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Potential effects of
phytoplankton community
composition on Vibrio
parahaemolyticus
Subtitle if needed
on one to three lines
Rebecca Eliasson
Degree project for Master of Science (One Year) in
Biology
30 hec
Department of Marine Ecology
University of Gothenburg
Contribution number
ABSTRACT
Vibrio parahaemolyticus is a potential human pathogen capable of inducing gastroenteritis
and it occurs naturally in the marine environment. Infection is usually associated with
consumption of seafood, or with exposure of wounds to seawater. In temperate waters the
abundance of Vibrio sp. has generally been correlated with a lower salinity and higher water
temperature, but in tropical waters the picture is not as clear. In this study I hypothesize that
the composition of phytoplankton community could be a potentially important factor for the
number of vibrios in tropical marine areas. The effect of the taxonomic composition of the
phytoplankton community and different environmental variables on Vibrio parahaemolyticus,
as well as other Vibrio sp. was investigated during March-May 2009 in Mangalore, India. The
presence and densities of naturally occurring vibrios was detected by enumerating colonies of
Vibrio species on plates of selective medium. The plankton community composition was
simultaneously examined. The abundance of naturally occurring vibrios was found to follow
the same trend as the proportion of dinoflagellates in the phytoplankton community. Also
when investigating the associations between labeled V. parahaemolyticus and different genera
of phytoplankton by epifluorescence microscopy, it was found that significantly more V.
parahaemolyticus were associated with dinoflagellates compared to diatoms.
KEYWORDS: Vibrio, phytoplankton, association, growth, Arabian Sea
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INTRODUCTION
Pathogenic bacteria are natural constituents of the marine environment and contribute to
decomposition of organic matter. One commonly occurring genus is Vibrio which includes
more than 30 marine species whereof at least 11 are pathogenic to humans, for example
Vibrio vulnificus and some strains of V. cholera, and -V. parahaemolyticus (e. g. Kaper et al.
1995; Deepanjali et al. 2005). They are widespread in estuaries and coastal waters all over the
world (Wright et al. 1996). Despite their important role as decomposers, some are also
harmful when occurring in high numbers, due to their ability to induce disease in humans. The
common source of infection is through ingestion of raw or poorly cooked fish and shellfish
(Thompson et al. 2005). Especially filtrating organisms like bivalves can accumulate high
concentrations of bacteria, which is an inconvenience and potential threat to the fish- and
shellfish industry. For instance, oysters can accumulate Vibrio spp. in concentrations that are
in 100-folds compared to the surrounding water (DePaola et al. 2003a). Swimming in Vibrioinfested waters can also lead to infection via cuts and wounds in the skin (Wright et al. 1996;
Andersson & Ekdahl, 2006).
The most notorious species of Vibrio is V. cholera which has caused eight pandemics since
1817 (Kaper et al. 1995). Cholera-outbreaks in Asia are often seasonal and start in several
locations simultaneously, indicating environmental factors as possible triggers (Kaper et al.
1995). However, the knowledge of what causes proliferation of vibrios in their natural
environment is limited.
The last few decades two other species of Vibrio have attracted attention, namely V. vulnificus
and V. parahaemolyticus, which are commonly occurring in fish and shellfish from coastal
waters (Wright et al. 1996; DePaola et al. 2003a; 2003b; Deepanjali et al. 2005). V.
vulnificus, which is always pathogenic, is responsible for the highest number of deaths in all
food-related bacterial infections (Todd, 1989), with mortality exceeding 50% (Wright et al.
1996).
The pathogenecity of V. parahaemolyticus varies depending on strain, but there are at least
two pathogenic strains that contain the major virulence factors; thermostable direct hemolysin
TDH and the related hemolysin TRH (DePaola et al. 2003b). In Japan, V. parahaemolyticus
accounts for about 70% of the gastroenteritis cases associated with seafood (Deepanjali et al.
2005). Gastroenteritis by V. parahaemolyticus is usually caused by ingestion of raw oysters
and most people recover after 3 days, but it can be severe for those with weakened immune
systems (CDC, 2008). A new pathogenic strain, O3:K6 possessing only the tdh-gene, has
emerged since 1996 and is thought to be responsible for the increased number of infection
cases by V. parahaemolyticus worldwide (DePaola et al. 2003b; Deepanjali et al. 2005).
There have been many studies focussing on what causes proliferation of V. parahaemolyticus
and other pathogenic Vibrio spp. in the environment (e.g. Wright et al. 1996; DePaola et al.
2003a; Thompson et al. 2004; Deepanjali et al. 2005). The abundance of potentially
pathogenic Vibrio spp. have been correlated with environmental variables like salinity
(Wright et al. 1996; DePaola et al. 2003a; Eiler et al. 2006), water temperature (Wright et al.
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1996; DePaola et al. 2003a; Thompson et al. 2004; Rehnstam-Holm & Collin, 2009), faecal
pollution (DePaola et al. 2003a), season (Wright et al. 1996; Thompson et al. 2004), location
(Wright et al. 1996; DePaola et al. 2003a) and living hosts, such as oysters (DePaola et al.
2003a) and copepods (Huq et al. 2005).
Many studies conducted in temperate coastal waters suggest that high water temperature
(Thompson et al. 2004) as well as low salinity favour the growth of Vibrio (Wright et al.
1996; DePaola et al. 2003a), although it might vary depending on the Vibrio species.
Thompson et al. (2004) found distinct warm-water and year-round populations in temperate
water, with a higher relative abundance of Vibrio during summer. This was due to correlations
between temperature and the year-round populations, and also because the warm-water
populations were only detected between 19 and 27.5 °C. The warm-water populations were
closely related to pathogenic vibrios of humans and marine fauna. This in accordance with
findings that Vibrio spp. causing disease in humans generally grow poorly in temperatures
below 17°C and die in temperatures exceeding 45°C (Rehnstam-Holm & Collin, 2009),
indicating that the risk of infection is greater in warmer water.
