for whole cell and nuclear were established to detect this alga. The results showed
that the rRNA-targeted probe hybridized with cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, whereas
cells labelled by rDNA-targeted probes exhibited exclusively a fluorescent nucleus. The probes were specific, without cross-reacting to
other testing algae. The method was also applied successfully to distinguish H. akashiwo from the mixture field samples. Consequently,
the established FISH method was proved promising for specific, rapid, precise and semi-quantitative detection of the cultured alga as
well as the field samples.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:43 PM Page 268
PCR-SSCP: AN ALTERNATIVE MONITORING TOOL
FOR MARINE DINOFLAGELLATE SPECIES
IN COASTAL BAJA CALIFORNIA SUR
Angelica Herrera, Thelma Castellanos-Cervantes, Norma Y. Hernandez-Saavedra
Centro de Investigaciones Biologicas del Noroeste, Mar Bermejo 195, La Paz, BCS 23096, Mexico.
E-mail: [email protected]
Molecular techniques applied to the marine environment for rapid, specific, and sensitive detection of target toxic microalgae have been
developed and used in many laboratories worldwide. Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used
in both mutation and soil bacterial community analyses, was used to analyze and differentiate cultivated pure-culture harmful algal bloom
(HAB) microorganisms and phytoplankton communities. By PCR with universal primers, two fragments of the LSU ribosomal RNA gene
(138 and 180 pb, D9 region) were amplified from 10 species of dinoflagellates (genera Akashiwo, Alexandrium, Ceratium, Coolia,
Cochlodinium, Gonyaulax, Gymnodinium, Lingulodinium, Procentrum, and Protoceratium (stored in the CODIMAR collection). These dinoflagellates are commonly present along the southern coast of the Baja California Peninsula. The tested species could be differentiated
from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 dinoflagellate strains, most of the single strains
could be detected in model communities after PCR and SSCP analyses. This demonstrates the potential of the selected PCR singlestranded DNA approach for HAB community analyses.
268
A BEAD-BASED IMMUNOASSAY FOR DOMOIC
ACID DETECTION: APPLICATION OF LUMINEX XMAP®
TECHNOLOGY AND TRANSITION TO A MICRO-FLOW
CYTOMETER
Lisa R. Hilliard1, George P. Anderson2, Jason S. Kim2, Jeffery S. Erickson2, Peter B. Howell2, Joel P. Golden2,
Lisa Shriver-Lake2, Frances S. Ligler2, Christopher T. Elliott3, Gregory J. Doucette1
1
Marine Biotoxins Program, NOAA/National Ocean Service, Charleston, SC, USA. E-mail: [email protected]
2
Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC, USA
3
Institute of Agri-Food and Land Use, Queen’s University, Belfast, UK
The paucity of methods for rapid, field-based detection of harmful algae and their toxins is a widely-acknowledged gap in the effective monitoring
and management of bloom events. A commercial, bead-based assay technology (Luminex xMAP®) is capable of rapid, multiplexed detection of target analytes via antibody- or nucleic acid-based recognition elements. Assays developed on this system can be transitioned to a micro-flow cytometer
that was designed with the aim of producing field-portable methods. Domoic acid (DA) was selected for development of a model competitive immunoassay on the Luminex 100 system followed by its transition to the micro-flow cytometer. The toxin was attached covalently to polymeric microspheres, and the immobilized DA competed with DA in a sample/standard for phycoerythrin-labeled antibody. A calibration curve was linear from
0.8-30 ng DA/mL. Assay performance was evaluated in natural seawater and aqueous methanolic extracts of algae and shellfish, all showing lower
limits of quantification (in extract) from ~3-12.5 ng DA/mL. Initial trials of the bead-based DA immunoassay were conducted on a micro-flow cytometer, which employs a novel, groove-based flow design allowing sheath fluid to completely envelope the core (or sample) stream, minimizing the
potential for clogging and permitting recycling of sheath fluid. Preliminary results showed that the micro-flow cytometer was capable of detecting
DA in buffer. Moreover, the system could discriminate different fluorescent bead sets, which will permit multiplexed detection of DA and other phycotoxins. Ultimately, our aim is to integrate the micro-flow cytometer into hand-held and potentially automated in-water platforms.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 269
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
DYNAMIC CYTOTOXIC RESPONSE TO SAXITOXIN
ON MICROELECTRONIC SENSOR ARRAYS
Dorothy Yu Huang1, Li Xie1, Stephan Gabos2, David Kinniburgh1
1
Alberta Centre for Toxicology, University of Calgary, HM-B19, 3330 Hospital Dr. NW, Calgary, Alberta, Canada.
E-mail: [email protected]
2
Alberta Health and Wellness, Edmonton, Alberta, Canada
Certain species of freshwater cyanobacteria and marine dinoflagellates can produce saxitoxin and its analogues, also known as paralytic
shellfish poisoning (PSP). Saxitoxin is a neurotoxin that acts on the voltage-gated sodium channels of nerve cells, resulting in the perturbation of normal neural functions. Saxitoxin and its analogues typically accumulate through the food chain. The presence of saxitoxin
and its analogues in cyanobacteria and dinoflagellate blooms has caused the contamination of freshwater resources and the death of fish,
livestock and humans. The benchmark toxicity assay for saxitoxin is the mouse bioassay. It has several limitations including lack of sensitivity (400 ng/mL), poor precision, and the sacrifice of large numbers of animals. The neuroblastoma cell bioassay was developed as
an alternative and has a greater sensitivity of 10 ng/mL. However, it only provides an endpoint measurement and it is labor-intensive. In
this study, we investigated the potential of optimizing the neuroblastoma cell bioassay using a novel electronic sensing technology, which
records the real-time cell physiological changes and generates two-dimension response curves of time vs cell index. By monitoring the
dynamic cellular response to saxitoxin, we were able to establish an assay with increased sensitivity and reduced labor requirement and
the potential for high sample throughput.
269
CONTAMINATION AND MONITORING PROGRAM
OF PARALYTIC SHELLFISH POISONING AND DIARRHETIC
SHELLFISH POISONING IN THE PEARL RIVER ESTUARY
Tianjiu Jiang, Feng Wu
Research Center for Harmful Algae and Aquatic Environment, Jinan University, Guangzhou 510632, China
This study collected and determined 181 samples from 17 sampling sites in Pearl River estuary and its adjacent sea quarterly from December 2007 to May 2009 to understand the consistency of temporal, spatial and species distribution of PSP and DSP toxins. Conclusions are drawn as follows: 1) the positive rate of PSP in shellfish collected from Pearl River estuary and its adjacent sea areas was
up to 20.4%. The concentrations of PSP toxins were generally under the public health safety standard (400 MU/100g). 2) The positive
occurrence of PSP toxins showed a markedly seasonal differentiation, the rates reached the highest in winter, and the lowest in autumn.
Comparing other bays, PSP pollution was more serious in Pearl River estuary, Dapeng Bay and Daya Bay. 3) The contaminate shellfish
species with PSP included Ostrea cucullata, Atrina pectinata, Chlamys nobilis, Ostrea plicatula, Crassostrea rivularis. dcGTX3, C1,
dcGTX2 and C2 were the principal components. 4) The positive rate and over-standard rate of DSP toxins were 36.5% and 25.4%, respectively. Generally, both the positive rate and the over-standard rate showed gradually increase from the winter of 2007 to winter of
2008 with a slight decrease at the spring of 2009. The spatial distribution of DSP showed a decreasing tendency from Daya Bay to Pearl
River estuary to Dapeng Bay. 5) The contaminate shellfish species with DSP included Perna viridis, Chlamys nobilis, Paphia undulata,
Atrina pectinata, Ruditapes philippinara, Meretrix meretrix, Corbicula fluminea, Ostrea cucullata, Pteria martensii, Ostrea plicatula, Crassostrea rivularis, Tegillarca granosa, Arcidae, Mytilus coruscus.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 270
CYANOBACTERIA BLOOM DEVELOPMENT IN THE BALTIC
SEA OBSERVED USING REMOTE SENSING, AUTOMATED
IN SITU MONITORING AND WATER SAMPLING
Bengt Karlson, Martin Hansson, Cia Hultcrantz, Marie Johansen, Ann-Turi Skjevik, Jörgen Öberg
Swedish Meteorological and Hydrological Institute, Sven Källfelts gata 15, 426 71 Västra Frölunda, Sweden.
E-mail: [email protected]
The Baltic Sea is a brackish water area with recurrent summer blooms of cyanobacteria. During warm summers with calm weather surface accumulations of i.e. Aphanizomenon sp., Nodularia spumigena (toxic) and Anabaena spp. is a nuisance to the public and may also
pose a health problem. To monitor the development of blooms a combination of tools are used. A FerryBox system on the route Gothenburg-Kemi-Oulo-Lübeck-Gothenburg provides data from a large part of the Baltic Sea twice a week. Automated measurements are made
every 20 seconds. This provides real time data on chlorophyll- and phycocyanine- fluorescence, proxies for the biomass of total phytoplankton and the biomass of certain cyanobacteria respectively. Every other week automated water sampling for microscopic phytoplankton analysis is made at a handful of sampling locations using the FerryBox system. Water sampling is also made from research vessels
at least monthly. Subsequent microscopic analysis provides information about species composition and identification of HAB species. Satellite monitoring of surface accumulations of cyanobacteria is carried out using images from MERIS (EnviSAT), and MODIS (Aqua). Data
from the extensive bloom in the summer of 2010 will be presented and the ecology and oceanography of the bloom will be discussed
together with the advantages and limitations of the different monitoring approaches.
270
BIOLUMINESCENCE A TOOL TO DETECT HARMFUL ALGAE
Anniina Lindroos, Seppo Kaitala, Anke Kremp, Stefan Simis
Finnish Environment Institute SYKE, Marine Research Centre, Erik Palmanin Aukio 1, 00560 Helsinki, Finland.
E-mail: [email protected]
Dinoflagellates occur in waters around the world. A wide range of dinoflagellate species produce toxic substances that can affect higher
trophic levels. Many of these harmful species are bioluminescent. Bioluminescence is a night-time optical phenomenon where cells emit
flashes of light induced by pressure on the cell wall. Virtually all bioluminescence in surface waters of marine environments originates from
dinoflagellates. The luminescent light of dinoflagellates can, under bloom conditions, be visible at the water surface. In the Baltic Sea,
bloom-forming dinoflagellates occur annually during the spring and summer seasons. Summer blooms contain toxin-producing bioluminescent dinoflagellate species, particularly Alexandrium ostenfeldii. Our aim is to record the bioluminescence of dinoflagellates in different environments of the Baltic Sea. The optical properties of the water body, e.g. scattering, absorption and reflectance are measured,
to estimate in what type of waters the bioluminescent dinoflagellates occur and in what light conditions bioluminescence can be detected
by the human eye or with the aid of optical sensors. The luminescent radiation is strongly attenuated in the blue spectral area caused by
a high load of coloured dissolved organic mater in the Baltic Sea. We present an optical model for the detectability of bioluminescence
in highly attenuating Baltic Sea waters. This will enable us to evaluate the added value of recording bioluminescence in environmental monitoring for potentially toxic blooms. The preliminary results from this summer will be presented.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 271
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
COMBINING IN-WATER AUTONOMOUS SENSORS, MOLECULAR ANALYTICAL TECHNIQUES AND OCEAN MODELING
TO DETECT THE ONSET OF A PSEUDO-NITZSCHIA BLOOM
ALONG THE COAST OF SOUTHERN CALIFORNIA
Roman Marin III1, Dave Caron2, Burt Jones2, Yi Chao3, Gaurav Sukhatme6, Greg Doucette4,
Raphe Kudela5, James Birch1, Chris Scholin1
1
Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USA. E-mail: [email protected]
2
Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA
3
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91125, USA
4
Marine Biotoxins Program, NOAA/National Ocean Service, Charleston, SC 29412, USA
5
Oceans Sciences Department, University of California Santa Cruz, Santa Cruz, CA 95064, USA
6
Computer Science Department, University of Southern California, Los Angeles, CA 90089, USA
Many harmful bloom events in California are associated with the presence of intoxicated marine birds and mammals that do not always correlate with
increases in potentially harmful species or toxins at regularly monitored near shore stations. These bloom events may initiate offshore or in the subsurface where routine phytoplankton monitoring is sparse. In a step toward developing an autonomous observing system to assist with detecting such blooms
and assessing their fate and transport, we combined the use of gliders, moored Environmental Sample Processors (ESPs) and ocean modeling. Here,
we focused on the San Pedro shelf region off Southern California, one of the biggest wildlife intoxication hotspots in California. In early April 2010, the
glider and ESP moorings showed relatively warm water with low chlorophyll, and only trace concentrations of potentially toxic Pseudo-nitzschia as determined by the ESP DNA probe arrays at depths ranging from 15-20m several km offshore. Later, consistent with predictions of upwelling favorable
conditions based on ROMS forecasting, a pulse of cold water was observed at the study site and was followed by an increase in chlorophyll. Over the
course of the next several days, the abundance of several Pseudo-nitzschia species (including P. cf. australis and P. cf. multiseries) as well as the concentration of domoic acid increased sharply at the ESP mooring locations. This is the first time operational ocean observing systems and ocean modeling have been combined with autonomous systems that utilize molecular probe technologies to detect specific species and toxins they produce.
