Download Interactions between harmful algae and calanoid copepods

WALTER AND ANDRÉE DE NOTTBECK FOUNDATION
SCIENTIFIC REPORTS
No. 35
Interactions between harmful algae
and calanoid copepods
in the Baltic Sea
SANNA SOPANEN
Academic dissertation in Aquatic Sciences,
to be presented, with permission of
the Faculty of Biosciences of the University of Helsinki,
for public criticism in the Lecture Room 12,
University Main Building, Fabianinkatu 33, Helsinki,
on December 11th 2009, at 12 noon.
HELSINKI 2009
This thesis is based on the following papers, which are referred to by their Roman numerals:
I
Sopanen, S., Koski, M., Kuuppo, P., Uronen, P., Legrand, C. & Tamminen, T. 2006: Toxic haptophyte
Prymnesium parvum affects grazing, survival, egestion and egg production of the calanoid copepods
Eurytemora affinis and Acartia bifilosa. – Mar. Ecol. Prog. Ser. 327: 223-232.
II
Sopanen, S., Koski, M., Uronen, P., Kuuppo, P., Lehtinen, S., Legrand, C. & Tamminen, T. 2008:
Prymnesium parvum exotoxins affect the grazing and viability of the calanoid copepod Eurytemora
affinis. – Mar. Ecol. Prog. Ser. 361: 191-202.
III
Sopanen, S., Uronen, P., Kuuppo, P., Svensen, C., Rühl, A., Tamminen, T., Granéli, E. & Legrand,
C. 2009: Transfer of nodularin to the copepod Eurytemora affinis through the microbial food web. –
Aquat. Microb. Ecol. 55: 115-130.
IV
Setälä, O., Sopanen, S., Autio, R. & Erler, K. 2009: Grazing and food selection of the calanoid
copepods Eurytemora affinis and Acartia bifilosa feeding on plankton assemblages containing
Dinophysis spp. – Boreal Env. Res. 14: 837-849.
The printed research articles included in this thesis are reproduced with the kind permission of InterResearch (papers I, II & III) and Boreal Environment Research Publishing Board (paper IV).
CONTRIBUTIONS
I
II
III
IV
FATE
FATE
FATE
OS, SS
Study design and methods
MK, PK,
SS, TT
SS, MK,
PK, TT, CL
SS, PU, PK,
TT
OS, SS
Data collection
MK, SS,
PU, CL
MK, SS, SL
PU, CL
PU, SS, PK,
CS, AR
OS, SS, KE
Original idea
Data analysis
SS, MK
SS, MK
PU, PK, SS
OS, SS
Contribution to interpretation of the
results and manuscript preparation
MK, PK,
TT, CL
MK, PK,
TT, CL
PK, TT, CL,
AR, EG
SS, RA, KE
Responsible for interpretation of the
results and manuscript preparation
SS
SS
SS, PU
OS
AR = Alexander Rühl, CL = Catherine Legrand, CS = Camilla Svensen, EG = Edna Granéli, FATE = the
working group of the EU project “Transfer and Fate of Harmful Algal Bloom (HAB) Toxins in European
Marine Waters”, KE = Katrin Erler, MK = Marja Koski, OS = Outi Setälä, PK = Pirjo Kuuppo, PU =
Pauliina Uronen, RA = Riitta Autio, SL = Sirpa Lehtinen, SS = Sanna Sopanen, TT = Timo Tamminen.
Supervised by
Doc. Pirjo Kuuppo
Neste Oil Corporation
Finland
Prof. Harri Kuosa
University of Helsinki
Finland
Reviewed by
Doc. Elena Gorokhova
Stockholm University
Sweden
Doc. Marko Reinikainen
University of Helsinki
Finland
Examined by
Prof. Peter Tiselius
University of Gothenburg
Sweden
Interactions between harmful algae and calanoid
copepods in the Baltic Sea
SANNA SOPANEN
Sopanen, S. 2009: – Interactions between harmful algae and calanoid copepods in
the Baltic Sea – W. & A. de Nottbeck Foundation Sci. Rep. 35: 1-57. ISBN 978-95299673-6-0 (paperback), ISBN 978-952-10-5867-7 (PDF)
Zooplankton have a central role in marine food webs as mediators of energy to higher
trophic levels. Eutrophication favours harmful algal blooms and may induce changes in
the phytoplankton community. These changes, in turn, may affect grazers, because the
quality of food, such as the content of toxic substances, may have effects on feeding,
growth and reproduction, and may influence both the species composition and the
production of copepods. This will affect the food availability of planktivorous animals,
such as Baltic herring. The aim of the studies reported in this thesis was to examine the
feeding interactions between calanoid copepods and toxic algae in the Baltic Sea. The
central questions in this research concerned the feeding, survival and egg production of
copepods exposed to toxic algae. Furthermore, the importance of copepods as vectors
in toxin transfer was examined.
These questions were examined experimentally using various harmful algal species
occurring in the Baltic Sea. The haptophyte Prymnesium parvum, which produces extracellular toxins, was the only studied species that directly harmed copepods. Beside this,
it had allelopathic effects (cell lysis) on non-toxic Rhodomonas salina. Copepods that
were exposed to P. parvum filtrates died or became severely impaired, although filtrates
were not haemolytic (indicative of toxicity in this study). Monospecific Prymnesium
cell suspensions, in turn, were haemolytic and copepods in these treatments became
inactive, although no clear effect on mortality was detected. These results suggest that
haemolytic activity may not be a good proxy of the harmful effects of P. parvum, and
this algal species may produce other chemical compounds that have not been identified,
but are harmful to grazers. In addition, P. parvum deterred feeding, and low egestion
and suppressed egg production were consequently observed in monospecific suspensions of Prymnesium. Similarly, ingestion and faecal pellet production rates were suppressed in high concentration P. parvum filtrates and in mixtures of P. parvum and R.
salina. These results indicate that the allelopathic effects of P. parvum on other algal
species together with lowered viability as well as suppressed production of copepods
may contribute to bloom formation and persistence. Furthermore, the availability of
food for planktivorous animals may be affected due to reduced copepod productivity.
A plankton community that contained the cyanobacterium Nodularia spumigena was
size-fractionated in order to follow nodularin transfer from different size fractions to
the copepod Eurytemora affinis. Nodularin produced by N. spumigena was transferred
to copepods via grazing on filaments of small N. spumigena and by direct uptake from
the dissolved pool. Copepods also acquired nodularin in fractions where N. spumigena
6
filaments were absent. Thus, the importance of microbial food webs in nodularin transfer should be considered. However, the mechanism behind nodularin accumulation in
these fractions remained unclear. Copepods were able to remove particulate nodularin
from the system, but at the same time a large proportion of the nodularin disappeared.
This indicates that copepods may possess effective mechanisms to remove toxins from
their tissues. The importance of microorganisms, such as bacteria, in the degradation
of cyanobacterial toxins could also be substantial.
Our results were the first reports of the accumulation of diarrhetic shellfish toxins
(DSTs) in copepods. The PTX2 content in copepods after feeding experiments corresponded to the ingestion of <100 Dinophysis spp. cells. However, no DSTs were
recorded from field-collected copepods. Neither of the copepod species selected the
dinoflagellate Dinophysis spp. and consumption remained low (up to 226 Dinophysis
spp. cells ind-1 d-1). Instead, the dinoflagellate Heterocapsa triquetra and ciliates were
selected, irrespective of their abundance. Although not selected, Dinophysis spp. formed
an important part of the copepod diet when the food concentration was low. It seems
likely that copepods are an unimportant link in the transfer of DSTs in the northern
Baltic Sea. However, at times when other food sources are scarce, or if copepods
encounter a Dinophysis spp. bloom with high cell densities typical for the Baltic Sea,
these dinogflagellates may form a significant part of the copepod diet.
Sanna Sopanen, Tvärminne Zoological Station, J. A. Palménin tie 260, FI-10900
Hanko and Finnish Institute of Marine Research, Erik Palménin aukio 1, P.O. Box 2,
FI-00561 Helsinki, Finland.
Present address: Environment Centre, Environmental Protection and Research, City
of Helsinki, P.O. Box 500, FI-00099 City of Helsinki, Finland.
7
CONTENTS
1. INTRODUCTION..................................................................................................... 9
1.1. Harmful algal blooms (HABs) and reasons for their magnitude ....................... 9
1.2. Role of copepods in planktonic food webs ...................................................... 10
1.3. Algalt oxins.......................................................................................................11
1.4. Feeding and food selection .............................................................................. 12
1.5. Interactions between harmful algae and copepods .......................................... 13
1.5.1. Grazing interactions are highly variable............................................... 13
1.5.2. The impact of zooplankton grazing on HABs ........................................ 15
2. OBJECTIVES OF THE STUDY ............................................................................ 17
3. INTRODUCTION TO HARMFUL ALGAL SPECIES IN THIS STUDY ............ 20
3.1. Prymnesium parvum C arter ............................................................................. 20
3.2. Nodularia spumigena Mertens ex Bornet & Flathault 1886 ........................... 20
3.3. Dinophysis spp. ................................................................................................ 21
4. MATERIAL AND M ETHODS ............................................................................... 22
4.1. Experiments ..................................................................................................... 22
4.1.1. Experiments with Prymnesium parvum (papers I & II) ........................ 22
4.1.2. Experiments with Nodularia spumigena and Dinophysis spp.
(papers III & IV) .............................................................................................. 23
4.2. Analytical and statistical methods ................................................................... 25
5. RESULTS AND D ISCUSSION .............................................................................. 26
5.1. Instantaneous toxic effects of ichtyotoxic Prymnesium parvum ..................... 26
5.1.1. Haemolytic activity cannot be used as a predictor of P. parvum
toxicity to copepods ......................................................................................... 26
5.1.2. P. parvum impairs the feeding of copepods ........................................... 30
5.1.3. Low ingestion is linked to suppressed egg production .......................... 32
5.2. Accumulating toxins of Nodularia and dinoflagellates ................................... 33
5.2.1. Transfer of nodularin to copepods through feeding interactions .......... 33
5.2.2. Processes in the microbial food web: importance in nodularin
loss and transfer .............................................................................................. 37
5.2.3. Feeding behaviour of Eurytemora affinis and Acartia bifilosa
on plankton communities containing Dinophysis spp. .................................... 40
6. SUMMARY AND CONCLUDING REMARKS ................................................... 43
ACKNOWLEDGEMENTS .......................................................................................... 45
REFERENCES ............................................................................................................. 47
8
9
1. INTRODUCTION
1.1. Harmful algal blooms (HABs)
and reasons for their magnitude
Phytoplankton blooms usually refer to mass
developments of microscopic algae and are
natural events in all marine food webs.
However, during past decades, the occurrence
of “exceptional”, often toxic or otherwise
harmful algal blooms1 has increased globally
(GEOHAB 2001). The definition “bloom”
is not unambiguous. For non-toxic species,
biomass is the most often used parameter
to define the status of a bloom. However,
harmful effects on co-occurring marine biota
as well as human health may accompany very
different levels of cellular abundance. Thus,
not only the abundance of a particular species
but also whether its occurrence has harmful
consequences is of importance. Generally, the
presence of harmful species or measurable
toxin levels is increasingly defined as a
bloom occurrence (Smayda 1997). For
example, the high-abundance, low-biomass
bloom of the haptophyte Chrysochromulina
polylepis in Scandinavian coastal waters
during 1988 had deleterious effects on
large parts of the heterotrophic components
of the plankton community (Nielsen et al.
1990). Another example is the high-biomass
blooms of diazotrophic cyanobacteria.
These filamentous, nitrogen fixing algae
dominated by toxic Nodularia spumigena and
potentially toxic Aphanizomenon flos-aquae
and Anabaena sp. are commonly present
in the Baltic Sea plankton community, but
regularly form late-summer blooms in the
Baltic Sea (Kahru et al. 1994, Kononen et al.
1996). At the other extreme are blooms with
a low abundance and biomass. Filter feeders,
such as oysters, can accumulate diarrhetic
shellfish toxins, which has led to human
poisoning despite the low density of the
dinoflagellate Dinophysis spp. in the water
(Belin 1993). In some cases, the ingestion of
small quantities of toxic algae can be lethal
to grazers. For example, the ingestion of only
a few cells of the dinoflagellate Alexandrium
tamarense has found to be lethal to firstfeeding larvae of capelin (Mallotus villotus)
and herring (Clupea harengus harengus)
(Gosselin et al. 1989). For this reason, the
International Council for the Exploration of
the Sea (ICES) has implemented an annually
updated list of potentially harmful algae in
the Baltic Sea. In 2007, the list contained
about 60 species with effects connected to
toxicity, mechanical disturbance, anoxia or
hypoxia and bloom formation (ICES 2007).
Anthropogenic nutrient loading to coastal
waters has led to eutrophication2 in the Baltic
Sea (HELCOM 2009). Nutrient loading has
caused a corresponding increase in nutrient
concentrations and changes in the ratios of
dissolved inorganic nutrients, nitrogen and
phosphorus (Kangro et al. 2007, Tamminen
& Andersen 2007, HELCOM 2009). It has
been suggested that the eutrophication of
the Baltic Sea has increased phytoplankton primary production and consequently
led to higher biomasses, caused changes
in phytoplankton community structure and
increased the incidence of harmful algal
blooms (Cloern 2001, HELCOM 2009).
Consequently, water transparency has become lower and higher sedimentation rates
2
1
Harmful algal bloom (HAB): A mass occurrence
of algae that can cause fouling, oxygen deficiency,
shading, clogging of fish gills, poisoning of various
organisms and other harmful effects.
Eutrophication arises when excessive amounts
of nutrients, mainly nitrogen (N) and phosphorus
(P) but also organic matter, build up in aquatic ecosystems and cause accelerated growth of algae and
plants, often resulting in undesirable effects.
10
have caused bottom water anoxia, resulting
in kills of bottom-dwelling fish and invertebrates (Cloern 2001, HELCOM 2009).
The Baltic Sea, like other low saline estuaries, is considered an area at high risk of
species invasions (Carlton & Geller 1993,
Leppäkoski & Olenin 2000). Species that
have resistant cysts or resting eggs in their
life-cycle have a high invasive potential (e.g.