In contradiction, DePaola et al. (2003a) found that there was an inverse relationship between
water temperature and the occurrence of pathogenic strains of V. parahaemolyticus, with
pathogenic ones being more common in Alabama oysters during the winter, although this was
suggested to be due to a decreased total abundance of V. parahaemolyticus. During warm
weather V. parahaemolyticus multiplies in oysters (DePaola et al. 2003a) and it is possible
that it overwinters in a viable but nonculturable state in temperate water, either in association
with marine fauna, like oysters or copepods (Kaneko & Colwell 1975; Thompson et al. 2004),
or associated with sediments (Thompson et al. 2004). Wright et al. (1996) found a significant
correlation between salinity and concentration of V. vulnificus, but not during warmer months.
In tropical waters, Deepanjali et al. (2005) found that water temperature and salinity did not
significantly influence the abundance of V. parahaemolyticus. They detected V.
parahaemolyticus throughout the year, with higher levels during the dry, premonsoon season
(Jan-May) and lower abundance during postmonsoon months (July-November). Furthermore,
DePaola et al. (2003b) found no relationship between the pathogenic V. parahaemolyticus
population of the Pacific Coast and those of either the Atlantic or Gulf Coast, suggesting that
most V. parahaemolyticus strains are regional. This was based on a combination of serotyping
and ribotyping different V. parahaemolyticus isolates. Influences of environmental factors,
e.g. salinity and water temperature, on the abundance of V. parahaemolyticus are mainly
relevant in temperate waters. Therefore there must be additional factors that influence the
abundance of this bacterium in marine environments.
It has earlier been suggested that zooplankton blooms can trigger an outbreak of V. cholera
(Kaper et al. 1995). Many microorganisms associate with particles, depending on them as
sources of organic nutrients. Like V. cholera, V. parahaemolyticus is also known to adsorb to
the surface of copepods, where it degrades and utilizes the chitinous material by way of an
active chitinase enzyme (Kaneko & Colwell, 1975). Its association with copepods is
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considered to be one of the most important relationships determining its natural habitat. In an
ecological study it was found that association with chitin and copepods was essential for the
continuation of the annual cycle of V. parahaemolyticus in the Chesapeake Bay. Its capacity
in colonizing copepods could be due to competition with other chitinoclastic bacteria over an
ecological niche. Its adsorbtion capacity is inversely correlated with both pH and salinity
(Kaneko & Colwell, 1975).
One aspect that has been less considered in previous studies is potential associations with
phytoplankton, which might benefit the growth of marine Vibrio. The dynamics between
phytoplankton and bacteria are thought to be closely linked in coastal marine environments,
but little is known about the interaction between the two communities. Phytoplanktons excrete
extracellular organic carbon (EOC) into their immediate surroundings, creating phycospheres
(Grossart, 1999), thereby attracting heterotrophic bacteria. The EOC will be utilized by the
bacteria and then recycled into inorganic nutrients in the microbial loop (Azam et al. 1983).
Bacterial mineralization alone can supply algae with nutrients, but more important are
protozoans, like ciliates and flagellates, which graze upon the bacteria, regenerating nutrients
for the phytoplankton. Both phytoplankton and bacterial growth can be significantly higher
when incubated together (Grossart, 1999), but bacteria are, theoretically due to their larger
surface to volume ratio, stronger competitors for inorganic nutrients. Bacteria require
additional uptake of inorganic nutrients to grow on EOC. They can thus inhibit phytoplankton
growth when nutrients are scarce (Bratbak & Thingstad, 1985; Grossart, 1999).
Phytoplanktons have actually been found to excrete more EOC during nutrient depletion,
thereby stimulating their competitors while being stressed for nutrients (Bratbak & Thingstad,
1985). When EOC is the main carbon/energy source for the bacteria, a combination of
competition and commensalism might be expected. The bacteria are dependent on EOC from
the algal cells, therefore they cannot outcompete the algae altogether, and equilibrium is
established (Bratbak & Thingstad, 1985; Grossart, 1999). However, when incubated together
in an organic nutrient-rich medium, no equilibrium was observed, but a higher ratio of
heterotrophs/autotrophs (Grossart et al. 1999). There were no bacterivorous organisms
enabling rapid nutrient recycling present, which could further explain the decline in
autotrophs (Sherr et al. 1988).
Bacteria can be loosely or tightly associated with phytoplankton and this enables many
possible interactions. It has been found to range from mutualism, commensalism to parasitism
(Grossart, 1999). Some macroalgal genera are known to have a negative effect on bacterial
growth, possibly possessing some antibacterial trait to prevent biofouling (Hellio et al. 2000).
Phytoplanktons can also produce antibiotics, except they use it as a method of preventing
bacterial parasitism (Grossart, 1999). Stressed algae have a reduced capability of producing
these substances, with a following consequence of increased bacterial colonization (Grossart,
1999). For example, it has been demonstrated that the number of bacteria increases towards
the latter stages of an algal bloom (Rehnstam-Holm et al. accepted). The phytoplanktonbacteria interactions of the same species are thus not constant, but change in response to
environmental conditions. The nature of the interactions between these two communities can
4
therefore more or less control primary production, depending on the environmental factors
(Grossart, 1999; Grossart et al. 2005).
Because of these interactions, changes in the composition of the phytoplankton community
could influence the composition and density of the bacterial community (Rooney-Varga et al.