271
MIDTAL(MICROARRAYS FOR THE DETECTION OF TOXIC ALGAE)
Linda K. Medlin1, Wiebe Kooistra2, Edna Graneli3, Beatriz Reguera4, Robin Raine5, Bente Edvardsen6,
Jane Lewis7, Chris Elliott8, Yolanda Pazos9, Lucie Maranda10
1
Marine Biological Association of UK, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK. E-mail: [email protected]
2
Laboratory of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
3
Marine Ecology Department, Linnaeus University, SE-39182 Kalmar, Sweden
4
Instituto Espan~ol de Oceanografίa, Centro Oceanogra΄fico de Vigo, Aptdo 1552, 36200 Vigo, Spain
5
Martin Ryan Institute, National University of Ireland, University Road 1, Galway, Ireland
6
Department of Biology, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway
7
School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
8
Institute of Agri-Food and Land Use, Queen’s University of Belfast, Belfast BT7 1NN, UK
9
Instituto Tecnoloxico Para o Control do Medio Marino de Galicia – Intecmar, Peirao De Villaxoan S/N, Villagarcia
de Arousa, 36611, Spain
10
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882-1197, USA
Microalgae in marine and brackish waters of Europe regularly cause harmful effects, considered from the human perspective, in that they cause economic damage to fisheries and tourism. Cyanobacteria cause similar problems in freshwaters. These episodes encompass a broad range of phenomena
collectively referred to as «harmful algal blooms» (HABs). For adequate management of these phenomena, monitoring of microalgae is required. However, the effectiveness of monitoring that it is time consuming and morphology as determined by light microscopy may be insufficient to give definitive
species and toxin attribution. In the EU FP7 project MIDTAL (microarrays for the detection of toxic algae) we will target rapid species identification using
rRNA genes as the target. These regions can be targeted for probe design to recognize species or even strains. We are also include antibody reactions
to specific toxins produced by these microalgae because even when cell numbers are low, toxins can be present and can be accumulated in the shellfish. Microarrays are the state of the art technology in molecular biology for the processing of bulk samples for detection of target RNA/DNA sequence.
Existing rRNA probes and antibodies for toxic algal species/strains and their toxins will be adapted and optimized for microarray use. The pose of MIDTAL is to support the common fisheries policy, aid the national monitoring agencies by providing new rapid tools for the identification of toxic algae and
their toxins so they can comply with ECC directive 91/1491/CEE that can be converted to cell numbers and reduce the need for the mouse bioassay.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 272
DIRECT FLUORESCENCE POLARIZATION ASSAY FOR THE
QUANTIFICATION OF SPIROLIDES IN MUSSEL SAMPLES
Paz Otero1, Amparo Alfonso1, Paula Rodrίguez1, Rόmulo Ara΄oz2, Jordi Molgό2, Mercedes R. Vieytes3, Luis M. Botana1
1
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
E-mail: [email protected]
2
CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire – FRE3295,
1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
3
Departamento de Fisiologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
Spirolides are a novel class of macrocyclic polyether neurotoxins that belong to the cyclic imine group. At present, legislated limits for spirolides
toxins do not exist, but its great recurrence in numerous countries and the toxicological information obtained in the mouse bioassay indicate that
these compounds constitute a group of toxins that should be regulated worldwide. For this reason, the search for methods for the detection of
spirolides is a priority in many laboratories. In the present work, a new fluorescence polarization (FP) method that detects and quantifies spirolides
in mussel extracts is described. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labeled with a fluorescein derivative was used. The spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were
extracted and purified from cultures of Alexandrium ostenfeldii. After 10 incubation minutes, data showed the decrease of FP when toxin concentration was increased. Thus, a direct relationship between the FP units and the spirolides present in a sample was obtained. The procedure
was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. This non-competitive assay
is a reproducible, simple and very sensitive method able to quantify 13-desMeC in the range of 50-350 μg kg-1 meat. Other liposoluble toxins
did not interfere with the assay, proving a specific method. Thus, the functional assay here described constitutes a solid choice to measure
spirolides concentrations in various samples, including contaminated mussels matrix for human consumption.
272
WIDE DISTRIBUTION OF KARLODINIUM VENEFICUM
IN KOREAN SEDIMENTS AS DETECTED BY REAL-TIME PCR
Tae-Gyu Park, Eung-Kwon Kim
National Fisheries Research and Development Institute, Tongyeong 650-943, Korea. E-mail: [email protected]
The toxic dinoflagellate Karlodinium veneficum has been implicated in numerous fish kill events around world. This organism commonly
co-occurs with other closely related dinoflagellates, such as Pfiesteria and Stoeckeria algicida. In this study, a TaqMan based real-time
PCR assay was developed on the internal transcribed spacer 2 (ITS2) ribosomal DNA region for species-specific detection in environmental samples. The K. veneficum-specific real-time PCR probe was used in conjunction with S. algicida-, Pfiesteria piscicida-, Pseudopfiesteria shumwayae-, Cryptoperidiniopsis brodyi-specific real-time PCR probes to investigate the geographic distribution of these
dinoflagellates in sediments collected from East, South, and Yellow Seas in Korea. A nested real-time PCR assay was also developed
for specific detection of K. veneficum in the sediment and showed approximately 105-fold increased sensitivity than conventional realtime PCR. Total 79 sediment samples were analyzed by the species-specific real-time PCR probes. The field survey showed that S. algicida and K. veneficum may be more widely distributed than P. shumwayae and P. piscicida in Korean sediments. These results also
indicate that “seed beds” of these dinoflagellates may exist in the sediment.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 273
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
CHEMILUMINESCENT, FLUORESCENT
AND COLORIMETRIC DETECTION OF 13-DESMETHYL
C SPIROLIDE USING A RECEPTOR-BASED ASSAY
Laura Perez1, Natalia Vilariao1, Jordi Molgo2, Romulo Araoz2, Luis M. Botana1
1
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario,
27002 Lugo, Spain. E-mail: [email protected], [email protected]
2
CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Moleculaire UPR9040, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
The spirolides are marine phycotoxins included in the group of cyclic imines. The threat that these toxins pose to human health is still unknown. However, these toxins display a high toxicity by intraperitoneal injection in rodents, being a source of false positives during marine
toxin detection by mouse bioassay. A method based on the competition of the 13-desmethyl C spirolide with biotin-labeled -bungarotoxin
for binding to nicotinic acetylcholine receptors from membranes of the electric organ of Torpedo marmorata was developed for the detection of spirolides using three alternative readouts: chemiluminescence, fluorescence and colorimetry. The technique consists of an inhibition
assay where receptor binding takes place in solution and subsequently, the receptors bound to the biotin-labeled -bungarotoxin are immobilized on the streptavidin coated surface of a 96 well plate and detected by specific antibodies. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, highly sensitive substrates of this enzyme generate a chemiluminescent, fluorescent or colorimetric
signal. The assay has similar sensitivity for the three approaches and performs well in shellfish extracts. The sensitivity is in the range of 5200 nM in shellfish extracts, approximately four times higher than the existing fluorescence polarization assay. The detection of spirolides
with microplate assays is an alternative to the mouse bioassay for sample screening purposes. This study improves and expands the choices
of spirolide detection methods, making these alternative techniques more accessible to a high number of laboratories.
273
STUDY OF THE ADSORPTION OF PARALYTIC SHELLFISH
POISONING TOXINS (PSP) ONTO A SYNTHETIC
ADSORBENT RESIN (SP700)
Paula Rodrίguez1, Amparo Alfonso1, Paz Otero1, Elizabeth Turrell2, Jean-Pierre Lacaze2, Luis M. Botana1
1
Departamento de Farmacologίa, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
E-mail: [email protected]
2
Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB, UK
The development and application of a passive solid-phase adsorption toxin tracking (SPATT) system is a relatively new field of research. Since
2004, several resins have been evaluated and their ability to adsorb a wide range of marine toxins was investigated. In this study, a synthetic resin (SEPABEADS® SP700) which previously demonstrated its ability to adsorb amnesic shellfish poisoning toxins (ASP), diarrheic
shellfish poisoning toxins (DSP) and lipophilic shellfish toxins (LSTs) from seawater was further investigated with regards to its capacity to
adsorb paralytic shellfish poisoning toxins (PSP). The resin, held within dialysis bags, was deployed in PSP toxins producer cultures of
Alexandrium tamarense and in mixed cultures of Alexandrium tamarense and Prorocentrum lima (DSP producer). Extraction of the PSP and
DSP toxins from the resin was carried out with water and methanol and the subsequent extracts were analysed using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) respectively. Final results showed that PSP and DSP
were both adsorbed by the resin after short exposure periods (3-7 days). Even though PSP toxins could partially be removed from the resin
by rinsing water or by desorption in a toxin-free medium, the extractable quantity of PSP toxins still attached to the resin was high enough
to be detected by a sensitive method (HPLC, LC-MS). The ability of the SP700 resin to adsorb a vast range of marine biotoxins found around
the European coasts could potentially be used as a useful early warming system to monitor the toxicity of algal blooms.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 274
NEW PALYTOXIN-LIKE MOLECULES IN MEDITERRANEAN
OSTREOPSIS OVATA (DINOFLAGELLATES) BY LC/TOF/MS
Rachele Rossi1, Olga Arace1, Teresa Bruno1, Daniela Capozzo1, Vincenzo Castellano1, Eleonora Scalco2, Luigi Serpe1,
Adriana Zingone2, Vittorio Soprano1
1
2
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute 2, 80055 Portici, Italy. E-mail: [email protected]
Stazione Zoologica Anton Dorhn, via Villa Comunale, 80121, Naples, Italy
Ostreopsis ovata is a benthic dinoflagellate that colonises macroalgae and seagrasses or attaches directly to the substrate. In recent years,
massive blooms of this species have become a threat along the Mediterranean coasts, where the toxins produced by the species (ovatoxin-a and putative-palytoxin) can affect human health through aerosol, render seafood toxic and affect benthic resources. A rapid, high
resolution liquid chromatography coupled with ElectroSpray Ionization Time-of-Flight Mass Spectrometry (LC/TOF/MS) method was
developed for the determination of the toxin pattern in cultured cells of O. ovata from the Mediterranean Sea. The samples were separated on a Phenomenex Luna 3μ HILIC 200A (150 x 2.00 mm) and analysed by LC/TOF/MS with electrospray ionization (ESI) interface in positive ion mode. The method was successfully applied for the determination of four new palytoxins (ovatoxin-b; ovatoxin-c,
ovatoxin-d and mascarenotoxin-C) and mascarenotoxin-A in O. ovata. These new molecules pose questions as to their actual contribution to the toxicity of this species, highlighting a possible underestimation of toxicity in analyses on marine organisms contaminated by O.
ovata that have been conducted so far. Indeed, the total concentration of the new analogues in the analyzed samples is comparable to
that of ovatoxin-a, which doubles the amount of palytoxin-like substances in O. ovata. Therefore, the analysis of shellfish contaminated
by O. ovata toxins should take into account the presence of ovatoxin-b, ovatoxin-c, ovatoxin-d and mascarenotoxin-A and mascarenotoxin-C in addition to the previously known toxins.
274
DETERMINATION OF DOMOIC ACID IN SHELLFISH AND
PSEUDO-NITZSCHIA NATURAL AND CULTURED MATERIAL
BY LIQUID CHROMATOGRAPHY-ELECTROSPRAY
IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY
Rachele Rossi1, Olga Arace1, Teresa Bruno1, Daniela Capozzo1, Vincenzo Castellano1, Anna Italiano2,
Luigi Serpe1, Adriana Zingone2, Vittorio Soprano1
1
2
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute 2, 80055 Portici, Italy. E-mail: [email protected]
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
A method coupling rapid resolution liquid chromatography (HPLC) with ElectroSpray Ionization Time-of-Flight Mass Spectrometry (ESITOF/MS) was developed for the determination of the neurotoxin domoic acid (DA). The method provides the capability for a fully automated analysis, does not require complicated sample preparation and gives clean and simple chromatograms. Shellfish, natural samples
and cultures of Pseudo-nitzschia species from the Gulf of Naples (Mediterranean Sea), the latter identified with morphological and molecular methods, were used for the analyses. Quantitative determination of DA in shellfish tissues was possible at concentrations as low
as 20 μg kg-1 tissue. Analyses of 15 strains belonging to 7 Pseudo-nitzschia species (P. galaxiae, P. multistriata, P. mannii, P. calliantha,
P. fraudulenta, P. cf. delicatissima and P. pseudodelicatissima) allowed the detection of domoic acid in two species. P. pseudodelicatissima, so far not known to be toxic in the Gulf of Naples, had DA concentrations ranging between 1.28 and 2.47 fg cell-1. P. multistriata
had DA concentrations ranging between 0.18 and 9.98 fg cell-1 In addition to DA, several DA isomers were present in the different cultures. No domoic acid was detected in strains of the other species. However, DA was detected in net samples collected during a bloom
of P. calliantha in July 2008. Traces of DA were also found in mussels. This study confirms the relevance of the analyses of multiple strains
and natural material in order to assess the actual toxicity of Pseudo-nitzschia species.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 275
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
QUANTITATIVE PCR ASSAYS
FOR SIX GAMBIERDISCUS SPECIES
Mark W. Vandersea1, R. Wayne Litaker1, Steven R. Kibler1, William C. Holland1, Mireille Chinain2, Patricia A. Tester1
1
2
National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA. E-mail: [email protected]
Laboratoire des Micro-Algues Toxiques, Institut Louis Malardé, BP 30 98713 Papeete, Tahiti
Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of ciguatoxins produced by species in the genus Gambierdiscus. This study describes the first molecular assays for the identification and relative quantification of six Gambierdiscus species and one uncharacterized Gambierdiscus ribotype. It has been difficult to ascribe toxicity to individual
Gambierdiscus species because species identifications are complicated. Rapid and sensitive identification and detection methods are
needed to make advances in ciguatera research. Consequently, SYBR green qPCR assays were developed that targeted unique regions
of the ribosomal gene complex including the SSU, the ITS and the D1/D3 LSU domains. Assay sensitivity and accuracy were tested
using DNA extracts containing multiple Gambierdiscus species. Standard curves were constructed using both known concentrations of
cultured cells and tenfold serial dilutions of PCR amplicons containing the target sequence for each species. The limits of detection were
less than a single cell in 100 μL. This corresponds to a minimum detection limit of 10–100 rDNA copies depending on the assay. Validation studies were completed using macroalgal samples containing Gambierdiscus collected from near shore areas at Ft. Pierce, Florida
USA. qPCR cell concentration estimates associated with the macroalgae were comparable to cell counts. These qPCR assays are suitable for use in a broad spectrum of detection, monitoring and research applications.