Cohen & Carlton 1998, Panov & Cáceres
2007, Sopanen 2008), and the ship-mediated
introduction of potentially harmful phytoplankton species or other aquatic organisms
to new areas can disturb the whole ecosystem. For example, the potentially toxic dinoflagellate Prorocentrum minimum spread to
the Baltic Sea in the early 1980s (Hajdu et
al. 2000). Since then, it has been reported to
bloom in many eutrophied coastal areas of
the Baltic (Hajdu et al. 2005). HABs have
become a recurrent phenomenon in the Baltic Sea and coastal regions globally (ICES
2007). Therefore, it is essential to examine
how these blooms are controlled and, on
the other hand, what effects harmful algae
can have on grazing zooplankton such as
copepods.
1.2. Role of copepods in planktonic
food webs
Calanoid copepods are abundant planktonic
crustaceans that have an important role in the
transfer of energy to higher trophic levels
in the planktonic food web. In general,
copepods obtain energy through two main
food pathways (Fig. 1). In the classical food
web (Steele 1974), copepods are the main
consumers of large phytoplankton and, in
turn, are eaten by planktivorous fish (Fig.
1, upper part). In the microbial food web,
carbon is transferred through several trophic
steps (Azam et al. 1983). Bacteria take up
dissolved organic matter that originates
from phytoplankton and other planktonic
organisms (Azam et al. 1983, Sherr & Sherr
1988, Sherr & Sherr 2000). On the next
level, heterotrophic flagellates consume
picoplankton (bacteria, picocyanobacteria,
picoeucaryotes) (González et al. 1990).
Ciliates, in turn, are considered to be an
important functional group in the microbial
food web as grazers of pico- and nanoplankton
(e.g. Sherr & Sherr 1987, Rassoulzadegan et
al. 1988). They are themselves controlled
Fig. 1. Schematic representation of the pelagial food
web (Steele 1974, Azam et al. 1983, Sherr & Sherr
1988), emphasizing the routes of toxin transfer to
copepods. Symbols: (light grey arrows) pathways of
dissolved organic matter, (dark grey arrows) particulate carbon flows, (I) bacteria, picocyanobacteria,
HNF, PNF, (II) microzooplankton and mixotrophic
organisms, (III) phytoplankton, (IV) mesozooplankton. Potential pathways of nodularin transfer to
copepods: (1) through direct grazing on Nodularia
spumigena filaments, (2) direct uptake from the dissolved pool, (3) through grazing on organisms in the
microbial food web.
11
by metazooplankton3, thus connecting the
microbial with the classical food web (e.g.
Stoecker & Capuzzo 1990, Lenz 2000) (Fig.
1, lower part). Both food webs co-exist, but
their relative significance varies regionally
and temporally.
In the northern Baltic Sea, the classical
food web prevails during the spring bloom
(Lignell et al. 1993). Due to thermal stratification, nutrients are locked in deep water
and the spring bloom uses up nutrients from
the productive layer above the thermocline.
Much of the algal biomass settles to the bottom during this season (Lignell et al. 1993).
The reason for this is the low abundance
of zooplankton because of the long development time of resting eggs as well as the
longer generation time of copepods in cold
water (Katajisto et al. 1998, Mauchline 1998,
Hagström et al. 2001). The microbial food
web dominates in energy transfer during
the summer, and the summer community is
largely driven by the recycling of nutrients
(Hagström et al. 2001). The seasonal variation in food resources affects the growth
and reproduction rates of copepods, and ultimately the biomass available for the next
trophic level varies seasonally (Koski 1999).
It has been estimated that in the northern
Baltic Sea up to 70 % of the copepod biomass is consumed by planktivorous mysid
shrimps and fish, such as Baltic herring (Clupea harengus) (Rudstam et al. 1992, 1994).
Thus, the importance of copepods may also
be substantial in the context of toxin accumulation and transportation through pelagic food webs due to feeding interactions
(Granéli & Turner 2006).
1.3.
Algal toxins
Toxic phytoplankton species can either
produce intracellular toxins or excrete
dissolved toxins into the water (GEOHAB
2001). Intracellular toxins are transferred
along the food web through grazers that serve
as vectors for toxin transfer to higher trophic
levels (Granéli & Turner 2006, Turner 2006).
This is indicated by the mortality of sea
mammals and birds following the ingestion
of algal toxins or organisms that have
accumulated toxins (Andersson & White
1992, Scholin et al. 2000). Extracellular
toxins have detrimental effects on diverse
marine organisms (Richardson 1997),
affecting coastal marine ecosystems and
causing considerable economic losses for
commercial aquaculture.
The reason for algal toxin production is
not clear. It has been argued that the production of these toxins evolved to deter grazers (Turner 2006). However, many of these
toxins harm upper-level consumers, such as
marine mammals or humans, but are rather
harmless to primary grazers, zooplankton
and bivalves (Turner et al. 1998). Therefore,
it is possible that some toxins are secondary
metabolites associated with other processes
such as nitrogen storage, nucleic acid biosynthesis, chromosomal structural organization,
bioluminescence or bacterial endosymbiosis
rather that serving as a grazing deterrents
(Cembella 1998, 2003). Some toxins may
also be involved in allelopathy4, which primarily causes deleterious effects on other
phytoplankton (Fistarol et al. 2003, Legrand
et al. 2003, Fistarol 2004).
Powerful algal toxins have been found
from various aquatic environments. These
3
A category that includes multicellular phagotrophic organisms, but is not based on size. In the
Baltic Sea, copepods, cladocerans, rotifers and meroplankton are included in this category.
4
The chemical inhibition of one plant (or other
organism) by another, due to the release into the environment of substances acting as growth inhibitors.
12
toxins cause poisonings to humans who consume intoxicated seafood. The best known
examples are paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning
(DSP), amnesic shellfish poisoning (ASP)
and ciguatera fish poisoning (CFP) (FAO
2004, Granéli & Turner 2006). The harmful
consequences of different toxic algae and
species that are responsible for the effects
are summarized in Zingone & Enevoldsen
(2000). The paralytic shellfish toxins (PSTs)
are mainly produced by dinoflagellates belonging to the genus Alexandrium. During
the last 20 years, there seems to have been
an increase in PST intoxications (FAO 2004),
and at least 21 PSTs have been recorded.
These toxins are potential neurotoxins that
can cause symptoms such as cramps, signs
of paralysis and inhibition of respiration
by blocking the excitatory current in nerve
and muscle cells (reviewed by Luckas et al.
2005). The diarrhetic shellfish toxins (DSTs)
are mainly produced by the genus Dinophysis and Prorocentrum. These toxins can
be divided into three groups according to
their chemical structure: (i) acidic toxins,
including okadaic acid (OA) and its derivatives, dinophysis toxins (DTXs); (ii) neutral
toxins, which consist of polyether-lactones
of the pectenotoxin group (PTXs); and (iii)
sulphated polyether and its derivative, yessotoxin (YTX) (FAO 2004). The OA group
causes diarrhoea, while pectenotoxins are
hepatotoxic and may damage the liver, and
yessotoxin affects cardiac muscle cells (van
Egmond et al. 1993). Because of health problems to humans, these toxins are routinely
monitored in water worldwide and especially
in shellfish meat in countries with a commercial seafood farming industry (FAO 2003).
Cyanobacterial toxins, which are mainly
found from fresh- or brackish water environments, are broadly classified as causative of
either neurotoxic or hepatotoxic symptoms
(Carmichael et al. 1990). The most important hepatotoxins are cyclic pentapeptides,
nodularin and microcystin (e.g. Sivonen et
al. 1992, Sivonen & Jones 1999, Vaitomaa
2006), which are produced, for instance, by
Nodularia spumigena and species of the genus Microcystis and Anabena. These toxins
inhibit the eukaryotic serine-threonine protein phosphatases PP1 and PP2A (Yoshizawa
et al. 1990) and are potent tumor promoters
(Nishiwaki-Matsushima et al. 1992). Cyanobacterial neurotoxins, such as saxitoxins and
anatoxins, are produced by the genus Anabaena and Microcystis (e.g. Vaitomaa 2006).
Cyanobacterial toxins are regularly found in
drinking water supplies, and standardized
monitoring methods as well as safety limits
are thus provided by the WHO (WHO 2003).
1.4. Feeding and food selection
Selective feeding5 is an important factor
in the relationships between grazers and
their food. It is of particular interest in the
formation of harmful algal blooms, because
selection might enable harmful algae to gain
a competitive advantage (Huntley et al. 1986,
Uye & Takamatsu 1990, Solé et al. 2006).
Early studies suggested that suspensionfeeding copepods were non-selective feeders
with size-dependent particle selection based
on the morphology of the filtering mesh of
the second maxillae (e.g. Frost 1972, Boyd
1976). However, subsequent experimental
and behavioural studies have demonstrated
that copepods possess a complex suite of
behavioural responses to different stimuli.
These include the ability to generate and
alter the near-field flow patterns of feeding5
Specific prey items are consumed disproportionately to their numerical abundance in the food
medium.
13
currents around the appendages to entrain
and transport food items into the copepod’s
capture area (e.g. Koehl & Stickler 1981,
Greene 1988, Jiang et al. 1999, Malkiel et
al. 2003), to capture and manipulate different
shapes of food items, such as algal chains
(e.g. Paffenhöfer et al. 1982), to detect dissolved compounds in the water (Cowles et
al. 1988), and to actively select or reject
food particles (e.g. Paffenhöfer et al. 1982,
Schultz & Kiørboe 2009).
Several criteria may be involved in food
selection. Prey size defines the upper and
lower boundaries within which other criteria
are also involved. Copepods can exploit prey
over a wide range of sizes (approximately
5 to 200 μm), depending on the size of the
copepod itself (Hansen et al. 1994, Gasparini
& Castel 1997). They are also able to retain
the largest particles more efficiently than
the smallest ones (Frost 1972). Among other
criteria involved in selection are manageability (e.g. prey morphology), quality (e.g.
nutritional value, toxicity, physiological condition) or visual stimuli such as motility (e.g.
Frost 1972, Bergreen et al. 1988, Cowles et
al. 1988, Hansen et al. 1994, Kiørboe et al.
1996, Schmidt & Jónasdóttir 1997).
Numerous studies have demonstrated the
importance of food quality in selection. For
example, Poulet and Marsot (1978) reported
that marine copepods are able to select microcapsules enriched with phytoplankton
homogenate over untreated capsules. It has
also been shown that copepods can ingest
live cells in preference to dead cells, and that
the presence of senescent algae may inhibit
overall feeding rates (e.g. Cowles et al. 1988).
Selection has also been shown to occur under
natural conditions. Meyer-Harms et al. (1999)
demonstrated by analysing gut pigments that
copepods are able to utilise cyanobacteria
along with eukaryotic phytoplankton. Thus,
food selection in copepods seems to be com-
plicated and may have effects on the prey
species composition in the plankton community, as well as on the copepods themselves
if, through selective feeding, they can obtain
food with a higher quality.
1.5. Interactions between harmful
algae and copepods
1.5.1. Grazing interactions are highly
variable
The grazing responses of copepods to toxic
algae seem to vary considerably. Most
studies addressing selection against harmful
algae have been carried out in the laboratory
with high cell concentrations and only a few
food species, and may not therefore give an
ecologically relevant picture of the feeding
and food selection of grazers (e.g. Turner et al.
1998). An increasing number of zooplankton
grazing studies have been performed with
natural plankton assemblages during blooms
of harmful algae (e.g. Turner et al. 1998,
Teegarden et al. 2001, Koski et al. 2005,
Kozlowsky-Suzuki et al. 2006). Although
also these studies have yielded variable
results, it seems that under natural conditions
both micro- and mesozooplankton6 grazers
unselectively ingest toxic species together
with other phytoplankton. This might dilute
the potential adverse effects that toxins
have on grazers, i.e. those effects that are
very often seen in laboratory experiments
(reviewed in Turner 2006).
The effects of different harmful algae on
copepods in the Baltic Sea and adjacent areas
are summarised in Table 1. Toxicity is evi6
Micro & mesozooplankton: Heterotrophic
planktonic organisms that belong to the size group
of 20-200 μm (micro) and 0.2-20 mm (meso) (Sieburth et al. 1978).
HA (haemolytic
activity)
nd
P. parvum
E. huxleyi & Phaeocystis cf. pouchetii
NC
CULT
CULT
PTX2
GTX1-3
GTX2-3, STX
Dinophysis spp.
Alexandrium lusitanicum
A. ostenfeldii
N. spumigena
N. spumigena
N. spumigena
nodularin
nodularin
nodularin
nd
Nodularia spumigena
N. spumigena & Microcystis aeruginosa nd
CULT
NC + CULT
MC + CULT
NC + CULT
CULT
NC
PTX2, PTX2SA
D. norvegica, D. acuta &
D. acuminata
Cyanobacteria
NC
NC
DTX1
nd
Dinophysis norvegica
D. norvegica & Dinophysis spp.
Dinoflagellates
LDH (lactate dehydro- MC
genase leaking)
nd
MC
nd
CULT
Prymnesium. patelliferum (cf. parvum)
& Emiliania huxleyi
P. patelliferum (cf. parvum)
P. patelliferum (cf. parvum)
CULT
MC
nd
NC
Observation of toxins Data source
or other substances
Chrysochromulina polylepis
Haptophytes
Species
E. aff & A. bif
E. aff & A. bif
E. aff & A. bif
E. aff & A. bif
A. ton
E. aff & A. bif
A. cla
E. aff & A. bif
Cal. hel
Cal. hel, Cent. sp.,
T. lon & Acartia sp.
A. bif, T. lon, C. typ
E. aff & A. bif
Cal. fin
A. cla
E. aff
Acartia sp., T. lon &
C. ham
Cal. fin
Copepod species
IR
not detected
IR
IR
EPR, survival
not detected
IR (mixed species-specific
responses)
IR (mild effects when
feeding high densities of
D. acuta)
suppressed IR
EPR, activity
IR, activity
Nejstgaard & Solberg 1996
Koski et al. 1999b
IR, EPR
IR, EPR, hatching, activity,
survival
IR, EPR, activity, survival
IR
Engström et al. 2000
Koski et al. 2002
Sopanen et al. 2009
Sellner et al. 1994
Schmidt & Jónasdóttir 1997
Setälä et al. 2009
Dutz 1998
Setälä et al. unpublished
Kozlowsky-Suzuki et al. 2006
Wexels Riser et al. 2003
Jansen et al. 2006
Sopanen et al. 2006, 2008
Nejstgaard et al. 1994
Nejstgaard et al. 1995
Nielsen et al. 1990
Reference
IR
EPR, survival
Negative effects
Table 1. Summary of the effects of various harmful algae on copepods in the Baltic Sea and adjacent areas. Studies have been carried out
experimentally on cell cultures (CULT), mesocosm communities (MC) or on natural phytoplankton communities (NC). Abbreviations: EPR =
egg production rate, IR = ingestion rate, nd = no data, not detected = no negative effects found. Species: A. cla (Acartia clausi), A. bif (Acartia bifilosa), A. ton (Acartia tonsa), Cal. fin (Calanus finmarchicus), Cal. hel (Calanus helgolandicus),C. ham (Centropages hamatus), C. typ
(Centropages typicus), Cent. sp. (Centropages sp.), E. aff (Eurytemora affinis), T. lon (Temora longicornis).
