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Fish & Shellfish Immunology
April 2006; 20(4) : 536-547
http://dx.doi.org/10.1016/j.fsi.2005.07.003
© 2005 Elsevier Ltd All rights reserved
Archimer, archive institutionnelle de l’Ifremer
http://www.ifremer.fr/docelec/
Effects of temperature and salinity on haemocyte activities
of the Pacific oyster, Crassostrea gigas (Thunberg)
Beatrice Gagnairea, Heloise Frouina, b, Kevin Moreaua,
Helene Thomas-Guyonb and Tristan Renaulta*
aLaboratoire de Génétique et Pathologie (LGP), Ronce-les-Bains, IFREMER La Tremblade, 17390 La Tremblade,
France
bLaboratoire de Biologie et Environnement Marin (LBEM), Université de La Rochelle, FRE 2727, Avenue Michel
Crépeau, 17042 La Rochelle, France
*: Corresponding author : Tel.: +33 5 4676 2649; fax: +33 5 4676 2611. trenault@ifremer.fr
Abstract: The Pacific oyster, Crassostrea gigas, is extensively cultivated and represents an important
economic activity. Oysters are reared in estuarine areas, subjected to various biotic and abiotic
factors. One of the limiting factors in aquaculture is mortality outbreaks, which may limit oyster
production, and the causes of these outbreaks are not completely understood. In this context, the
effects of temperature and salinity on Pacific oyster, C. gigas, haemocytes, were studied. Haemocytes
are the invertebrate blood cells and thus have been shown to be involved in defence mechanisms.
Flow cytometry was used for monitoring several haemocyte parameters. An increase of temperature
induced an increase of haemocyte mortality, in both in vitro and in vivo experiments. Temperature
modulated aminopeptidase activity. An in vitro decrease of salinity was associated with cell mortality.
During the course of in vivo experiments, an increase of phagocytic activity was reported at 15‰ and
50‰. Environmental physical parameters may modulate haemocyte activities.
Keywords: Pacific oyster; Crassostrea gigas; Haemocyte; Temperature; Salinity; Flow cytometry;
Cellular activity
Introduction
Shellfish farming represents an important economic activity around the world. Among
shellfish, the Pacific oyster, Crassostrea gigas, is the most cultivated species. In France, C.
gigas was introduced in the 1970s to replace the Portuguese oyster, C. angulata (1). France
ranks fourth worldwide in the production of C. gigas with 150 000 tons produced annually.
However, oyster production may be subjected to various limiting factors including mortality
outbreaks. For several decades now, French Pacific oyster livestocks have presented abnormal
mortality outbreaks during the summer period. This phenomenon called summer mortality has
also been reported in North America and in Japan since the 1940s (2-4). Authors hypothesised
that summer mortality outbreaks are the result of multiple factors, including elevated
temperatures, physiological stress associated with sexual maturation, aquaculture practices,
pathogens or pollutants (5). The Pacific oyster, C. gigas, is mostly reared in estuaries which
are continually contaminated by pollutants (6). Estuaries are also subjected to important
variations of abiotic environmental factors, including temperature and salinity. C. gigas is an
osmo- and thermo-conformer species (7). In oysters natural habitat, salinity fluctuates with
tidal cycles, rainfall and with drainage from adjacent terrestrial sites (8). In summer period,
temperature can reach high values. Oysters are sessile benthic animals and as such are
continually exposed to physico-chemical modifications of the environment. Physical stress
such as tidal exposure, which modify temperature and salinity, can affect marine invertebrate
defence mechanisms (9).
Bivalve defence mechanisms involve circulating blood cells, the so-called haemocytes (10).
In C. gigas, two types of haemocytes can be differentiated on the basis of morphological
features: hyalinocytes and granulocytes (10). Haemocytes constitute one of the main line of
defence against non-self particles. They are involved in phagocytosis and encapsulation of
foreign material (10-12). They also contain hydrolytic enzymes and produce reactive oxygen
3
species (ROS), which play a key role in pathogen degradation (13-15). They have been used
as immune capacity indicators in many bivalve species (16-19).
