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Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations
Harmful Algae 4 (2005) 201–209
Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations Hélène Hégaret, Gary H. Wikfors∗ NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA Received 12 September 2003; received in revised form 10 December 2003; accepted 26 December 2003
Abstract Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum. © 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve mollusk; Dinoflagellate; HAB; Harmful algae; Innate immunity
1. Introduction Direct trophic interactions between harmful algal blooms (HAB’s) and suspension-feeding mollusks suggest not only the potential for intense exposure to toxins, but also the expectation that mollusks would, in evolutionary time, develop tolerance mechanisms to recurrent HAB’s or face local extinction where ∗ Corresponding author. Tel.: +1-203-882-6525; fax: +1-203-882-6517. E-mail address: [email protected] (G.H. Wikfors).
blooms recur (Bricelj et al., 2000). After valve closure, the next line of defense in molluscan shellfish to noxious, toxic, or pathogenic agents is the immune system (Cheng, 1996), mediated by chemical agents and specialized cells called hemocytes that circulate throughout the animal’s body. Hemocytes do recognize and attempt to eliminate non-self particles within the open vascular system and tissues; however, the innate immune system cannot “remember” a prior experience with a harmful agent and protect the individual from subsequent exposures. Accordingly, the only means by which increased tolerance to HAB’s
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can develop in molluscan populations is by selective survival and reproduction of individuals with a more effective defense against the recurrent HAB. Most evidence of effects of HAB’s upon molluscan shellfish is from laboratory experiments (Landsberg, 2002), and it is unclear in most cases if the same effects are seen in nature. Moreover, the evidence of immune response of bivalves to HAB’s is only circumstantial, based upon histology (Wikfors and Smolowitz, 1993, 1995). Modern methods for assessing immune function in oysters were developed only in the past few years (see refs. in Hégaret et al., 2004). The fundamental purpose of this field study was to determine if there is an effect of natural or simulated (by adding cultured algae to natural plankton) blooms of harmful dinoflagellates, especially Prorocentrum minimum (Pavillard, Schiller), upon the immune response system of eastern oysters, Crassostrea virginica (Gmelin). This subject has not been studied previously under natural conditions; however, for comparison, controlled laboratory experiments presented in a separate report were done to determine the immune response of oysters and bay scallops in the absence of environmental variability (Hégaret and Wikfors, 2004). The approach in the present field study was to plant oysters in three coastal ponds that had experienced prior HAB’s, analyze pre-bloom immune characteristics of oysters, monitor the ponds’ phytoplankton, and analyze oyster immune system characteristics during any blooms of algae with recognized effects upon shellfish (specifically P. minimum). In addition, oysters were exposed to a simulated P. minimum bloom at one site by adding cultured algae to the natural plankton and analyzed for immune response. We also compared responses of two oyster populations—one with prior exposure to recurrent P. minimum blooms and the other without—under identical conditions of bloom exposure to see if there may have been selection for certain immune system characteristics by repeated HAB exposures. 2. Materials and methods 2.1. Sites Three sites on the northeast US coast were chosen, where harmful algal blooms had been documented
(Wikfors, unpublished phytoplankton monitoring data), in particular, summer Prorocentrum blooms that may interfere with hatchery and nursery production of scallops, clams, and oysters: Lagoon Pond in Martha’s Vineyard, MA; Eagle Pond in Greenwich, Connecticut; and Playland Lake in Rye, NY. All three sites are coastal ponds in which exchange with surrounding coastal sea water is restricted and, thus, hydraulic residence time is relatively high (estimated to range from 17 days in Lagoon Pond to 4 days in Eagle Pond). In previous years, several studies of the phytoplankton of Eagle Pond and Playland Lake showed seasonal occurrence of dinoflagellates that are harmful to shellfish, including oysters (Wikfors, unpublished phytoplankton monitoring data). No harmful algal blooms were found in Eagle Pond or Playland Lake during the present study; therefore, it was not possible to conduct bloom-effects studies at these two sites. The third site, Lagoon Pond, experienced a P. minimum bloom. Staff at the Martha’s Vineyard Shellfish Group (MVSG) hatchery have observed potentially harmful microalgae in Lagoon Pond annually, and the 2002 summer dinoflagellate bloom in Lagoon Pond was documented by Milford researchers in a collaborative project (Wilcox and Grunden, 2003). Accordingly, Lagoon Pond was an excellent site for studying the effects of harmful algae upon shellfish. 2.2. Experimental design 2.2.1. Martha’s Vineyard On 19 May 2003, we placed 300 oysters in wire-mesh cages suspended from the dock of the Martha’s Vineyard Shellfish Group Hatchery in Lagoon Pond. Approximately half of these oysters were progeny of oysters that had spent many generations in Lagoon Pond (native), and the other half were imported from a population in Maine (non-native), supplied by the Muscungus Bay hatchery and grown in waters where Prorocentrum blooms have not been observed (Tonie Simmons, personal communication) Three weeks later, from 9 to 13 June 2003, we conducted an experiment to establish baseline immune status in oysters and to simulate a bloom by adding cultured P. minimum to natural plankton. We randomly put 24 oysters of each type (native and non-native) in six different basins (six oysters of the same provenance in each basin) of flowing sea water containing natural
Component 2 (21.7%)
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209 0.51 0.31 0.11 -0.09
Oxi SH Oxi SG Mort Gran Oxi Gran Oxi LH Mort Hyal Phago Perc Hyal Mort SG Nb Hyal Perc SG Nb SG Nb Gran Agg Hem Perc Gran
-0.29 -0.37
-0.17
0.03
0.23
0.43
Component 1 (41.0%) Fig. 1. PCA plot of selected hemocyte-function and characteristics measurements, after an artificial exposure to the harmful alga P. minimum. Oxi.: respiratory burst; Mort.: hemocyte mortality; Agg.: aggregation, Phago.: phagocytosis; Hem: hemocytes; Gran.: granulocytes; Hyal.: hyalinocytes; SG: small granulocytes; SH: small hyalinocytes, and LH: large hyalinocytes.