14
15
denced if grazers die by direct intoxication
after ingesting toxic algae or when exposed
to exudates (Turner et al. 1998). More often,
however, the effects of toxins on grazers may
be sublethal, such as changes in behaviour or
lowered ingestion, fecundity or egg hatching
rates (Turner 2006). For example, copepods
may reduce their filtering rates or refuse to
ingest toxic phytoplankton, which may lead
to low egg production and elevated mortality
due to starvation (e.g. Huntley et al. 1986,
Uye & Takamatsu 1990, Buskey & Stockwell 1993, Dutz 1998, Koski et al. 1999b,
Maneiro et al. 2000, Colin & Dam 2002,
Guisande et al. 2002, Barreiro et al. 2006).
This, in turn, may lead to reductions in the
biomass that is available to the next trophic
level. Other adverse effects have also been
demonstrated, such as regurgitation and loss
of motor control, which are generally related
to the physical condition of the copepods
(e.g. Ives 1985, Sykes & Huntley 1987).
Contradictory results with no apparent
negative effects have also been reported
(reviewed by Turner 2006). This variability
is probably related to the dose of particular
toxins (Turner & Tester 1997), because the
toxin content in algae may vary within a single strain and also be modified by the culture
age and nutrient conditions (e.g. TaroncherOldenburg et al. 1997, Béchemin et al. 1999,
Johansson & Granéli 1999, Hansen et al.
2003, Maclean et al. 2003, Frangópulos et
al. 2004, Uronen et al. 2005). Generally,
diets with higher toxin concentrations should
cause greater effects. However, grazers probably also have species- and site-specific responses or adaptations to different toxins.
It has been demonstrated that the ecological
relationships between zooplankton grazers and HABs are closely related to their
common history (Colin & Dam 2002, 2005,
2007, Hairston et al. 2002). For instance,
Colin and Dam (2002) found that copep-
ods from areas where they are constantly
exposed to blooms of toxic dinoflagellate
Alexandrium spp. were less affected by
ingesting toxic cells than copepods of the
same species from areas where they were
not naturally exposed to these toxins. This
was explained by the evolutionary adaptations and selection of grazers for resistance
to the toxic effects of these algae. However, interactions between grazers and algae
are not unidirectional and phytoplankton
can also respond to the presence of grazers, which suggests that the hypothesis of
grazing deterrence is not entirely incorrect.
For instance, Selander et al. (2006) demonstrated that copepods induced an increase in
the PST content of Alexandrium minutum,
and that toxin production correlated with
an increased resistance to grazing. Another
example of chemical interaction is a study
by Tang (2003), which showed that grazing,
and specifically grazing-related chemical
signals, induced an increase in colony size
of the prymnesiophyte Phaeocystis globosa.
Therefore, aspects such as genetic selection and physiological adaptations should be
taken account when examining interactions
between grazers and toxic algae.
1.5.2 The impact of zooplankton grazing
on HABs
Harmful algal blooms and phytoplankton in
general are controlled by bottom-up regulation,
which is related to factors that control the
availability of nutrients and nutrient uptake
by the phytoplankton (summarised in Uronen
2007), and by top-down control, which refers
to grazing and predation by organisms at
higher trophic levels.
Regulation by grazing is determined by
the balance between algal growth (μ) and
grazing rates (g) (e.g. Smayda 2008). This
16
Fig. 2. Schematic representation of the cascading
effects during an Aureococcus anophagefferens
bloom in Narraganset Bay, USA, illustrating the
grazing pressure by ciliates and subsequent zooplankton collapse during bloom initiation (I and II).
The reduction in the zooplankton grazing pressure
was responsible for the continued increase in A.
anophaggefferens abundance (μ > g). The greatest
breakdown in grazer control was linked to cessation of benthic grazing by filter feeders, especially
Mytilus edulis. The combined reduction in grazing
regulation by micro- and mesozooplankton and benthic grazers led to a huge excess of A. anophaggefferens net population growth (μ >>> g). It is possible
that viral infection and ingestion of A. anophaggefferens by phagotrophic flagellates contributed to the
decline of the bloom (IV) (Milligan & Cosper 1994,
Smayda 2008). During bloom senescence, grazers
recovered over time and were able to take control
of the declining A. anophaggefferens population (redrawn after Smayda 2008).
relationship has rarely been demonstrated in
natural conditions. A good example of this
relationship is a high-density low-biomass
brown tide bloom of the pelagophyte Aureococcus anophaggefferens, which persisted
for 5 months in Narraganset Bay, USA.
During this bloom, the authors were able to
separate several stages of grazer influence,
characterized by three grazer-breakdown
episodes (I-III, Fig. 2) and three grazerrestoration stages (V-VII) (Smayda 2008).
As this example demonstrates, the contribution of grazing by the zooplankton community (micro- and mesozooplankton) can
be significant during bloom initiation and
termination. However, during its peak, the
bloom escapes grazing control due to the
high biomass and population growth rate,
although individual growth rates usually
slow down (Turner 2006). Recent studies
indicate that the grazing impact of copepods
on HABs is variable, but in most situations
quite low (Campbell et al. 2005, Jansen et
al. 2006, Kozlowsky-Suzuki et al. 2006). In
contrast, the grazing impact of microzooplankton on blooms appears to exceed that
of mesozooplankton. For example, Calbet
et al. (2003) found that microzooplankton
grazing in the Mediterranean was similar to
or exceeded that of Alexandrium minutum
growth rates. Although the copepods Acartia
grani and Oithona sp. were able to graze on
Alexandrium, the daily consumption rate
was only 0.003-0.007 % of the A. minutum
standing stock due to the low standing stocks
of copepods. Such comparisons between
the grazing impact of mesozooplankton and
microzooplankton have not been carried out
in the Baltic Sea.
17
2. OBJECTIVES OF THE STUDY
Harmful algal blooms, especially if they
produce toxic substances, can have
widespread negative effects on aquatic food
webs. These effects include fish kills, kills
of marine mammals and birds as well as
lowered production and viability of grazers
exposed to toxic algae. Copepods have a
central role in the food web, because they
transfer energy to higher trophic levels.
The toxicity of food species may affect
their feeding, viability and reproduction,
reducing the biomass that is available to the
next trophic level. Additionally, if copepods
accumulate toxins, they may transfer them
to zooplanktivores. The aim of the studies
reported in this thesis was to advance our
understanding of the feeding interactions
between copepods and toxic algae.
Feeding interactions between copepods
and toxic algae are probably the main mechanisms that determine what kind of negative effects algae may have on copepods or
whether toxins accumulate in grazers. Papers
I and II examine the outcomes of the interaction between copepods and toxic algae. The
main objective in papers III and IV, however,
were to investigate feeding and food selection and their role in toxin transfer to copepods. This links the separate studies in this
thesis together. The experimental design and
objectives of all the studies are summarized
in Table 2.
In papers I and II the feeding-responses,
reproduction and behavioural changes in
copepods feeding on toxic Prymnesium
parvum were examined (potential negative
effects / responses and treatments are presented in Fig. 3). The aim was to investigate
what kind of feeding-related responses copepods could have when they are exposed to P.
parvum. Could there be changes in feeding
rates, and are these reflected in reproduction
or viability, which may alter the population
growth rates and decrease the grazer biomass
available to higher trophic levels? Are there
changes in the behaviour of copepods due to
P. parvum, such as lowered swimming activity, and are these changes linked to toxicity
or just an outcome of lowered feeding?
The importance of the microbial food web
in the transfer of nodularin has rarely been
considered, although the microbial community associated with the bloom-forming
cyanobacteria is presumably a valuable food
source for copepods (Sellner 1997, Engström-Öst et al. 2002, see Uronen 2007).
In experiments with Nodularia spumigena
(paper III), the transfer of nodularin to Eurytemora affinis was followed by testing three
potential pathways: (1) a direct pathway by
grazing on N. spumigena filaments; (2) a
direct pathway from the dissolved nodularin
pool; and (3) through grazing on organisms
in the microbial food web (see Fig. 1).
Paper IV deals with the feeding-interactions between Dinophysis spp. and copepods.
The aim was to test whether copepods are able
to feed on Dinophysis spp., and whether the
relative abundance of Dinophysis spp. compared to other species in the community could
influence feeding and selection. The importance of copepods as vectors in the transfer of
toxins derived from Dinophysis spp. to higher
trophic levels was also considered.
18
Table 2. Objectives and experimental design in the studies of this thesis. Grazing studies were carried out
with either Eurytemora affinis or Acartia bifilosa. Prymnesium parvum was grown in nitrogen-limited (-N),
phosphorus-limited (-P), and in nutrient-balanced (+NP) conditions. Different treatments were prepared
from these cultures (see also Fig. 3). Non-toxic Rhodomonas salina and filtered seawater (FW) were used
as controls (papers I & II). Copepods were incubated in different size fractions to estimate how much nodularin was transferred from these fractions (paper III). Feeding behaviour and toxin transfer were assessed in
plankton communities that contained Dinophysis spp. (paper IV).
Objectives
Experimental
unit
Responses in
grazing, faecal
pellet production,
egg production
and survival
Species
Cultures
Treatments
No. replicates /
grazing controls
Paper
Bottle incubation E. affinis,
3 × 24 h with the A. bifilosa
same animals
P. parvum
(+NP, -N,
-P), R. salina
(+NP)
Monospecific
Prymnesium (5
cell concentrations)
Rhodomonas control (5 cell concentrations)
FW control
6/4
I
See above
See above
E. affinis
See above
Low concentration
filtrate (Filt 1-2: 2
concentrations &
Rhodomonas)
High concentration filtrate
(Filt 3-4: 2 concentrations &
Rhodomonas)
Mixture 1 & 2
(1:1, 2 cell concentrations)
Rhodomonas
control
6/4
II
Nodularin transfer to copepods
through the microbial food web.
Grazing, food
selection, faecal
pellets, survival
Bottle incubation E. affinis
1 × 24 h; 2 experiments (Expt
1, Expt 2)
Mesocosm
water with
addition
of cultured
Nodularia
spumigena
5 different sizefractions (45-150,
20-10, 10-3, 3-0.2
and <0.2 μm)
5/3
III
Grazing, food
selection, survival, transfer of
Dinophysis spp.derived toxins to
copepods
Bottle incubation E. affinis,
1 × 24 h; 10 ex- A. bifilosa
periments (Exps
I to X)
Natural
plankton
community
The share of
Dinophysis spp.
compared other
plankton groups
was manipulated
by inverse filtration method
4 / 3 (130 ml units)
15 / 3 (30 ml units)
IV
19
Fig. 3. Experimental set-up in studies with Prymnesium parvum (papers I and II). Symbols: (P) P. parvum
cells, (R) R. salina cells, (-N, -P, +NP) N-deficient, P-deficient and NP-balanced cultures that were used for
the preparation of different treatments. The range of cell concentrations in different treatments is presented
and the potential effects of P. parvum are summarised.
20
3. INTRODUCTION TO HARMFUL
ALGAL SPECIES IN THIS STUDY
3.1. Prymnesium parvum Carter
The haptophyte Prymnesium parvum, which
is a common member of coastal plankton
communities worldwide, excretes toxins
into the surrounding water. It forms blooms
with deleterious effects on diverse marine
organisms, such as fish kills, affecting coastal
marine ecosystems and causing considerable
economic losses to commercial aquaculture
(Richardson 1997). P. parvum occurs in many
parts of the Baltic Sea (Hällfors 2004), but has
formed few blooms associated with high fish
mortality in Finnish coastal waters (Lindholm
& Virtanen 1992, Lindholm et al. 1999). The
toxicity of P. parvum blooms in the Åland
archipelago has been related, for instance,
to reduced water exchange and phosphorus
deficiency (Lindholm et al. 1999). Because
fish farms in Finland are usually located in
sheltered bays, they are vulnerable to noxious
P. parvum blooms. Natural concentrations
of P. parvum during blooms usually vary
between 50 × 103 and 100 × 103 cells ml-1,
but extreme cell concentrations of up to 8001600 × 103 cells ml-1, have been observed
(Edvardsen & Paasche 1998). The highest
P. parvum concentrations in our experiments
were approximately 350 × 103 cells ml-1, thus
representing a normal bloom event.
Several compounds have been described
as causing the haemolytic effect7 of P.
parvum, such as proteolipids, glycolipids
(haemolysins) and polyethers (Prymnesin 1
and 2) (Shilo & Rosenberger 1960, Igarashi
et al. 1996, Morohashi et al. 2001, Edvard7
Haemolytic effect, haemolytic activity (HA):
the potential to induce rupture or destruction of red
blood cells. Describes the toxicity of Prymnesium
parvum in this study.
sen & Imai 2006). P. parvum has also been
reported to cause ichtyotoxic and cytotoxic8
effects (Shilo 1971, Igarashi et al. 1998).
Both nitrogen and phosphorus limitation has
been found to enhance the toxicity of P. parvum (Shilo 1971, Johansson & Granéli 1999,
Uronen et al. 2005). Therefore, we used both
nutrient-limited and nutrient-balanced cultures in our experiments (Fig. 3).