Studies have previously been conducted on the effects of temperature and salinity on bivalve
haemocytes (20-23). Since bivalves are both osmo- and thermo-conformers, haemolymph
readily acquires salinity and temperature of the external environment (7). In fact, haemocytes
found in haemolymph and in tissue sinuses are exposed to temperature and salinity variations
that occur in the environment. High water temperatures inhibit haemocyte spreading and
locomotion in the eastern oyster, C. virginica (Gemlin) (24) while variations of temperature
can also affect haemocyte counts and phagocytic activity in Ostrea edulis and Ruditapes
philippinarum (25, 26). On the other hand, elevated salinity increased the time for spreading
and reduced haemocytes locomotion towards target particles and may therefore pose an
additional stress (27) and may also reduce oyster defence capacities and leave them more
susceptible to parasites (27). Moreover, the susceptibility of C. virginica to the protozoan
parasites Perkinsus marinus and Haplosporidium nelsoni is influenced by temperature and
salinity (24, 28, 29).
In this study, the effects of temperature and salinity on Pacific oyster, C. gigas, haemocyte
parameters were investigated. Haemocytes were subjected in vitro to a range of temperatures
and salinity. In vivo experiments were also carried out by placing oysters in waters at defined
salinities or in incubators at controlled temperatures. Haemocyte parameters were monitored
using flow cytometry. This emerging tool has often been used in marine bivalve research to
describe haemocyte population characteristics (30, 31) or changes associated with pathology
or environmental stress (32, 33). Cell mortality, esterase, aminopeptidase activities and
phagocytic activity were monitored.
4
Material and Methods
Oysters
Eighteen month-old Pacific oysters, C. gigas, 7-10 cm in shell length, were produced in the
IFREMER hatchery in La Tremblade (Charente-Maritime, France). Temperature experiments
were conducted in April and May 2002 and salinity experiments were undertaken in July
2004. For both experiments, oysters were held in tanks receiving a constant flow of external
seawater.
Haemocyte collection
For temperature experiments, haemolymph was withdrawn from the pericardial cavity while
for salinity experiments, haemolymph was collected from the posterior adductor muscle sinus.
In both cases, a 1-mL syringe equipped with a needle (0.9x25 mm) was used. Haemolymph
samples were filtered on a 60 µm mesh to eliminate debris and were maintained on ice to
prevent aggregation. In order to reduce interindividual variation and to provide enough
haemocytes for experiments, haemolymph samples were pooled.. Haemocytes counts were
performed using a Malassez cell and the cell concentration was adjusted to 106 cells per mL
with artificial seawater (ASW: 23.4 g NaCl, 1.5 g KCl, 1.2 g MgSO4 4 H2O, 0.15 g CaCl2 2
H2O, 0.1.5 g CaCl2 anhydrous; H2O qsp 1L).
Effect of temperature on haemocyte parameters
Before each experiments, the oysters were acclimated in tanks receiving external seawater
(temperature and salinity were 11.6°C-17.7°C and 31.5 ‰-32.3 ‰).
In vitro experiments
After collection of haemocytes and resuspension in ASW, antibiotics (kanamycin,
erythromycin, oxolinic acid, 0.1 mg.mL-1) were added. Haemocytes were incubated for 2 h
5
and 4 h at varying temperatures (4°C, 11°C, 20°C, 25°C, 35°C, 40°C, 50°C and 60°C). Cell
mortality, phagocytic activity , esterase and aminopeptidase activities were monitored by flow
cytometry as described below. Experiments were carried out in triplicates.
In vivo experiments
Five oysters were emersed during 4 hours in incubators at different temperatures (4°C, 11°C,
20°C, 25°C, 35°C, 40°C, 50°C and 60°C). Haemolymphs were then withdrawn and pooled
from five oysters without any treatment. Cell mortality, phagocytosis, esterase and
aminopeptidase activities were analysed by flow cytometry as described below. Experiments
were carried out in triplicates.
Effect of salinity on haemocyte parameters
Before each experiments, the oysters were acclimated in tanks receiving external seawater
(temperature and salinity of external seawater were 18.7°C-19°C and 33.9 ‰-34.5 ‰).