2005). It has been reported that different diatom species harbour distinct bacterial
communities and that phytoplankton- associated bacteria are significantly different from freeliving (Grossart et al. 2005). Bacterial abundance and community structure is dependent on
the species, growth and physiological state of even closely related algae. An algal species can
therefore be associated with a succession of different bacteria, adapted to the specific
environmental condition created by the alga at that particular stage of growth (Grossart et al.
2005). It is not known if Vibrio has a preference for any specific type of microalga, but Eiler
et al. (2006) have reported dinoflagellates as possible candidates. On the other hand it has
been reported that peaks in Vibrio abundance can be preceded by elevated numbers of
diatoms (Rehnstam-Holm et al. accepted).
Fisheries products can accumulate pathogenic bacteria, and an increased knowledge of the
coupling between bacterial/phytoplankton communities could be of large interest for local
fisheries to predict when it is unfavourable to harvest fisheries products from the marine
environment. By not harvesting during harmful bacterial blooms, the risk of humans being
infected through ingestion is diminished.
The aim of the present study, carried out in Mangalore, India, was to investigate if there are
any effects of phytoplankton community composition from the Arabian Sea on V.
parahaemolyticus. The purpose was to examine 1) if there in situ are any correlations between
environmental variables (such as temperature, salinity, pH, and nutrient concentrations) and
abundance of V. parahaemolyticus as well as other Vibrio spp. in water samples collected off
the coast of Mangalore. I wanted to test 2) a possible effect of the phytoplankton community
composition, from net samples collected at the same site, on the growth of an added V.
parahaemolyticus culture. Furthermore I sought to investigate 3) if V. parahaemolyticus
“prefer” to be associated with certain types of microalgae in a natural net sample community.
5
MATERIALS & METHODS
Sampling
Surface plankton net samples (mesh size 10µm) were collected with boat outside of the coast
of Bengre, Mangalore, India (Fig. 1) for six consecutive sampling dates during March-May
2009. At each sampling several hauls were taken and then pooled until the sample volume
reached a total volume of at least 100-150 cm3. These net samples were further used in the
experimental setup (see below). Water temperature was measured in field. Simultaneously
seawater was collected in a 5L container in order to enable investigation of the phytoplankton
community composition as well as the in situ hydrographic data of the sampling site. The
hydrographic data was also collected twice during the two weeks prior to the actual sampling.
Mangalor
e city
Bengr
e
Fig. 1. Samples were collected outside of the coast of Bengre.
In the laboratory
Measurements of in situ data
The following environmental variables were measured: pH (Digital pH Meter 335,
Systronics), salinity, concentrations of atomic phosphate-Phosphorus, ammonia-Nitrogen and
nitrate-Nitrogen. The salinity was measured using a refractometer. For phosphate-P and
ammonia-N measurements, subsamples of water was collected from the 5L container at the
laboratory, then fixated and further analyzed for their respective concentrations according to
the spectrophotometric method by Strickland & Parsons, 1972. The nitrate-N was measured
by a “Nitrate test in seawater” kit (Merck). The remains of the seawater was filtered (=FSW,
filter pore size 0.2µm) and then autoclaved to be used in the experiment described below.
To determine the phytoplankton community composition in the seawater samples a 100150mL of the sample was fixed with 1% Lugol. 25mL of Lugol-fixed samples (Throndsen et
al. 2003) were settled in sedimentation chambers (Utermohl, 1958) overnight, and the
phytoplankton community was characterized at 200× and 400× using a Zeiss Axiovert 135
6
inverted microscope. Species identification was based on Thomsen (1992), Throndsen et al.
(2003), Tomas (1997), and the Checklist of Phytoplankton in the Skagerrak–Kattegat,
available at www.smhi.se. Dimensions of all recorded dinoflagellate and diatom taxa were
measured, and biovolumes were calculated using formulae for the geometric shapes closely
approximating the taxa (Sun & Liu, 2003) in order to be able to normalize to the cell surface
of the algae. From these data the proportion of the biovolumes of each major group in
comparison to the total biovolumes of the major groups could be calculated. The observed
numbers of zooplankton were also recorded.
The collected net plankton sample was equally divided into 4 NUNC-flasks (20mL/flask) and
put on a rotary shaker until used in the experiment described below.
Experimental preparations
Labeling Vibrio parahaemolyticus with a CellTracking probe (CMFDA)
In order to study potential associations between Vibrio parahaemolyticus and naturally
occurring microalgae from the Arabian Sea, a CellTracking probe was used to be able to
visualize the living bacteria in an epifluorescent microscope. A green fluorescent
CellTracking probe (Invitrogen Molecular probes) that absorb and emit (abs. 492nm, em.
517nm) within the same spectral range as chlorophyll a was selected in order to visualize the
V. parahaemolyticus bacteria together with the phytoplankton. This made the bacteria appear
green and the phytoplankton red when observed in an epifluorescent microscope with the
filter sets used (Filter set 09, Zeiss Axiovert 40). The probe must be incorporated into the cell
membrane of a living cell in order to fluoresce. The V. parahaemolyticus cells were labeled
according to the following procedure: A stem solution (10mM) of the CellTracker was
prepared. The CellTracker was dissolved in DMSO and stored dark, aliquot 10µL in 20 pcrtubes. A culture of Vibrio parahaemolyticus (environmental isolate 24ST16 belonging to
serotype O4:K37, identified at College of Fisheries, Mangalore) was grown in LB-medium
(Luria Bertani Broth, Miller, HiMedia Laboratories Pvt. Ltd) with 3.5% NaCl at 35˚C in a
shaking incubator overnight (14-16h). The log-phase culture was transferred to 15mL Falcon
tubes and centrifuged 10min at 4300rpm 25°C (cooling centrifuge CPR 24, Remi). The
culture was washed once with 10mL filtered (filter pore size 0.2µm) seawater (FSW) and
vortexed thoroughly until the pellet was dissolved. The culture was centrifuged again, at the
same speed and temperature as above. A new 10mL of FSW was added, the tube was
vortexed and bacteria were let to acclimatize for one hour in room temperature (RT).