Several studies have indicated that P. parvum has allelopathic effects on other algae
(e.g. Legrand et al. 2003) and is able to feed
on other algae and bacteria (Nygaard & Tobiesen 1993, Tillmann 1998, Legrand et al.
2001). Mixotrophy9 is stimulated by nutrient
limitation (Skovgaard et al. 2003). In addition, noxious effects on ciliates (Fistarol et
al. 2003, Granéli & Johansson 2003), rotifers
(Barreiro et al. 2005) and copepods (Nejstgaard & Solberg 1996, Koski et al. 1999b,
Sopanen et al. 2006, Sopanen et al. 2008)
have been reported. Thus, a bloom of this
species is likely to have adverse effects on
large parts of the plankton community. Since
these chemicals are rapidly degraded in water
(e.g. Fistarol et al. 2003), the possibility of
deleterious effects is most pronounced during the bloom.
3.2. Nodularia spumigena Mertens ex
Bornet & Flathault 1886
Blooms of diazotrophic cyanobacteria10
naturally occur in the Baltic Sea (Bianchi
et al. 2000). However, the frequency and
8
Toxic to fish and cells, respectively.
9
Mixotrophy: organisms that are able to combine
phototrophy (i.e. assimilate dissolved CO2 from the
water) and heterotrophy (i.e. use only carbon from
organic sources for biosynthesis).
10
Cyanobacterial species that have an ability to
fix dissolved atmospheric nitrogen gas.
21
intensity of these blooms, dominated by
Nodularia spumigena and Aphanizomenon
flos-aquae, have increased during recent
years (Kahru et al. 1994, Finni et al. 2001,
Poutanen & Nikkilä 2001), and have been
related to eutrophication in the Baltic Sea
(Vahtera 2007). The average bloom biomass
of N. spumigena in the Gulf of Finland,
Baltic Sea, usually ranges between 0.08 and
0.28 mg WW l-1, but values of up to 2.91
mg WW l-1 have been recorded (Kanoshina
et al. 2003). The biomasses during our
grazing experiments (0.25 to 0.99 WW l-1)
represented normal bloom values.
Increased toxin production by N. spumigena has been related to a high biomass as
well as an increase in production and growth
(Lehtimäki et al. 1997). Nodularin is mainly
intracellular during optimal growth, but is
released into the water when the bloom is decaying (e.g. Codd et al. 1989, Lehtimäki et al.
1997). N. spumigena has caused fatal poisonings of livestock, dogs and ducks (reviewed
in Karjalainen 2005). Because nodularin is a
stable molecule, it can be transferred through
the food web. Food-chain experiments have
demonstrated that zooplankton can act as
vectors for nodularin transfer to consumers
at higher trophic levels (Engström-Öst et
al. 2002). In the Baltic Sea, nodularin has
also been found to accumulate, for example,
in mysids (Engström-Öst et al. 2002), fish
(Sipiä et al. 2001) and eiders (Sipiä et al.
2003).
Copepods are able to graze on Nodularia
spumigena and accumulate toxins in their
body tissues and faecal pellets (Sellner et
al. 1994, Koski et al. 1999a, Lehtiniemi et
al. 2002, Kozlowsky-Suzuki et al. 2003).
In general, however, cyanobacteria are considered to be ‘poor’ food for zooplankton
(reviewed by Lampert 1987) due to the low
manageability of large filaments or colonies,
or nutritional inadequacy and toxicity (e.g.
Lampert 1987, Ahlgren et al. 1990, Schmidt
& Jónasdóttir 1997, Kozlowsky-Suzuki et al.
2003). Meyer-Harms et al. (1999) demonstrated that copepods are also able to ingest
cyanobacteria under natural conditions, and
they considered that a cyanobacterial bloom
in the late phase may actually provide nutritious food for copepods because of the
accompanying diverse fauna of microzooplankton. Therefore, it is essential to evaluate the importance of the different routes by
which nodularin is transferred to grazers.
3.3. Dinophysis spp.
An increasing trend in dinoflagellate stocks
has been observed in the Baltic Sea (Wasmund
& Uhlig 2003), and incidences of massive
late-summer blooms of potentially toxic
species, such as Alexandrium ostenfeldii,
have been reported (Hajdu et al. 2006,
Kremp et al. 2009). If the frequency and
intensity of potentially toxic dinoflagellate
blooms increases, grazers will encounter
toxic dinoflagellate blooms more frequently.
Three toxic Dinophysis species – D.
acuminata, D. norvegica and D. rotundata
– commonly occur in the northern Baltic
Sea. The abundances of Dinophysis spp. in
the Baltic Sea are typically quite low (about
1-5 cells ml-1), but high concentrations of
up to 150 cells ml-1 have also been found
(Carpenter et al. 1995, Kuuppo et al. 2006).
The concentrations of Dinophysis spp.
during our study (40-77 cells ml-1) were thus
higher than is typically seen in non-bloom
conditions.
Dinophysis-derived toxins, such as pectenotoxins (PTXs) and dinophysistoxins
(DTXs), have been detected in the northern
Baltic phytoplankton communities (Goto
et al. 2000, Kozlowsky-Suzuki et al. 2006,
Kuuppo et al. 2006). Dinoflagellate-derived
22
toxins accumulate in consumers, such as
shellfish, and are vectored to higher trophic
levels through trophic interactions, causing
diseases to humans though the consumption
of seafood (reviewed in Hallegraeff 1993).
In the Baltic Sea, these problems are not entirely relevant, since brackish water mussels
are not commercially exploited. However,
animals that feed on mussels are exposed to
toxins. For example, OA has been detected
in blue mussels (Mytilus edulis), and in the
common flounder (Platichthys flesus) in the
northern Baltic Sea (Pimiä et al. 1998, Sipiä
et al. 2000). Therefore, it is important to
investigate the dinoflagellate-grazer interactions and toxin transfer between species
naturally occurring in the Baltic Sea.
4. MATERIAL AND METHODS
4.1. Experiments
4.1.1. Experiments with Prymnesium
parvum (papers I & II)
We were interested in determining whether
toxic Prymnesium parvum is able to harm
the copepods Eurytemora affinis and Acartia
bifilosa (Fig. 3), which are important grazers
in the coastal areas of the northern Baltic
Sea (Kivi et al. 1996, Uitto 1997, Koski
et al. 1999a, 1999b, Engström et al. 2000)
and valuable prey for zooplanktivores such
as mysids and fish. Adult females were
collected from the Tvärminne sea area on
the SW coast of Finland (59º 49’ N, 23º 17’
E). For a detailed description of the area, see
Niemi (1975).
To illustrate the effects of P. parvum, the
survival, feeding (clearance- and weightspecific ingestion rates), faecal pellet production rate and egg production rate were
examined in copepods exposed to P. par-
vum. Copepods were incubated with: (1) cell
suspensions of Rhodomonas salina, which
represented a non-toxic control treatment
(Rhodomonas control); (2) filtered seawater
(FW, as starvation control); and (3) cell suspensions of P. parvum (monospecific Prymnesium) (paper I). P. parvum excretes toxic
substances into the water. These exudates
could have direct effects on copepods, and
also allelopathic effects on other algae. In
order to examine these effects, copepods
were exposed to: (1) cell-free P. parvum
filtrates in the presence of a saturated concentration of non-toxic R. salina cells (high
and low concentration filtrates)11; (2) 1:1 cell
mixtures of P. parvum and R. salina (mixture 1 & 2); and (3) R. salina in a saturated
food concentration (Rhodomonas control)
(paper II). P. parvum cell concentrations that
were used during our studies were selected
to represent normal bloom conditions. The
study design, treatments, the range of cell
concentrations and responses are presented
in Figure 3. The study aims, experimental
set-ups and methods are presented in Tables
2 and 3. Because the haemolytic activity
of P. parvum, which represented toxicity
in this study, has been found to increase in
nutrient-limited conditions (e.g. Johansson
& Granéli 1999), all cell suspensions and
cell-free P. parvum filtrates were prepared
from P. parvum cultures, grown under NPbalanced conditions or under nitrogen (-N)
or phosphate (-P) deficiency (for detailed
culture methods and P. parvum growth and
toxicity, see Uronen et al. 2005). Non-toxic
control food, Rhodomonas salina, was grown
under NP-balanced conditions.
11
Filtrates were prepared by gently GF/C filtering
the cell suspensions taken from P. parvum cultures
and diluting them with 0.2 μm filtered seawater to
obtain appropriate concentrations.
23
4.1.2. Experiments with Nodularia
spumigena and Dinophysis spp. (papers
III & IV)
The aim of these studies was to examine
selective feeding and the role of copepods
in toxin transfer (see Table 2). Nodularin
transfer experiments were conducted with
cultured females of Eurytemora affinis
to ensure that the copepods were free of
nodularin. The phytoplankton community,
collected from the Baltic proper and grown
in mesocosms, was gently fractionated into
5 size fractions12 in order to create different
plankton communities, and animals were
allowed to feed on the size-fractionated
plankton community to follow nodularin
transfer to copepods via these fractions (see
Table 2 and Fig. 1). Studies were carried
out in two different situations regarding N.
spumigena biomass and the phase of the
bloom. The first experiment (Expt 1) was
performed during the initial growth phase
of an N. spumigena bloom with low chl-a
(1.3 μg C l-1), and the second one (Expt 2)
during stationary growth conditions with
high chl-a (9.4 μg C l-1) and decaying
filaments of N. spumigena. Since the initial
N. spumigena concentration was low in the
mesocosm community in Expt 1, cultured
Nodularia spumigena were inoculated to the
mesocosms. This resulted in the presence
of two types of filaments in Expt 1: longchained filaments, with a cell size of 3.5-6 ×
10-13 μm and a short morphotype13, with a
12
<150 μm size fractions represented the total community in Expt 1 and 2. These fractions were further
divided into <45, <20, <10, <3 and <0.2 μm fractions.
13
Our aim was to remove Nodularia spumigena
filaments in all but the largest size fractions. However, in Expt 1, small filaments of N. spumigena
passed filtration all the way into <45, <20 and
<10 μm size fractions (see Table 5), while in
cell size of 2-4 × 4-5 μm. These filaments are
referred to in this text as “large” and “small”
N. spumigena. In the second experiments
(Expt 2), only long-chained filaments were
present.
In the experiments with Dinophysis spp.
(paper IV), selective feeding of the copepods
Acartia bifilosa and Eurytemora affinis was
evaluated in plankton communities that differed regarding the proportion of Dinophysis
spp. in phytoplankton mixtures. The aim was
to examine whether the copepods fed on
Dinophysis spp. and may thus accumulate
toxins (Table 2). Experiments were conducted with a natural plankton community that
was collected below the thermocline from
Tvärminne Storfjärd, SW coast of Finland.
Experimental water was filtered through
a 76 μm mesh net to remove zooplankton
and larger phytoplankton cells. During the
procedure, some of the largest Dinophysis
spp. cells could have been removed. The
proportion of Dinophysis spp. in the food
suspensions was adjusted by inverse filtration through a 20 μm net. Due to filtration,
food communities differed regarding the
total cell concentration and the proportion
of different algae or algal groups. Because
of variation in total food concentrations,
experiments I – VI are referred to as “high
food experiments” and experiments VII – X
as “low food experiments”14. The initial cell
Expt 2, only few small pieces of large N. spumigena
were found in the <45 μm fraction.
14
Expts I – VI consisted almost entirely of the dinoflagellate Heterocapsa triquetra, which often forms
blooms during late summer, and the total cell concentrations in the plankton communities were relatively
high (up to 1300 cells ml-1). In Expts VII – X, the total
cell concentrations were up to 20 fold lower and the
community had shifted towards Dinophysis spp. dominance (the initial proportions of different species/
species groups are presented in the respective papers).
24
Table 3. Summary of the methods used in this thesis.
Measurement
Sampling and methods
P. parvum & R.
salina counts
Cell concentrations were
Electronic particle counter
analyzed from fixed samples (Elzone, Particle Data Inc.)
(100 ml sample, acid Lugol’s
solution, 4% final conc.)
Instruments and equipment
Microprotozoans
and phytoplankton counts
50 ml sample, acid Lugol’s
solution
Zeiss inverted microscope (205×
and 806× magnification)
or Leica DMIL (100-400×
magnification)
Pico- and
nanoplankton
20 ml sample (<0.2 μm
filtered glutaraldehyde, 1
% final conc.) flagellates,
picocyano: proflavine stain;
heterotrophic bacteria: DAPI
stain
Olympus DP50, Leiz Diaplan
III
(500× and 1 250× magnification)
using blue (proflavine) or
ultraviolet (DAPI) excitation
wavelengths
Kuuppo-Leinikki & Kuosa
1989,
Porter & Feig 1980, Turley
1993
Carbon
conversions for
plankton
Biovolumes were calculated
from cell dimensions with
species-specific formulas
and calculated further to
carbon
Carbon conversion factors:
III, IV
bacteria 0.35 pg C μm-3,
picocyano 0.22 pg Cμm-3,
dinoflagellates pg cell-1 = 0.706 ×
vol0.819, other algal groups 0.11 pg
Cμm-3, ciliates 0.19 pg Cμm-3
Bjørnsen 1986, Børsheim
& Bratbak 1987, MendenDeuer & Lessard 2000,
Edler 1979, Putt & Stoecker
1989
Faecal pellet
production
100 ml sample, acid Lugol’s
solution, length and width
measurements, carbon
conversion: 0.057 × 10-9 mg
C μm-3
Microscopy with preparation
I, II, III Utermöhl 1958, Gonzalez
microscope or inverted
et al. 1994
microscope (100× magnification)
Egg production
E. affinis: 48 h incubation in
FW after the experiments
A. bifilosa: Sample fixed
with acid Lugol’s solution
Microscopy counts: eggs and
nauplii after incubation
Microscopy counts after each
24-h incubation
Haemolytic
activity (HA)
Haemolytic activity on horse Absorbance at 540 nm was read
blood cells was measured
on a Fluorstar 403 microplate
and expressed as saponin
reader
equivalents (SnEq pg cell-1)
Nodularin
Dissolved nodularin,
particulate nodularin and
nodularin in copepods was
measured and expressed as
ng l-1 or pg individual-1
DSP toxins
The copepod toxin content
IV
HPLC system equipped with a
was measured and expressed PE series 200 autosampler and
as pg individual-1
pump coupled to an Applied
Biosystems API 165 SCIEX mass
spectrometer. The electrospray
ionization operated in positive
mode
HPLC-MS with electrospray
ionization (LC–ESI–MS)
(PE series 200 autosampler
and pump coupled to an
Applied Biosystems API 165
mass spectrometer), which
was operated in selective ion
monitoring mode using positive
ionization of nodularin
Paper
References
I, II
Utermöhl 1958
III, IV
Utermöhl 1958
I, II
Utermöhl 1958, Gonzalez
et al. 1994
I, II
Igarashi et al. 1998,
Johansson & Granéli 1999
III
Dahlmann et al. 2003
(Dissolved nodularin
sample preparation:
ISO 20179:2005, water
quality. Determination
of microcystins: Method
using solid phase extraction
[SPE] and HPLC with UV
detection)
McNabb et al. 2005
25
concentrations were in the range of natural
concentrations of nano- and microplankton
reported in the study area (Kononen 1988,
Setälä & Kivi 2003, Suikkanen et al. 2007).