275
SPIROLIDES AND GYMNODIMINE TARGET MUSCARINIC
ACETYLCHOLINE RECEPTORS
Natalia Vilario, Carolina B. Wandscheer, Laura Perez, Luis M. Botana
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario,
27002 Lugo, Spain. E-mail: [email protected], [email protected]
Spirolides and gymnodimines belong to a group of recently described cyclic imine marine toxins. Although no human intoxication has yet
been unequivocally related to their presence in shellfish, the possible toxicological consequences to human health are unknown. The mechanism of action of these toxins is an important piece of information in the evaluation of the toxic threat. Currently, compounds of both
groups have been reported to antagonize nicotinic acetylcholine receptors. In this study, we explored the effects of 13-desmethyl C
spirolide, 13,19-didesmethyl C spirolide and gymnodimine on muscarinic acetylcholine receptor (mAChR)-mediated responses in neuroblastoma cells. Modifications of mAChR expression by this group of toxins were also studied. Both spirolides inhibited the acetylcholineinduced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The effect of the spirolides
persisted after toxin removal. In contrast, the inhibition of the acetylcholine-elicited response by gymnodimine did not lower the maximum
response to the agonist and was readily reversible after toxin removal. The inhibition of [3H]QNB binding by the spirolides, which disappeared in the presence of the reversible mAChR antagonist atropine, indicated that the effect of the spirolides occurred at the receptor
level. Protein levels of M3 mAChR were not altered by 13-desmethyl C spirolide, although the properties of membrane bound receptors
changed after long incubations with the toxin. Gymnodimine behaves as a reversible competitive antagonist of mAChRs, whereas the
spirolides are virtually irreversible competitive antagonists in a relatively short time frame.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 276
MAXIMUM QUANTUM YIELD OF PHOTOSYSTEM II
PHOTOCHEMISTRY AS A DIAGNOSTIC TOOL OF NUTRIENT
STRESS IN THE COAST SEAWATER OF QINGDAO, CHINA
Jiangtao Wang, Zhaoyu Wang, Hongju Qi
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao,
266100, People’s Republic of China. E-mail: [email protected]
In aquatic sciences, it has been widely accepted that the maximum quantum yield of photosystem II photochemistry ( Fv/Fm) is influenced by
nutrient stress. Some scholars put forward that Fv/Fm may be a good indicator of nutrient stress. The method involves placing water samples
in containers supplied with one or a number of nutrients, and measuring Fv/Fm after 24h. If a particular nutrient addition significantly enhances
Fv/Fm relative to the control treatment, it is considered that the nutrient is a limiting factor. On the other hand, If Fv/Fm in a particular nutrient
treatment is similar to that in the control treatment, it is deduced that the nutrient is not limiting. Because it is rapid and non-destructive, the
method is potentially very useful for exploring the nutrient status of phytoplankton. To verify whether this method can be applied in coastal water
of Qingdao, China, seven nutrient enrichment (nitrate, phosphate or silicate) experiments were conducted in the coast of Qingdao from June
to September during 2009. Fv/Fm, species composition and dissolved inorganic nutrients were measured every 24h. In addition, two more conventional techniques for determining nutrient limitation were also used to test the accuracy of this method. In general, Fv/Fm following P addition was markedly higher than the control treatment after 24h. Conversely, Fv/Fm in N and Si treatments varied little. P thus appears to be the
limiting nutrient in most experiments. Results of the three approaches were consistent. Accordingly, if there is no change in species composition, Fv/Fm following resupply of nutrients can be used as a quick diagnostic tool for P stress in the coast of Qingdao. However, physical enclosure of a natural population has some deleterious effects, such as relieving grazing pressure and isolating samples from nutrient loading and
resupply. Besides, the addition of one nutrient may induce limitation of other nutrients. Therefore, the conclusion drawn from this method may
not be true, especially when nutrient concentrations are close to the threshold values (DIN=1 μmol/L, P=0.l μmol/L).
276
DEVELOPMENT OF A REAL-TIME PCR METHOD
FOR RAPID DETECTION AND QUANTIFICATION
OF PROROCENTRUM DONGHAIENSE
Jian Yuan1, Tiezhu Mi2, Yu Zhen2, Qingzhen Yao3, Zhigang Yu3
1
College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, 266003 Qingdao, China.
E-mail: [email protected]
2
College of Environmental Science and Engineering, Ocean University of China, 238 Songling Road, 266100 Qingdao, China
3
College of Chemistry and Chemical Engineering, Ocean University of China, 238 Songling Road, 266100 Qingdao, China
Prorocentrum donghaiense is commonly seen in red tides which occur in the East China Sea. It is urgent and necessary to study this dinoflagellate in the monitoring of HABs. In this research, we designed a pair of specific primers based on the sequence of Internal Transcribed Spacer (ITS) of Prorocentrum donghaiense. After examination of their specificity by PCR against a range of other related algae,
the amplification fragments were cloned into plasmids. Then the recombinant plasmids were diluted into a 10x series in order to construct
a standard curve in a real-time PCR method. In this method, a 10x dilution series of DNA were also used, which were carefully extracted
from Prorocentrum donghaiense whose cell numbers were calculated in advance. Another regression curve was also built according to
the cell numbers and their corresponding plasmid copy numbers indicated by the standard curve, respectively, to indicate their relationship. Finally, we checked the accuracy of it with quantified Prorocentrum donghaiense cells mixed with several other algae and some samples collected from East China Sea this April. As a result, this method showed good potential in the detection and quantification of
Prorocentrum donghaiense both in cultured and field samples.
12. ABSTRACT BOOK (257-277)_Layout 1 10/22/10 2:44 PM Page 277
NOVEL SENSOR TECHNOLOGIES
FOR BIO-SENSING APPLICATIONS IN HAB
RESEARCH AND MONITORING
POSTER PRESENTATIONS
COMPARATIVE PROTEOMIC ANALYSIS OF DIFFERENTIAL
EXPRESSED PROTEINS IN CELL CYCLE
OF PROROCENTRUM DONGHAIENSE
Ying-Jiao Zhang, Yue Gao, Hua-Sheng Hong, Da-Zhi Wang
State Key Laboratory of Marine Environmental Science/Environmental Science Research Center, Xiamen University, 182 Daxue
Road, 361005 Xiamen, People’s Republic of China. E-mail: [email protected], [email protected]
Prorocentrum donghainese is a key harmful algal blooms (HABs) species in the coast of China. The mechanism of regulating the cell growth
and proliferation is critical for us to understand the blooms of HABs. This study compared 2-DE protein profiles of P. donghainese collected at
different cell cycle phases (G1, S, G2/M) and identified differentially expressed proteins using 2D-PAGE and MALDI-TOF-TOF mass spectrometer. The results showed that the cell cycle length of synchronized P. donghainese was approximately 24 hours, G1 duration was about
18 hrs, S duration was 4 hrs and G2/M was 2 hrs. Eighty differentially expressed proteins in G1, S and G2/M phases were identified and these
proteins were involved in various biological functions, such as cell cycle regulation, DNA replication and cellular metabolisms. Among these proteins, a subset of the periodical variation proteins, the cyclins were identified. Subsequently, validations of these proteins were conducted using
western blotting method, and cyclin A, cyclin B, cyclin D1, cyclinE and PCNA were detected in P. donghainese and presented the similar variation trends to these proteins in eukaryotic cells. This work represents the first attempt to investigate cell cycle regulating proteins of dinoflagellates, which provides new insights into cell cycle regulation of dinoflagellate and blooming mechanism.
277
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 278
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 279
HARMFUL ALGAE
CULTURE
COLLECTIONS
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 280
POSTER PRESENTATIONS
GROWTH RATES OF SOME ALGAL BLOOM SPECIES
IN KUWAITI WATERS
Wafa’a Al-Rashed, Faiza Al-Yamani, Hadeel Al-Mansouri, Graceykutty Varghese
Kuwait Institute for Scientific Research, Mariculture and Fisheries Department, P.O. Box 1638, 22017 Salmiya, Kuwait.
E-mail: [email protected]
Five phytoplankton species were cultured using Guillard’s media. They have been recultured several times to reach the final volume of
1.5 L. Non-continuous enrichment with fresh media was used and exposed to continuous light for 26 days. Chemical measurements
were done for all cultures. On days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 23 and 25, samples were collected. Chlorophyll-a concentration
and phytoplankton density were measured for all cultures. Highest chlorophyll-a concentrations for filamentous blue green algae, picoplankton (Prochlorococcus sp.), flagellates (Pyramimonus sp.), green algae (Dunaliella salina) and dinoflagellate (Prorocentrum sp.) were
56.88 μg/l (day 4), 83.22 μg/l (day 4), 59.06 μg/l (day 2), 819.56 μg/l (day 4) and 55.20 μg/l (day 2), respectively. Highest phytoplankton densities for the same previous sequence of species were as follows: 1.18x1010 cells ± 2.79x109 (day 16), 7.56 x 109 cells/l
± 1.74 x 109 (day 2), 4.2x108 cells/l ± 1.17x108 (day 16), 2.39 x 109 cells/l ± 5.7 x 108 (day 14) and 4.4 x 107 cells/l ± 1x 107 (day
2), respectively. Growth rates were calculated for all phytoplankton species during the 26 days.
280
GROWTH AND BIOCHEMICAL CHARACTERIZATION
OF RAPHIDOPHYTE STRAINS
FROM THE GULF OF CALIFORNIA
Christine J. Band-Schmidt1, Aίda Martίnez-Lόpez1, José J. Bustillos-Guzma΄ n2, Laura Carreόn-Palau2,
Lourdes Morquecho-Escamilla2, Norma O. Olguίn-Monroy2, Tania Zenteno-Savίn2,
Francisco E. Herna΄ndez-Sandoval2, Carmelo Tomas3
1
Centro Interdisciplinario de Ciencias Marinas, A.P. 592, La Paz, B.C.S. 23000, Mexico. E-mail: [email protected]
2
Centro de Investigaciones Biolόgicas del Noroeste, A.P. 128, La Paz, B.C.S. 23000, Mexico
3
University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, N.C. 28409, USA
Raphidophytes have been reported in the Gulf of Mexico and the Mexican Pacific coast. Six strains of raphidophytes from different regions of
the Gulf of California have been established for identification and biochemical description. The six strains identified consisted of five Chattonella
subsalsa and one Fibrocapsa japonica strains. All strains were cultivated in modified f/2 media at 23°C with a light dark cycle (12h:12h) and
150 μmol/m2 s illumination. Exponential growth rates and highest cell density of C. subsalsa strains varied between 0.209 - 0.354 div/day,
and 3168 - 4592 cells/ml, respectively. Fibrocapsa japonica had a growth rate of 0.250 div/day with a maximum cell density of 5589 cells/ml.
For HPLC pigment composition, brevetoxin, superoxide radical production, and fatty acid analyses, cultures were harvested in late exponential growth phase. In all strains, the main pigment was chlorophyll a (61.49-67.03%), followed by fucoxanthin (16.89-23.55%), chlorophyll c1
and c2 (7.88-10.21%), violaxanthin (2.1-4.82%), β-carotene (1.99-2.97%), and diadinoxanthin (0.39-1.04%). Strains were tested for PbTx1, 2, 3, 6, 9, PbTx-carboxylic acid, brevenal, and brevisin; only PbTx-1 and PbTx-2 were detected. Superoxide radical production varied between 0.009 and 0.058 nmol/min mg protein. Lipid peroxidation, quantified as the tissue content of thiobarbituric acid reactive substances,
presented significant differences between strains from 2.99 to 138.10 nmol/mg protein. The main fatty acids were18:4 (n-3) and 20:5 (n-3).
All strains had typical fatty acid composition for raphidophytes, produced brevetoxin-like compounds, the superoxide radical, and caused lipid
peroxidation. Ongoing studies should be carried out to confirm toxic effects on other organisms.
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 281
HARMFUL ALGAE CULTURE COLLECTIONS
THE CULTURE COLLECTION OF ALGAE AND PROTOZOA:
AN ESSENTIAL SUPPORT SERVICE FOR HAB RESEARCH
Christine N. Campbell, John G. Day, Keith Davidson
Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll, PA37 1QA, UK.