In vitro experiments
After collection, haemocytes were divided into eight tubes, and centrifuged (10 min, 100 g,
4°C; Microfuge Beckman). The cells were resuspended in haemolymph only in tube 1. The
cells from tubes 2 to 7, were resuspended in a haemolymph-distilled water mixture in order to
obtain a range of decreasing salinity (tube 2: 29 ‰, tube 3: 25.5 ‰, tube 4: 22.5 ‰, tube 5:
16 ‰, tube 6: 6.5 ‰, tube 7: 3 ‰). The cells from tube 8 were resuspended in distilled water
(0 ‰).
Cell mortality was monitored using flow cytometry after 2 h and 18 h at 15°C. Experiments
were carried out four separate times.
6
In vivo experiments
In the first experiment, 40 oysters were placed in three tanks at 15 ‰ (hyposalinity), 35 ‰
(control) and 45 ‰ (hypersalinity), respectively. Hyposalinity was obtained by mixing 25 L
of seawater and 15 L of freshwater. Hypersalinity was obtained by the addition of 516 g of
aquarium sea salts (Instant Ocean Aquarium Systems, synthetic sea salts without nitrate and
phosphate, Haurit, Saintes, France) in 40 L of seawater. Oysters were fed with Chaetoceros
gracialis (3.109 cells per tank). Water and food were provided every day. Temperature of
external seawater was maintained between 18.7°C-19°C during the experiments which lasted
7 days. Sampling of ten oysters per tank were then performed at day 1, 3 and 7. Ten oysters
were also analysed at the beginning of the experiment. At each time, the oysters were divided
in three pools. Cell mortality and phagocytosis were analysed by flow cytometry as described
below.
A second experiment was conducted using the same protocol as previously described with the
following differences: :oysters were maintained at 5 ‰ (hyposalinity), 35 ‰ (control) and 60
‰ (hypersalinity). Hyposalinity was obtained by mixing 35 L of freshwater and 5 L of
seawater. Hypersalinity was obtained by adding 1219 g of aquarium sea salts (Instant Ocean
Aquarium Systems, synthetic sea salts without nitrate and phosphate, Haurit, Saintes, France)
in 40 L of seawater.
Flow cytometry analysis
The protocols used in the present study were previously described (34). For each sample, 3
000 events were counted using an EPICS XL 4 (Beckman Coulter). Results were depicted as
cell cytograms indicating cell size (FSC value) and cell complexity (SSC value) and the
fluorescence channel(s) corresponding to the marker used. Recorded fluorescence depended
on the monitored parameters: enzymatic activities and phagocytosis were measured using
7
green fluorescence while cell mortality was measured using red fluorescence. Mortality was
quantified using 200 µL of haemocyte suspension. Haemocytes were incubated in the dark for
30 min at 4°C with 10 µL of propidium iodide (PI, 1.0 mg.L-1, Interchim). Esterase and
aminopeptidase activities were evaluated with commercial kits (Cell Probe TM Reagents,
Beckman Coulter). Each analysis required 200 µL of haemolymph and 20 µL of the
corresponding kit (FDA•Esterase and A•Aminopeptidase M). Haemocytes were incubated in
the dark at ambient temperature 15 min for esterases and 30 min for aminopeptidases.
Phagocytosis was measured by ingestion of fluorescent beads. Two hundred µL of haemocyte
suspension were incubated 1 h in the dark at ambient temperature with 10 µL of a 1/10
dilution of Fluorospheres ® carboxylate-modified microspheres (diameter 1 µm, Interchim).
Statistical analysis
Results were expressed as percentage of positive cells. In order to detected an effect of tested
conditions, an ANOVA was performed using Statgraphics ® Plus version 5.1 software. To
ensure respect of a priori assumptions for normality and homogeneity, values were converted
into r angular arcsinus √ (% of positive cells) before analysis and .in the case of rejection of
H0, an a posteriori test was used. Significance was concluded at p ≤ 0.05.