4.2. Analytical and statistical methods
Descriptions of the incubations, sampling
procedures and methods are presented in
Tables 2 and 3. The species abundances
in all studies were converted to carbon by
using volume-based carbon conversion
factors (Table 3). Both phytoplankton and
protists were grouped in the further analyses
of grazing and food preference (papers III &
IV). Grazing (clearance and ingestion rates)
was calculated according to Frost (1972)
from the disappearance of food cells from the
experimental bottles containing copepods in
comparison with the control bottles without
copepods (papers I–IV). The Chesson
selectivity index α15 was estimated as the
ratio of the grazed units to the availability
of particular food in the incubation bottles
(Chesson 1983) (papers III & IV). The faecal
pellet production rate (PPR) quantifies the
part of the food that has not been absorbed
in the digestive tract of the animal and
can be used as an estimate of ingestion. In
order to estimate faecal pellet production
in different studies, conversion factors that
were based on volume were used (Gonzales
& Smetacek 1994) (papers I, II & III). To
estimate reproduction, the egg production
rate (EPR) for the egg-carrying copepod E.
affinis was measured, taking into account the
15
Selectivity indices are used to estimate food selection. In the Chesson selectivity index, a particular
food type is neither selected nor avoided (i.e. each
food type is grazed proportionally to its availability) if α = m-1 (m = number of food types available).
When α > m-1 the food type is selected, and when α
< m-1 the selection is negative.
temperature-dependent development time
of the eggs (Andersen & Nielsen 1997),
whereas the eggs of A. bifilosa were counted
after each incubation (papers I & II). Gross
growth efficiency16 (GGE, % day-1) in each
treatment was calculated from the weightspecific egg production rate divided by the
average weight-specific ingestion.
The toxicity of P. parvum, measured by
haemolytic activity, was analysed daily from
the P. parvum cultures and cell-free P. parvum filtrates, and on the first experimental
day from monospecific Prymnesium treatments (papers I & II, see Table 3). The allelopathic effect of P. parvum on R. salina
was estimated by comparing the control
treatments without copepods in the filtrate
and mixture treatments with the Rhodomonas
controls (paper II). Nodularin in particulate
and dissolved17 forms was analyzed before
and after the experiments from the size fractions and after the incubations in copepods
(paper III, Table 3). The toxin content (DSP
toxins) in copepods was measured after the
incubations (paper IV, Table 3).
Differences in grazing, egestion, egg production and survival were tested with 2-way
ANOVA18 (papers I & II). The differences
16
Gross growth efficiency expresses the quantitative relationship between growth and ingestion.
17
Dissolved nodularin was measured from the
<0.2 and <3 μm fractions in Expt 1 and <0.2, <3
and <150 fractions in Expt 2 after the incubations.
Initial dissolved nodularin was measured from the
mesocosms. Detailed descriptions of the toxin analyses and preparation of samples are provided in the
respective papers.
18
In paper 1 (experiments with E. affinis, grazing, survival and PPR), cell concentration and algal treatment (R. salina and P. parvum -N, -P, +NP)
were used as independent variables,while in experiments with A. bifilosa examining grazing, PPR and
EPR, the day and algal treatment were used as inde-
26
among treatments in the EPR of E. affinis
were tested with 1-way ANOVA (papers I
& II). To identify which groups were grazed
on by the copepods, the calculated clearance or weight-specific ingestion rates were
compared using 1-way ANOVA (papers III
& IV). Pairwise multiple comparisons were
made if significant differences between
treatments were found. After calculating
the Chesson selection index α, Dunnet’s
method or a non-parametric Mann-Whitney
rank sum test was used to distinguish those
food groups that were significantly preferred
in the copepod’s diet (Papers III & IV).
The relative importance of the microbial
food web in nodularin transfer to copepods
was estimated by comparing the average
amount of nodularin in copepods from the
dissolved size fraction with those from other
size fractions using 1-way ANOVA. Linear
regression was performed to examine which
variables (ingestion of different plankton
groups) could explain the variance in the
nodularin content of the copepods (paper
III)19, and to examine the dependence of
clearance and ingestion rates of the copepods
on the initial prey cell concentration in different experiments (paper IV). All analyses
were performed with the statistical software
Sigma Stat for Windows 3.0.1. (SPSS).
pendent variables. In paper II (experiments with E.
affinis, grazing and PPR), the day and filtrate concentration / cell concentration in mixtures were used
as independent variables.
19
Variables: small & large ciliates, dinoflagellates, N. spumigena and HNF (rate of ingestion of
these groups).
5. RESULTS AND DISCUSSION
5.1. Instantaneous toxic effects of
ichtyotoxic Prymnesium parvum
The feeding, egestion, reproduction and
mortality of copepods exposed to toxic
Prymnesium parvum cells, filtrates that
were prepared from P. parvum cultures or
mixtures containing P. parvum and nontoxic Rhodomonas salina were examined
and responses compared to R. salina and
filtered water (FW) controls. These studies
were carried out because it has been found
that P. parvum has deleterious effects on
many organisms including phytoplankton,
microzooplankton, mesozooplankton and
fish. Thus, it is crucial to assess what effects
P. parvum may have on copepods, which are
important as grazers in Baltic Sea food webs
and as prey for zooplanktivorous animals.
5.1.1. Haemolytic activity cannot be used
as a predictor of P. parvum toxicity to
copepods
Prymnesium parvum negatively affected
the feeding, survival, behaviour and
reproduction of the calanoid copepods
Eurytemora affinis and Acartia bifilosa.
The cell concentrations20 used in this study
represented the normal bloom levels of P.
parvum (Edvardsen & Paasche 1998). All
the observed responses are summarized
in Figure 3 and Table 4. The P. parvum
cultures were in a steady state and all were
haemolytic during the experiments (papers I
& II) (Uronen et al. 2005). Thus, P. parvum
20
The cell concentration of P. parvum in blooms
usually varies between 50 to 100 × 103 cells ml-1, but
densities of up to 1600 × 103 cells ml-1 have been
recorded (Edvardsen & Paasche 1998).
27
has the potential to cause adverse effects
on grazers, irrespective of the nutrient
conditions.
Substances causing the HA appeared to
be quite labile. In this study, HA decreased
by up to 70 % during the 24 h incubations
in all Prymnesium treatments (paper I), and
was below detection limits in the filtrates
(paper II). The survival of copepods was
related to the presence of P. parvum or compounds released by P. parvum, but not necessarily to HA (papers I and II). This was
indicated by the finding that filtrates were
toxic to copepods, although they were not
haemolytic. Survival rates were inversely
related to the initial concentration of filtrates.
All animals died during the first incubation
period in the highest filtrate concentration
(Filt 4, -P and +NP) (Fig. 4). Exposure to
high concentration filtrates21 resulted in significantly lower survival compared to low
concentration filtrates22 and the Rhodomonas
control (Fig. 4). Despite the absence of HA,
the negative effect on survival was stronger
in the filtrates in comparison to monospecific Prymnesium suspensions or mixtures.
Copepods survived well in the mixtures (>90
%, paper II), and the differences between
monospecific Prymnesium, the Rhodomonas
control and FW were not significant (paper
I). Survival percentages in the Rhodomonas
control and FW (43-86 %) were only slightly
better compared to the monospecific Prymnesium suspension (26-66 %), and varied irrespective of the cell concentration or nutrient
treatment (paper I). We observed relatively
high mortality once in the first incubation
period in the Rhodomonas control due to
an unknown reason, which could explain
21
Filt 3-4: corresponding to 50-354 cells × 103
cells ml-1.
22
Filt 1–2: corresponding to 5 and 10 × 103 cells
ml .
-1
Fig. 4. Survival (%) of Eurytemora affinis in
Rhodomonas (Rho) control and in high (Filt 3-4)
and low (Filt 1-2) concentration Prymnesium parvum filtrates on day 3 (average ± S.E.). Different
symbols indicate P. parvum nutrient treatments (redrawn from paper II).
the lack of significant differences. The visually observed condition of copepods in the
monospecific Prymnesium suspension was,
however, noticeably worse than the actual
survival rates indicated. The copepods were
swimming actively in the Rhodomonas control and FW, whereas copepods incubated in
the monospecific Prymnesium suspension or
in high concentration filtrates were inactive23
and showed reduced pipette avoidance. This
observation was consistent with the video observations of Koski et al. (1999b), according
to which copepods feeding solely on Prymnesium patelliferum were inactive. Acartia
bifilosa showed relatively high survival in
the monospecific Prymnesium suspension
and differences among treatments (monospecific Prymnesium 71-84 %; FW 89 %;
23
In this study means decreased swimming activity (i.e. copepods were completely motionless). The
expression is based on visual observation and is thus
only qualitative.
28
Table 4. Summary of the observed relationships between copepods and Prymnesium parvum. Monospecific
Prymnesium denotes cell suspensions with only P. parvum cells, filtrates were prepared from P. parvum
cultures, and mixtures were prepared by mixing cell suspensions of P. parvum and Rhodomonas salina.
Treatments of R. salina and filtered seawater (FW) were used as non-toxic and starvation controls, respectively. (-N) nitrogen-limited, (-P) phosphorus-limited, (+NP) nutrient-balanced P. parvum cell suspensions
or filtrates. (–) negative effect, (0) weak negative effect, (☺) no effect, (ns) not studied.
Treatment
HA
Allelopathy
Swimming
behaviour
Survival
Feeding
Egestion
Egg
production
GGE
monospecific
Prymnesium
-N
-P
+NP
–
–
–
ns
ns
ns
–
–
–
0
0
0
–
–
–
–
–
–
–
–
–
–
–
–
Rhodomonas
control
☺
☺
☺
0
☺
☺
☺
☺
FW control
☺
☺
☺
0
ns
–
–
-
Low filtrate
-N
-P
+NP
☺
☺
☺
–
–
–
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
High filtrate
-N
-P
+NP
☺
☺
☺
–
–
–
–
–
–
–
0
–
–
–
0
–
–
0
–
–
–
–
–
–
Mixture 1
-N
-P
+NP
ns
ns
ns
–
–
–
–
–
–
☺
☺
☺
ns
ns
ns
☺
☺
☺
☺
☺
☺
☺
☺
☺
Mixture 2
-N
-P
+NP
ns
ns
ns
–
–
–
☺
☺
☺
☺
☺
☺
ns
ns
ns
0
–
–
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
☺
Rhodomonas
control
Rhodomonas control 100 %) were not significant (paper I). The different survival rates
of these copepods suggest that responses to
Prymnesium are species-specific and could
also reflect the life history of the animals,
such as age, feeding history, condition or
physiology. For example, Valkanov (1964),
who examined the toxic effects of P. parvum,
reported that copepods (Cyclops sp. and Calanipeda sp.) were unaffected even after a
3-day exposure to high cell densities (1.1 ×
105 cells ml-l). Similarly, Calanus finmarchicus and Acartia clausi were unaffected after
a 2-day exposure to P. patelliferum (106 cells
29
ml-1) in a study by Nejstgaard et al. (1995).
In contrast, Koski et al. (1999b) observed
the mortality of E. affinis to increase when
copepods were fed with P. patelliferum at
a cell density of 13 to 29 × 103 cells ml-1.
Additionally, P. parvum strains in distinct
growth stages or nutrient conditions may
simply vary in their toxicity. Therefore, the
results obtained with different Prymnesium
strains may not be comparable.
What could have caused the different responses in survival that were particularly apparent between filtrates and cell suspensions
(mixtures and monospecific Prymnesium)?
Since filtrates were not haemolytic, they may
have contained other chemical compounds
that have not been recognized with the available methods. So far, only qualitative indirect
measurements of toxicity such as haemolytic
activity, or lethal effects on Artemia salina
nauplii are available (Meldahl et al. 1994).
These may not be as accurate as direct measurements of toxicity, and the ultimate factor
that caused the observed responses thus remains unclear. In addition, the mechanisms
underlying the activation or promotion of
toxicity have remained obscure (Edvardsen
& Imai 2006). Therefore, HA is not always
a good proxy for the toxicity of P. parvum.
Other factors could also explain the acute
toxicity of P. parvum. One possible explanation is linked to a general failure of cell membrane function (changes in permeability).
For example, Meldahl and Fonnum (1995)
showed that substances extracted from P.
patelliferum increase the permeability to
Na+, Ca2+ and K+ in the synaptosomal membranes as well as causing membrane depolarization and inhibition of the net uptake of
L-glutamate. This type of general effect on
cell membranes may cause symptoms that
were apparent in monospecific Prymnesium
treatments as well as in filtrates. In addition,
many ichtyotoxic phytoplankton species, in-
cluding P. parvum, produce superoxides, also
known as reactive oxygen species, which
are strong oxidants (Marshall et al. 2005).
Reactive agents such as superoxide (O2-) and
hydrogen peroxide (H2O2) are well-known
toxigenic products in biological systems, and
have been linked to fish gill tissue injury,
alteration of chloride cells and physiological responses such as hypoxia, among other
effects (Marshall et al. 2005).