phytoplankton, and 24 oysters of each type (native and non-native) in six different basins (six oysters of the same provenance in each basin) in flowing sea water containing natural phytoplankton to which we added a culture of P. minimum (strain JA-98-01) at a concentration 104 cells/ml (equivalent to previous natural blooms). Replicate basins containing oysters exposed to natural plankton, or natural plankton to which cultured P. minimum was added, were supplied with the same water from a reservoir with flow-controlled, gravity-fed silicone tubing inserts (‘octopus buckets’ depicted in Fig. 1 of Hégaret and Wikfors, 2004). After 2 days of exposure, we measured the immune status of the oysters using flow-cytometric methods. In early August, 2003, a natural bloom of P. minimum occurred in Lagoon Pond. Approximately 7 days after bloom initiation, we placed 24 oysters of each provenance (native and non-native) for 2 days (after grinding shell notches for later hemolymph extraction) in basins of flowing sea water containing the natural P. minimum bloom in the MVSG Hatchery. We analyzed the immune parameters of these oysters (four replicates for each provenance containing six oysters each) and compared results to oysters in pre-bloom conditions. 2.3. Monitoring of phytoplankton To ensure that detection of any dinoflagellate blooms, we monitored phytoplankton in water sam-
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ples collected at least weekly at each of the sites using light and epifluorescence microscope. Simultaneously, survival of the oysters in cages suspended in the ponds was evaluated weekly. 2.4. Feeding analyses Oyster feeding was measured by flow-cytometric measurements of algal cells in water samples withdrawn from basins containing feeding oysters; these measurements showed the clearance of algae by oysters in the basins. We also analyzed the feces and pseudofeces to determine the ingestion and digestion of the food. 2.5. Immunological analysis Analyses of hemocyte morphology and function were done with hemolymph extracted from the oysters. For extraction of hemolymph, a notch was ground in the shells’ ventral edges, and oysters were put back into sea water flow for 1 or 2 days to clear shell grit. Then the hemolymph was withdrawn from the adductor muscle of each individual oyster, using a needle and a 1 ml syringe. Hemolymph was stored temporarily in an Eppendorf micro-centrifuge tube on ice to retard cell clumping. For each experiment, hemolymph from four to six oysters was pooled (Hégaret et al., 2003b) for the flow cytometer analyses. Procedures for characterization of hemocytes were those of Hégaret et al. (2003a) and for function (mortality, phagocytosis, aggregation and oxidative burst) according to the methods described in Hégaret et al. (2003b). We used a FACScan (BD Biosciences, San Diego, CA; use of trade names does not imply endorsement by the National Marine Fisheries Service) flow cytometer for all hemocyte analyses. For these field experiments, the FACScan was transported to the experimental sites, i.e., the MVSG hatchery in Martha’s Vineyard and the Edith Read Wildlife Sanctuary near Playland Lake. Hematological parameters measured were: relative counts of the three or four sub-populations of hemocytes that could be resolved from cytometer plots, and numbers of cells detected during a set sampling time (as an estimate of hemocyte counts per ml). The five immune functions we measured were: (a) hemocyte characterization, which compared the size and the internal complexity of each type of
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(b)
(c)
(d) (e)
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hemocyte (small granulocytes, granulocytes, small hyalinocytes and large hyalinocytes); hemocyte viability/health, expressed as percentage of dead hemocytes (small granulocytes, granulocytes and hyalinocytes); aggregation and adherence ability of hemocytes (small granulocytes, granulocytes and hyalinocytes); phagocytosis of fluorescent beads by hemocytes; respiratory burst response in hemocytes (small granulocytes, granulocytes, small hyalinocytes and large hyalinocytes).
2.6. Statistical analysis Results of individual hemocyte analyses were analyzed statistically using ANOVA and multifactor analysis of variance (MANOVA). Data were further evaluated using principal components analysis (PCA) models developed in our previous research on effects of nutritional and temperature stresses upon oyster immune response (Hégaret et al., 2004). Statgraphics Plus statistical software (Manugistics Inc., Rockville, MD, USA) was used for statistical analyses.
3. Results
Table 1 Main effects of P. minimum “artificial bloom” or origin of the oysters upon each of the dependent variables tested by multifactor ANOVA Main effect
Types of hemocytes
Characterization with formolin Size Hemocytes Small granulocytes Granulocytes Hyalinocytes Complexity Hemocytes Small granulocytes Granulocytes Hyalinocytes Number Hemocytes Small granulocytes Granulocytes Hyalinocytes Percentage in Small each pop granulocytes Granulocytes Hyalinocytes Functions Aggregation
Viability
3.1. Simulated bloom of P. minimum Phagocytosis of hemocytes Respiratory burst
Small granulocytes Granulocytes Hyalinocytes Small granulocytes Granulocytes Hyalinocytes Hemocytes
Nutrition
Origin of oysters
∗
∗
NS
NS
∗
NS
∗
∗∗
∗
∗
NS
∗
∗
NS
∗ ∗ ∗∗ ∗ ∗
∗
NS ∗∗
NS NS
NS
∗
∗
NS
∗
NS
∗
NS
∗
∗
NS NS
NS ∗ ∗
NS NS NS
NS NS
NS
∗
3.1.1. Feeding analysis There was no mortality of oysters during this experiment. The feeding analysis demonstrated that the oysters fed on P. minimum seemed to ingest, but not digest the cells very well. Water analysis showed a high clearance in percentage of P. minimum, but whole cells were present in the feces and pseudofeces when observed on the flow-cytometer and by microscope, indicating poor digestion.
Non-significant effects are reported here with the initials NS. ∗ P < 0.05. ∗∗ P > 0.01.
3.1.2. Hemocyte analysis The first null hypothesis tested by ANOVA was “no effect of added P. minimum culture or of the provenance of the oysters upon each of the dependent variables”. Results of the main effects for these analyses are presented in Table 1. P. minimum significantly decreased the mortality and the aggregation of
the hemocytes and increased phagocytosis. Some immune function parameters were different depending upon oyster origin, with a lower phagocytosis and a higher aggregation and hemocyte mortality for the native oysters, as compared with non-native oysters. The effect of P. minimum on the respiratory burst of hemocytes was increase respiratory burst in native oysters;
Small granulocytes Granulocytes Small hyalinocytes Large hyalinocytes
NS ∗
NS NS
∗
NS
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whereas, the oysters from Maine were not affected. Analysis of Hemocyte morphology shows that numbers and percentages were highly affected by both the provenance of the oysters and the treatment to which they had been subjected. P. minimum tended to increase the number of hemocytes, especially granulocytes, resulting in a significant increase in percentage of granulocytes over hyalinocytes. Moreover, P. minimum triggered an increase in the size and the complexity of each population of hemocyte in both types of oysters. 3.1.3. Interactions of P. minimum with origin of oysters To test somewhat different null hypotheses concerning possible interdependence of origin of oysters and effects of P. minimum upon the same hemocyte measurements, we ran a multifactor ANOVA to analyse first-order interactions between P. minimum exposure and provenance of the oysters. Effect of P. minimum in this experiment had no statistical-significance (P > 0.05) interactions with oyster origin. 3.1.4. Principal components analysis The above ANOVAs yielded several significant differences for individual hemocyte functions, and additional, non-significant trends were apparent from plotted data. These observations suggested development of a “profile” of immune response in oysters exposed to P. minimum. We wanted to explore differences in hemocyte characteristics and function attributable to the exposure to P. minimum, and differences in response associated with source of oysters, by examining correlations between individual hemocyte measurements. Therefore, we ran a PCA for most hemocyte measurements, followed by a multifactor ANOVA with P. minimum exposure and origin of the oysters as factors and PCA Component 1 as the dependent variable. This approach was taken to determine if the combined hemocyte measurements could detect an effect of the provenance of the oysters or on what they were fed, to define a profile of P. minimum stress in the oysters, and to define this profile according to the origin of the oysters. The following immune functions were included in the PCA and are visualized on a plot (Fig. 1) defined by the first two principal components:
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(a) the numbers and percentages of each population of hemocytes (small granulocytes, granulocytes and hyalinocytes), (b) viability of small granulocytes, granulocytes and hyalinocytes, (c) aggregation, (d) phagocytosis of all hemocytes, (e) respiratory burst of small granulocytes, granulocytes and small and large hyalinocytes. We did not include the morphology (size and complexity) of the different hemocyte populations because we do not know what factors (oyster size, season, etc.) may alter these basic hematological characteristics. The PCA plot in Fig. 1 indicates that increases in the numbers of hemocytes and phagocytosis were associated with decreases in mortality and aggregation of hemocytes. A MANOVA was run with PCA Component 1 as the dependent variable, to compare profiles of hemocytes according to feeding and the origin of the oysters. Differences in combined immune functions attributed to P minimum (P < 0.01) or to the origin of the oysters (P < 0.05) were significant (Fig. 2). We can summarize the effect of the simulated bloom exposure to P. minimum as: an increase in the numbers of hemocytes, with the numbers of small granulocytes and granulocytes increasing more than the numbers of hyalinocytes (explaining the variation in the percentages). This exposure to P. minimum also resulted in an increase in hemocyte phagocytosis and decreases in aggregation and mortality of the several populations of hemocytes. We also show a difference between the native oysters and the oysters from Maine. Before the bloom, the oysters from Maine had a higher number of hemocytes, a higher phagocytosis index and a lower aggregation and mortality levels. First-order interactions between P. minimum and provenance of the oysters, with Component 1 from the PCA as dependant variable (Fig. 2), showed no significant combined effects. Most of the trends were similar for both types of oysters exposed to P. minimum, except for respiratory burst. Indeed, analyzing the two types of oysters individually with the same statistical method (PCA-MANOVA), revealed no significant (P > 0.05) effect of P. minimum on the respiratory burst of the non-native oysters (from Maine); whereas, it trig-
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209 Component 1 from the previous PCA
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5.4
Algae fed Natural plankton P. minimum
3.4 1.4 -0.6 -2.6 -4.6 Maine
Native
Oysters
Fig. 2. Interaction plot of nutrition/origin of the oysters with Component 1 from PCA of hemocyte characteristics as a dependant variable (±95% confidence interval). Effects of P minimum (Natural plankton > P. minimum, ANOVA P < 0.01) and origin of the oysters (Native > Maine, ANOVA P < 0.05) are significant.
gered an increase of 35% of the respiratory burst in the native oysters. 3.2. Natural bloom of P. minimum in Martha’s Vineyard In this analysis, we compared immune profiles of oysters held for 2 days (after grinding shells for hemolymph extraction) in basins receiving flowing sea water containing natural plankton, including a natural bloom of P. minimum, with oysters analyzed in June (before the bloom). This bloom had been present for approximately 7 days prior to the analysis reported here. 3.2.1. Phytoplankton analysis Samples of particles retained by a 5 m bag-filter were collected daily by MVSG staff, preserved in formalin, and mailed to the Milford Laboratory for phytoplankton analysis. These samples were not quantitative; particle concentration was not related to volume of water filtered, and smaller particles were less likely to be retained than larger particles. Nevertheless, the high frequency of sampling and high particle quantity offered an excellent opportunity to monitor the dominant and larger phytoplankton. In addition, on a weekly basis, whole-water samples preserved in formalin were collected as well, to relate bag-filter samples with water to which experimental oysters were exposed. On 7 July, 2003, P. minimum was present in essentially all bag samples at less than 20% of the phytoplankton community (generally much less). On 2 August, P. minimum became
the co-dominant phytoplankton taxon, accounting for 60% of the community retained by the bag-filter. The pinnate diatom, Rhizosolenia setigera (Brightwell), was co-dominant, representing about 40% of the phytoplankton captured. From 6–13 August, P. minimum and R. setigera remained co-dominant, with the percentage of P. minimum increasing to 70% during the final 2 days of the experiment (12–13 August). Counts of settled, whole-water samples collected on 6 and 13 August were 6.25 × 106 /l and 1.22 × 107 /l, respectively. Accordingly, P. minimum densities to which oysters were exposed in the natural bloom were in the same range as the cell density used in the previous, simulated bloom experiment. 3.2.2. Oyster analysis Mortalities of each type of oyster, native and non-native, in the cages sampled were 13% and 38%, respectively. All oysters placed in basins following shell grinding produced pseudofeces and fecal pellets containing whole cells of P. minimum, indicating both partial ingestion and digestion. 3.2.3. Hemocyte analyses PCA was used to establish combined immune profiles of the oysters. Indeed, we saw in the previous section that the PCA was a good way of summarizing all hemocyte characteristics and functions. This approach allowed us to determine if the combined hemocyte measurements could detect a difference according to the provenance of the oysters or the bloom, to (1) define an oyster immune system profile of stress caused by a natural bloom of P. minimum,
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209
Component 2 (23.3%)
Plot of Component Weights 0.53
Oxi Gran Oxi LH Oxi SG Oxi SH
0.33 0.13 -0.07
Nb Hyal Nb Gran Phago Perc Gran
Mort SG Mort Gran Perc Hyal Mort Hyal
Nb SG Perc SG
Agg Hem -0.27 -0.36
-0.16
0.04
0.24
0.44
Component 1 (43.3%) Fig. 3. PCA plot of selected hemocyte-function and characteristics measurements, after a natural bloom of the harmful alga P. minimum. Oxi.: respiratory burst; Mort.: hemocyte mortality; Agg.: aggregation; Phago.: phagocytosis; Hem: hemocytes; Gran.: granulocytes; Hyal.: hyalinocytes; SG: small granulocytes; SH: small hyalinocytes, and LH: large hyalinocytes.
Component 1 from the previous PCA
and (2) determine if this profile varied according to the origin of the oysters. Therefore, we ran a PCA, including all individual hemocyte parameters (numbers, percentages, viability, aggregation, phagocytosis, and respiratory burst) of both types of oysters (native and non-native from Maine). This PCA was followed by a multifactor ANOVA, with P. minimum exposure and origin of the oysters as treatment factors and the PCA Component 1 as the dependent variable. The PCA plot (Fig. 3) indicates that an increase in the numbers of hemocytes and of phagocytosis index was associated with decreases in mortality and aggre-
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gation of hemocytes. A MANOVA was run with Component 1 of this PCA as the dependent variable, to compare profiles of hemocytes according to the quality of the natural plankton and the origin of the oysters. Differences in combined immune characteristics attributable to P minimum (P < 0.01) or to the origin of the oysters (P < 0.05) were significant (Fig. 4). The natural bloom of P. minimum tended to increase the number of hemocytes. The numbers of small granulocytes and granulocytes increased more than the number of hyalinocytes, thereby explaining the variation in the percentages. The bloom of P. minimum was associated with an increase in hemocyte phagocytosis and decreases in aggregation and mortality of the several types of hemocytes. The immune response was also different according to the origin of the oysters. The oysters from Maine had a higher number of hemocytes, a higher phagocytosis and a lower aggregation and percentage of dead hemocytes than the native population. We also analysed first-order interactions between the origin of the oysters and the effect of the bloom with Component 1 from the PCA as dependant variable; no significant interaction was found.