8
Results
Temperature effects
In vitro experiments
After a 2 h incubation period, cell mortality significantly increased at 40°C (Figure 1,
p<0.001),while after a 4 h incubation period, mortality was significantly higher at 50°C and
60°C (Figure 1, p<0.001). Percentages of aminopeptidase positive cells were significantly
lower for both incubation periods at 50°C and 60°C (Figure 2). Percentages of esterase
positive cells were significantly lower after a 2h incubation period at 50°C and after a 4h
incubation period at 50°C and 60°C (Figure 3).
Figure 1: Haemocyte mortality percentage of oysters evaluated by flow cytometry after an in vitro 2 h or 4 h
incubation period at several temperatures (4°C, 11°C, 20°C, 25°C, 35°C, 40°C, 50°C and 60°C). Values are
mean of three replicates. Bars represent standard deviation. ***: p<0.001.
0
10
20
30
40
50
60
70
80
90
100
4°C 11°C 20°C 25°C 35°C 40°C 50°C 60°C
Temperature
Mortality (percentage)
2 h
4 h
***
***
***
*** ***
9
Figure 2: Percentage of positive cells for aminopeptidases of oysters evaluated by flow cytometry after an in
vitro 2 h or 4 h incubation period at several temperatures (4°C, 11°C, 20°C, 25°C, 35°C, 40°C, 50°C and 60°C).
Values are mean of three replicates. Bars represent standard deviation. *: p<0.05, ***: p<0.001.
0
10
20
30
40
50
60
70
80
90
100
4°C 11°C 20°C 25°C 35°C 40°C 50°C 60°C
Temperature
Aminopeptidases (percentage of
positive cells)
2 h
4 h
*
***
***
*
Figure 3: Percentage of positive cells for esterases of oysters evaluated by flow cytometry after an in vitro 2 h or
4 h incubation period at several temperatures (4°C, 11°C, 20°C, 25°C, 35°C, 40°C, 50°C and 60°C). Values are
mean of three replicates. Bars represent standard deviation. *: p<0.05, ***: p<0.001.
0
10
20
30
40
50
60
70
80
90
100
4°C 11°C 20°C 25°C 35°C 40°C 50°C 60°C
Temperature
Esterases (percentage of positive
cells)
2 h
4 h
***
***
***
10
In vivo experiments
Cell mortality significantly increased at 40°C, 50°C and 60°C (Figure 4, p<0.001). The
percentage of esterase positive cells was significantly lower at 4°C and 60°C compared to
other temperatures (Figure 4, p<0.001) and phagocytosis activity decreased at 60°C (Figure 4,
p<0.001).
Figure 4: Haemocyte mortality percentage, phagocytosis percentage and percentage of positive cells for
esterases of oysters evaluated by flow cytometry after an in vivo 4 h incubation period at several temperatures
(4°C, 11°C, 20°C, 25°C, 35°C, 40°C, 50°C and 60°C). Values are mean of three replicates. Bars represent
standard deviation. **: p<0.01, ***: p<0.001.
0
10
20
30
40
50
60
70
80
90
100
4°C 11°C 20°C 25°C 35°C 40°C 50°C 60°C
Temperature
Percentage
Mortality
Phagocytosis
Esterases
***
*** ***
***
***
***
Salinity
In vitro experiments
After a 2 h incubation period, cell mortality was significantly higher for lower salinities (6.5
‰, 3 ‰ and 0 ‰) (Figure 5, p<0.001) in contrast with the percentage of esterase positive
cells which was significantly lower (Figure 6, p<0.001). After a 18 h incubation period,
mortality was significantly higher for 3 ‰ and 0 ‰ (Figure 5, p<0.001) and esterase
11
percentage of positive cells was significantly lower for the same salinities (Figure 6,
p<0.001).
Figure 5: Haemocyte mortality percentage of oysters evaluated by flow cytometry after an in
vitro 2 h or 18 h incubation period at different salinities (32 ‰, 29 ‰, 25.5 ‰, 22.5 ‰, 16
‰, 6.5 ‰, 3 ‰ and 0 ‰). Values are mean of four replicates. Bars represent standard
deviation. ***: p<0.001.