E-mail: [email protected], [email protected]
The Culture Collection of Algae and Protozoa (CCAP) located in the Scottish Association for Marine Science (SAMS), European Centre
for Marine Biotechnology (ECMB) is a National Facility funded by the UK Natural Environmental Research Council. It acts as a service
culture collection, providing biological materials, bioinformatics data, advice and services to the scientific community world-wide. It holds
a uniquely diverse collection of live protists, multi-cellular algae and cyanobacteria, originating from freshwater, terrestrial, marine and hypersaline niches. Algal culture collections offer an essential support service to harmful algal bloom research by providing: a depository for
novel toxic isolates, especially where there is a requirement to lodge in culture collections, strains described in publications; a source for
reference toxic strains; a facility to isolate and characterise HAB strains; a unique service to act as a patent strain depository; input to HAB
strain genomic studies; cryopreservation storage to conserve genetic and phenotypic stability; and training in all of the relevant skills.
CCAP currently holds a range of HAB strains, including Alexandrium spp., Prorocentrum lima, Pseudo-nitzschia spp. and microcystin producing freshwater cyanobacteria. Within the SAMS Institute, CCAP also supports HAB research and biotoxin producing phytoplankton
monitoring.
281
CCVIEO. THE CULTURE COLLECTION
OF HARMFUL MICROALGAE OF IEO
Santiago Fraga, Isabel Bravo, Francisco Rodrίguez, Amelia Ferna΄ndez-Villamarίn, Pilar Rial, Isabel Ramilo
Centro Oceanogra΄fico de Vigo, IEO (Instituto Espan~ol de Oceanografίa), Subida a Radio Faro 50, 36390 Vigo, Spain.
E-mail: [email protected]
The Culture Collection of Harmful Microalgae of IEO in Vigo (CCVIEO) is the result of multiple research projects since 1984 when the
first strain of this collection, Alexandrium tamarense PE1V, was isolated. Today it holds about 500 strains. At the beginning, most of the
strains were isolated from Galician waters at the Spanish Atlantic coast, but now it holds strains from all over the world, obtained from
other collections or isolated by the staff. Planktic and benthic dinoflagellates are the most abundant but also holds some diatoms, raphydophytes, and haptophytes. This collection only provides strains on friendship collaboration as it has not commercial character. Some
Alexandrium spp. and Prorocentrum lima strains are very popular and have been used by many researchers from different countries as
a source of toxins or for molecular studies.
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 282
EFFECT OF DIFFERENT NITROGEN/PHOSPHORUS
RATIOS ON GROWTH AND TOXIC CONTENT IN FOUR
ALEXANDRIUM CATENELLA (DINOPHYCEAE)
STRAINS FROM SOUTHERN CHILE
Ma΄ ximo Frangόpulos1, Cristia΄ n Garrido2, Soraya Moraga1, Claudia Zamora2, Gemita Pizarro2, Daniel Varela3
1
Centro de Estudios del Cuaternario de Fuego-Patagonia (Fundaciόn CEQUA), Av. Bulnes 01890, Postal Code 6213029,
Punta Arenas, Chile. e-mail: [email protected]
2
Instituto de Fomento Pesquero (IFOP) Base Punta Arenas, Enrique Abello 0552, P.O. Box 101, Punta Arenas, Chile
3
Centro “i~mar”, Universidad de Los Lagos, P.O. Box 557, Puerto Montt, Chile
Among the environmental factors controlling the development of blooms in toxic phytoplankton species, nutrients are considered between
the most important. Here we investigate the growth and toxic content in four strains of Alexandrium catenella (Whedon et Kofoid) Balech
cultures at high (N:P=24:1), balanced (N:P=16:1) and low (N:P=3:1) nitrogen ratios. The strains of A. catenella used in this study were
collected from cyst from a recurring blooms area in southern Chile (42°S to 56°S). The cultures were run as batch cultures for 30 days
and the toxin profile of A. catenella was determinate by HPLC. The results showed that AY2 strain presented the faster growth (μ=0.49
d-1) at high N:P medium, although the maximum abundance was registered in ACC02 strain at the low N:P medium (8400 cell ml-1 at
day 12). The toxic content in the strains showed that the higher amount of toxins was observed in AY2 strain, in low N:P medium (24.56
fmol cell-1, at day 12), and at high and balanced N:P ratios, reaching values of 21.28 and 23.20 fmol-1cell-1 respectively. In the four strains,
GTX3 toxins were the predominant component. The results suggested that under these conditions, N/P ratios possess no synergic effect on the growth and toxicity of this toxic algae, resulting in a stimulation of growth by nitrogen (24:1 and 16:1 ratios), and an increase
of toxin content under limiting conditions (3:1 ratios), reflecting the importance of these nutrient on the dynamics of toxic algae. Financial support: Fondecyt Project No 1080548.
282
CURRENT SITUATION AND PERSPECTIVES OF THE
COLLECTION OF MARINE DINOFLAGELLATES (CODIMAR)
Lourdes Morquecho, Amada Reyes-Salinas
Centro de Investigaciones Biolόgicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Sta. Rita, La Paz,
B.C.S. 23096, Mexico. E-mail: [email protected]
To meet the goals of international scientific research agencies related to the study of harmful microalgae, we proposed to the Consejo
Nacional de Ciencia y Tecnologίa (CONACYT) a project: “Towards the establishment of the first taxonomic and biological bank of dinoflagellates in Mexico”. The main objective of the project was to establish the first live collection of marine dinoflagellates in Mexico. The
project was approved. From October 2000, we started the Collection of Marine Dinoflagellates (CODIMAR) at the Centro de Investigaciones Biolόgicas del Noroeste (CIBNOR). Our official opening occurred in February 2004 with an Internet website (http://www.cibnor.mx/en/research/biological-collections/codimar). CODIMAR currently contains 120 strains isolated mainly from the southern Gulf of
California. The collection includes strains of the main harmful species worldwide, such as: Akashiwo sanguinea, Cochlodinium polykrikoides,
Cochlodinium fulvescens, Coolia monotis, Gymnodinium catenatum, Prorocentrum lima, Prorocentrum minimum, Protoceratium reticulatum, Pyrodinium bahamense, among others. The collection also includes some strains of raphidophytes of the genus Chattonella and
Fibrocapsa. The strains had been used in national and international research projects on Harmful Microalgae, ongoing studies on growth
kinetics, toxicity, taxonomy, phylogeny, and harmful effects on marine fauna. Investigations using strains from our collection have been
used by students in masters and doctoral theses, and have led to 12 scientific publications. Strains have also been used in taxonomy and
biotechnology training courses. Examples of the use of the strains will be shown and plans and prospects for the short and medium-term
are explained.
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 283
HARMFUL ALGAE CULTURE COLLECTIONS
POSTER PRESENTATIONS
OPTIMIZATION OF CULTURE CONDITIONS FOR MASS
PRODUCTION OF MICROALGAE, CHLORELLA SCHROTERI,
DUNALIELLA SALINA AND DUNALIELLA SP.
USING THE RESPONSE SURFACE METHOD
Jang Min Park, Seong Eun Bang, Hee Gon Kim, Si Wouk Kim
Dept. of Environmental Engineering, Pioneer Research Center for Controlling of Harmful Algal Bloom, Chosun University,
Gwangju 501-759, Korea
In our study, culture conditions for mass production of Chlorella schroteri, Dunaliella salina and Dunaliella sp. were optimized using the
response surface method. A central composite design, which was developed to facilitate the RSM, was applied to investigate the effects
of initial pH, nitrogen sources and phosphate concentration on the mass production of the microalgae. The optimum growth conditions
estimated from the design were: Chlorella schroteri (pH 7.2, ammonium 17mM, phosphate 1.2 mM), Dunaliella salina (pH 7, nitrate
3.3mM, phosphate 0.0375mM) and Dunaliella sp. (pH 8, nitrate 3.7mM, phosphate 0.17mM). Culturing of the microalgae with the optimized conditions confirmed that the maximum growth rates were attained from the parameters. The specific growth rates (μmax) of
Chlorella schroteri, Dunaliella salina and Dunaliella sp. were 0.99, 3.45 and 4.33/day, respectively. The biomass productivity of Chlorella
schroteri, Dunaliella salina and Dunaliella sp. were 0.244, 0.237 and 0.1 dry cells weight g·L-1·day-1, respectively.
283
STEROL BIOSYNTHESIS IN THE MARINE
DINOFLAGELLATES, KARENIA BREVIS PINEY ISLAND
AND KARLODINIUM VENEFICUM MD5
Shannon A. Roche1, Nicole M. Porter1, Allen R. Place1, Jeffrey D. Leblond2
1
Institute of Marine and Environmental Technology, Univ. of Maryland Center for Environmental Sciences, Baltimore, MD, USA.
E-mail: [email protected]
2
Department of Biology, Middle Tennessee State Univ., Murfreesboro, TN, USA
The primary objective of this study was to elucidate steps in the biosynthesis of brevesterol and gymnodinosterol, the primary sterols produced
by Karenia brevis and most other members of the Kareniaceae, by determining structures of selected intermediates that accumulate during exposure of K. brevis to particular fungicides known to inhibit sterol biosynthesis. The fungicides utilized include econazole, an inhibitor of cytochrome
P-450-dependent 14-demethylation of lanosterol and obtusifoliol, intermediates in fungal and plant pathways, respectively, and fenpropidine, an
inhibitor of 14-reductase and 8(9)‚7(8) isomerase. Our data suggest that a combination of fungal- and plant-like pathways is present in K. brevis. Exposure of K. brevis to econazole produced a C29:2 intermediate that resembles lanosterol, and exposure to fenpropidine produced two
tri-unsaturated intermediates (C28:3 and C29:3) with one having the same side-chain as ergosterol, a key fungal sterol. Characterization of these
intermediates and their potential placement within a brevesterol/gymnodinosterol biosynthetic pathway will be presented. Also discussed will be
the discovery of stigmasterol, a common plant sterol, in K. brevis, independent of fungicide treatment. As stigmasterol is a major component of
haptophytes, it is possible that production of stigmasterol in K. brevis is the result of sterol biosynthesis genes that have been incorporated from
its haptophyte endosymbiont. A secondary objective of the study was to identify the C27:1 sterol produced by Karlodinium veneficum MD5 as
its sole sterol. Unlike other strains of K. veneficum, the MD5 isolate was not observed to produce brevesterol and gymnodinosterol. Exposure of
K. veneficum MD5 to econazole produces a C28:1 intermediate; characterization of it will also be discussed.
13. ABSTRACT BOOK (278-284)_Layout 1 10/22/10 2:45 PM Page 284
HARMFUL ALGAE CULTURE COLLECTIONS
POSTER PRESENTATIONS
ALISU - THE CULTURE COLLECTION
AT THE UNIVERSITY OF LISBON
Vera Veloso, Ana Amorim
Instituto de Oceanografia, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. E-mail: [email protected]
The Algae Culture Collection at the University of Lisbon (ALISU) was started in 1997 and is dedicated to marine phytoplankton from Portuguese waters with emphasis on Harmful Algae (HABs). In addition, several freshwater and estuarine microphytobenthos species are
kept in culture for teaching or research purposes. The main objectives of the collection are: (1) ex situ conservation of regional representatives of HAB species and other biological and ecological relevant groups, (2) contribute to national and international research on the
biology, ecology and taxonomy of marine and estuarine microalgae, and (3) support undergraduate, graduate, MSc and PhD research
projects at the University of Lisbon. Currently the collection hosts a total of 48 species and 92 strains assigned to Cyanobacteria, Bacillariophyceae, Chlorophyceae, Cryptophyceae, Dinophyceae, Haptophyceae and Raphidophyceae. Some strains are still in the process
of being described or studied.
284
MICROALGAL BLOOM - AN INNOVATIVE TOOL
FOR SUSTAINING THE BIOSPHERE
Yee-Keung Wong1, Kin-Chung Ho1, Tat-Shing Tang2
1
2
The Open University of Hong Kong, Hong Kong, SAR, China. E-mail: [email protected]
Christian Alliance S W Chan Memorial College, China
Nowadays, development of renewable energy becomes a hot topic in scientific research because of the potential shortage of fossil fuel
and the global warming resulted from the over-consumption of fossil fuel. Algal fuel, which is the third generation of biomass fuel, may
be one of the promising green alternatives to replace traditional non-renewable fuel, such as diesel or petroleum oil. To maintain the sustainability of biosphere, chemical and biological treatments should be taken before the effluents were discharged. Interestingly, the various algal bloom triggering factors can be synthetically controlled to produce huge amount of phytoplankton which in turn can lower the
harmful effects of global warming. This research attempts to investigate the optimal conditions to cultivate the maximum algal biomass
by in-house method and algae oil was successfully extracted from the algal cells.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:47 PM Page 285
MANAGEMENT,
MITIGATION AND PUBLIC
OUTREACH FOR HABS
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 286
ORAL PRESENTATIONS
AUTOMATED IMAGING-IN-FLOW ANALYSIS
FROM IMAGING FLOWCYTOBOT PROVIDES SUCCESSFUL
EARLY WARNING OF HARMFUL ALGAL BLOOMS
IN THE GULF OF MEXICO
Lisa Campbell1, Heidi M. Sosik2, Robert J. Olson2
1
2
Department of Oceanography, Texas A&M University, College Station, Texas, USA. E-mail: [email protected]
Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA
Monitoring programs for HABs typically rely on manual microscopy for phytoplankton identification; however, the level of effort required
makes it difficult to obtain results with sufficient temporal resolution. Continuous automated methods, such as imaging-in-flow by the Imaging FlowCytobot (IFCB), have the potential to solve this problem. The effectiveness of IFCB for HAB early warning has been documented at a coastal Texas site in the Gulf of Mexico. In February-March 2008 the imaging-in-flow approach revealed an unexpected and
harmful bloom of Dinophysis cf. ovum; bloom levels of this dinoflagellate and oyster toxicity due to okadaic acid had never been reported
for the Gulf of Mexico. Early warning led to evaluation of toxicity, closure of oyster harvests, and a swift recall of harvested oyster. A recurrence of Dinophysis in 2010 was detected early enough so that the shellfishery remained closed and no recalls were necessary. In
fall 2009, early warning of a Karenia brevis bloom was also successful. In mid-August, images of Karenia were recorded by IFCB and
by the beginning of September images provided confirmation of the developing bloom. Intermittent appearances (apparently influenced
by weather/ rainfall) might have been missed or overlooked by routine, less frequent sampling. Continuous automated analysis provided
data at sufficient temporal resolution to provide state agencies the information needed for appropriate response. Subsequent analyses
of archived IFCB data also permitted growth rate calculations (maximum 0.18 d-1). Each of these examples demonstrates that monitoring can be guided by continuous automated methods to provide real-time detection of a HAB event.