4. Discussion There are no previous literature reports of immune system analyses of shellfish sampled during natural harmful algal events, and we discussed previously the literature relevant to the physiology of HAB effects
6.1
Oysters Maine Native
4.1 2.1 0.1 -1.9 -3.9 Natural plankton
P. minimum
Algae fed
Fig. 4. Interaction plot of bloom/origin of the oysters with Component 1 from PCA of hemocyte characteristics as a dependant variable (±95% confidence interval). Effects of P minimum (Natural plankton < P. minimum, ANOVA P < 0.01) and origin of the oysters (Native < Maine, ANOVA P < 0.05) are significant.
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upon molluscs (Hégaret and Wikfors, 2004). Changes in hemocyte profiles of oysters exposed to P. minimum in the natural and simulated blooms described in the present report are consistent with changes found in our previous, controlled laboratory experiments after several days of exposure (Hégaret and Wikfors, 2004), i.e., numbers of hemocytes, especially granulocytes, increased, as did phagocytosis and aggregation, while mortality of hemocytes decreased. The unique question addressed in the present study was whether or not the native and non-native oyster populations would react differently to P. minimum exposure under identical conditions. Populations of oysters from two sources, (1) native to the Lagoon Pond waters experiencing annual P. minimum blooms and (2) non-native and from waters with no recently-documented P. minimum blooms, were used in these field experiments to explore the possibility that the native oyster population had been selected for resistance to P. minimum. Examples of selection for locally-resistant grazer populations by repeated exposure to toxic algae have been reported for Daphnia feeding on cyanobacteria (Hairston et al., 2001), copepods feeding on the dinoflagellate Alexandrium (Colin and Dam, 2002), and soft-shell clams, Mya, feeding on Alexandrium (Bricelj et al., 2000). Survival and reproduction of Lagoon Pond oysters, following recurring P. minimum blooms, could be based upon more effective defense mechanisms against harmful effects of P. minimum. If these defense mechanisms involve hemocyte activity, we postulated that our measurements would detect this difference. These results show that Maine oysters constitutively had a hemocyte profile more similar to a P. minimum-stressed oyster than do the native, Martha’s Vineyard oysters (Fig. 4), i.e., circulating hemocyte counts were higher, especially granulocytes, aggregation and phagocytosis were higher, and mortality was lower in extracted hemocytes (Fig. 3). Challenging oysters from both populations with the same P. minimum concentration, whether as a natural or simulated bloom, resulted in essentially the same change in immune profile, seen as similar slopes in Fig. 4. From this, it appears that the different immune profile of Maine oysters in the absence of a bloom offers no adaptive advantage to P. minimum stress. The one parameter that differed significantly between native and non-native oysters is that an increase in respi-
ratory burst is stimulated by P. minimum in native oysters. This difference can be interpreted as a more effective defense response in native oysters, assuming the induced respiratory burst is effective in killing and disintegrating P. minimum cells that enter the tissues. Previous histological observations have shown the presence of P. minimum cells within the open vascular system of oysters (Wikfors and Smolowitz, 1995) and possible hemocyte responses (aggregation and phagocytosis) to these cells. Thus, our finding of increased respiratory burst in native oysters exposed to P. minimum, but not in non-native oysters, supports the hypothesis that the native oyster population has evolved a more effective defense response against P. minimum stress by increasing respiratory burst.
5. Conclusions 1. In this experiment, hemocyte-function profiles, incorporating several individual measurements in multivariate statistical models, were developed for oysters exposed to natural and simulated blooms of P. minimum as compared to oysters with no P. minimum. 2. The numbers of hemocytes per volume of hemolymph withdrawn from an oyster exposed to a natural or simulated bloom of P. minimum increased significantly. The numbers of cells from each sub-population of hemocytes increased, but the increase was less for hyalinocytes than for granulocytes and small granulocytes, explaining the decrease of the percentage of hyalinocytes while the percentages of the other sub-populations increased. The numbers of hemocytes and the change in their percentages seem to be the main parameters affected by the harmful alga P. minimum; however, changes in percentages of different hemocyte types were also associated with shifts in overall hemocyte function. 3. These changes in hemocyte profiles attributable to the natural and simulated P. minimum blooms in the present study and are in agreement with findings of controlled, laboratory experiments reported previously (Hégaret and Wikfors, 2004). 4. Concentrations of P. minimum that the oysters were subjected to, during the simulated and natural exposure, were approximately the same (104 cells/ml).
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Thus, we saw that the immune response of the oysters was affected the same way by both blooms. The simulated bloom of P. minimum seemed to be a good proxy for the natural bloom. 5. Maine oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance to the non-native oysters. 6. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.
Acknowledgements Tonie Simmons, Muscungus Bay hatchery, Maine; Ed Stillwagon, Greenwich Cove hatchery, CT; and Rick Karney, Martha’s Vineyard Shellfish Group hatchery, Oak Bluffs, Massachussetts, kindly donated animals used in these experiments, for which we are grateful. Denise Savageau, Town of Greenwich, Jason Klein, Edith Read Wildlife Sanctuary, Rye, NY, and Rick Karney helped us during these experiments and allowed us to use their facilities; we are very grateful for these contributions. We also want to thank Jennifer Alix, Barry Smith and Mark Dixon of the Milford Laboratory for their help during these experiments. This work was supported by the US–France Bilateral Agreement in Oceanography element, “Domestication of Bivalve Molluscan Shellfish,” through NOAA, OAR, International Activities; we thank Eileen Casabianca, Rene Eppi, and James McVey for their administrative and financial support of this program. References Bricelj, V.M., MacQuarrie, S., Twarog, B.M., 2000. Differential sensitivity and uptake of PSP toxins within and between softshell clam (Mya arenaria) populations from Atlantic
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Canada. In: Abstract Book, Harmful Algal Blooms. 9th Conference, Tasmania, Australia, 6–11 February. Cheng, T.C., 1996. Hemocytes: forms and functions. In: Kennedy, V.S., Newell, R.I.E., Eble, A.F. (Eds.), The Eastern Oyster Crassostrea virginica. Maryland Sea Grant, College Park, MD, USA, pp. 299–333. Colin, S.P., Dam, H.G., 2002. Latitudinal differentiation in the effects of the toxic dinoflagellate, Alexandrium spp. On the feeding and reproduction of populations of the copepod, Acartia hudsonica. Harmful Algae 1 (1), 113–125. Hairston Jr., N.G., Holtmeier, C.L., Lampert, W., Weider, L.J., Post, D.M., Fischer, J.M., Caceres, C.E., Fox, J.A., Gaedke, U., 2001. Natural selection for grazer resistance to toxic cyanobacteria: evolution of phenotypic plasticity. Evolution 55 (11), 2203–2214. Hégaret, H., Wikfors, G.H., Soudant, P., 2003a. Flow-cytometric analysis of hemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation. I. Haemocyte types and morphology. J. Exp. Mar. Biol. Ecol. 293, 237–248. Hégaret, H., Wikfors, G.H., Soudant, P., 2003b. Flow-cytometric analysis of hemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation. II. Hemocyte functions aggregation, viability, phagocytosis and respiratory burst. J. Exp. Mar. Biol. Ecol. 293, 249–265. Hégaret, H., Wikfors, G.H., 2004. Time-dependent changes in hemocytes of eastern oysters, Crassostrea virginica, and northern bay scallops, Argopecten irradians irradians, exposed to a cultured strain of Prorocentrum minimum. Harmful Algae, in press. Hégaret, H., Wikfors, G.H., Soudant, P., Delaporte, M., Alix, J.H., Smith, B.C., Dixon, M.S., Quéré, C., Le Coz, J.R., Paillard, C., Moal, J., Samain, J.-F., 2004. Immunological competence of eastern oysters, Crassostrea virginica, fed different microalgal diets and challenged with a temperature elevation. Aquaculture, in press. Landsberg, J.H., 2002. The effects of harmful algal blooms on aquatic organisms. Rev. Fish. Sci. 10 (2), 113–390. Wikfors, G.H., Smolowitz, R.M., 1993. Detrimental effects of a Prorocentrumisolate upon hard clams and bay scallops in laboratory feeding studies. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier, New York, pp. 447–452. Wikfors, G.H., Smolowitz, R.M., 1995. Experimental and histological studies of four life-history stages of the eastern oyster, Crassostrea virginica, exposed to a cultured strain of the dinoflagellate Prorocentrum minimum. Biol. Bull. 188, 313– 328. Wilcox, W.M., Grunden, D.W., 2003. Initial investigation of an annual Prorocentrum bloom in Lagoon Pond, Martha’s Vineyard. J. Shellfish Res. 22 (1), 303–304.
Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations Hélène Hégaret, Gary H. Wikfors∗ NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA Received 12 September 2003; received in revised form 10 December 2003; accepted 26 December 2003
Abstract Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum. © 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve mollusk; Dinoflagellate; HAB; Harmful algae; Innate immunity
1. Introduction Direct trophic interactions between harmful algal blooms (HAB’s) and suspension-feeding mollusks suggest not only the potential for intense exposure to toxins, but also the expectation that mollusks would, in evolutionary time, develop tolerance mechanisms to recurrent HAB’s or face local extinction where ∗ Corresponding author. Tel.: +1-203-882-6525; fax: +1-203-882-6517. E-mail address: [email protected] (G.H. Wikfors).
blooms recur (Bricelj et al., 2000). After valve closure, the next line of defense in molluscan shellfish to noxious, toxic, or pathogenic agents is the immune system (Cheng, 1996), mediated by chemical agents and specialized cells called hemocytes that circulate throughout the animal’s body. Hemocytes do recognize and attempt to eliminate non-self particles within the open vascular system and tissues; however, the innate immune system cannot “remember” a prior experience with a harmful agent and protect the individual from subsequent exposures. Accordingly, the only means by which increased tolerance to HAB’s
1568-9883/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2003.12.005
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can develop in molluscan populations is by selective survival and reproduction of individuals with a more effective defense against the recurrent HAB. Most evidence of effects of HAB’s upon molluscan shellfish is from laboratory experiments (Landsberg, 2002), and it is unclear in most cases if the same effects are seen in nature. Moreover, the evidence of immune response of bivalves to HAB’s is only circumstantial, based upon histology (Wikfors and Smolowitz, 1993, 1995). Modern methods for assessing immune function in oysters were developed only in the past few years (see refs. in Hégaret et al., 2004). The fundamental purpose of this field study was to determine if there is an effect of natural or simulated (by adding cultured algae to natural plankton) blooms of harmful dinoflagellates, especially Prorocentrum minimum (Pavillard, Schiller), upon the immune response system of eastern oysters, Crassostrea virginica (Gmelin). This subject has not been studied previously under natural conditions; however, for comparison, controlled laboratory experiments presented in a separate report were done to determine the immune response of oysters and bay scallops in the absence of environmental variability (Hégaret and Wikfors, 2004). The approach in the present field study was to plant oysters in three coastal ponds that had experienced prior HAB’s, analyze pre-bloom immune characteristics of oysters, monitor the ponds’ phytoplankton, and analyze oyster immune system characteristics during any blooms of algae with recognized effects upon shellfish (specifically P. minimum). In addition, oysters were exposed to a simulated P. minimum bloom at one site by adding cultured algae to the natural plankton and analyzed for immune response. We also compared responses of two oyster populations—one with prior exposure to recurrent P. minimum blooms and the other without—under identical conditions of bloom exposure to see if there may have been selection for certain immune system characteristics by repeated HAB exposures. 2. Materials and methods 2.1. Sites Three sites on the northeast US coast were chosen, where harmful algal blooms had been documented
(Wikfors, unpublished phytoplankton monitoring data), in particular, summer Prorocentrum blooms that may interfere with hatchery and nursery production of scallops, clams, and oysters: Lagoon Pond in Martha’s Vineyard, MA; Eagle Pond in Greenwich, Connecticut; and Playland Lake in Rye, NY. All three sites are coastal ponds in which exchange with surrounding coastal sea water is restricted and, thus, hydraulic residence time is relatively high (estimated to range from 17 days in Lagoon Pond to 4 days in Eagle Pond). In previous years, several studies of the phytoplankton of Eagle Pond and Playland Lake showed seasonal occurrence of dinoflagellates that are harmful to shellfish, including oysters (Wikfors, unpublished phytoplankton monitoring data). No harmful algal blooms were found in Eagle Pond or Playland Lake during the present study; therefore, it was not possible to conduct bloom-effects studies at these two sites. The third site, Lagoon Pond, experienced a P. minimum bloom. Staff at the Martha’s Vineyard Shellfish Group (MVSG) hatchery have observed potentially harmful microalgae in Lagoon Pond annually, and the 2002 summer dinoflagellate bloom in Lagoon Pond was documented by Milford researchers in a collaborative project (Wilcox and Grunden, 2003). Accordingly, Lagoon Pond was an excellent site for studying the effects of harmful algae upon shellfish. 2.2. Experimental design 2.2.1. Martha’s Vineyard On 19 May 2003, we placed 300 oysters in wire-mesh cages suspended from the dock of the Martha’s Vineyard Shellfish Group Hatchery in Lagoon Pond. Approximately half of these oysters were progeny of oysters that had spent many generations in Lagoon Pond (native), and the other half were imported from a population in Maine (non-native), supplied by the Muscungus Bay hatchery and grown in waters where Prorocentrum blooms have not been observed (Tonie Simmons, personal communication) Three weeks later, from 9 to 13 June 2003, we conducted an experiment to establish baseline immune status in oysters and to simulate a bloom by adding cultured P. minimum to natural plankton. We randomly put 24 oysters of each type (native and non-native) in six different basins (six oysters of the same provenance in each basin) of flowing sea water containing natural
Component 2 (21.7%)
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209 0.51 0.31 0.11 -0.09
Oxi SH Oxi SG Mort Gran Oxi Gran Oxi LH Mort Hyal Phago Perc Hyal Mort SG Nb Hyal Perc SG Nb SG Nb Gran Agg Hem Perc Gran
-0.29 -0.37
-0.17
0.03
0.23
0.43
Component 1 (41.0%) Fig. 1. PCA plot of selected hemocyte-function and characteristics measurements, after an artificial exposure to the harmful alga P. minimum. Oxi.: respiratory burst; Mort.: hemocyte mortality; Agg.: aggregation, Phago.: phagocytosis; Hem: hemocytes; Gran.: granulocytes; Hyal.: hyalinocytes; SG: small granulocytes; SH: small hyalinocytes, and LH: large hyalinocytes.