It was interesting that copepods in the
mixtures were active and survived well (paper II). The cell concentrations24 of P. parvum
in mixtures were comparable to concentrations of the P. parvum suspensions from
which the filtrates and monospecific Prymnesium treatments were prepared, and in which
adverse effects (swimming behaviour, survival) were apparent. This indicates that the
presence of R. salina in mixtures somehow
buffered the harmful effects of P. parvum on
copepods. Exudates released by P. parvum
have a tendency to bind to particles such
as other phytoplankton cells (discussed in
Uronen et al. 2005). The total cell numbers in
the mixtures were higher than in the filtrates,
which mean that if exudates were bound to
other particles, this may have diluted the
negative effects (paper II). Furthermore, P.
parvum is mixotrophic and able to ingest
other organisms, such as bacteria and other
phytoplankton (Legrand et al. 2001, Skovgaard et al. 2003). Mixotrophy may release
P. parvum from cellular stress, reducing its
toxicity (Legrand et al. 2001, Uronen et al.
2007). Our results revealed a strong allelopathic effect in the mixtures (see Fig. 5),
which may have promoted mixotrophy and
consequently led to lower toxicity (paper II).
In addition, the presence of copepods may
reduce toxicity. Mechanisms such as sloppy
24
approx. 15 and 50 × 103 cells ml-1 in mixture 1
and 2, respectively.
30
Fig. 5. Growth rate (d-1, average
± S. E.) of Rhodomonas salina in
Rhodomonas control (Rho, dark grey
bar), in different concentrations of
Prymnesium parvum filtrates (Filt
-N, -P, +NP, white bars) and in 2
mixture concentrations (Mix 1 &
2 -N, -P, +NP, grey bars) (redrawn
from paper II).
feeding25, excretion by copepods or leakage of nutrients from dead copepods release
nutrients and could relieve P. parvum from
nutrient deficiency. Responses under natural
conditions could be expected to resemble
those found in the mixtures. It is, however,
possible that copepods may become inactive
and stop feeding during a mass occurrence
of P. parvum, especially in situations with
minimal water turbulence. This, together
with allelopathic effects, may contribute to
bloom formation and persistence.
5.1.2. P. parvum impairs the feeding of
copepods
The experiments revealed that both
Eurytemora affinis and Acartia bifilosa
avoided feeding on Prymnesium, but
readily consumed non-toxic Rhodomonas in
monospecific treatments (Fig. 6 A & B, paper
I). However, clearance rates at the lowest
Prymnesium concentration indicated that E.
affinis is able to consume P. parvum cells when
the cell abundance is low. Filtrates clearly
interfered with feeding. This was especially
apparent in the high concentration filtrates
(Filt 3 and 4), in which reduced ingestion
rates were recorded26, while the clearance
and ingestion rates in the low concentration
filtrates (Filt 1 and 2) were comparable to
the Rhodomonas control. Results from other
studies have been controversial. Higher
ingestion compared to this study or low
grazing rates have been observed in grazing
experiments with Prymnesium patelliferum
(Nejstgaard et al. 1995, Koski et al. 1999b).
The lack of similarity in feeding responses
may thus be linked to the species, sites and
common history of grazers and prey.
The faecal pellet production rate (PPR)
supported the observed ingestion rates.
The average PPR of E. affinis and A. bifilosa reflected feeding, being high in the
Rhodomonas controls in comparison with
the FW and monospecific Prymnesium treat-
25
Sloppy feeding refers to inefficient ingestion of
large cells by zooplankton, which can produce small
ungrazed particles.
26
Two-way ANOVA results are presented in Table
2 in article 2.
31
Fig. 7. Average (± S.E.) weight-specific pellet production of Eurytemora affinis in filtered seawater
(FW) and at different concentrations of Rhodomonas
salina (Rho) and Prymnesium parvum (Pry –N, -P,
+NP) in monospecific cell suspensions (redrawn
from paper I).
Fig. 6. Average (± S.E.) weight-specific ingestion of (A) Eurytemora affinis and (B) feeding on
Rhodomonas salina (Rho) or Prymnesium parvum
(Pry -N, -P, +NP) in monospecific cell suspensions
(redrawn from paper I).
ment (Fig. 7). Similarly, the PPR in the high
concentration filtrates (Filt 3) suggested that
ingestion rates were lower in these treatments27. Interestingly, we found that the PPR
was significantly higher in mixture 1 compared to mixture 2, where the P. parvum
concentration as well as the total food concentration was higher, indicating that feeding
was suppressed in these treatments. In the
mixtures, the ingestion rate was roughly es27
Two-way ANOVA results for filtrates and mixtures are presented in Table 3 in article 2.
timated based on the regression between the
PPR and ingestion in the filtrates (ingestion
= -0.032 × PPR; R2 = 0.64, p < 0.05). According to the estimations, the ingestion rate
in mixture 2 was roughly 40 % lower than
in mixture 1. However, these estimations are
only indicative, because palatability in the
mixtures could have differed from that in
filtrates, or Prymnesium may have affected
the assimilation efficiency of the copepods.
Both ingestion and PPR revealed that E.
affinis fed in filtrates and mixtures. However,
in the high concentration filtrates, exudates
produced by P. parvum interfered with E.
affinis feeding. Interestingly, the PPR in Mixture 2 suggested that feeding was reduced.
Thus, it is possible that P. parvum exudates
negatively affected the overall activity of the
copepods, as in the filtrates. However, since
copepods were active in the mixtures, other
explanations should also be considered. If
an alga produces allelochemicals that harm
other algae, it may reduce the quantity of
32
competitive algae, which may in turn lead
to a reduced availability of alternative food
for grazers. P. parvum showed strong allelopathic effects on Rhodomonas salina (Table
4, Fig. 5). In the mixtures, the cell density
decreased approximately 30 % during the
incubations, both in controls without copepods and in replicates with copepods (paper
II). The effect was weaker in the filtrates,
in which only 5 to 10 % of the cells disappeared. The stronger allelopathic effect in
the mixtures compared to the filtrates may be
due to the continuous release of allelopathic
chemicals by P. parvum cells. Uronen et al.
(2007) observed that practically all R. salina
cells lysed within a short time-span with P.
parvum densities (64 × 103 cells ml-1) corresponding to bloom conditions. However,
the allelopathic effect during our study was
not as strong and the copepods were probably not food-limited. Thus, it is possible
that compounds released by P. parvum had
sublethal effects on copepods that were seen
in lowered grazing.
5.1.3. Low ingestion is linked to
suppressed egg production
Our results demonstrated a reduced fecundity
of both E. affinis and A. bifilosa on a diet
of Prymnesium. The EPRs of E. affinis and
A. bifilosa in monospecific Prymnesium
suspensions were lower compared to the
Rhodomonas control (paper I). Additionally,
the EPR of A. bifilosa increased over time
when copepods fed on Rhodomonas, but
remained low or even decreased in the
monospecific Prymnesium treatment
and in FW (Fig. 8). In the filtrates and
mixtures, variation in EPR was unrelated
to the treatment and was at the same level
as in the Rhodomonas control (paper II).
Gross growth efficiencies (GGEs) were
Fig. 8. Average (± S.E.) egg production rate of
Acartia bifilosa (eggs female-1 d-1) in filtered seawater (FW), Rhodomonas salina control (RHO) and in
monospecific Prymnesium parvum cell suspensions
(PRY –N, -P, +NP) during a 3-day experiments.
generally higher in the Rhodomonas controls
(mean ± S.D. = 15 ± 7 % d-1) compared
to monospecific Prymnesium or filtrates (5
± 6 % d-1). In accordance with this, other
studies have also shown a suppressed EPR
due to the avoidance of Prymnesiophytes P.
patelliferum (Nejstgaard & Solberg 1996)
or Chrysochromulina polylepis (Nielsen et
al. 1990). Thus, the reduced EPR was most
probably caused by the low feeding rates
together with the inactivation of copepods
exposed to Prymnesium. In the filtrates and
mixtures, copepods were able to feed on
non-toxic R. salina, which diminished the
effect. This finding is relevant, because
monospecific blooms could be rather rare
in nature. Thus, the toxic effects may be
diluted if grazers ingest small amounts of
harmful taxa along with other non-toxic
phytoplankton (Turner 2006). This type of
selection, with a wide food-spectrum, was
apparent in studies with Nodularia spumigena
(paper III) and Dinophysis spp. (paper IV),
in which the phytoplankton community was
33
diverse. Furthermore, no negative effects,
such as increased mortality, were observed
in these experiments. Unfortunately, the EPR
was not estimated in these studies.
Reproduction is linked to factors that influence the quality or quantity of the diet.
Thus, if copepods avoid feeding on algae
because of taste, toxicity (Nielsen et al. 1990,
Nejstgaard & Solberg 1996, Dutz 1998),
shape/size (Infante & Abella 1985, Bergreen
et al. 1988) or low nutritional value (Jónasdóttir 1994, Schmidt & Jónasdóttir 1997),
reproduction may fail. During bloom events,
the proportion of unsuitable food compared
other prey species could be high. In such
situations, energy reserves may be used for
maintenance rather than allocated to reproduction.
5.2. Accumulating toxins of Nodularia
and dinoflagellates
Nodularin as well as Dinophysis-derived
toxins are among those toxins that can be
vectored in the food web. The aim of these
studies was to examine selective feeding
and the role of copepods in toxin transfer. In
experiments with Nodularia spumigena we
were especially interested in the pathways
of nodularin transfer. Do copepods gain
nodularin by direct ingestion of filaments,
can they take up dissolved nodularin, or
could nodularin perhaps be transferred via the
microbial food web? These two experiments
aimed to address these questions.
(e.g. Engström-Öst et al. 2002, Karjalainen
et al. 2006), but also directly in the dissolved
form (Karjalainen et al. 2006). However,
very little is known about the importance of
the microbial food web in nodularin transfer,
and we thus aimed to focus especially on
the importance of this pathway. Potential
nodularin sources were estimated by
calculating the ingestion and food selection
of copepods incubated in different size
fractions. Pre-filtration produced relatively
different planktonic communities in the size
fractions in terms of both total biomass and
species composition. Dinoflagellates and
ciliates comprised an important group in this
study in terms of biomass. The contribution of
nano- and pico-sized organisms (flagellates,
pikocyanobacteria, and bacteria) varied from
moderate to high28.
The initial biomass of N. spumigena (27
μg C l-1, 0.25 mg WW l-1) during Expt 1 was
in the range of average bloom biomasses in
the Gulf of Finland, whereas the biomass
during Expt 2 (109 μg Cl-1, 0.99 mg WW l-1)
fitted within the range of maximum values29.
The initial nodularin concentrations in our
experiments (Table 5) were in the range of
values naturally observed in the Baltic Sea
(80-18 700 ng l-1) (Kononen et al. 1993). The
initial concentration of dissolved nodularin
(Table 5) in both experiments corresponded
to approximately one-fifth of the total nodularin in the <150 μm fraction. Nodularin was
found in the copepods incubated in all the
plankton size fractions in both experiments,
indicating that all pathways presented in
Figure 1 are possible (Table 5). The im28
5.2.1. Transfer of nodularin to copepods
through feeding interactions
In the Baltic Sea, copepods mainly obtain
nodularin via the ingestion of toxic filaments
Composition of the planktonic community, see
Table 2 in article III.
29
The average bloom biomass in the Gulf of Finland ranges between 0.08-0.28 mg WW l-1, while
maximum values are in the range of 0.23-2.91 mg
WW l-1 (Kanoshina et al. 2003).
34
Table 5. Initial Nodularia spumigena biomass and nodularin concentration in particulate and dissolved
forms. Nodularin (pg ind-1) in Eurytemora affinis after 24-h incubations, proportion of initial nodularin
channelled to copepod total biomass (n = 5 replicate bottles with 35 copepods in each; mean ± S.D.). * Initial
dissolved nodularin concentrations were measured from mesocosms.
Particulate pool
<150 μm
<45μm
<20 μm
Dissolved pool
<10 μm
<3 μm
<0.2 μm
Expt 1
Initial N. spumigena biomass (μg C l-1)
Initial nodularin (ng l )
-1
Nodularin in copepods (pg ind )
-1
% copepod biomass
27
13
20
5
0
0
387
257
85
50
111*
111*
14.3 ± 11.6
0.1 ± 0.1
5.7 ±1.3
3.8 ± 2.6
4.3 ± 2.6
1.3 ± 2.8
1.7 ± 3.8
0.08 ± 0.02
0.2 ± 0.1
0.3 ± 0.2
0.04 ± 0.09
0.05 ± 0.1
Expt 2
Initial N. spumigena biomass
109
2
-
0
0
0
Initial nodularin
199
3
-
2
42*
42*
Nodularin in copepods
6.6 ± 0.7
3.5 ± 3.3
-
4.2 ± 4.7
3.7 ± 1.6
1.6 ± 3.5
% copepod biomass
0.1 ± 0.01
4.1 ± 3.4
-
6.5 ± 7.3
0.3 ± 0.3
0.1 ± 0.3
portance of possible pathways will next be
discussed by presenting questions and evaluating whether our data are able to support
these assumptions.
Do copepods obtain nodularin directly by
grazing on toxic filaments? We found that
some of the nodularin was obtained through
direct grazing on small N. spumigena filaments in the <150, <45, <20, and <10 μm
fractions in Expt 1 (Fig. 1, pathway 1, Fig. 9
A-D). These filaments were grazed on in the
proportion they were available, without significant preference or avoidance (Dunnett’s
test p > 0.05 compared to α), while large
filaments were avoided in both experiments.
Grazing on the small filaments correlated
with the amount of nodularin found in the
copepods in Expt 1. However, the correlation was not strong enough (r2 = 0.39, p <
0.05) to exclude other possible pathways
for nodularin transport to copepods. In the
smaller fractions (<45 to <0.2 μm), the nodularin content of the copepods was up to 52
% lower compared to copepods incubated
in the <150 μm community, indicating that
a relatively large part of the nodularin was
obtained through grazing on filaments.