286
EVOLUTION OF EUROPEAN MONITORING SYSTEMS
FOR SHELLFISH TOXINS – AN INCREASED NEED
FOR THE SURVEILLANCE OF PHYTOPLANKTON
Philipp Hess1, Zouher Amzil2, Catherine Belin1, Chantal Compère3, Patrick Lassus2, Alain Ménesguen4
1
Ifremer, Département Environnement, Microbiologie et Phycotoxines, Rue de l’ le d’Yeu, 44311 Nantes Cedex 03, France.
E-mail: [email protected]
2
Ifremer, Laboratoire Phycotoxines, Rue de l’Ile d’Yeu, 44311 Nantes Cedex 03, France
3
Ifremer, Service Interfaces et Capteurs, Technopole de Brest-Iroise, 29280 Plouzané, France
4
Ifremer, Département Dynamiques de l’Environnement, Technopole de Brest-Iroise, 29280 Plouzané, France
Monitoring systems for shellfish toxins in Europe have constantly evolved over the last 20 years. An overview of these systems, as implemented
by various member states, is given through the 2007 survey of national reference laboratories. The survey reports on management practises used
for the opening and closing of production areas, pre- and post-harvest official control, the status of the shellfish industry’s own checks and official
agency involvement in the organisation of own checks. Legislative changes proposed in 2009 have resulted in the need for further evolution of
sanitary monitoring systems. The switch from mouse bioassay to chemical testing for lipophilic toxins is a major development and requires increased vigilance in the monitoring of toxicity in shellfish. Experiences from comparative surveillance systems are outlined at the example of the
French surveillance system over a 6-year period (2003 to 2008) and its recent implementation of chemical testing for lipophilic toxins. Beside the
requirement for novel techniques in the detection of emerging and unknown toxins, the increased need for vigilance also puts a stronger emphasis on possible changes in the composition and toxicity of micro-organisms occurring in the coastal environment surrounding shellfish growing areas.
The paper reviews recent developments in the detection of harmful algal blooms and the description of the phytoplankton community, including
satellite imagery, modeling approaches, automated shape recognition in microscopy, bio-molecular techniques and flow cytometry. The use of integrating data from environmental surveillance with those from sanitary programs is suggested for the detection of changes in coastal ecosystems.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 287
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
ONE HAB, MANY ANGLES: THE FLORIDA STORY
Barbara Kirkpatrick1, Cynthia Heil2, Leanne Flewelling2
1
2
Mote Marine Laboratory, 1600 Ken Thompson Parkway, 34236, Sarasota, FL, USA. E-mail: [email protected]
Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Ave SE, 33701
St. Petersburg, FL, USA
As Harmful Algal Blooms (HABs) are spreading in intensity and duration around the world, stakeholders are increasing pressure on the
scientific community to identify potential control strategies and mitigation techniques. The State of Florida allocated funds from 2007 to
2009 specifically for the exploration of environmentally acceptable approaches to control the harmful algae, Karenia brevis, which blooms
almost annually in Florida coastal waters, or to mitigate the effects of K. brevis blooms. The Red Tide Control and Mitigation Grant Program administered by the Florida Fish and Wildlife Conservation Commission (FWC) was the first US-funded program specifically for HAB
Control and Mitigation research. A 2 and ½ day public workshop was held in February, 2010 at Mote Marine Laboratory in Sarasota, FL,
to present the results of 12 projects funded through the FWC Red Tide Control and Mitigation Grant Program. The workshop was sponsored by the Gulf of Mexico Alliance, Solutions to Avoid Red Tide (START) and the Marine Policy Institute at Mote Marine Laboratory. In
addition to the 12 principal investigators from the FWC program, workshop participants included scientists with previous control and mitigation experience, advocacy groups, government officials, and resource managers. A summary of workshop sessions and discussions
will be presented.
287
MODIFIED LOCAL SANDS FOR THE MITIGATION
OF HARMFUL ALGAL BLOOMS
Gang Pan1, Jing Chen1, Donald M. Anderson2
1
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 10085, China.
E-mail: [email protected]
2
Biology Department, Woods Hole Oceanographic Institution, MS 32, Woods Hole 02543, MA, USA
Harmful algal bloom (HAB) suppression or control using clay as a flocculating agent has been studied extensively, and is used in some
countries for HAB management. One concern that is frequently raised relates to the environmental impacts and costs of using clays from
other regions in the treatment. Here we describe a new method for HAB mitigation using local sands modified with chitosan and polyaluminum chloride (PAC). 100 mg L-1 beach sand or silica sand were ineffective in flocculating algal cells in seawater alone, but 80% removal efficiency was achieved for Amphidinium carterae Hulburt and a Chlorella sp. in 3 min (t80 = 3 min) using 120 mg L-1 modified
sands that contained 10 mg L-1 polyaluminum chloride (PAC) and 10 mg L-1 chitosan. 92-96% removal was achieved after several
hours. The t80 for removing A. carterae using the modifiers only (PAC and chitosan) was 60 min and that for Chlorella sp. was 120 min
- much slower than when modified sands are added as well. Therefore, sands were critical for speeding up the kinetic processes of flocculation and the sedimentation of algal flocs, presumably because of the mass they provide for sinking. Chitosan was important in the
formation of flocs between algal cells and sands and PAC was effective in maintaining the netting and bridging function of chitosan under
high ionic strength in seawater. Chitosan was more effective in inhibiting the motility of the cells in the flocs than PAC. The algal floc diameter (d50) for PAC increased from 600 μm to 860 μm through the addition of chitosan. Larger and denser algal flocs were formed by
charge neutralization, adsorption, binding and bridging interactions to prevent the escape of motile cells from the flocs. Chitosan and PAC,
used together as modifiers, make it possible to use local beach sands for HAB mitigation in seawater. Environmental concerns are thus
greatly reduced through the use of local sands and biodegradable chitosan, but the potential impacts of PAC need further study.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 288
ORAL PRESENTATIONS
BIOFOULING TUNICATES ON AQUACULTURE GEAR
AS POTENTIAL VECTORS OF HARMFUL ALGAL
INTRODUCTIONS
Sandra E. Shumway1, Maria Rosa1, Gary H. Wikfors2, Bridget A. Holohan1
1
Department of Marine Sciences, University of Connecticut, 1080 Shennecosset Road, Groton, CT, USA.
E-mail: [email protected]
2
NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT, USA
Biofouling tunicates are ubiquitous in coastal systems and among the main overgrowers of aquaculture gear. Our study tests the hypothesis
that the proliferation of abundant tunicate fouling species and their subsequent transport, removal and transfer by aquaculturists provide
mechanisms for concentration and distribution of harmful algal cells to new areas. Wild-caught species of common, biofouling ascidians
(Styela clava, Ciona intestinalis, Molgula manhattensis, Bottryloides violaceus, Didemnum sp., and Botryllus schlosseri) were exposed individually to cultured strains of harmful algae (Prorocentrum minimum, Alexandrium fundyense, Alexandrium monilatum, Karenia mikimotoi,
Aureococcus anophagefferens, or Heterosigma akashiwo) at simulated bloom densities of each HAB. The ability of algal cells to pass
intact through the digestive system and remain viable was assessed. After feeding, ascidians were transferred and kept for 48 h in ultrafiltered seawater. After 24 and 48 h, biodeposits were collected and observed microscopically for the presence of intact and possibly
viable cells. Subsamples of biodeposits were transferred into algal culture medium and monitored for algal growth. Results and potential
mitigation strategies will be discussed regarding the risks of transporting harmful algal species through movement of shellfish aquaculture gear and disposal of biofouling material.
288
RISK MANAGEMENT OF FARMED MUSSEL HARVEST BANS
DUE TO HAB INCIDENTS IN GREECE
John A. Theodorou1,3, Ioannis Tzovenis1, Patrick Sorgeloos2, Jacques Viaene3
1
Department of Fisheries & Aquaculture, Technological Educational Institution (TEI) of Epirus, GR 46100 Igoumenitsa, Greece.
E-mail: [email protected]
2
Laboratory of Aquaculture & Artemia Reference Centre, University of Gent, Rozier 44, B-9000 Gent, Belgium
3
Faculty of Applied Biological Sciences, University of Gent, Coupure Links 653, B-9000 Gent, Belgium
The severity and the consequences of site closures to shellfish commercial harvesting, a protection measure for public health against toxicity inflicted by harmful algal blooms, has been estimated for the Mediterranean mussel (Mytilus galloprovincialis) farming in Greece. Estimations were carried out in a semi-quantitative manner at the farm level. Results showed that the financial losses depended on the season
and duration of the harvest ban. Since the product becomes marketable from late spring to early autumn site-closures longer than 6 weeks
within that period could be catastrophic for a farm. Consequences include yield losses due to extended stocking of ready to harvest mussels in the farm and ex-farm price reduction due to oversupply after the harvest ban. Moreover, mussel seed collection and placement
within the farm is delayed due to lack of space as the bulk of mussels remain un-harvested putting in danger next season’s production.
Proposed strategies to minimise losses consisting of differential handling of the marketable mussels and of extraordinary spatial extension of farm facilities due to harvest bans caused by HABs were discussed.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 289
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
INTERNATIONAL ATOMIC ENERGY AGENCY ASSISTANCE
FOR THE MONITORING OF PSP AND CFP
IN THE CARIBBEAN AND LATIN AMERICA REGIONS
Florence Boisson1, Jane Gerardo-Abaya2
1
Marine Environment Laboratories, International Atomic Energy Agency, 4 Quai Antoine Ier, MC98000 Monaco, Principality of
Monaco. E-mail: [email protected]
2
Department of Technical Cooperation, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria
In response to the growing interest expressed by its Member States in early detection and managing the effects of Harmful Algal Blooms,
the International Atomic Energy Agency is supporting technology transfer activities to address the impacts of PSP and CFP on seafood
safety in the Caribbean and in the Latin America regions. The Receptor Binding Assay (RBA), a cost-effective technology involving radioactive isotopes, is being transferred based on the experience gained under previous similar IAEA Technical Cooperation projects in the
SE Asia and in Africa. The RBA is a simple and high throughput method for detecting and quantifying neurotoxins at very low levels in
phytoplankton and shellfish. In the case of PSP, it is considered as an early warning method for the management and mitigation of HABs
events. The IAEA is currently enhancing the capabilities of laboratories in Chile, Colombia, Costa Rica, Cuba, El Salvador, Mexico, Dominican Republic, Uruguay and Venezuela to use the RBA for PSP & CFP toxins. In addition, to support the acceptance of the method
for export requirements and ensure its sustainability, the IAEA is supporting the AOAC certification of the Receptor Binding Assay and
the assured supply of radiolabelled-toxins required for the assay, through collaborations established with the US Food and Drug Administration and the Center for Coastal Environmental Health and Biomolecular Research of NOAA. Technical backstopping for these activities is provided by the IAEA’s Marine Environment Laboratories in Monaco.