phytoplankton, and 24 oysters of each type (native and non-native) in six different basins (six oysters of the same provenance in each basin) in flowing sea water containing natural phytoplankton to which we added a culture of P. minimum (strain JA-98-01) at a concentration 104 cells/ml (equivalent to previous natural blooms). Replicate basins containing oysters exposed to natural plankton, or natural plankton to which cultured P. minimum was added, were supplied with the same water from a reservoir with flow-controlled, gravity-fed silicone tubing inserts (‘octopus buckets’ depicted in Fig. 1 of Hégaret and Wikfors, 2004). After 2 days of exposure, we measured the immune status of the oysters using flow-cytometric methods. In early August, 2003, a natural bloom of P. minimum occurred in Lagoon Pond. Approximately 7 days after bloom initiation, we placed 24 oysters of each provenance (native and non-native) for 2 days (after grinding shell notches for later hemolymph extraction) in basins of flowing sea water containing the natural P. minimum bloom in the MVSG Hatchery. We analyzed the immune parameters of these oysters (four replicates for each provenance containing six oysters each) and compared results to oysters in pre-bloom conditions. 2.3. Monitoring of phytoplankton To ensure that detection of any dinoflagellate blooms, we monitored phytoplankton in water sam-
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ples collected at least weekly at each of the sites using light and epifluorescence microscope. Simultaneously, survival of the oysters in cages suspended in the ponds was evaluated weekly. 2.4. Feeding analyses Oyster feeding was measured by flow-cytometric measurements of algal cells in water samples withdrawn from basins containing feeding oysters; these measurements showed the clearance of algae by oysters in the basins. We also analyzed the feces and pseudofeces to determine the ingestion and digestion of the food. 2.5. Immunological analysis Analyses of hemocyte morphology and function were done with hemolymph extracted from the oysters. For extraction of hemolymph, a notch was ground in the shells’ ventral edges, and oysters were put back into sea water flow for 1 or 2 days to clear shell grit. Then the hemolymph was withdrawn from the adductor muscle of each individual oyster, using a needle and a 1 ml syringe. Hemolymph was stored temporarily in an Eppendorf micro-centrifuge tube on ice to retard cell clumping. For each experiment, hemolymph from four to six oysters was pooled (Hégaret et al., 2003b) for the flow cytometer analyses. Procedures for characterization of hemocytes were those of Hégaret et al. (2003a) and for function (mortality, phagocytosis, aggregation and oxidative burst) according to the methods described in Hégaret et al. (2003b). We used a FACScan (BD Biosciences, San Diego, CA; use of trade names does not imply endorsement by the National Marine Fisheries Service) flow cytometer for all hemocyte analyses. For these field experiments, the FACScan was transported to the experimental sites, i.e., the MVSG hatchery in Martha’s Vineyard and the Edith Read Wildlife Sanctuary near Playland Lake. Hematological parameters measured were: relative counts of the three or four sub-populations of hemocytes that could be resolved from cytometer plots, and numbers of cells detected during a set sampling time (as an estimate of hemocyte counts per ml). The five immune functions we measured were: (a) hemocyte characterization, which compared the size and the internal complexity of each type of
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(b)
(c)
(d) (e)
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hemocyte (small granulocytes, granulocytes, small hyalinocytes and large hyalinocytes); hemocyte viability/health, expressed as percentage of dead hemocytes (small granulocytes, granulocytes and hyalinocytes); aggregation and adherence ability of hemocytes (small granulocytes, granulocytes and hyalinocytes); phagocytosis of fluorescent beads by hemocytes; respiratory burst response in hemocytes (small granulocytes, granulocytes, small hyalinocytes and large hyalinocytes).
2.6. Statistical analysis Results of individual hemocyte analyses were analyzed statistically using ANOVA and multifactor analysis of variance (MANOVA). Data were further evaluated using principal components analysis (PCA) models developed in our previous research on effects of nutritional and temperature stresses upon oyster immune response (Hégaret et al., 2004). Statgraphics Plus statistical software (Manugistics Inc., Rockville, MD, USA) was used for statistical analyses.
3. Results
Table 1 Main effects of P. minimum “artificial bloom” or origin of the oysters upon each of the dependent variables tested by multifactor ANOVA Main effect
Types of hemocytes
Characterization with formolin Size Hemocytes Small granulocytes Granulocytes Hyalinocytes Complexity Hemocytes Small granulocytes Granulocytes Hyalinocytes Number Hemocytes Small granulocytes Granulocytes Hyalinocytes Percentage in Small each pop granulocytes Granulocytes Hyalinocytes Functions Aggregation
Viability
3.1. Simulated bloom of P. minimum Phagocytosis of hemocytes Respiratory burst
Small granulocytes Granulocytes Hyalinocytes Small granulocytes Granulocytes Hyalinocytes Hemocytes
Nutrition
Origin of oysters
∗
∗
NS
NS
∗
NS
∗
∗∗
∗
∗
NS
∗
∗
NS
∗ ∗ ∗∗ ∗ ∗
∗
NS ∗∗
NS NS
NS
∗
∗
NS
∗
NS
∗
NS
∗
∗
NS NS
NS ∗ ∗
NS NS NS
NS NS
NS
∗
3.1.1. Feeding analysis There was no mortality of oysters during this experiment. The feeding analysis demonstrated that the oysters fed on P. minimum seemed to ingest, but not digest the cells very well. Water analysis showed a high clearance in percentage of P. minimum, but whole cells were present in the feces and pseudofeces when observed on the flow-cytometer and by microscope, indicating poor digestion.
Non-significant effects are reported here with the initials NS. ∗ P < 0.05. ∗∗ P > 0.01.