The optical density (OD, absorbance at 600nm) of the culture-sea water mixture was
measured and then diluted with FSW until OD 1 (UV-1601, Shimadzu). The bacteria were
further transferred to 2 x 1mL eppendorf tubes per treatment, totalling of 8 x 1mL per each
experiment. The tubes were centrifuged 3min at 15000g at 25°C (Biofuge stratus, Heraeus),
after which the supernatant was discarded and then mixed with 400µL FSW. 1µL of the
CellTracker-stemsolution was added to each tube. The tubes were incubated for 3h in
darkness at RT.
7
Following the labeling of the bacteria, they were washed with 1mL FSW and centrifuged four
times for 3min at 15000g at 25°C in between washings, after which they were pooled together
in one 15mL Falcon tube.
Experimental setup
Investigation of Vibrio-phytoplankton associations and Vibrio growth
An experiment was setup in order to investigate if the growth of V. parahaemolyticus was
affected by the composition of phytoplankton community and whether V. parahaemolyticus
was associated with any specific microalgae. In the experiment a total of three different
treatments was used: one with the net samples with natural plankton community from the
Arabian Sea together with the CellTracker probe labeled Vibrio parahaemolyticus
(V+P=Vibrio+Plankton) and two control treatments; one with only the net sample
(CP=CPlankton) as control for plankton development during the experiment and one with only
the fluorescent labeled Vibrio (CV=CVibrio) in autoclaved FSW as control for the Vibrio
development during the experiment. All treatments were duplicated. As mentioned above,
20mL of the collected net sample was added to the 4 NUNC-flasks used for the V+P and CP
treatments. In contrast, 20 mL of FSW was added to the 2 NUNC-flasks used for the CV
treatment.
At the start of the experiment 2 mL of the CellTracker fluorescent labeled Vibrio was added
to each NUNC-flask, except for the CP treatment where 2mL FSW was added. The treatments
were kept in RT, 12h of daylight (28.8 µEm -2s-1 constant, light intensity measured with lux
meter) and 12h night on a rotary shaker. At the start-point (t=0h) of each experiment, when
fluorescent labeled Vibrio was added, a sample was
taken from the NUNC-flasks by collecting 1.5mL from
each flask. The samples were fixed with 1% formalin.
After 24h (t=24h) the procedure was repeated. These
start- and stop samples, respectively, were then
examined in epifluorescence microscope (Zeiss
Axiovert 40, filter set 09, 200× and 400×
magnification). In addition, 0.1mL was collected from
each NUNC-flask at the start- (t=0h) and stop point
(t=24h) to be diluted for culturing on Vibrio-specific
TCBS-plates (Thiosulfate Citrate Bile salts Sucrose,
HiMedia Laboratories Pvt. Ltd). The following types
of dilutions were prepared in arranged eppendorf tubes
each containing 900 µL 3.5% NaCl: Undiluted, 10-1,
10-2 and so forth. 0.1 mL from each dilution was
Fig. 2. The TCBS agar plates were
spread on two replicates of TCBS agar plates, these
incubated for 20-24 h in RT.
were then incubated for 20-24h in RT awaiting
enumeration (Fig. 2). Two types of CFU were expected; green CFU, recorded as V.
parahaemolyticus and yellow ones, which were other Vibrio spp.
8
The number of colony forming units per mL (CFU/mL) of each type was calculated by
multiplying the number of CFU on the TCBS agar plate by 10 (for dilution N), 100 (for
dilution 10-1), 1000 (for dilution 10-2) and so on. This would give the number of CFU/mL of
V. parahaemolyticus as well as other Vibrio spp. The start- and stop samples from the NUNCflasks were further treated as described for the respective treatments below.
Treatment: CPlankton=CP
The number of algal cells was estimated in an epifluorescence microscope and the dominating
phytoplankton groups, i.e. dinoflagellates and diatoms, in the samples were recorded using a
1mL Sedgewick-rafter chamber containing 1000 squares (Fig. 3). The concentration of
phytoplankton cells per mL was calculated by counting
200 squares and multiplying the number of each group
by 5. The phytoplankton community composition and
abundance in the start sample (t=0h) was expected to be
proportional to the phytoplankton community
composition and cell concentration in the Lugol-fixed
sample.
The number of CFU/mL was decided from the plate
counts on TCBS-plates, which gave the naturally
occurring number of culturable Vibrio spp. in the
plankton samples that could be compared with the
added ones in the other two treatments.
Fig. 3. A diatom of the genus Odontella
observed in the Sedgewick rafter chamber.
Treatment: ControlVibrio=CV
The number of fluorescent free-living bacteria per mL was estimated in an epifluorescence
microscope using a 1mL Sedgewick-rafter chamber. 10 squares were counted. The number of
chains- and clusters per mL was also estimated in the same squares. A chain was defined as
≥three bacteria in a row and a cluster as a multitude of fluorescent bacteria packed so tightly
that they were hard to count individually.
The number of CFU/mL was decided from the plate counts on TCBS-plates and gave the
number of culturable added V. parahaemolyticus (glowing and non-glowing) in the FSW.
Treatment: Vibrio parahaemolyticus +Plankton = V+P
The number of algal cells was estimated in an epifluorescence microscope and the dominating
phytoplankton groups in the sample were recorded using a 1mL Sedgewick-rafter chamber.