289
NUMERICAL SIMULATION OF COAGULATION AND
REMOVAL OF RED TIDE ALGAE WITH CLAY PARTICLES
Xihua Cao, Hongliang Wang, Zhiming Yu, Xiuxian Song, Yongquan Yuan
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, People’s Republic of China. E-mail: [email protected]
Clay coagulation is a promising method for red tide emergencies management and has been successfully used in several countries, e.g.,
Japan, Korea, China. However, the algae removal efficiencies (ARE) with clays are still far from perfect, the mechanisms of algae removal need to be further explored and the critical factors on algae removal need to be clarified. In this study, all particles in the system
were firstly characterized based on fractal theories; numerical simulation was then carried out using a FORTRAN program to elucidate
the processes and the key factors during red tide algae coagulation and settlement with clay particles. Our results showed that the ARE
increased from 16% to 78% as collision efficiencies (CE) of the clay particles with algae adding from 0.01 to 0.20, from 38% to 82%
as the clay concentrations adding from 2.0x10-4 g•cm-3 to 1.0x10-3 g•cm-3, and from 56% to 88% as the fractal dimensions of clay particles reducing from 2.80 to 2.00. The CE of clay particles with algae, the fractal dimensions and the concentrations of clay particles were
the most significant factors which influenced on the ARE. Clays modified with polyaluminium chloride had higher ARE because this improved the CE of clay particles with algae and decreased the fractal dimensions of clay particles. The transparent exopolymer particles in
algae culture would increase the CE of clay particles with algae and lead to higher ARE.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 290
NOVEL DERIVATIVES OF THIAZOLIDINEDIONE ARE SELECTIVE
ALGICIDES AGAINST RED-TIDE HARMFUL ALGAE
Hoon Cho1, Si Wouk Kim2, EonSeon Jin3
1
Dept. of Polymer Science & Engineering, Chosun University, Gwangju, Korea
Dept. of Environmental Engineering, Chosun University, Gwangju, Korea
3
Dept. of Life Science, Hanyang University, Seoul, Korea
2
Thiazolidinedione (TD) derivatives exhibited algicidal activity against harmful algae in our previous report. In this study efficacy and selectivity of newly synthesized various TD derivatives were tested with analyzing structure-activity relationship of various TD derivatives. To investigate the structure-activity relations for growth inhibition of harmful algae, we added methylene group between cyclohexyl ring and
oxygen of 5-(3-chloro-4-hydroxybenzylidene)-TD, which decreased inhibitory potency of compound 17. Interestingly, further addition of
methylene group increased significantly the inhibitory potency against C. polykrikoides as indicated for compound 20. The addition of 1μM
compound 17 resulted in rupture of harmful algae tested less than 10 hours. Compound 17 applied to both harmful and non-harmful
algae showed drastic reduction of Photosystem II efficiency which resulted in reduced photosynthetic oxygen evolution. Compound 17
destroyed all the harmful algae at the 5μM concentration while the algicidal activity against non-harmful algae did not exceed 30% of control within the concentration range tested. In contrast, herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) exhibited 40-70% algicidal activity of control against both harmful and non-harmful algae tested at 5μM concentration. Compound 17 can thus be considered
a promising lead compound for the development of algicides addressing the universal need for effective, harmful algae management.
290
THE EFFECTS OF YELLOW DUST ON THE GROWTH
OF DINOFLAGELLATES AND DIATOMS
Joong Ki Choi, Young Ju Lee, Jae Young Park, Man Ho Yoo
Department of Oceanography, Inha University, Incheon 402-751, Korea. E-mail: [email protected]
To investigate the possibility of mitigating the deleterious effect of harmful algal blooms, the inhibitions of yellow dust on the growth of 3
common harmful dinoflagellates and 4 diatoms were tested in the laboratory. In the inhibition effect experiment with the yellow sand from
the deserts in China and Mongol on the growth of phytoplankton, Cochlodinium polykrikoides was stressed by the low and high concentration of yellow sand, showing the negative variance of gross growth rate on that of control bottle, while the growths of Navicula sp.
was stimulated by adding the yellow sand, showing the positive (10-25%) variance of gross growth rate on that of control experiment.
In the aeorian yellow dust experiment, Heterocapsa triguetra, Cochlodinium polykrikoides and Prorocentrum minimum showed negative
variance of gross growth rate by adding the low concentration of yellow dust, but Skeletonemea costatum, Pleurosigma angulatum and
Thalassiosira pacifica showed positive variance of gross growth rate (10-100%). These experiments suggest that the growths of dinoflagellates, as HAB species, were inhibited by adding the yellow dusts, while the growths of diatoms were stimulated positively. The
yellow dust from deserts in China and Mongol has a possibility as mitigation material.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 291
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
INTERNET-BASED INFORMATION SYSTEM
FOR RED TIDE IN KOREAN WATER
Sang-Ok Chung, Sung-Hwan Yang, Bo-Ra Kim, Joon-Yong Yang
Fishery and Ocean Information Division, National Fisheries Research and Development Institute, 152-1 Haean-ro, Gijang-eup,
Gijang-gun, Busan 619-705, Republic of Korea. E-mail: [email protected]
We introduce an integrated information system for monitoring red tide and minimizing its damages to fisheries and aquaculture. During
the first stage of development from 2006 to 2009, the National Fisheries Research and Development Institute of Korea (NFRDI) established a Geographic Information System for compiling reports of red tide occurrences together with oceanographic (e.g., water temperature, salinity, and chlorophyll) and meteorological data (e.g., wind direction, wind speed, and precipitation). The data were assembled
and analyzed to assess and inform the state of red tide to the public. In 2010, the second stage of development was initiated with the
aim of integrating real-time information on red tide outbreaks with subsequent mitigation activities. This new internet-based information
system is expected to facilitate communications and decision making by local and central governments for controlling harmful algal blooms
in Korean coastal waters.
291
TROUBLESHOOTING IN REAL-TIME PCR
QUANTIFICATION OF THE TOXIC CYANOBACTERIUM
PLANKTOTHRIX AGARDHII
Catarina Churro1, Paulo Pereira1, Elisabete Valério2
1
Laboratόrio de Biologia e Ecotoxicologia, Instituto Nacional de Saude Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016
Lisboa, Portugal. E-mail: [email protected]
2
Centro de Recursos Microbiolόgicos, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,
2829-516 Caparica, Portugal
Real-time PCR analysis has become an emergent and attractive technique for the detection and quantification of harmful algae in the
environment. Combining PCR chemistry with fluorescent probe detection, this method allows the monitoring of the amount of a target
sequence as it is amplified in real-time and absolute quantification can be achieved using a standard curve constructed by amplifying known
amounts of target DNA. However, accurate target DNA quantification from environmental samples remains challenging and several factors in qPCR development have to be taken in to account. The aim of this study was the development of a suitable preparation method
for standards and environmental samples that allows the quantification of the cyanobacterium Planktothrix agardhii in spiked culture experiments and field samples. For that purpose, all the steps required for the analysis were studied in detail, such as: 1) molecular marker
choice and primer design, 2) optimization of PCR conditions, 3) accurate cell quantification in cultures to be used as standards, 4) preparation and preservation of the standards, 5) standard curve construction from known dilution factors in the proper assay range, 6) cell
harvesting concentration in field samples, 7) effects of biological matrix and samples diluents in spiked cultures and field samples. Furthermore, during the development of the method several approaches were applied to overcome the shortcomings that appeared during
the experiments. The critical factors and troubleshooting for a successful quantification of Planktothrix agardhii are discussed.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 292
PARALYTIC SHELLFISH TOXINS PROFILES IN FOUR
SHELLFISH SPECIES EXPOSED TO BLOOMS OF
GYMNODINIUM CATENATUM IN THE PORTUGUESE COAST
Pedro R. Costa, Maria J. Botelho, Ana Monteiro, Susana Rodrigues, Teresa Quental, Maria Teresa Moita
IPIMAR - National Institute of Biological Resources, INRB I.P., Av. Brasilia s/n, 1449-006 Lisbon, Portugal.
E-mail: [email protected]
Paralytic shellfish toxins (PST) have been detected in bivalve molluscs from the Portuguese coast since 1950s, however after 1995
no evident blooms of PST-producing dinoflagellates have been observed. In 2005 blooms of Gymnodinium catenatum occurred for the
first time in 10 years. Toxin composition in shellfish is dependent of the ingested toxic algae, shellfish metabolism and toxin kinetics of
uptake-elimination. This study aims to investigate the toxin profile in four shellfish species and to assess changes during and after
blooms of Gymnodinium catenatum. The AOAC-approved HPLC-FLD pre-column oxidation method for PSP determination in shellfish was used. A hydrolysis step for estimation of C3+4 and B2 toxins was included. During blooms in Aveiro Lagoon (NW coast) and
the corresponding periods of high toxin concentration, sulfocarbamoyl toxins were dominant in mussels (Mytilus galloprovincialis). A significant (p<0.05) decrease of the ratio of sulfocarbamoyl:decarbamoyl toxins was observed with the decrease of the total toxin burden.
For cockles (Cerastoderma edule) the average ratio between the two groups of PST (15:1) did not significantly change from highly to
residual contamination phase. In the case of cockles collected in Formosa Lagoon (S coast) a sulfocarbamoyl:decarbamoyl ratio of 4:1
was determined. Donax clam (Donax spp.) also collected in S coast presented an average ratio of 5:1. C3+4 and B2 were detected
in these 3 shellfish species. Their contribution to the total toxicity can reach 42%. A unique profile constituted only by decarbamoyl toxins was determined in Spisula solida (offshore the NW coast). This study provides reliable information for effective risk assessment and
environmental management.
292
FISH DISEASE DIAGNOSIS IN MALDIVES FOLLOWING
MASSIVE FISH KILL OF 2007
Ahmed Fahmi
UNESCO South Asia Cluster Office, 110029 Delhi, India. E-mail: [email protected]
In line with the Mauritius Strategy Implementation for Small Islands Developing States (SIDS), UNESCO organized a training course
for researchers from the Marine Research Centre, Maldives, on conventional and molecular fish disease diagnosis, at the UNESCOMIRCEN for Marine Biotechnology, Department of Microbiology, College of Fisheries, Mangalore, India. This project was prompted
by the massive fish kill that was seen in several islands of the Maldivian archipelago in 2007. Some islands reported as many as 4000
dead fish accumulated on the beach on a daily basis, causing a major public concern. The maintenance of a healthy fish population in
the Maldives is important because the Maldivian economy is heavily dependent on fisheries sector, which is the country’s second
largest industry after tourism. In addition, local communities depend on reef fish, in particular, as a source of food and livelihood. We
considered two causes for this fish kill; poor management of sewage disposal which in turn alters the nutrient balance in the sea and
promotes the growth of blue-green algae and other harmful algal blooms. In addition, changes in oceanographic hydrodynamics have
also been associated with promoting the growth of these algal blooms as well as affecting growth and spread of bacterial and viral
pathogens and their vectors leading to mass fish kill incidents. Training was on specific staining methods and antibody-based techniques of fish organs. Moreover, DNA-based technologies were also used in quick diagnosis of fish diseases which assists in controlling the spread of fish diseases, particularly, in efforts to limit trans-boundary movement of pathogens and reduce the economic
and socioeconomic impact of disease in mariculture.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 293
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
LEVELS OF PSP TOXINS IN BIVALVES EXPOSED
TO NATURAL BLOOMS OF ALEXANDRIUM MINUTUM
IN CATALAN HARBOURS
~
Gemma Giménez1, Amandine Caillaud1, Elisabeth Can
ete1, Pablo de la Iglesia1, Helena Eixarch1, Margarita
1
2
2
1
Ferna΄ndez , José Franco , Pilar Riobό , Jorge Diogène
1
2
IRTA Sant Carles de la Ràpita, Ctra. Poble Nou Km. 5.5, 43540 Sant Carles de la Ràpita, Spain. E-mail: [email protected]
Grupo de Fitoplancton Tόxico, Instituto Investigaciόns Marin~as (CSIC), Eduardo Cabello 6, 36208 Vigo, Galicia, Spain
The development, validation, comparison and evaluation of analytical methods for marine toxins rely on the availability of toxic material. Within the project JACUMAR PSP, our interest is mainly focused on autochthonous bivalve species with the toxic profile of Alexandrium minutum, since this is the principal species involved regionally in PSP outbreaks. In order to obtain large amounts of toxic material,
we exposed different species of bivalves to A. minutum blooms identified in harbors. Mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) were exposed during 3-8 days in the harbor of Vilanova i la Geltru΄, to blooms reaching a maximum A. minutum concentration of 200000 cells L-1 in 2008, 40000 cells L-1 and 800000 cells L-1, in 2009. Mussels, oysters and clams (Ruditapes sp.)
were exposed to one bloom of 22000 cells L-1 in the harbor of Cambrils in 2009. In all situations higher toxic levels analyzed by HPLCFLD with postcolumn oxidation were observed in mussels (i.e. 1200-2500 equivalents of saxitoxin per kg, eqSTX kg-1) than in oysters (i.e. 60-800 μeqSTX kg-1) exposed to the same bloom in Vilanova. Blooms with higher concentrations of A. minutum did not
correspond to higher levels of PSP toxins in bivalves. These differences may be explained by differences in A. minutum population dynamics, toxin production or in the physiological state or behaviour of shellfish. These results evidence that mussels concentrate more
PSP toxins from A. minutum than oysters and clams. More information about the factors involved in toxin intake is needed.