3.1.2. Hemocyte analysis The first null hypothesis tested by ANOVA was “no effect of added P. minimum culture or of the provenance of the oysters upon each of the dependent variables”. Results of the main effects for these analyses are presented in Table 1. P. minimum significantly decreased the mortality and the aggregation of
the hemocytes and increased phagocytosis. Some immune function parameters were different depending upon oyster origin, with a lower phagocytosis and a higher aggregation and hemocyte mortality for the native oysters, as compared with non-native oysters. The effect of P. minimum on the respiratory burst of hemocytes was increase respiratory burst in native oysters;
Small granulocytes Granulocytes Small hyalinocytes Large hyalinocytes
NS ∗
NS NS
∗
NS
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whereas, the oysters from Maine were not affected. Analysis of Hemocyte morphology shows that numbers and percentages were highly affected by both the provenance of the oysters and the treatment to which they had been subjected. P. minimum tended to increase the number of hemocytes, especially granulocytes, resulting in a significant increase in percentage of granulocytes over hyalinocytes. Moreover, P. minimum triggered an increase in the size and the complexity of each population of hemocyte in both types of oysters. 3.1.3. Interactions of P. minimum with origin of oysters To test somewhat different null hypotheses concerning possible interdependence of origin of oysters and effects of P. minimum upon the same hemocyte measurements, we ran a multifactor ANOVA to analyse first-order interactions between P. minimum exposure and provenance of the oysters. Effect of P. minimum in this experiment had no statistical-significance (P > 0.05) interactions with oyster origin. 3.1.4. Principal components analysis The above ANOVAs yielded several significant differences for individual hemocyte functions, and additional, non-significant trends were apparent from plotted data. These observations suggested development of a “profile” of immune response in oysters exposed to P. minimum. We wanted to explore differences in hemocyte characteristics and function attributable to the exposure to P. minimum, and differences in response associated with source of oysters, by examining correlations between individual hemocyte measurements. Therefore, we ran a PCA for most hemocyte measurements, followed by a multifactor ANOVA with P. minimum exposure and origin of the oysters as factors and PCA Component 1 as the dependent variable. This approach was taken to determine if the combined hemocyte measurements could detect an effect of the provenance of the oysters or on what they were fed, to define a profile of P. minimum stress in the oysters, and to define this profile according to the origin of the oysters. The following immune functions were included in the PCA and are visualized on a plot (Fig. 1) defined by the first two principal components:
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(a) the numbers and percentages of each population of hemocytes (small granulocytes, granulocytes and hyalinocytes), (b) viability of small granulocytes, granulocytes and hyalinocytes, (c) aggregation, (d) phagocytosis of all hemocytes, (e) respiratory burst of small granulocytes, granulocytes and small and large hyalinocytes. We did not include the morphology (size and complexity) of the different hemocyte populations because we do not know what factors (oyster size, season, etc.) may alter these basic hematological characteristics. The PCA plot in Fig. 1 indicates that increases in the numbers of hemocytes and phagocytosis were associated with decreases in mortality and aggregation of hemocytes. A MANOVA was run with PCA Component 1 as the dependent variable, to compare profiles of hemocytes according to feeding and the origin of the oysters. Differences in combined immune functions attributed to P minimum (P < 0.01) or to the origin of the oysters (P < 0.05) were significant (Fig. 2). We can summarize the effect of the simulated bloom exposure to P. minimum as: an increase in the numbers of hemocytes, with the numbers of small granulocytes and granulocytes increasing more than the numbers of hyalinocytes (explaining the variation in the percentages). This exposure to P. minimum also resulted in an increase in hemocyte phagocytosis and decreases in aggregation and mortality of the several populations of hemocytes. We also show a difference between the native oysters and the oysters from Maine. Before the bloom, the oysters from Maine had a higher number of hemocytes, a higher phagocytosis index and a lower aggregation and mortality levels. First-order interactions between P. minimum and provenance of the oysters, with Component 1 from the PCA as dependant variable (Fig. 2), showed no significant combined effects. Most of the trends were similar for both types of oysters exposed to P. minimum, except for respiratory burst. Indeed, analyzing the two types of oysters individually with the same statistical method (PCA-MANOVA), revealed no significant (P > 0.05) effect of P. minimum on the respiratory burst of the non-native oysters (from Maine); whereas, it trig-
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209 Component 1 from the previous PCA
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5.4
Algae fed Natural plankton P. minimum
3.4 1.4 -0.6 -2.6 -4.6 Maine
Native
Oysters
Fig. 2. Interaction plot of nutrition/origin of the oysters with Component 1 from PCA of hemocyte characteristics as a dependant variable (±95% confidence interval). Effects of P minimum (Natural plankton > P. minimum, ANOVA P < 0.01) and origin of the oysters (Native > Maine, ANOVA P < 0.05) are significant.
gered an increase of 35% of the respiratory burst in the native oysters. 3.2. Natural bloom of P. minimum in Martha’s Vineyard In this analysis, we compared immune profiles of oysters held for 2 days (after grinding shells for hemolymph extraction) in basins receiving flowing sea water containing natural plankton, including a natural bloom of P. minimum, with oysters analyzed in June (before the bloom). This bloom had been present for approximately 7 days prior to the analysis reported here. 3.2.1. Phytoplankton analysis Samples of particles retained by a 5 m bag-filter were collected daily by MVSG staff, preserved in formalin, and mailed to the Milford Laboratory for phytoplankton analysis. These samples were not quantitative; particle concentration was not related to volume of water filtered, and smaller particles were less likely to be retained than larger particles. Nevertheless, the high frequency of sampling and high particle quantity offered an excellent opportunity to monitor the dominant and larger phytoplankton. In addition, on a weekly basis, whole-water samples preserved in formalin were collected as well, to relate bag-filter samples with water to which experimental oysters were exposed. On 7 July, 2003, P. minimum was present in essentially all bag samples at less than 20% of the phytoplankton community (generally much less). On 2 August, P. minimum became
the co-dominant phytoplankton taxon, accounting for 60% of the community retained by the bag-filter. The pinnate diatom, Rhizosolenia setigera (Brightwell), was co-dominant, representing about 40% of the phytoplankton captured. From 6–13 August, P. minimum and R. setigera remained co-dominant, with the percentage of P. minimum increasing to 70% during the final 2 days of the experiment (12–13 August). Counts of settled, whole-water samples collected on 6 and 13 August were 6.25 × 106 /l and 1.22 × 107 /l, respectively. Accordingly, P. minimum densities to which oysters were exposed in the natural bloom were in the same range as the cell density used in the previous, simulated bloom experiment. 3.2.2. Oyster analysis Mortalities of each type of oyster, native and non-native, in the cages sampled were 13% and 38%, respectively. All oysters placed in basins following shell grinding produced pseudofeces and fecal pellets containing whole cells of P. minimum, indicating both partial ingestion and digestion. 3.2.3. Hemocyte analyses PCA was used to establish combined immune profiles of the oysters. Indeed, we saw in the previous section that the PCA was a good way of summarizing all hemocyte characteristics and functions. This approach allowed us to determine if the combined hemocyte measurements could detect a difference according to the provenance of the oysters or the bloom, to (1) define an oyster immune system profile of stress caused by a natural bloom of P. minimum,
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209
Component 2 (23.3%)
Plot of Component Weights 0.53
Oxi Gran Oxi LH Oxi SG Oxi SH
0.33 0.13 -0.07
Nb Hyal Nb Gran Phago Perc Gran
Mort SG Mort Gran Perc Hyal Mort Hyal
Nb SG Perc SG
Agg Hem -0.27 -0.36
-0.16
0.04
0.24
0.44
Component 1 (43.3%) Fig. 3. PCA plot of selected hemocyte-function and characteristics measurements, after a natural bloom of the harmful alga P. minimum. Oxi.: respiratory burst; Mort.: hemocyte mortality; Agg.: aggregation; Phago.: phagocytosis; Hem: hemocytes; Gran.: granulocytes; Hyal.: hyalinocytes; SG: small granulocytes; SH: small hyalinocytes, and LH: large hyalinocytes.