The concentration of phytoplankton cells per mL was calculated in the same manner as for CP
above. The phytoplankton community composition and abundance in the start sample (t=0h)
was expected to be proportional to the total phytoplankton community composition and cell
concentration in the Lugol-fixed sample.
9
The number of glowing free-living bacteria was calculated in the same manner as for CV
above, whereof Z1 was associated with a phytoplankton cell of group 1 (i.e. dinoflagellates)
and Z2 was associated with a phytoplankton cell of group 2 (i.e. diatoms). Associated bacteria
were counted as the ones being within a 3 bars radius (7.5µm) on the ruler or appeared to be
within or on the surface of the algal cell in 400 × enlargements. The number of bacteria chains
and -clusters were counted in the same squares.
The number of CFU/mL was decided from the plate counts on TCBS-plates and gave the
naturally occurring number of culturable Vibrio spp. + the added number of culturable V.
parahaemolyticus in the plankton sample.
Calculations and statistics
The relationships between in situ abundances of Vibrio spp. and environmental variables were
analyzed by performing two-tailed Pearson correlation tests. The data was visually examined
for homogeneity of variances in boxplots and variables with skewed variances were either
square-root transformed or log transformed. Before the test, the correlations were carried out
with both the untransformed and transformed variables, although the transformations did not
affect the results. From the phytoplankton data in the water samples the proportion of the
biovolume of major groups were calculated and their in situ succession during the six
sampling dates were visually compared to the in situ succession of the natural occurring
Vibrio.
The Vibrio growth in the treatments was calculated as the percentage change in number of
Vibrio bacteria between the stop (t=24) and start (t=0) for both the colony forming units from
the plate counts on TCBS and for the free-living labeled bacteria in the epifluorescence
microscopy. To evaluate a possible effect of phytoplankton community composition, a paired
two-sided T-test was done to compare the growth of Vibrio spp. in the two treatments
containing added V. parahaemolyticus. A paired two-sided T-test was also made to
investigate a potential difference in preference for association with certain types of
phytoplankton by V. parahaemolyticus. The start- (t=0h) and stop (t=24h) concentrations of
clusters and chains of labeled V. parahaemolyticus observed in epifluorescence microscopy
were compared by performing paired two-sided T-tests. All calculations and statistics were
performed in Microsoft Office Excel 2007 and in the statistical software SPSS 17.0.
10
RESULTS
In situ data
The environmental variables measured in the surface seawater samples (Fig. 4) collected
outside of Bengre, Mangalore, between March and May 2009 showed the following
hydrographic configurations. The mean water temperature was 30.9°C during time of study.
The mean salinity was 35.2 psu, the highest value (38 psu) being measured on March 17. The
salinity generally declined throughout the course of the study. The mean pH was 8.04 with
min- and max values of 7.21 to 8.75, occurring less than two weeks apart. The ammonia-N
levels ranged from 1.10 to 8.21 with a mean of 3.74 µM atomic nitrogen. The minimum value
for ammonia-N occurred simultaneously as the maximum value for pH and vice versa.
Phosphate-P ranged from 0.06 to 0.11 µM atomic phosphorus during time of study. The
nitrate-N ranged from a high value of 42.84 µM atomic nitrogen on March 17 where after it
declined to below detection level during end of the study.
The abundance of Vibrio spp. (other than V. parahaemolyticus) ranged from approximately
2.2 x 103 to 1.2 x 105 CFU/ml in the surface water. The number of V. parahaemolyticus in the
surface water ranged from 1.8 x 103 to 4.0 x 104 CFU/ml, constituting between 2-53% of the
total number of culturable vibrios during time of study. The highest abundance of V.
parahaemolyticus as well as other Vibrio spp. was recorded in the beginning of April. The
total abundances of vibrios (i.e. V. parahaemolyticus + Vibrio sp.) and V. parahaemolyticus,
respectively estimated from six sampling occasions, were tested for correlation with each
individual environmental variable. The total abundance of vibrios was inversely significantly
correlated with ammonia-N (r=-0.87, Pearson correlation test) and there may also be a
positive correlation with pH (r=0.65, Pearson correlation test), although not statistically
shown in these results. The abundance of V. parahaemolyticus was significantly correlated
with pH (r=0.87, Pearson correlation test) and also ammonia-N may have a negative influence
(r=-0.51, Pearson correlation test) on the Vibrio parahaemolyticus abundance, although not
statistically significant (Table. 1).
Table 1. Correlations between measured hydrographic factors, total Vibrio abundance (V. parahaemolyticus+Vibrio sp.) as
well as V. parahaemolyticus abundance. Also observe the mean values and standard deviation for Vibrio and V.
parahaemolyticus.
*Correlation is significant at the 0.05 level (2-tailed).
Vibrio, Mean=71316,67
total Sd=63981.88
Pearson correlation
Sig. (2-tailed)
N
V. parahaemolyticus
Mean=16679.17
Sd=18182.62
Pearson correlation
Sig. (2-tailed)
N
Water
temp.
Salinity
-.255
.626
6
.124
.876
4
.646
.166
6
-.429
.396
6
.275
.725
4
.867*
.025
6
pH
AmmoniaN
PhosphateP
NitrateN
-.868*
.025
6
-.253
.629
6
-.399
.601
4
1
-.513
.298
6
.126
.812
6
-.371
.629
4
.792
.060
6
Vibrio
6
V. parahaemolyticus
.792
.060
6
1
6
11
Fig. 4. The hydrographic data from outside the coast of Bengre, Mangalore measured between March-May
2009. a) Mean water temperature=30.9 °C, sd=.611, n=8. b) Mean salinity=35.2 psu, sd=2.714, n=6. c) Mean
pH=8.04, sd=.518, n=8. d) Mean ammonia-N=3.74 µM at.N, sd=2.749, n=8. e) Mean phosphate-P=.08 µM
at.P, sd=.020, n=8. f) Mean nitrate-N=17.85 µM at.N, sd=14.099, n=6.