0
10
20
30
40
50
60
70
80
90
100
32 ‰ 29 ‰ 25,5 ‰ 22,5 ‰ 16 ‰ 6,5 ‰ 3 ‰ 0 ‰
Salinity
Mortality (Percentage)
2 h
18 h
***
*** ***
*** ***
Figure 6: Percentage of positive cells for esterases of oysters evaluated by flow cytometry after an in vitro 2 h or
18 h incubation period at different salinities (32 ‰, 29 ‰, 25.5 ‰, 22.5 ‰, 16 ‰, 6.5 ‰, 3 ‰ and 0 ‰).
Values are mean of four replicates. Bars represent standard deviation. ***: p<0.001.
12
032 ‰ 29 ‰ 25,5 ‰ 22,5 ‰ 16 ‰ 6,5 ‰ 3 ‰ 0 ‰
Salinity
E
10
20
30
40
50
60
70
80
90
100
sterase (Percentage of positive cells)
2 h
18 h
*** *** *** ******
In vivo experiments
No mortality was noted during the first experiment. After one day, phagocytosis activity was
significantly lower in oysters placed in hyposalinity compared to the two other conditions
(Figure 7, p<0.05). After three and seven days, phagocytosis activity was significantly lower
in control oysters than in the two other conditions (Figure 7, p<0.01). Two-way analysis of
variance showed that phagocytosis activity was significantly lower in control oysters than in
oysters placed in hypersalinity (p<0.05). Cell mortality showed no variation in relation with
salinity condition (data not showed).
In the second experiment, a high daily mortality was reported in hypo- and hypersalinity
conditions (Figure 8). Oyster mortality appeared on day 3 for both conditions (15 % of
mortality in hyposalinity, 25.9 % of mortality in hypersalinity, Figure 8). Highest mortality
levels were observed on day 6 (55.5 % of mortality in hyposalinity, 66.6 % of mortality in
hypersalinity, 0 % of mortality in the control, Figure 8). Cell mortality and phagocytosis
activity showed no effect related to salinity conditions during the first three days of
experiment (data not shown).
Figure 7: Phagocytic activity of oysters evaluated by flow cytometry after an in vivo exposure to 15 ‰
(hyposalinity), 35 ‰ or 45 ‰ (hypersalinity) during seven days. Values are mean of three pools. Bars represent
standard deviation. *: p<0.05; **: p<0.01.
0
10
20
30
40
50
60
70
80
90
100
01234567
Days of experiment
Phagocytosis (percentage)
Hyposalinity
Hypersalinity
Control
**
*
*
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Figure 8: Daily mortality of oysters after an in vivo exposure to 5 ‰ (hyposalinity), 35 ‰ or 60 ‰
(hypersalinity) during seven days.
0
10
20
30
40
50
60
70
01234567
Days of experiment
Mortality (percentage)
Hyposalinity
Hypersalinity
Control
Discussion
In the present study, flow cytometry was used to demonstrate effects of two abiotic factors
(temperature and salinity) on haemocyte parameters of the Pacific oyster, C. gigas. Flow
cytometry has been shown to be an efficient tool for analysis of haemocytes in various
mollusc species (31, 33-35). However, relatively few studies have used this tool for
monitoring effect of temperature and salinity on haemocyte parameters (22, 36).
Pacific oysters were exposed to varying regimes of temperature and salinities in order to
assess their sensitivity to abiotic factors including temperature and salinity. In in vitro
experiments, we have demonstrated that while high temperatures induced hemocyte mortality,
hemocytes can tolerate a temperature of 35°C without presenting any mortality. In contrast, a
4 h in vivo incubation period at 40°C, 50°C and 60°C increased cell mortality of oysters.
Esterase activity was decreased at 4°C and 60°C and phagocytosis was decreased only at
60°C. This was consistant with the decreased phagocytosis activity of Ostrea edulis
haemocytes (25) and of C. virginica haemocytes (22, 36) as well as with the increased cell
mortality of C. virginica haemocytes (22, 36) following temperature elevation. However, the
14
decrease of enzymatic activities was predictable, as hemocytes presented mortality which is a
consequence of morbidity. In Marennes-Oleron Bay (Charente-Maritime, France), 4 hours
correspond to the time of exondation between low tide and high tide. The temperature in the
field may often reach 40°C during summer period (37). Since oysters often encountered these
extreme conditions, they may have become tolerant and thus the effect of high temperature
may have been masked. In addition, oysters are thermo-conformers and our results confirm
that oyster hemocytes can adapt to elevated temperatures.