293
THE RELATIVE ABUNDANCE AS A TOOL TO INCREASE
THE CERTAINTY OF TEMPORAL AND SPATIAL
DISTRIBUTION OF HARMFUL ALGAL SPECIES
Leonardo Guzma΄n1, Ximena Vivanco1, Gemita Pizarro3, Gastόn Vidal1, Victoria Arenas1, Luis Iriarte2,
Susana Mercado2, Cesar Alarcόn3, Herna΄n Pacheco3, Mauricio Palma1
1
Instituto de Fomento Pesquero, Balmaceda 252, Puerto Montt, Chile. E-mail: [email protected]
Instituto de Fomento Pesquero, Camino Ensenada Baja s/n, Puerto Chacabuco, Chile
3
Instituto de Fomento Pesquero, Enrique Abello 0552, Punta Arenas, Chile
2
Due to the harmful microalgae patchiness distribution, traditional quantitative phytoplankton samplings show biases in its results. Relative abundance (RA) can be utilized to minimize these failures, since represents more appropriately the species distributions and provides a good insight of species relative abundances, especially when densities are low or very low. RA is a semi-quantitative estimator
of the abundance for a given species in a specific site and sampling date, based on net samples. It is defined for an ample geographic
sector and it is valid for an annual cycle. RA is referred to a species in particular and not to its relationship to the other species assemblage. Data on RA for five harmful species or potentially noxious species from Southern Chile (Alexandrium catenella, Dinophysis
acuminata, D. acuta, Protoceratium reticulatum and A. ostenfeldii), in comparison to density estimations (Utermöhl method) for each
species, are presented. Data are from May 2006 to May 2010 and encompassing a geographic area from 41° to 55° S. along 151
fixed sampling sites, which were periodically visited in an almost monthly sampling scheme. Although both variables are statistically correlated, there is not a straight forward relationship between RA and density. These results have practical meanings for harmful species
in their border areas of distribution. Previous data for A. catenella, have shown that RA can be utilized as an early warning of PSP shellfish increases. Also RA for other mentioned species, excluding A. ostenfeldii, is statistically correlated to DSP mouse bioassay results.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 294
METHODOLOGICAL STRATEGIES FOR ACHIEVING
BEHAVIORAL CHANGES IN KEY TARGET GROUPS
IN COMMUNITIES AFFECTED BY HABS ASSOCIATES
TO PSP IN SOUTHERN CHILE
Leonardo Guzma΄n1, Viviana Baza΄n2
1
2
Instituto de Fomento Pesquero, Balmaceda 252, Puerto Montt, Chile. E-mail: [email protected]
Instituto de Fomento Pesquero, Enrique Abello 0552, Punta Arenas, Chile
The Magellan region in Southern Chile, affected by PSP since 1972, presents high economical and social impacts in activities such
as fisheries, aquaculture and tourism. In public health the greatest impacts are human beings losses, existing a risk of contaminated
shellfish consumption regardless the actions taken by the authority to protect public health, consequently there is a need of a systematic
cognitive empowerment aimed at establishing responsible behavior by those in charge of the captures, marketing and consumption.
At the local level, knowledge about HABs topics and its effects exists, due to the messages given by the Sanitary Authority, but the
operational activities initiated in 1997 constitute a landmark about the methodological treatment of the HAB topics to target groups.
The strategies include, i) the importance recognizing of performing preventive interventions aimed at establishing responsible behaviour in local groups, ii) the subject has been treated with a constructivist approach and iii) methodologies, means and activities are treated
according to target groups. The constant reinforcing activities envisaged: i) workshops for direct intervention with knowledge measurements (pre-test and post-test), ii) development of support materials iii) public exhibitions, iv) activities with fishermen in landing
points and v) activities with groups that due to its profession or activity should clarify concepts on HAB. In this contribution we emphasize
the criteria for choosing the pertinent methods and strategies, so that through the technical knowledge are attainable the changes in
attitudes and behaviours of target groups and the local community in general.
294
ALGAL MICROCYSTINS REMOVAL DURING ARTIFICIAL
GROUNDWATER RECHARGE UNDER AEROBIC
AND ANAEROBIC CONDITIONS
Talis Juhna1, Ieva Barda2, Ingrida Purina3, Maija Balode2,3
1
Riga Technical University, 1 Kalku St., LV-1658, Riga, Latvia
Faculty of Biology, University of Latvia, 4 Kronvalda Blvd., LV-1586, Riga, Latvia
3
Latvian Institute of Aquatic Ecology, 8 Daugavgrivas St., LV -1048, Riga, Latvia. E-mail: [email protected]
2
Artificial recharge of groundwater (AGR) is becoming more and more frequently used for drinking water production because it is a simple, cost effective water treatment method and it provides safety barrier against pathogens. However there is no enough evidence for
how efficient this method is for removal of algal Microcystin LR, which has been found in many drinking water resources in Europe. It is
generally accepted that Microcystin LR removal occurs in aerobic conditions in unsaturated zone (vadose zone) below the infiltration
basins which are used for AGR. However since water retention time in this zone is short, it is not clear whether bacterial degradation rate
is sufficient to remove high concentrations of Microcystin LR. In this study with the column experiments we clearly show that removal of
Microcystin LR occurs effectively, from 1 mg/l to under detection limits, in anaerobic condition after about one week of biomass adaptation in columns. This finding implies that Microcystin LR is removed in vadose and saturated aquifer because both aerobic and anaerobic bacteria are able to degrade this toxic substance providing that the sufficient time of biomass adaptation is achieved. Further studies
will be directed towards identifying the species of anaerobic bacteria responsible for the phenomena observed.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 295
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
A COMPARISON OF DIFFERENT HAB MITIGATION
METHODS WITH PARTICULAR REFERENCE
TO AGGLOMERATION PRECIPITATION
Virginia Yuk-kam Kai, K. C. Ho
Environmental Studies Programme, School of Science & Technology, The Open University of Hong Kong, Hong Kong SAR,
China. E-mail: [email protected]
Due to its complicate properties, harmful algal bloom (HAB) is difficult to be prevented, controlled and mitigated. Researchers have
attempted different methods to reduce the harms of red tide including the use of ozone in killing the harmful algal cells, use of clay in
precipitate the causative dinoflagellates and injection of oxygen to increase dissolved oxygen in seawater near fish culture zones. Yet,
these mitigation measures could hardly be effectively applied due to economic, ecological and environmental reasons. For example,
the use of aluminum salt and iron salt to agglomerate the algae aluminum salt was criticized for causing environmental impacts and
relatively high in cost. The present research also studied an innovative method - agglomeration precipitation by using the coagulants PEI and Type IV polymer. Comparatively, those coagulants are relatively cheap in price and could be easily purchased in the market.
Therefore, it could be widely used in water industry. Experimental results showed that PEI and Type IV polymer could effectively precipitate nearly all of the harmful algae and improve water quality which is considered an added benefit.
295
ASSESSING ECOLOGICAL QUALITY IN GREEK
FRESHWATERS USING PHYTOPLANKTON-BASED INDICES
Matina Katsiapi1, Maria Moustaka-Gouni1, Konstantinos Ar. Kormas2, Evangelia Michaloudi3, Elias Dimitriou4
1
Department of Botany, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
E-mail: [email protected]
2
Department of Ichthyology & Aquatic Environment, School of Agricultural Sciences, University of Thessaly, 384 46 Nea Ionia,
Volos, Greece
3
Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
4
Hellenic Centre for Marine Research, Institute of Inland Waters, 46.7 km of Athens- Sounio Ave., 19013, Anavissos Attikis,
Greece
In the EU Water Framework Directive (WFD) (2000/60/EC), phytoplankton is one of the biological quality elements required for the ecological status/potential assessment of surface waters. A number of assessment methods and indices based on phytoplankton have already been applied in
European countries or are still under development, to fulfil the WFD requirements. The ecological water quality of 13 Greek lakes (Volvi, Doirani,
Kastoria, Megali Prespa, Mikri Prespa, Zazari, Xeimaditis, Pamvotis, Vegoritis, Petron, Trichonis, Yliki, Koronia) and seven reservoirs (Kerkini,
Marathonas, Tavropos, Polyphytos, Thesaurus, Platanovrisi, Aisimis) was assessed on the basis of phytoplankton. Data were obtained during the
warm period of the years 2007-2010. Harmful algal blooms were observed and were dominated often by toxin-producing species, mainly of
cyanobacteria. Species composition, abundance and biomass metrics were applied as outlined by the WFD. Additional ecological features, i.e.
species richness and the Q Index modified for Mediterranean freshwaters, along with the presence of microcystin-producing genes and cyanobacterial 16S rRNA gene libraries were used for ecological water quality assessment. A comparison of phytoplankton-based indices used in Greek and
other Mediterranean freshwaters is also presented. Only Tavropos Reservoir out of the twenty freshwater bodies studied showed a good ecological
potential. Lake Yliki phytoplankton metrics revealed a good water quality. However, this contradicts with the lake’s phytoplankton history in the
1990s, i.e blooms of Peridimium gatunense, and recent water column physical-chemical features. Lake Koronia, an extreme environment with
mass kills of fish and birds, ranked at the worst ecological water quality.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 296
SUPPRESSING OF DINOFLAGELLATE NOCTILUCA
SCINTILLANS BY ALGICIDAL BACTERIA FOR IMPROVING
SHRIMP AQUACULTURE
Teeyaporn Keawtawee1, Kimio Fukami1, Putth Songsangjinda2, Pensri Muangyao3
1
Graduate School of Kuroshio Science, Kochi University, Monobe otsu 200, Nankoku, 783-8502, Kochi, Japan.
E-mail: [email protected]
2
Trang Coastal Aquaculture Station, Mai Fad, Sikao, 92150, Trang, Thailand
3
Coastal Aquaculture Research Institute, Kao Seng, Muang, 90000, Songkhla, Thailand
It is often reported that the blooming of dinoflagellates Noctiluca scintillans causes the occurrence of diseases in shrimp culture and results in the economic losses due to increase in shrimp mortality. For suppressing the growth of N. scintillans, we tried to use algicidal bacteria and to assess the possibility for improving shrimp aquaculture. As a result, some algicidal bacteria were isolated from shrimp culture
pond. Among these, a strain BS2 showed the strongest killing activity on N. scintillans, and then was used for the following laboratory
experiments with two species of shrimp (Penaeus monodon and Litopenaeus vannamei). When BS2 was not added to the shrimp culture with N. scintillans, nearly 80-90% of shrimp was died within 7 days, probably due to N. scintillans. Shrimps of smaller size (body
length of 1.5-1.8 cm) were more strongly affected by N. scintillans than shrimps of bigger size (3.5-4.0 cm). On the contrary, a strain
BS2 could inhibit the N. scintillans growth in shrimp culture within 48 hours of incubation time and shrimp survival rates increased from
23% to 87% and 13% to 87% of P. monodon and L. vannamei, respectively. However, a bacterium BS2 did not impact on shrimp condition at all. From the results of this study, it was suggested that application of algicidal bacteria for suppressing N. scintillans (harmful
algal bloom) in the field would be promising.
296
MIGRATION OF DOMOIC ACID BETWEEN TISSUES DURING
FREEZING/THAWING OF SCALLOPS
Angeles Moron~o1, Fabiola Arévalo1, Jorge Correa1, Arabela G. Sanchez2, Adoraciόn R. Sanchez2
Instituto Tecnolόxico para o Control do Medio Marin~o de Galicia (INTECMAR). Peirao de Vilaxoa΄n s/n, 36611, Vilagarcίa de
Arousa, Pontevedra, Spain
2
Centro de Investigaciόns Marin~as (CIMA), Apto. 13, 36620 Vilanova de Arousa, Pontevedra, Spain
1
Scallops (Pecten maximus) were monthly sampled, from March to September 2010, in Rίa de Vigo (NW, Spain). Previously to domoic
acid (DA) analyses all the scallops were eviscerated separating three anatomical parts: adductor muscle, gonad and remainder tissues.
With each sample four different pre-treatments were studied: 1) immediate freezing and evisceration after thawing; 2) immediate freezing and after thawing evisceration over a filter paper (draining the tissues) 3) evisceration and freezing of each anatomical part separately;
4) evisceration and processing in fresh for DA analyses. Frozen samples were stored for a month at -20°C and thawed at ambient temperature. The DA concentration in the entire body ranged between 38.5 and 148.6 μg·g-1. Freezing of scallops decreased an average
30% in the DA content of remainder tissues. An average increase in DA concentration of 40% was observed for gonads of those samples without draining of the tissues after thawing. For adductor muscle the increase of DA content, during freezing of the entire body,
would even prevent its commercialization below the EU Commission Decision 2002/226/EEC.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 297
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
HAB-MITIGATING CLAY APPLICATION: EFFECTS
ON NON-TARGET BENTHIC AND PELAGIC ORGANISMS
Iris S. Orizar, Maria Lourdes San Diego-McGlone, Peter Paolo L. Rivera, Rhodora V. Azanza
Marine Science Institute, University of the Philippines, Diliman, Quezon City, 1101, Philippines. E-mail: [email protected]
An important consideration in the application of clay as Harmful Algal Bloom (HAB) mitigation strategy is the effect of clay addition on
non-target benthic and pelagic organisms. This study examined the effect of ball clay (1 g/L) on a) the phytoplankton Isochrysis galbana,
b) Perna viridis commonly known as Asian Green Mussel, and c) commercially cultured fish Chanos chanos. Results showed that the effective concentration of clay at 50% mortality of I. galbana was 7.99 g/L and 7.80 g/L of clay after 24-hr and 48-hr of application, respectively. This implies that higher concentrations of clay are needed before levels toxic to the plankton are reached. Results on the
effect of ball clay on P. viridis showed that they were able to accumulate clay particles <
_ 0.5 hr after clay addition. Two hours after application, the accumulated clay was released from the mussel through the pseudofeces/feces as seen by a color change of the feces from
black to light brown. The amount of pseudofeces/feces from the treated mussels and the control were similar. There was no death of
P. viridis during the acute (6-hr) experiment. The treated mussels had a faster rate of feeding compared to the control. The chronic (30day) experiment showed no significant difference on the shell length and dry weight of the treated mussels and control. The effect of ball
clay on the juvenile C. chanos showed 25% mean mortality within 48-hour exposure to the clay. No additional fish mortalities were observed after 96 hours of clay addition.