12
The proportion of dinoflagellate biovolume out of the total biovolume of both diatoms and
dinoflagellates together was also measured and displayed great similarities with the total
abundance of vibrios during time of sampling (Fig. 5). That is, the maximum and minimum
values, respectively, for the two graphs occur almost at the same time and overall are similar
in trend. The same trend was not visible for the proportion of diatoms out of the total
biovolume of these two groups of phytoplankton (data not shown).
A B Fig. 5. Graph A illustrating the natural abundance of vibrios in the net samples as the colony forming units per ml in
the start sample of the CP treatment (plankton control) plated on TCBS. Graph B displays the relative dinoflagellate
biovolume from the Lugol-fixed water samples during time of sampling. Observe that max- and min-values of the
respective graphs occurs almost simultaneously.
The zooplankton community composition was enumerated simultaneously as the
phytoplankton community. It was found that the abundance of naupliis correlated with
ammonia-N, although not statistically significant (r=0.65, Pearson correlation test, p=0.16).
The abundance of naupliis was inversely correlated with the total Vibrio abundance, but not
statistically shown in this study (r=-0.75, Pearson correlation test, p=0.09). No correlation was
found between the abundances of naupliis and V. parahaemolyticus (r=-0.28, Pearson
correlation test, p=0.60) (data not shown).
13
Growth
A possible difference in growth was tested between the control for V. parahaemolyticus, CV,
and the treatment containing both plankton sample and V. parahaemolyticus, V+P. It was
tested both by culturing on TCBS agar plates as well as by observations in epifluorescence
microscopy. For the TCBS agar plates, all but one sample of the CV treatment displayed a
negative growth; i.e. the bacterial count had generally declined after the 24h incubation. The
mean value of bacterial decline for CV was by 48% (sd=0.40%, n=6), with the only one
sample exhibiting a 24% increase in growth. The V+P samples all exhibited a negative
growth, with a mean value of 91% declination (sd=0.09%, n=6). No statistically significant
difference in growth was detected between the TCBS agar plates of the CV- and V+P
treatments (p=0.35, paired two-sided T-test). Also when analyzing with the epifluorescence
microscopy method, all investigated samples of CV and V+P displayed a negative growth. The
mean declines for CV and V+P were by 91% (sd=0.07%, n=6) and 94% (sd=0.05%, n=6),
respectively. No statistically significant difference in growth was detected (p=0.40, paired
two-sided T-test).
The chains and clusters of fluorescent V. parahaemolyticus in the CV- and V+P treatments
were also enumerated by epifluorescence microscopy. No significant difference was found in
neither the number of chains nor clusters between the CV and V+P from the different
sampling dates when analyzing the start samples (t=0h) of both treatments (chains: p=0.29
n=6; clusters: p=0.26 n=6). When analyzing the stop samples (t=24h) the number of clusters
(Fig. 6) in the two treatments were significantly different in respect of p<0.05 (p=0.04 n=6),
but there were no significant difference in the number of chains (p=0.93 n=6). The abundance
of clusters in the V+P stop samples exhibit similarities to the proportion of dinoflagellate
biovolume (out of the total biovolume of both diatoms and dinoflagellates together) and the
total abundance of vibrios during time of sampling (Fig 7, compare to fig. 5).
Fig. 6. Mean number of clusters in the stop samples of V+P (sd=568.38) and CV
(sd=165.54). p=0.04, difference is significant at p<0.05.
14
Fig. 7. The number of clusters in the V+P stop sample (t=24h). Notice
the similarities to the graphs depicted in Fig. 5).
Association with V. parahaemolyticus
The majority of phytoplankton enumerated was either dinoflagellates or diatoms. Diatoms
were dominant in all samples in respect to biovolume. The results from enumeration by
epifluorescence microscopy demonstrate that labeled V. parahaemolyticus show a
significantly higher affinity for dinoflagellates (p=0.035, paired two-sided T-test) over
diatoms when both genera were represented in the sample (Fig. 8). The declines of individual
dinoflagellates, enumerated by epifluorescence microscopy, were compared between the V+P
and CP samples. It was indicated that dinoflagellates incubated with V. parahaemolyticus in
the V+P treatment were declining more rapidly than the ones in the CP treatment during the
24h period, although not statistically significant (p=0.17, paired two-sided t-test).
Fig. 8. The proportion of phytoplankton cells within the two dominant genera associated
with V. parahaemolyticus out of the total (dinoflagellates+diatoms) recovered in the V+P
treatment.
15
DISCUSSION
In this study it was demonstrated that V. parahaemolyticus show a significantly higher
association to dinoflagellates over diatoms from the Arabian Sea. It was found that the
in situ abundance of vibrios follow the same trend as the proportion of dinoflagellates.
The environmental variables in the surface water outside Bengre, Mangalore was measured
during the Indian summer, just before the monsoon starts. Precipitation occurs more
frequently closer to monsoon, which was also the case this year (Fig. 9)
(www.wunderground.com/history/station/43284). This might explain why the latter salinity
values, during the sampling period, were lower. Water of less salinity could also have drifted
by. There is no data on salinity for the last two sampling dates; I can only speculate that those
values would have continued the trend of decreasing salinity. Water temperature generally
increased throughout the study, consistent with same period in 2002 measured by Deepanjali
et al. (2005). Precipitation could also have influenced the varying pH- and nutrient-records,
since the sampling location were quite close to shore and probably strongly influenced by
runoff. There could also have been raining in the north or at sea, and then this water could
have drifted by. Why the minimum level of ammonia-N occurred simultaneously as the
maximum value of pH and vice versa is yet to be explained. The correlation between
ammonia-N and pH was found to be inverse (r=-0.48, Pearson correlation test).