Could copepods obtain nodularin directly
in the dissolved form? Our data suggest that
part of the nodularin was directly obtained
from the dissolved pool in the <0.2 μm
fraction (Fig. 1, pathway 2; Table 5). This
accounted for up to 24 % of the E. affinis
nodularin content compared to the <150 μm
fraction. Consistently with this, Karjalainen
et al. (2003) found that E. affinis was able
to incorporate similar amounts of labelled
dissolved nodularin (1.8 ± 0.5 pg ind-1), although the initial nodularin concentration
was 45 to 120-fold higher than in our study.
Thus, the possibility that copepods took up
dissolved nodularin has to be considered.
It is important to point out, however, that
nodularin is a hydrophobic compound, which
easily attaches onto surfaces (Hyenstrand et
al. 2001). If nodularin attaches to particles,
it is thus likely that some of the nodularin
was attached to the surface of the copepods
35
Fig. 9. Average (± S.D.) clearance rate (ml ind-1 h-1) of Eurytemora affinis on different plankton groups during Expt 1 (upper
panel) and Expt 2 (lower panel).
The differences between plankton communities in different size
fractions are presented schematically. * The few large ciliates
(>23 μm diameter) initially
found in the <10 μm fraction
were grazed to zero during the
experiments. These bottles were
excluded from the clearance and
ingestion rate calculations; thus,
the grazing for large ciliates is
underestimated (redrawn from
paper III).
36
rather than incorporated in their tissues. In
such a case the amount of adsorbed nodularin
could be quite constant, irrespective of the
initial amount of dissolved nodularin. The
toxin would not, however, interfere with the
metabolism of the copepods and the degradation of the toxin would not occur in their tissues. The copepods would, nevertheless, act
as a vector of adsorbed nodularin to higher
trophic levels. To estimate the relevance of
adsorption, dead and living animals should
be incubated in the dissolved nodularin and
their nodularin content compared.
The possibility that nodularin is attached
onto particles should also be kept in mind
when discussing the role of microbial organisms in nodularin transfer. The <0.2 and <3
μm fractions in our study only contained
dissolved nodularin. However, the <0.2 μm
fraction contained bacteria and the <3 μm
fraction bacteria and nanoflagellates. Thus,
part of the nodularin may have been bound to
the microbial biomass at low concentrations,
below the detection limit of the nodularin
analysis. In Expt 2, copepods obtained as
much nodularin in the <3 μm fraction as
in the <10 μm fraction (Table 5). Hence,
it is possible that part of the nodularin was
accumulated through grazing on nanoflagellates in this fraction. The production of
faecal pellets in the <3 μm fractions support
this suggestion, because it was at the same
level as in the < 10 μm fractions (Fig. 10),
although the clearance rates of heterotrophic
nanoflagellates (HNF) and photosynthetic
nanoflagellates (PNF) were low or negative
(Fig. 9, E & I). Low clearance rates could
be explained by the high growth potential
of nanoflagellates, which probably masked
feeding. For example, Uitto (1996) found
that copepods are not efficient in removing
nano-sized organisms. In her study, copepods were able to clear only 2 to 6 % of the
nanoprotozoan biomass daily. Therefore, we
cannot exclude the possibility that some nodularin was vectored through these organisms
to copepods. Thus, based on this study, it is
not possible to exclusively conclude whether
copepods obtained nodularin directly from
the water, via adsorbtion or via ingestion of
bacteria or nanoflagellates.
What could be the role of the microbial
food web? Could copepods obtain part of
the nodularin through feeding on microbial organisms? The answer is that we do
not know. Unfortunately, we were unable
to verify this pathway because we failed
to remove cyanobacteria filaments from
the smaller size fractions in Expt 1. Therefore, results gathered during Expt 2 will be
highlighted here, since in this experiment
toxic filaments were scarce in the <45 μm
fraction and not present in the <10 μm or
smaller fractions. To estimate the relative
importance of the microbial food web in
nodularin transfer, we compared the average
amount of nodularin in copepods from the
dissolved size fraction (<0.2 μm) with those
from the other size fractions. Copepods that
were incubated in the <45, <10 and <3 μm
fractions obtained roughly 2 to 3 times more
nodularin compared to the <0.2 μm fraction
in Expt 2 (Table 5), although the differences
were not statistically significant (ANOVA, p
> 0.05). However, these differences indicate
that nodularin was probably transferred from
other planktonic organisms to the copepods
through grazing (Fig. 1, pathways 3 & 4).
Based on this study, dinoflagellates and
ciliates could be the most promising candidates for nodularin transfer. The clearance
rates on these organisms in the <150, <45,
<20 and <10 μm fractions were the highest
in both experiments (Fig. 9), and they were
an important carbon source for copepods
(Expt 1: dinoflagellates 33-64 %, ciliates
27-57 %; Expt 2: dinoflagellates 65-69 %,
ciliates approximately 23 %). Grazing on
37
Fig. 10. Average (± S.D.) weightspecific faecal pellet production of
Eurytemora affinis during Expt 1
(open bars) and Expt 2 (grey bars).
dinoflagellates explained 47 % of the variance in the copepod nodularin content (r2
= 0.47, p = 0.01) in Expt 1. Recent studies
have revealed that increasing numbers of
dinoflagellate species are mixotrophic, with
the ability to utilise organic matter for their
growth (reviewed by Hansen 1998, Ptacnik
et al. 2004). Thus, they may be able to take
up nodularin, and nodularin transfer via mixotrophic dinoflagellates consuming bacteria
and small phytoplankton cannot therefore be
excluded. However, further studies on this
are required. In Expt 2, copepods selected
ciliates (Dunnett’s test, p <0.05 compared
with α), and they were an important carbon
source for the copepods. These results are
important because ciliates graze on organisms at lower trophic levels, but also because
they are able to take up nodularin directly in
the dissolved form (Karjalainen et al. 2003).
On the other hand, as pointed out earlier,
nodularin may attach onto the surfaces of
particles. This would mean that nodularin
could be transferred through overall grazing
on any particles.
5.2.2. Processes in the microbial food
web: importance in nodularin loss and
transfer
We found that copepods were able to remove
particulate nodularin from the system. This
was not apparent in the <150 μm fraction,
where the relative change in the particulate
nodularin concentrations was similar between
the treatments with copepods and the controls
in both experiments (Fig. 11). In the smaller
size fractions, the presence of copepods
lowered particulate nodularin concentrations
compared to the controls (t-test, p < 0.001
for <20 and <45 μm fractions, Fig. 11).
However, at the end of the experiments, the
copepods contained less nodularin than the
amount that had been lost. Up to 0.3 % of
the initial particulate nodularin in Expt 1
and up to 6.5 % in Expt 2 was found in
copepods (Table 5). Of the initial dissolved
nodularin in the <3 and <0.2 fractions, up
to 0.3 % ended up in copepods. Similarly,
Kozlowsky-Suzuki et al. (2003) estimated
that only 0.1 % of ingested nodularin was
present in copepod tissues.
38
Part of the ingested nodularin was probably metabolized by the copepods (Karjalainen et al. 2006). It has been found that
animals originating from areas with Nodularia spumigena blooms may have the ability
to cope with ingested toxins (KozlowskySuzuki et al. 2003). Consistently with this,
nodularin had no direct adverse effects on
the copepods, as observed in the studies with
Prymnesium parvum, and their survival remained above 98 %. Toxins with differing
chemical composition clearly have variable
modes of action in food webs. Some toxic
species, such P. parvum, which excretes toxic
compounds into water, could harm organisms rather rapidly, using chemical defence
to repel grazing (e.g. Granéli & Johansson
2003, papers I and II). On the other hand,
if grazers are continuously able to feed on
toxic species because they are not negatively
affected, they could transfer toxins to other
organisms. Copepods may also possess effective mechanisms, such as detoxification
enzymes, to remove toxic compounds from
their tissues (Sipiä et al. 2002, KozlowskySuzuki et al. 2009). Therefore, the amount of
nodularin that was transferred to copepods
through ingestion was probably higher than
we estimated. Due to the low toxin retention,
a smaller proportion was detected in the
copepods. However, although the proportion of nodularin in the copepods appears
low, they have the potential to act as a link
in the transfer of nodularin to planktivorous
fish and mysids, because of the high feeding
rates of these animals (Viherluoto & Viitasalo
2001, Karjalainen et al. 2005, this study).
Besides direct grazing, other loss factors
are also involved in nodularin dynamics.
Nodularin concentrations declined more during Expt 2 than during Expt 1 (Fig. 11). The
conditions varied between these experiments.
During Expt 1, the cyanobacterial community was at initial growths stage, while Expt 2
represented stationary conditions with decaying filaments. This discrepancy could explain
the observed variation in nodularin dynamics
between these experiments. Interactions in
the microbial food web can be important in
nodularin dynamics. There could therefore
have been changes in the microbial community in our study that promoted degradation during the aging of the cyanobacterial
communities.
Cyanobacterial toxins are considered intracellular, but are released into the water
during cell decay. Cyclic cyanobacterial toxins, such as nodularin, are relatively stable
due to their chemical structure, and are thus
not easily degraded by physical factors such
as light or temperature (e.g. Twist & Codd
1997, Mazur & Plinski 2001). However,
biological factors, including sloppy feeding
by copepods and processes in the surface
community of the cyanobacterial cells, could
lead to toxin release. For example, studies
by Teegarden et al. (2003) suggested that
regurgitation and sloppy feeding seem to
be important mechanisms that release PSP
toxins in a dissolved form, which are then
available for uptake or degradation. Furthermore, it has been shown that specialized
bacteria are responsible for the degradation
of cyanobacterial toxins (Mazur & Plinski
2001, Hagström 2006). Bacteria can cause
disruption of toxic cyanobacteria (Nakamura
et al. 2003), and some bacterial strains are
able to metabolize cyanotoxins such as the
cyclic pentapeptide microcystin-LR and use
it as a carbon and nitrogen source (Bourne
et al. 1996). Furthermore, Lahti et al. (1998)
isolated several bacterial strains from freshwater sediments and water that are able to
degrade microcystins and nodularin.
Although bacteria are presumably essential in nodularin degradation, the role of
other members of the microbial food web
(e.g. ciliates, mixotrophic organisms, HNF)
39
Fig. 11. Change (%, average ± S.D.)
in particulate and dissolved (diss)
nodularin in different size fractions
compared with initial concentrations
in (A) Expt 1 and (B) Expt 2. Open
bars represent controls and grey bars
represents replicates with copepods.
In Expt 2, additional measurements
of dissolved nodularin were made in
<150 and <3 μm size fractions (redrawn from paper III).
in removing and transferring toxins is still
largely unknown. In this study, ciliates were
largely removed due to grazing by copepods.
Bacterial growth rates, on the other hand,
were strongly controlled by HNF, as has also
been shown in other studies (e.g. KuuppoLeinikki 1990). This was indicated by the
differences in the specific growth rates of
bacteria in the <0.2 μm (no grazers) and
<3 μm (small HNF) size fractions (0.5 and
0.3 d-1 in Expt1; 0.5 and 0.2 d-1 in Expt 2).
Thus, some of the nodularin could have disappeared as a result of trophic interactions
within the microbial food web.
As this study demonstrated, it is relatively
easy to show that grazers accumulate toxins,
but more challenging to follow the pathways
of toxin transfer. According to this study,
direct grazing on filaments of small Nodularia spumigena was an important pathway
40
in nodularin transfer. Copepods also directly
acquired nodularin from the dissolved pool,
although the mechanism remained obscure.
Whether the microbial food web was important in nodularin transfer is questionable.
However, this study provided some indications that nodularin could be transferred to
copepods via grazing on ciliates, dinoflagellates or HNF, and trophic cascades may
actually be important in the transfer of algal
toxins in aquatic environments.
5.2.3. Feeding behaviour of Eurytemora
affinis and Acartia bifilosa on plankton
communities containing Dinophysis spp.
The aim of this study was to test whether
copepods feed on Dinophysis spp., and could
thereby transport toxins to higher trophic
levels in the food web. The rates of ingestion
of Dinophysis spp. by E. affinis ranged from
no grazing to 226 cells ind-1 d-1 (up to 0.5
μg C ind-1 d-1). The ingestion rates of A.
bifilosa feeding on Dinophysis spp. varied
from 43 to 64 cells ind-1 d-1 (up to 0.14 μg
C ind-1 d-1). Although the ingestion rate
varied irrespective of the Dinophysis spp.
proportion in the planktonic community,
Dinophysis formed a relatively large part of
the copepod diet in situations where the food
concentration was low (Fig. 12 B & C). Total
ingestion rates increased with an increase in
the food concentration (r2 = 0.62, p = 0.007).
Thus, in the low food experiments (Exp
VII-X), ingestion accounted for only about
8-20 % of the copepods’ carbon demand, but
in the high food experiments (Exp I-VI) the
ingestion rates corresponded to about 2570 % of the body carbon d-1. Heterocapsa
triquetra formed the main part of the E.
affinis diet at high food concentrations (Fig.
12 A), while at low food concentrations other
food groups also contributed to the copepod’s
diet (Fig. 12 B & C).
Neither of the copepod species selected
Dinophysis spp., while H. triquetra and
ciliates were the most preferred food in all
experiments (Fig. 13). The feeding interactions between copepods and Dinophysis spp.
have been shown to vary. Different studies have reported grazing rates that vary
from no grazing up to 1 128 cells ind-1 d-1
(Maneiro et al. 2000, Jansen et al. 2006,
Kozlowsky-Suzuki et al. 2006). The rates of
Dinophysis spp. ingestion in our study (up
to 0.5 μg C ind-1 d-1) were at the lower end
of those reported by Kozlowsky-Suzuki et
al. (2006) for Acartia bifilosa feeding on D.
norvegica (0.3 to 3.6 μg C ind-1 d-1, Baltic
Sea). The low feeding rates on Dinophysis
spp. at both high and low food concentrations suggest that grazing was not related
to the relative concentration of Dinophysis
spp. in the plankton community. Several
criteria may be involved in food selection.
Factors that contribute to selection have been
presented earlier in the introduction (chapter
1.4). Both Eurytemora affinis and Acartia
bifilosa are relatively small (adults 1.0-1.5
mm) compared to oceanic species such as
Calanus helgolandicus, which have been
found to feed efficiently on D. norvegica
(Jansen et al. 2006). Acartia bifilosa is considered to be more selective compared to E.
affinis because it is able to switch between
filtering-type and more active ambush-type
feeding modes (Kiørboe et al. 1996). However, in this study, the selection patterns of
these two copepods were similar (Fig 13).