Component 1 from the previous PCA
and (2) determine if this profile varied according to the origin of the oysters. Therefore, we ran a PCA, including all individual hemocyte parameters (numbers, percentages, viability, aggregation, phagocytosis, and respiratory burst) of both types of oysters (native and non-native from Maine). This PCA was followed by a multifactor ANOVA, with P. minimum exposure and origin of the oysters as treatment factors and the PCA Component 1 as the dependent variable. The PCA plot (Fig. 3) indicates that an increase in the numbers of hemocytes and of phagocytosis index was associated with decreases in mortality and aggre-
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gation of hemocytes. A MANOVA was run with Component 1 of this PCA as the dependent variable, to compare profiles of hemocytes according to the quality of the natural plankton and the origin of the oysters. Differences in combined immune characteristics attributable to P minimum (P < 0.01) or to the origin of the oysters (P < 0.05) were significant (Fig. 4). The natural bloom of P. minimum tended to increase the number of hemocytes. The numbers of small granulocytes and granulocytes increased more than the number of hyalinocytes, thereby explaining the variation in the percentages. The bloom of P. minimum was associated with an increase in hemocyte phagocytosis and decreases in aggregation and mortality of the several types of hemocytes. The immune response was also different according to the origin of the oysters. The oysters from Maine had a higher number of hemocytes, a higher phagocytosis and a lower aggregation and percentage of dead hemocytes than the native population. We also analysed first-order interactions between the origin of the oysters and the effect of the bloom with Component 1 from the PCA as dependant variable; no significant interaction was found.
4. Discussion There are no previous literature reports of immune system analyses of shellfish sampled during natural harmful algal events, and we discussed previously the literature relevant to the physiology of HAB effects
6.1
Oysters Maine Native
4.1 2.1 0.1 -1.9 -3.9 Natural plankton
P. minimum
Algae fed
Fig. 4. Interaction plot of bloom/origin of the oysters with Component 1 from PCA of hemocyte characteristics as a dependant variable (±95% confidence interval). Effects of P minimum (Natural plankton < P. minimum, ANOVA P < 0.01) and origin of the oysters (Native < Maine, ANOVA P < 0.05) are significant.
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upon molluscs (Hégaret and Wikfors, 2004). Changes in hemocyte profiles of oysters exposed to P. minimum in the natural and simulated blooms described in the present report are consistent with changes found in our previous, controlled laboratory experiments after several days of exposure (Hégaret and Wikfors, 2004), i.e., numbers of hemocytes, especially granulocytes, increased, as did phagocytosis and aggregation, while mortality of hemocytes decreased. The unique question addressed in the present study was whether or not the native and non-native oyster populations would react differently to P. minimum exposure under identical conditions. Populations of oysters from two sources, (1) native to the Lagoon Pond waters experiencing annual P. minimum blooms and (2) non-native and from waters with no recently-documented P. minimum blooms, were used in these field experiments to explore the possibility that the native oyster population had been selected for resistance to P. minimum. Examples of selection for locally-resistant grazer populations by repeated exposure to toxic algae have been reported for Daphnia feeding on cyanobacteria (Hairston et al., 2001), copepods feeding on the dinoflagellate Alexandrium (Colin and Dam, 2002), and soft-shell clams, Mya, feeding on Alexandrium (Bricelj et al., 2000). Survival and reproduction of Lagoon Pond oysters, following recurring P. minimum blooms, could be based upon more effective defense mechanisms against harmful effects of P. minimum. If these defense mechanisms involve hemocyte activity, we postulated that our measurements would detect this difference. These results show that Maine oysters constitutively had a hemocyte profile more similar to a P. minimum-stressed oyster than do the native, Martha’s Vineyard oysters (Fig. 4), i.e., circulating hemocyte counts were higher, especially granulocytes, aggregation and phagocytosis were higher, and mortality was lower in extracted hemocytes (Fig. 3). Challenging oysters from both populations with the same P. minimum concentration, whether as a natural or simulated bloom, resulted in essentially the same change in immune profile, seen as similar slopes in Fig. 4. From this, it appears that the different immune profile of Maine oysters in the absence of a bloom offers no adaptive advantage to P. minimum stress. The one parameter that differed significantly between native and non-native oysters is that an increase in respi-
ratory burst is stimulated by P. minimum in native oysters. This difference can be interpreted as a more effective defense response in native oysters, assuming the induced respiratory burst is effective in killing and disintegrating P. minimum cells that enter the tissues. Previous histological observations have shown the presence of P. minimum cells within the open vascular system of oysters (Wikfors and Smolowitz, 1995) and possible hemocyte responses (aggregation and phagocytosis) to these cells. Thus, our finding of increased respiratory burst in native oysters exposed to P. minimum, but not in non-native oysters, supports the hypothesis that the native oyster population has evolved a more effective defense response against P. minimum stress by increasing respiratory burst.
5. Conclusions 1. In this experiment, hemocyte-function profiles, incorporating several individual measurements in multivariate statistical models, were developed for oysters exposed to natural and simulated blooms of P. minimum as compared to oysters with no P. minimum. 2. The numbers of hemocytes per volume of hemolymph withdrawn from an oyster exposed to a natural or simulated bloom of P. minimum increased significantly. The numbers of cells from each sub-population of hemocytes increased, but the increase was less for hyalinocytes than for granulocytes and small granulocytes, explaining the decrease of the percentage of hyalinocytes while the percentages of the other sub-populations increased. The numbers of hemocytes and the change in their percentages seem to be the main parameters affected by the harmful alga P. minimum; however, changes in percentages of different hemocyte types were also associated with shifts in overall hemocyte function. 3. These changes in hemocyte profiles attributable to the natural and simulated P. minimum blooms in the present study and are in agreement with findings of controlled, laboratory experiments reported previously (Hégaret and Wikfors, 2004). 4. Concentrations of P. minimum that the oysters were subjected to, during the simulated and natural exposure, were approximately the same (104 cells/ml).
H. H´egaret, G.H. Wikfors / Harmful Algae 4 (2005) 201–209
Thus, we saw that the immune response of the oysters was affected the same way by both blooms. The simulated bloom of P. minimum seemed to be a good proxy for the natural bloom. 5. Maine oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance to the non-native oysters. 6. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.
Acknowledgements Tonie Simmons, Muscungus Bay hatchery, Maine; Ed Stillwagon, Greenwich Cove hatchery, CT; and Rick Karney, Martha’s Vineyard Shellfish Group hatchery, Oak Bluffs, Massachussetts, kindly donated animals used in these experiments, for which we are grateful. Denise Savageau, Town of Greenwich, Jason Klein, Edith Read Wildlife Sanctuary, Rye, NY, and Rick Karney helped us during these experiments and allowed us to use their facilities; we are very grateful for these contributions. We also want to thank Jennifer Alix, Barry Smith and Mark Dixon of the Milford Laboratory for their help during these experiments. This work was supported by the US–France Bilateral Agreement in Oceanography element, “Domestication of Bivalve Molluscan Shellfish,” through NOAA, OAR, International Activities; we thank Eileen Casabianca, Rene Eppi, and James McVey for their administrative and financial support of this program. References Bricelj, V.M., MacQuarrie, S., Twarog, B.M., 2000. Differential sensitivity and uptake of PSP toxins within and between softshell clam (Mya arenaria) populations from Atlantic
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