Fig. 9. Precipitation in Mangalore occurs more frequently closer to monsoon season.
Two environmental factors that earlier have been closely connected to abundance of Vibrio
are salinity and temperature, at least in temperate waters. In this study I did not find a
significant correlation between the total abundances of vibrios or V. parahaemolyticus with
neither salinity nor water temperature. This is consistent with results from Deepanjali et al.
(2005); water temperature and salinity do not significantly influence the abundance of V.
parahaemolyticus in tropical water. They studied the abundance of V. parahaemolyticus in
oysters along the SW coast of India, and the bacteria could be detected throughout most of the
year. Their sampling frequency was comparative to ours. According to their results the levels
16
were high during the dry season, highest in May, the levels then decreased during the
postmonsoon months. During this study, the highest abundance of total Vibrio and V.
parahaemolyticus in the seawater samples occurred in April and then declined towards
monsoon season. This study further supports the indications from Deepanjali et al. (2005);
that the abundance of V. parahaemolyticus displays a seasonal cycle in tropical water
different from that in temperate waters, where higher temperature and decreased salinity seem
to favour Vibrio spp. (Wright et al. 1996; DePaola et al. 2003a; Thompson et al. 2004;
Rehnstam-Holm & Collin, 2009). For example, DePaola et al. (2003a) only detected V.
parahaemolyticus in oysters wintertime (Dec-Mar) after enrichment, the levels were even
below detection (10 CFU per g oyster meat) at times. Additionally, Wright et al. (1996) found
declining numbers of V. vulnificus during the colder months in Chesapeake Bay, USA, using a
DNA probe specific for V. vulnificus.
Although there were no relationships between salinity and temperature with the abundance of
Vibrio, I did find correlations between Vibrio and other environmental parameters. The
abundance of V. parahaemolyticus displayed a significant correlation with pH (r=0.87,
Pearson correlation test) and the total abundance of vibrios also indicated a correlation
(r=0.65, Pearson correlation test), although not significant. According to Kaneko & Colwell
(1975), V. parahaemolyticus adsorb to the surface of copepods and the adsorbtion capacity is
inversely correlated with pH, that is; the adsorption of bacteria to copepods is higher when the
pH is lower. Since I found a positive correlation between the bacteria and pH, it makes sense
that the culturable bacteria were free-living, since the pH did not favour adsorbtion. However,
this is only speculation; the number of copepods was estimated, but I did not count the
number of copepods with associated V. parahaemolyticus.
The total abundance of vibrios was inversely correlated with ammonia-N (r=-0.87, Pearson
correlation test), a result opposite from that reported by Rehnstam-Holm et al. (accepted),
who found a positive correlation between ammonia and Vibrio spp. abundance. An
explanation to this could be because ammonia-N is consumed quickly when present.
The abundance of naupliis showed a tendency, not statistically significant, to correlate with
ammonia-N (r=0.65, Pearson correlation test, p=0.16). The reason for this possible correlation
is because nauplii faecals can dramatically raise the ammonia-N in the sample. Since naupliis
have a chitinous exoskeleton they would theoretically correlate with V. parahaemolyticus,
since this bacterium is chitinoclastic (Kaneko & Colwell, 1975), but there was no correlation
between the abundances of naupliis and V. parahaemolyticus in this study (r=-0.28, Pearson
correlation test, p=0.60). A possible inverse correlation was found between the abundance of
naupliis and the total Vibrio abundance, although not statistically significant (r=-0.75, Pearson
correlation test, p=0.09).
The in situ results from the plankton control sample CP, using TCBS-plates, showed that the
total abundance of vibrios (V. parahaemolyticus+Vibrio sp.) and the abundance of V.
parahaemolyticus follow the same trend as the proportion of dinoflagellates during time of
study. A delay of 3-14 days is often observed in coastal waters between the bloom of
17
phytoplankton and the peak of bacterial abundance (sited in Rehnstam-Holm et al. accepted).
This can explain why the maximum abundance of vibrios occurred one sampling (8 days)
after the peak of dinoflagellate abundance. In accordance with Eiler et al. (2006), I found that
the total abundance of vibrios and also the abundance of V. parahaemolyticus are correlated
with the abundance of dinoflagellates. Eiler et al. (2006) even found that it was correlated
with distinct groups of dinoflagellates.
Given that I only found indications that the dinoflagellates in the V+P treatment were
declining faster compared to the plankton control CP, it is difficult to speculate about the
nature of the relationship between the vibrios and dinoflagellates. Rehnstam-Holm et al.
(accepted) demonstrated that the number of bacteria increases towards the latter stages of an
algal bloom, suggesting that Vibrio were growing on the increased release of organic carbon
by decaying planktonic cells.
There was no statistically significant difference in growth between the treatments CV,
containing only V. parahaemolyticus and FSW, and V+P, containing both V.
parahaemolyticus, net sample and FSW. Irrespective of method of testing it was found that
the bacteria generally declined. In CV, containing only bacteria and FSW, the bacteria were
not growing well, possibly because of the lack of organic nutrients. In V+P the bacteria were
probably eaten by bacterivorous ciliates and flagellates, since I did not filter these samples to
avoid losing long chains of diatoms. Besides, there would have been obvious difficulties in
removing these protozoans, since diatoms, ciliates and bacterivorous flagellates are the same
size; filtering would have deprived us of all phytoplankton cells. Studies have indicated that
contact-feeding flagellates are never more than a few seconds away from a potential prey like
a bacterium (Sherr et al. 1988). I therefore assume that the bacteria were being eaten quite
effectively.