The dinoflagellate H. triquetra is considered to be high quality food for copepods.
It is of a suitable size and known to support
high egg production rates in copepods (Uye
& Takamatsu 1990). Similarly, ciliates are a
nutritionally adequate and suitable sized food
for copepods (Stoecker & Egloff 1987, Klein
41
Breteler et al. 1999). However, it should be
mentioned that opposite observations have
also been reported. Ciliates cannot synthetize sterols and HUFAs (highly unsaturated
fatty acids), which are essential for copepods
(Klein Breteler et al. 2004). Thus, the nutritional quality of ciliates may vary depending
on their diet. Copepods selected H. triquetra
and ciliates irrespective of their abundance
in the community (Fig. 13). This implies that
copepods selected food that was of high quality and of a suitable size, and hence easier to
manage than the large Dinophysis spp. cells.
Similarly, Jansen et al. (2006) suggested that
Dinophysis cells may be simply too large to
be grazed on by smaller species. Although
Dinophysis spp. was not the preferred food
for the copepods, the weight-specific ingestion rates were reasonably high in some
experiments due to the large cell volume
(Olenina et al. 2006).
Diarrhetic shellfish toxin (PTX2) was
found from the copepods after incubation in
two combined samples (Expt I & II: 142 pg
ind-1, Expt III & IV 53 pg ind-1). The ingestion rate in the replicates in these experiments
ranged from no grazing to 226 cells ind-1 d-1.
Kuuppo et al. (2006) reported that PTX2
concentrations in the Baltic Sea area vary
between 1.6 and 19.2 pg cell-1. Based on their
results, we calculated that the measured toxin
content in the copepods corresponded to the
ingestion of less than 100 cells. The detection
of PTX2 in copepods suggests that the toxins
were not instantaneously metabolized, as
they usually are in filter-feeding shelfish such
as mussels and scallops (e.g. Draisci et al.
1996, Suzuki et al. 2001). However, feeding
on Dinophysis does not necessarily indicate
digestion or toxin accumulation in the grazer.
This assumption is supported by observations
in which the ingested Dinophysis cells have
remained intact in the faecal pellets (Maneiro
et al. 2000, 2002, Wexels Riser et al. 2003).
Fig. 12. Average (± S.D.) weight-specific ingestion
rates of copepods in (A) high food concentrations
(Exp I-VI, Eurytemora affinis), (B) low food experiments (Exp VII & IX, E. affinis), and (C) low
food experiments (Exp VIII & X, Acartia bifilosa).
Experiments were pooled in order to compress the
original number of figures. (Dinoph) Dinophysis
spp., (Htri) Heterocapsa triquetra, (Auto) phototrophic dinoflagellates, (Cil) ciliates, (open bars)
total ingestion (redrawn from paper IV).
42
Fig. 13. Pooled (average + S.E.) Chesson selectivity
coefficients (α) for Eurytemora affinis (grey bars)
and Acartia bifilosa (black bars). Dashed lines indicate α = m-1 (i.e. neither selection nor avoidance).
Other abbreviations as in Fig. 12 (redrawn from paper IV).
Since the faecal pellets were not examined
in our experiments, it is not clear whether the
Dinophysis cells were actually digested or
simply passed through the copepods. Nevertheless, whether the copepods accumulate
toxins or not, they may become toxic after
ingesting toxin-containing cells, or produce
toxic faecal pellets.
Kuuppo et al. (2006) examined the transfer of DSTs to sediments in the Gulf of Finland. Their sediment trap material contained
PTX2 and DTX1. The authors suggested that
copepod faecal pellets might be the source of
these toxins, since no Dinophysis spp. cells
or cell remnants were found. They estimated
that only 0.01 % of the PTX2 and 0.1 % of
the DTX1 that was found suspended in the
water column had settled in the trap material.
These low estimates are in agreement with
our results of low feeding rates on Dinophysis spp. cells by the copepods. Consistently with this, no DSTs were found from
animals that were individually collected in a
field study from the same area30 (Setälä, Sopanen, Autio, Erler, unpublished data). The
high loss rate implies that toxins could have
passed along the food web, decomposed in
the sediment or transformed into other toxin
derivatives (Kuuppo et. al. 2006). In accordance with this, we found PTX-2 seco acid31 in
zooplankton net samples at 9 of 13 locations
in the northern Baltic Sea. However, weak
positive signals of PTX-2 seco acid probably originated from disrupted Dinophysis
spp. cells (Setälä, Sopanen, Autio, Erler,
submitted manuscript). These observations
imply that similar mechanisms to those involved in nodularin degradation (see chapter
5.2.2.) could also apply in the degradation
of other toxins, such as those derived from
toxic dinoflagellates.
On the basis of this study and the observations of other authors, it seems likely
that copepods are an unimportant link in the
transfer of DSTs in the northern Baltic Sea.
On the other hand, at times when other food
sources are scarce, or if copepods encounter a
Dinophysis spp. bloom with high cell densities typical for the Baltic Sea (Kuosa 1990,
Carpenter et al. 1995), these dinoflagellates
may form a significant part of the copepod
diet.
30
Tvärminne sea area, SW coast of Finland, Baltic Sea.
31
A metabolic product of PTX2 and produced, for
instance, by shellfish that have been exposed to algae containing PTX2 (Suzuki et al. 2001).
43
6. SUMMARY AND CONCLUDING
REMARKS
We found that algal species producing
extracellular toxins could have acute
negative effects on grazers, such as elevated
mortality and suppressed feeding and egg
production. In contrast, other algal toxins,
especially those that grazers are able to
accumulate, may not be potent enough to
induce immediate adverse effects. However,
they could have sublethal effects that may be
seen, for example, as suppressed production
after a longer time period due to low food
quality. These changes may influence both
species composition and production in the
long term and affect the food availability
of planktivorous organisms, such as fish.
Grazers, in turn, could regulate HABs
especially during bloom initiation and
termination. Microzooplankton grazers
could be more effective in this, but no
comparative studies have been carried out
in the Baltic Sea.
These results revealed that the ichtyotoxic haptophyte Prymnesium parvum has
negative effects on copepods. Although not
haemolytic, P. parvum filtrates clearly reduced the viability of copepods. In contrast,
even though the monospecific Prymnesium
treatments were haemolytic during the experiments, the toxicity was not reflected in
the mortality of the copepods. The copepods
in these treatments were, however, severely
impaired and reacted slowly to mechanical disturbance. This effect was not due to
starvation, as copepods in the FW control
were active. Therefore, high mortality and
inactivation might not be dependent on HA
alone, but also on other unknown substances
that P. parvum releases into the water. Thus,
HA is not always a good proxy for algal toxicity. The results indicated that P. parvum is
able to deter grazing. Due to the low feeding
rates, suppressed pellet production and egg
production rates were observed. Harmful effects on the well-being of potential grazers,
together with severe allelopathic effects on
other phytoplankton species, may accelerate the formation of P. parvum blooms and
contribute to bloom persistence.
Nodularin was transferred to copepods
through three pathways: by feeding on Nodularia spumigena filaments, by direct uptake
from the dissolved pool, and via organisms
in the microbial food web. According to this
study, direct grazing could have been a relatively important pathway in nodularin transfer. However, we were unable to conclude
whether the copepods obtained nodularin
directly in the dissolved form, via adsorption
or via ingestion of bacteria or nanoflagellates
in the smallest fractions. Whatever the mechanism, copepods could still act as vectors of
nodularin transfer to higher trophic levels.
However, the role of the microbial food web
remained unclear. While our results indicated
that copepods obtained nodularin through
grazing on microbial organisms, nodularin
accumulation could have been a result of
overall grazing on particles if the toxin had
been adsorbed onto the particles. Nodularin
was not so potent in either a particulate or
dissolved form to deter grazers such as P.
parvum, and no acute effects, such as high
mortality, were detected. Thus, algal toxins
of this kind have the potential to accumulate
in grazers and further along the food web.
We found that copepods were able to remove nodularin from the system. However,
only a small proportion of the initial nodularin was channelled into the copepods, while
most of the toxin disappeared. It may have
partly been metabolized by the copepods, but
several other processes might also have been
responsible for the degradation of nodularin.
This implies that processes in the microbial
food web, such as bacterial degradation,
44
probably contribute to the biodegradation
of toxins. Because nodularin is chemically
closely related to microcystins, which are
produced, for example, by the freshwater
species Microcystis and Anabaena, they
probably share common mechanisms of toxin
transfer. Thus, trophic cascades could also be
important in the transfer of cyanobacterial
toxins in freshwater environments.
Neither of the copepod species in our
experiments selected the dinoflagellates
Dinophysis spp., which may simply be
too large to be grazed on by these smaller
copepod species. Instead, the dinoflagellate
Heterocapsa triquetra and ciliates were the
preferred food in all experiments. This implies that copepods selected food that was
of high quality and a suitable size. However,
in experiments where the food concentration was low, the large Dinophysis spp. cells
formed a relatively large component of the
copepod diet. PTX2 toxin was found in the
animals after the incubations. However, no
DSTs have been detected in field-collected
animals from the same area. According to
these observations, copepods seem to be an
unimportant link in the transfer of DSTs in
the Baltic Sea.
This study examined the interactions between copepods and toxic algae. Comparisons with various other studies, especially
when considering P. parvum – copepod interactions, revealed that the tolerance of algal
toxins, as well as feeding and reproduction
responses, are species-specific. Moreover,
dissimilar responses due to different algal
toxins, such as P. parvum-derived substances
and nodularin or DSTs, could shape zooplankton communities in different ways. A
P. parvum bloom could have detrimental
effects on a large part of the community if
water turbulence is low. However, nodularin
and DSTs could be rather harmless to grazers, although due to their transport along the
food web they may harm organisms at higher
trophic levels. Despite the variable results,
experimental studies provide valuable information on potential interactions between
grazers and harmful algal species, leading
to better understanding of the importance
of grazers in the regulation of harmful algal
blooms, and processes that control the accumulation and degradation of algal toxins.
45
ACKNOWLEDGEMENTS
The studies included in this thesis have been financed
by Academy of Finland, the European Commission
(project FATE: “Transfer and Fate of Harmful Algal
Bloom (HAB) Toxins in European Marine Waters”
as part of the EC-EUROHAB cluster), and by the
Walter and Andrée de Nottbeck Foundation. I would
like to thank Prof. Peter Tiselius for acting as an
opponent in the public examination of my thesis.
Prof. Jorma Kuparinen has kindly helped me with
university bureaucracy and the final preparation of
the dissertation. I greatly acknowledge the reviewers,
Elena Gorokhova and Marko Reinikainen, for their
valuable comments on my thesis. Roy Siddall checked
the English language and Carl-Adam Hæggström
edited my thesis, thank you.
My most sincere gratitude goes to my supervisors.
Without Pirjo Kuuppo this thesis would have been
a never-ending story. Her way of giving advice and
support as well as ideas when I was struggling with
manuscripts or became otherwise stuck with the writing was essential. I am grateful to Harri Kuosa, who
commented on the pre-mature text and believed that
someday I would get this work finished.
Marja Koski taught me how to carry out experimental zooplankton studies. I will always remember
endless nights in the clima-room picking and transferring copepods to a new food solution (luckily music
by Työväen Tyttäret also gave strength to keep on
picking at 2 o’clock in the morning). Marja also provided numerous critical comments on manuscripts, as
well as the thesis. My humble thanks belong to Timo
Tamminen (inventor of the great copepod spoon!)
for his innovative way of planning and carrying out
studies and constructive comments and ideas during
all phases of my work.
Deepest thanks to Outi Setälä for all the support
and friendship through all these years. Our discussions of work and life have been brilliant and we
have shared unforgettable moments when running
experiments. I want to thank my dear colleagues Sirpa
Lehtinen, Pauliina Uronen and Anke Kremp for those
moments we have shared together both during and
outside of work: our discussions, for instance, in the
“lukupiiri” have been relaxing and cheerful.
Catherine Legrand from the University of Kalmar
has given me constructive criticism on my work,
which greatly improved the quality of the papers
included in thesis. A bunch of wonderful people built
up the “FATE team” and carried out some excellent
studies at Tvärminne Zoological Station and the University of Kalmar. These people created a warm and
inspiring atmosphere and taught me a lot about team
work, which I greatly appreciated. I would also like
to thank all other authours in the individual papers.
I first visited Tvärminne as a young hydrobiology student. Since then, my summers at Tvärminne
have included not only work, but also unforgettable
football games, coffee breaks on the balcony and
evenings together with friends; thanks Ripa, Pirjo,
Kaitsu, Ansti, Jukka, Purjo, Timo, Outi, Tarja, Riitta,
Kristian, Jonna, Laura and Sanna, among others. My
warmest thanks go to the whole Tvärminne staff for
all the help one can just imagine and for excellent
facilities. Special thanks to Mervi and Elina, Totti,
Ulla, Bebbe and Lallu. Your work has been the backbone for all studies that have been have carried out
in the Tvärminne lab. Experiments with Nodularia
were done in the laboratories of the University of
Kalmar. Our stay there was joyful and I want to thank
you for the excellent facilities and preparation of the
experiments.
The working atmosphere at the Finnish Environment Institute (SYKE) in the former ITO (Research
Programme in the Baltic Sea) and later in SYKE
Marine Research Centre was warm, and I would like
to thank you all for great company and cooperation.
Special thanks to Code, Antti, Hexi and Seppo for
providing good company during lunch breaks (I really have gained all my knowledge about strategies in
football as well as ice-hockey during these moments!).
I want to thank Riitta and Maiju & Milja, Ansku and
Sari for friendship through all these years.
Science is not everything and there is also life
beyond the microscope. I wish to thank all my dear
friends for sharing my life outside research. I want to
thank my parents Raimo and Merja for all the support
46
you have always provided me. I would also like to
thank Tommi’s father Eikka and his life-companion
Ritva, and Tommi’s sister and brother with families
for all those happy moments we have shared together.
Finally, I would like to give an extra big hug for my
loving husband Tommi and our beloved children Iida
and Tuure. You have been so patient and brought so
much joy to my life, which has helped to carry this
project to the end.
47
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