In vitro decrease of salinity also induced an increase of cell mortality. However, as mentioned
before, the decrease of enzymatic activities was predictable, as hemocytes presented
mortality. Salinity variations also reduced haemocyte activities of C. virginica haemocytes in
other studies (24, 27). However, oyster haemocytes are able to synthesize osmotic shock
protein therefore protecting themselves from acute salinity variations (8). The relationships
between in vitro measurements of haemocyte activities and the ability of marine bivalves to
develop accurate defence in the field have not been established. In vitro measurements do not
represent in vivo or in situ conditions (25).
In vivo experiments involving salinity were also conducted. In the first experiment,
phagocytosis activity was enhanced at high both salinity (45 ‰) and low salinity (15 ‰).
Another study reported a decrease of phagocytosis in Ruditapes philippinarum associated
with an increase of salinity (38). These results indicate that salinity may modulate phagocytic
activity. In the second experiment, oysters were reared at 5 ‰ and 60 ‰, and both conditions
induced high mortality. However, those values are distant from the range of salinity reported
in Marennes-Oleron Bay (21-34 ‰), where C. gigas are reared (37). Oysters may not be able
to acclimate to high salinities. However, a part of oysters are reared in “claires” (oyster
pounds), a confined zone, where salinity may decrease to 15 ‰ after rainfalls (Soletchnik,
personal communication).
15
C. gigas have been successfully introduced in many countries over the world since the 1950s
(1, 39). We can therefore conclude that this species is naturally subjected to important
variations of environmental conditions and can acclimate to them. Moreover, our results
clearly demonstrated that only extreme values of temperature and salinity can modify
haemocyte activites of C. gigas. Haemocytes are apparently resistant cells because only high
values of temperature and salinity kill them. This phenomenon has already been observed
with pollutant exposure: only high concentrations of mercury chloride were able to kill
haemocytes after 4 h of in vitro contact (40).
These results could lead us to study possible interactions between the effects of abiotic factors
and pollutants or susceptibility to infections. Most of the studies conducted on abiotic factors
including temperature and salinity on bivalve defence functions pointed on to the
relationships between abiotic factors and diseases. The virulence of infectious agents in the
field has been correlated with high salinities and temperatures (41). Prevalence and intensity
of Perkinsus marinus in C. virginica is positively correlated to salinity (29) and to
temperature (42), suggesting that parasite virulence may be increased or oyster resistance may
be decreased at high salinities (43) and high temperatures.
Viral infection may also be influenced by temperature and salinity. In the hard clam, Meretrix
lusoria, birnavirus has proved to be more pathogen to young stages when a rapid increase of
water temperature occurs (44). Moreover, herpes virus affecting the Pacific oyster, C. gigas,
may exist as a latent form or low productive infection and temperature elevation can declare
the disease into the whole organism for larvae and spat (45, 46).
In vivo acute variations of temperature and salinity increase could temporarily affect the
ability of shellfish haemocytes to resist foreign invasion (25). Temperature and salinity appear
to be key factors modulating the host immune defence in invertebrates and influences the
16
severity of disease in several bivalve species, particularly during young stages (47). Their role
in massive mortalities affecting different invertebrate species of economic interest must be
taken into account. The interactions between temperature, salinity, pollutants and pathogens,
added with all other environmental factors (pH, dissolved oxygen) could represent scraps of
explanation of summer mortality phenomenon in C. gigas. In this context, experiments
studying relationships between modulation of abiotic factors and infectious agents (including
bacteria or OsHV-1) could be conducted in the future.
Acknowledgements
The authors are thankful to P. Goulletquer for allowing the work at the IFREMER station in La
Tremblade (Charente-Maritime, France). Thanks to Dr Thierry Burgeot for his advices. The
authors are grateful to Dr Sylvie St-Jean (Fisheries and Oceans Canada, Burlington, Ontario,
Canada) for improving the English of this manuscript. This research was supported in part by
the Poitou-Charentes Region.
17
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