Apoptosis of the protozoan oyster pathogen Perkinsus marinus in vivo and in vitro in the Chesapeake Bay and the Long Island Sound.

ABSTRACT Perkinsus marinus, a protozoan pathogen of the eastern oyster, Crassostrea virginica, infects oysters at high prevalences along the east coast of the United States. P. marinus was previously reported to be frequently apoptotic among the intestine epithelial cells in oysters collected from Long Island Sound. In this work, we study whether apoptotic activity of P. marinus cells is consistent with the distribution patterns of the parasite in the field in Long Island Sound and Chesapeake Bay. Prevalences and intensities of P. marinus infections were compared between Chesapeake Bay and Long Island Sound oysters during a 5-year period, from 1997 to 2001. In situ hybridization for apoptosis was performed on archived oyster histological tissues to detect differences in apoptotic indices (% of apoptotic P. marinus cells) between Chesapeake Bay and Long Island Sound oysters. Parasite apoptotic indices in Chesapeake Bay oysters were compared between different oyster habitat salinities. Two different P. marinus in vitro isolates, Chesapeake Bay isolate ATCC 50439 and Long Island Sound isolate ATCC 50508 were grown in cell cultures and exposed to different temperatures and salinities for 24 h. In situ hybridization assays for apoptosis were performed on cytospin preparations of the exposed cell cultures. During the five-year-period, the prevalences and intensities of P. marinus infections were significantly higher in Chesapeake Bay oysters. There was a significant increase in the prevalences and mean intensities of P. marinus infections in Chesapeake Bay oysters between the periods 1997-1998 and 1999-2001. This was largely because of increases in infection prevalences and mean intensities in Chesapeake Bay oysters from the low-salinity zone, where actual salinities and P. marinus associated disease in oysters were elevated by extended drought conditions during 1999-2001. Such a trend was not observed in Long Island Sound or in the higher-salinity zones of Chesapeake Bay. There was significantly more apoptosis of P. marinus in oysters from lower salinities than in those from higher salinities in Chesapeake Bay. Although temperature and salinity during a 24-h in vitro exposure affected apoptosis in both strains of P. marinus, the apoptosis dynamics significantly differed between the two P. marinus isolates with changes in salinity (11.6 [per thousand] to 37.8 [per thousand]), but not temperatures (4[degrees]C to 35[degrees]C). The Chesapeake Bay isolate had an immediate decline in apoptosis at salinities above 11.6 [per thousand], and its apoptotic indices were low throughout the tested salinity range. The Long Island Sound isolate had high apoptosis at all salinities except 28 [per thousand], which is the approximate salinity where the Long Island Sound oysters are grown. We conclude that parasite apoptosis is an important factor regulating the distribution of P. marinus infections in the field. Our results suggest that Chesapeake Bay and Long Island Sound P. marinus strains may have evolved distinct genetic or phenotypic traits. The Long Island Sound strain reflects its adaptation to high-salinity oyster hosts, whereas the Chesapeake Bay strain possibly reflects adaptation to oyster hosts from low and variable estuarine salinities.

KEY WORDS: apoptosis, eastern oyster, Crassostrea virginica, salinity, temperature, Long Island Sound, Chesapeake Bay, Perkinsus marinus

INTRODUCTION

The population of eastern oysters, Crassostrea virginica, on the east coast of the United States has been severely reduced by 2 protozoan parasites, Perkinsus marinus and Haplosporidium nelsoni (MacKenzie 1996, Andrews 1988, Andrews & Ray 1988, Burreson & Calvo 1996). The Long Island Sound and the Chesapeake Bay are two major historical areas for harvesting the eastern oyster. In the early 1900s, the Long Island Sound produced 2 million bushels of seed and market oysters annually, and the Chesapeake Bay nearly 20 million bushels of oysters, which were about 60% of North America's total oyster harvest (MacKenzie 1996). The population of the eastern oyster in the Chesapeake Bay has declined by 95% since 1980, because of the combined effects of diseases and overharvesting (Kennedy 1991, MacKenzie 1996).

Perkinsus marinus was first identified as a pathogen of the Gulf of Mexico oysters about 50 y ago (Mackin et al. 1950), and was quickly also found in oysters throughout the southeastern USA (Ray 1954). P. marinus was first reported in the Long Island Sound in the 1990s (Ford 1996). Temperature and salinity are key elements regulating the prevalence and intensity of P. marinus infections (Andrews 1996). Recently, unusual warming trends allowed the parasite to spread to a wider range, including the USA northeast coast (Ford 1996). In the Chesapeake Bay, P. marinus extended its distribution when salinities were higher than normal during 1980s drought periods (Burreson & Calvo 1996), and widespread infections increased in both prevalence and intensities during a 1999-2002 extended drought period (Ragone Calvo & Burreson 2003, Tarnowski 2003).

The disease presentation and the mortality associated with P. marinus infections in the Chesapeake Bay and Long Island Sound are quite different, with major oyster mortalities and associated poor meat condition in the Chesapeake Bay but not in Long Island Sound (Sunila 1998). In the Long Island Sound, most oyster meats were healthy by macroscopic examination even when they were infected with P. marinus (Karolus et al. 2000). Production statistics further support a possible reduced pathogenicity of P. marinus in Long Island Sound, with a record oyster production in 1995 (over 750,000 bushels) after P. marinus had already thoroughly established itself there (Sunila 1998). In 1995, Long Island Sound accounted for 94% of all cultured oysters in the United States northeast, whereas the Maryland portion of Chesapeake Bay only produced 1% (Spatz et al. 1996).