The TCBS agar plates are selective for the genus Vibrio, but there could have been other
bacteria growing on them, but only up to 5-10% (pers. comm. Ann-Sofi Rehnstam-Holm).
Wright et al. (1996) discovered that the recovery of V. vulnificus was found to be better on
nonselective medium compared to selective medium, such as TCBS agar. Perhaps this is part
of the explanation to the generally poor growth also for the V. parahaemolyticus on TCBS
agar plates during this study. Thompson et al. (2004) suggests that culture independent
molecular methods, like quantitative PCR (QPCR), are better suited to assess the true
abundance of coastal Vibrio populations, since the bacteria can shift to a viable but
nonculturable physiological state, possibly as a response to low temperature (Wright et al.
1996).
The significantly higher number of clusters in the stop samples of the V+P- compared to the
CV treatment is speculated to be due to some factor in the net plankton community that
favours clustering. Since the general appearance of fig. 7 is similar to the proportion of
dinoflagellates in fig. 5, dinoflagellates could be this factor. But, of course, there could also be
something else, like particles of organic matter.
18
The results from enumeration by epifluorescence microscopy demonstrate that V.
parahaemolyticus show a significantly higher association to dinoflagellates over diatoms from
the Arabian Sea. Eiler et al. (2006) found that the bacterium correlate with distinct groups of
dinoflagellates. Since I did not differ between different groups, I can only speculate that the
labeled V. parahaemolyticus were more prone to associate to some groups of dinoflagellates
over others.
Rehnstam-Holm et al. (accepted) found that the diatom genus Coscinodiscus was positively
correlated with Vibrio abundance while other diatoms were negatively correlated. Since I did
not differ between different diatom genera in this study, I can only speculate whether the
diatoms with associated labeled V. parahaemolyticus observed during this study might have
belonged to Coscinodiscus or any other diatom species that might favour Vibrio bacteria.
What I do know is that Coscinodiscus was represented in the collected seawater samples from
the two dates when I found diatoms with associated V. parahaemolyticus. Grossart et al.
(2005) demonstrated that free-living and phytoplankton- associated bacteria are significantly
different and dominated by distinct phylogenetic groups. They found that the diatomassociated bacteria were dominated by members of the Flavobacteria-Sphingobacteria group
of the Bacteroidetes phylum, whereas free-living bacteria predominantly belonged to the
Roseobacter group of α-Proteobacteria. The genus Vibrio belongs to the γ-Proteobacteria
(Rehnstam-Holm & Collin, 2009) which has been found to be one of the dominant groups of
free-living bacteria during a dinoflagellate bloom (Fandino et al. 2001). In this study
however, I found V. parahaemolyticus to be associated with dinoflagellates and I also found a
connection between high abundance of vibrios and high relative abundance of dinoflagellates
from the Arabian Sea.
Since it has been found that populations of V. parahaemolyticus have distinct geographical
distributions (DePaola et al. 2003b), it is possible that the different strains have adapted to
different floras of plankton. This could be an explanation to the great diversity worldwide in
its preference for associating with diverse microalgae.
With the new pathogenic strain of V. parahaemolyticus O3:K6 and increasing surface water
temperatures it is essential to improve the techniques of enumerating vibrios in the
environment and seafood. Faster and more sensitive methods are needed. Nowadays, efficient
methods like PCR and DNA probes are used to identify strains of V. parahaemolyticus
carrying the virulence factor TDH, produced by the tdh-gene. These methods are faster than
what was used before, namely the Kanagawa test (DePaola et al. 2003b). Deepanjali and coworkers (2005) states that the advantage with PCR over conventional isolation is that it can
distinguish between virulent and avirulent strains. Additionally, it also saves time. There is no
phenotypic test for TRH, the other virulence factor (Deepanjali et al. 2005). Previous studies
(DePaola et al. 2003a; Deepanjali et al. 2005) have tested the total and pathogenic V.
parahaemolyticus abundance, using methods like colony hybridisation with DNA probes and
PCR. In this study, I only tested the total abundance of the bacteria by the conventional
method of TCBS-plating and cannot make any statements as to how large a proportion the
pathogenic strain/-s might have constituted.
19
The threshold for shellfish consumption should be less than 10,000 V. parahaemolyticus cells
per g (FDA guidelines reproduced in Deepanjali et al. 2005). This level was exceeded during
May, September and October in the study conducted by Deepanjali and colleagues (2005).
Deepanjali et al. also showed that colony hybridisation could be a useful method for
enumeration of total and pathogenic V. parahaemolyticus in tropical oysters.
Bacteria have been discovered to modify the surface of diatoms, thereby affecting aggregation
and sinking, but also the persistence and intensity of algal blooms (Azam, 1998; Grossart et
al. 2006). Bacterial action on EOC can thus change ocean basin-scale carbon fluxes:
microbial loop, sinking, grazing food chain and carbon storage as well as carbon fixation.
Changes in bacteria-phytoplankton interactions due to environmental conditions such as
eutrophication and pollution might have huge impacts on global carbon cycling and also, in
the case of Vibrio, on human health.
20
ACKNOWLEDGEMENTS
Thanks to my supervisors: Ph D student Maria Asplund and Dr. Anna Godhe.
Thanks to all the employees at the College of Fisheries in Mangalore, India, and especially
Dr. Vijay Atanur, Prof. Indrani Karunasagar, Prof. M.N. Venugopal, Laboratory technician
Tejapal Puthran, Ph D student Krishna Kumar, Dr. Malathi Shekar, as well as all the helpful
students. Also huge thanks to Latish and Jeethan for providing boat and help during sampling.
21
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