297
NETS AND MUSSELS AS CYSTS CAPTORS DURING
THE DECLINE PHASE OF AN ALEXANDRIUM CATENELLA
BLOOM
Gemita Pizarro1, Miriam Seguel2, Leonardo Guzma΄n3, César Alarcόn1, Herna΄n Pacheco1, Elias Ferna΄ndez1
1
Instituto de Fomento Pesquero, Enrique Abello 0552, Punta Arenas, Chile. E-mail: [email protected]
Centro Regional de Ana΄ lisis de Recursos y Medio Ambiente, Universidad Austral de Chile, Los Pinos s/No, Puerto Montt, Chile
3
Instituto de Fomento Pesquero, Balmaceda 252, Puerto Montt, Chile
2
In late summer 2009, an intense Alexandrium catenella bloom occurred in the outside of Aysén fjord, Chile (45° 06’ S, 73° 30’ W). The
maximum concentration found in the first 10 m depth during the end of the exponential phase was 396,100 cell l-1. During the bloom
decline, twenty days after, commercial size mussels (Mytilus chilensis) and nets (mean length 22.5 m and width 0.09 m; 3 cm mesh size)
taken from a place free of this dinoflagellate, were suspended in the water column to assess its capacity as captors of A. catenella cysts
in good physiological conditions. Three nets were suspended at 5, 10 and 15 m depth while the three net cages with 35 mussels each
were suspended at 5 m deep in each of three sampling stations. Both, the nets and mussels were moored for 30 days. Considering the
three stations, the net cyst mean number by depth was 2,761; 975 and 258 cysts at 5, 10 and 15 m, respectively. The cysts mean
content in the mussel digestive glands for three stations was 14 cysts g-1 (wet weigth). Since nets and mussels, are widely utilized in fishing and aquaculture activities, constitute a potential dispersant agents of A. catenella to free areas of this plague species in Chile. Measures to be taken by regulatory authorities to prevent this potential spread are discussed.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 298
THE REMOVAL OF MICROCYSTINS IN DRINKING WATER
BY CLAY MINERALS
Erik Prochazka1, Gab Soo Hwang2, Darryl Hawker3, Glen Shaw4, Wasa Wickramasinghe5
1
Smart Water Research Centre, Griffith University, Southport, QLD 4222, Australia
Department of Env. Engineering, Kunsan National University, Kunsan 573-701, Korea
3
School of Environment, Griffith University, Nathan, QLD 4111, Australia
4
School of Public Health, Griffith University, Gold Coast Campus, QLD 4222, Australia
5
National Research Centre for Environmental Toxicology, The University of Queensland, 39, Kessels Road, Coopers Plains,
QLD 4108, Australia. E-mail: [email protected]
2
Quality drinking water is a fundamental necessity for the wellbeing of all humans. During a bloom event, cyanotoxins pose a serious
threat to the quality of drinking water. Most of the major cities in the developed world have water purifications systems which can remove
the threat posed by these toxins. However, in rural and remote communities, where sophisticated, large-scale water purification systems
are not always available, the population is at risk of being exposed to cyanotoxins when toxic cyanobacteria are present in surface water
supplies. In continuing studies into removal of cyanotoxins from drinking water, we have been investigating a simple removal technique
that can be easily adapted by rural and remote communities using clay minerals. Such systems may be useful in the vast Australian outback. Laboratory scale experiments have shown that a significant amount of dissolved microcystins can be removed efficiently by the use
of Whangto or Korean Yellow Clay, an alumino silicate mineral high in iron. Korean yellow clay has been used to mitigate HABs in sea
around Korea since 1995. The microcystin content in drinking water, in most cases, can be brought down to below WHO guidelines (1
ug/L) by two sequential runs. The results have been compared to another clay mineral Bentonite. The removal process is by adsorption
and known adsorption isotherms could be fitted to the data.
298
EFFICACY OF VARIOUS CLAY MATERIALS IN CONTROLLING TROPICAL HARMFUL ALGAL BLOOM (HAB) FORMING
SPECIES
Peter Paolo Rivera, Iris Orizar, Maria Lourdes San Diego-McGlone, Rhodora V. Azanza
Marine Science Institute, University of the Philippines, Diliman, Quezon City, 1101 Philippines. E-mail: [email protected]
The ability of clay minerals to remove tropical harmful algal bloom (HAB) species was examined. Seawater slurries of brown bentonite,
zeolite, talc powder, ball clay and jombadsel bentonite were added into non axenic cultures for a final clay concentration of 1.0 g/L. Cell
removal efficiency (RE) was measured for 24 hours from initial application. Talc powder removed 95% of Pyrodinium bahamense var.
compressum (Pbc), followed by jombadsel bentonite (86.4%), ball clay (80.6%), zeolite (77.4%) and brown bentonite (74.4%). Ball clay
removed 71.7% of Alexandrium affine after 1 hour exposure, followed by talc powder (61.9%), jombadsel bentonite (55.3%), zeolite
(49.2%), and brown bentonite (30.8%). Ball clay also had the highest RE for Amphidinium carterae (90.2%) followed by talc powder
(75.8%), zeolite (62.8%), brown bentonite (49.2%), and jombadsel bentonite (31.8%). A. carterae showed almost full recovery after
24-hour exposure to all the clay minerals. Within 2-hour exposure, 63.2 % Chatonella marina cells were removed by ball clay, 51.4%
by talc powder, 35.7% by zeolite, 4.2% by jombadsel bentonite and 0.3% by brown bentonite. The RE of talc powder for Noctiluca scintillans was 74.7% after one hour, while for brown bentonite RE was 71.6% after 2-hour exposure. RE decreased 1 to 2 hours after addition of clay in all the test species except for talc powder in Pbc. Ball clay when reapplied to Pbc one hour after the first application showed
98-99% RE. Use of clay should be studied in the field prior to its utilization as a management option.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 299
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
MICROENCAPSULATION OF OKADAIC ACID AS A TOOL
FOR THE STUDY OF THE ACCUMULATION OF DSP TOXINS
IN BIVALVES
Araceli E. Rossignoli, David Ferna΄ndez, Carmen P. Acosta, Juan Blanco
Centro de Investigaciόns Marin~as (CIMA), Pedras de Corόn, s/n. Apdo. 13, 36620 Vilanova de Arousa (Pontevedra), Spain. Email: [email protected]
Experimental studies on the absorption, transformation and elimination of okadaic acid (OA) in bivalves are strongly needed in order to
design methods to mitigate the consequences of toxic blooms. Probably, the most important problems for these experimental approaches
are the difficulty to obtain toxic food for bivalves and the near impossibility of supplying single toxins or derivatives to those organisms during reasonably prolonged periods of time. With the objective of obtaining a technique which solves those difficulties, we have encapsulated okadaic acid (OA) in gelatin-acacia microcapsules. The encapsulation efficiency was ca. 33%. The microcapsules were very stable,
not leaching more than 9% of the OA in a 20-hour period. Mussels feed on them without any noticeable rejection as pseudofaeces when
they were supplied mixed with microplankton. OA was absorbed by mussels very efficiently, accumulating, after 3 days of feeding and
one of depuration, 65% of the OA in microcapsules and 22% of the total OA used at the beginning of the microencapsulation process.
These efficiencies and the possibility of encapsulating single DSP toxins and derivatives will constitute a valuable tool for the study of accumulation and biotransformation of DSP toxins in bivalves.
299
INVESTIGATION OF RED TIDE DEVELOPING
IN THE PERSIAN GULF AND OMAN SEA USING
REMOTELY SENSING DATA FROM MODIS SENSOR
Mortazavi Mohammad Seddiq, Motallebi A. A. Hamzei Samad
Persian Gulf and Oman Sea Ecological Research Institute, P.O. Box 1597, Bandar Abbass, Hormozgan, Iran.
E-mail: [email protected]
Remotely sensing with satellite is a beneficial way for the identification of phenomena occurring in surface waters and lands. A red tide
engenders duration early of autumn 2008 until early of spring 2009 in the Persian Gulf and Oman Sea and Strait of Hormuz. In this Harmful Algal Bloom (HAB) were observed many of unknown phenomena that there was not existing sufficient information. In this research
we utilize data and images from MODIS sensor of Aqua and Terra satellites. Analysis of this information provided temperature, chlorophyll-a and organic carbon images for Persian Gulf and Oman Sea during the red tide. The comparison of the satellite images with images from analogy measurement during red tide development gave attractive results. Results of this research show that decreasing water
temperature follow propagation HAB. During HAB it was observed regional increase of chl-a and organic carbon in waters. The density
of HAB in an area where an industrial city was situated was increased, whereas the major current of Persian Gulf moved HAB to the
west regional. The highest density of HAB was observed in the northern Strait of Hormuz according to satellite images.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 300
CLAY FLOCCULATION OF MICROCYSTIS AERUGINOSA
IN THE CHESAPEAKE: PRELIMINARY LABORATORY
AND SOCIO-ECONOMIC ASSESSMENT RESULTS
Kevin Sellner1, Rebecca Certner2, Haena Cho2, Alexander Gibbons2, Christine Kim2, Tina Liu2, Hannah Miller2, Neelam Parikh2, Natasha Savranskaya2, Ann Sun2, Matthew Wooten2, Taylor Throwe3, Holly Bowers4, Allen Place4, Catherine Wazniak5
1
Chesapeake Research Consortium, 645 Contees Wharf Road, Edgewater, MD 21037, USA. E-mail: [email protected]
2
GEMSTONE Program, University of Maryland, College Park, MD 20742, USA
3
Atholton High School, 6520 Freetown Road Columbia, MD 21044, USA
4
Center for Environmental Sciences, University of Maryland, 701 E. Pratt Street, Baltimore, MD 21202, USA
5
MD Department of Natural Resources, 580 Taylor Avenue, Annapolis, MD 21401, USA
The cosmopolitan and occasional microcystin-producing cyanobacterium Microcystis aeruginosa is a summer HAB in fresh and tidal-fresh regions of the Chesapeake Bay watershed. To develop a standard regional mitigation technology for use across the basin, a 3-year laboratory, modeling, and socio-economic analysis has been undertaken to identify likely clays (types, concentrations, flocculants) for routine treatment of these
recurrent blooms and public willingness to support field use of the technology. Following Pan et al. (2006 Environ. Pollut. 141:206), trials indicate that Georgia montmorillonite and kaolinite and local MD clays plus acidified chitosan solutions were effective in removing cultured M. aeruginosa from suspension, with no detectable resuspension or re-emergence from bottom sediments. The addition of seeds of the submersed
grasses Ruppia maritima and Potamogeton perfoliatus to the clay-chitosan mixture resulted in germination and significantly higher growth than in
samples with seeds plus the cyanobacterium, indicating that the technique fulfills a primary regional restoration goal. Further, no nutrient flux from
flocculated and settled M. aeruginosa was detected over 30 d. Water column microcystin content declined following flocculation, but bottom flux
and turnover remain to be determined. Mitigation costs appear to be very low, approximating $0.69 and $0.15/MD household, respectively, for
non-local and local clays. Finally, the public appears to support bloom mitigation in region-wide surveys. These initial results served as the foundation for a recently awarded 3 year grant, ‘Mitigating Microcystis in the Chesapeake (MMIC)’, to further refine the technique for field application.
300
NEW DEVELOPMENT OF HARMFUL ALGAE MANAGEMENT
USING ORGANOCLAYS
Hyun-Jae Shin1, Yeon-Mi Kim2, Si Wouk Kim3, EonSeon Jin2
1
Dept. of Chemical and Biochemical Engineering, Chosun University, Gwangju, Korea
Dept. of Life Science, Hanyang University, Seoul, Korea
3
Dept. of Environmental Engineering, Chosun University, BK21 Team for Biohydrogen Production, Gwangju, Korea
2
In order to investigate a new method of mitigating deleterious effect on harmful algal blooms (HABs), the algicidal effect of organoclays
on three common harmful algae, Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides was studied. The optimum
preparation condition for organoclays, the inhibition capability of organoclays and the interaction mechanism of organoclays on three
HABs species were investigated. Organoclay (Mg-AMP clay, Ca-AMP clay), consisting mostly of trioctahedral phyllosilicate (talc), displayed removal efficiencies greater than 90% against H. akashiwo and C. polykrikoides at a clay loading of 0.20 mg/ml. The optimum
concentration of 0.05 - 0.10 mg/ml organoclays inhibited the motility of about 90% of the tested harmful algae without recovery. In contrast, the algicidal activity of organoclays against non-harmful algal species (Amphidinium sp., Navicula pelliculosa, Nannochloropsis oculata and Phaeodactylum sp.) did not exceed 20% within the concentration range tested. The purchased mineral clay did not show
algicidal activity against the HABs species tested at maximum 1.0 mg/ml concentration. Microscopic observation revealed that organoclays induced the swelling of C. marina, C. polykrikoides and quick lysis of H. akashiwo within the five minute of clay treatment. Overall, these results indicated that organoclays are selective algicides to manage HABs.
14. ABSTRACT BOOK (285-303)_Layout 1 10/22/10 2:48 PM Page 301
MANAGEMENT, MITIGATION
AND PUBLIC OUTREACH FOR HABS
POSTER PRESENTATIONS
STUDIES ON THE MECHANISM OF HAB ORGANISM
COAGULATION BY MODIFIED CLAYS
Xiuxian Song1, Xihua Cao1, Zhiming Yu1, Rudolf Wu2
1
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,
7 Nanhai Road, Qingdao, 266071, People’s Republic of China. E-mail: [email protected]
2
School of Biological Sciences, University of Hong Kong, Pokfulam Road