Many P. marinus cells in infected oysters reside within hemocytes as intracellular parasites. Oyster death may ensue if abundant parasites deplete the host's energy reserves, cause tissue lysis, or occlude major hemolymph vessels (Ford & Tripp 1996). Recent P. marinus in vitro isolates from diverse geographic oyster sources afford tools to investigate pathogen virulence mechanisms, and to compare isolates for functional genetic and physiologic differences (LaPeyre et al. 1996). Such in vitro isolates include American type culture collection (ATCC) isolate 50439 from mesohaline Chesapeake Bay oysters (Dungan & Hamilton 1995) and ATCC 50508 from high-salinity Long Island Sound oysters (Reece et al. 2001). During their in vitro cell cycle, P. marinus merozoite cells subdivide internally by schizogony, producing multiple daughter cells, which enlarge until the mother cell ruptures releasing the daughter cells that enlarge to repeat the process (Sunila et al. 2001).

Apoptosis is a genetically regulated form of cell death (Cohen et al. 1992). It is functionally and morphologically different from necrosis, where cell membranes lose integrity with a resulting loss in ATP production. Necrotic cells are pale and swollen with disrupted nuclear membranes; whereas apoptotic cells undergo shrinkage, chromatin condensation, and nuclear blebbing (Kerr 1971, Sanderson 1982). The nuclear blebs are composed of 180 to 200 base pair DNA fragments that have been cleaved to that size by apoptosis-specific endonucleases (Arends et al. 1990). Apoptotic cells break into apoptotic bodies that are phagocytosed to protect surrounding tissues from intracellular contents (Cohen et al. 1992). Both hosts and pathogens--protozoa, bacteria and viruses-may use apoptosis. Many intracellular pathogens have evolved mechanisms to manipulate host cell apoptosis to their apparent advantage (Heussler et al. 2001). Pathogens may inhibit host cell apoptosis to create a supportive intracellular environment for enhanced replication, or may increase host cell apoptosis with the target of destroying immune cells and/or inducing systemic infections (Heussler et al. 2001, Moss et al. 1999).

There are few publications documenting apoptosis in bivalves or in their parasites. These include an ultrastructural description of apoptotic cell morphology in herpes-like virus-infected larval Crassostrea gigas and Ostrea edulis oysters (Renault et al. 2000). Terahara et al. (2003) induced apoptosis of C. gigas hemocytes with Arg-Gly-Asp (RGD) peptide, a peptide well known to induce apoptosis in many types of cells. Lacoste et al. (2002) induced apoptosis of C. gigas hemocytes by noradrenaline exposure, and also studied signaling pathways in C. gigas, which suggested that apoptosis was induced by a [beta]-adrenoceptor-mediated activation of adenylate cyclase, and a cAMP-dependent protein kinase. Sunila and LaBanca (2003) described high apoptotic indices of P. marinus cells in Long Island Sound C. virginica oysters by in situ hybridization assays. Sunila and LaBanca (2003) also documented a decrease in P. marinus- infected oyster hemocyte apoptosis, and suggested that the parasite may inhibit hemocyte apoptosis to provide more host cells for itself. Goedken et al. (2005a) infected C. virginica hemocytes in vitro with P. marinus and observed suppression of hemocyte apoptosis by flow cytometry. Using flow cytometry, Goedken et al. (2005b) studied the effects of temperature and salinity on apoptosis of C. virginica hemocytes and P. marinus, after in vitro incubations. Temperature did not significantly affect hemocyte or parasite apoptosis frequencies within the range studied, but apoptosis of P. marinus was significantly higher in cultures at the lower salinity than at the higher salinity.

Sunila and LaBanca (2003) observed high apoptotic indices of P. marinus in oysters sampled from Long Island Sound, where P. marinus infections are not associated with significant oyster mortalities. In this study we compared parasite apoptosis in Long Island Sound and Chesapeake Bay oysters during different seasons and from different salinities to determine whether parasite apoptosis effects under different environmental conditions are consistent with distribution patterns of P. marinus infections in the field, and with apparent differences in P. marinus virulence between these regions. Apoptotic indices among Chesapeake Bay and Long Island Sound P. marinus were calculated after propagating the parasites in vitro at different temperatures and salinities encountered in these geographical areas.

MATERIAL AND METHODS

Comparison of Intensity and Prevalence of Perkinsus marinus Infections in the Chesapeake Bay and Long Island Sound Oysters

Archived Perkinsus marinus infection prevalence and intensity data from Chesapeake Bay and Long Island Sound fall surveys from 1997 to 2001 were supplied by the State of Connecticut, Bureau of Aquaculture and the Maryland Department of Natural Resources (DNR), Cooperative Oxford Laboratory. Chesapeake Bay has a salinity gradient with high salinity seawater at the mouth of the Bay and the salinity gradually decreasing toward the upper Bay and turning into fresh water in the upper reaches of tributary rivers. Maryland DNR has divided the Maryland portion of the Bay into three geographic salinity zones. The zones are delineated spatially using 10-y average summer salinity data measured at a depth range of 10-20 feet: Zone 1, low-salinity 5 [per thousand] to 11 [per thousand]; Zone 2, medium-salinity 12-14 [per thousand]; and Zone 3, high-salinity 15 [per thousand] to 18 [per thousand] (P. Genovese, MDDNR pers. comm.). Long Island Sound does not have a salinity gradient and the oyster beds lie in uniform 26 [per thousand] to 28 [per thousand], high-salinity waters. There are 43 oyster disease sampling stations in the Maryland Chesapeake Bay and 30 sampling stations in the Long Island Sound. P. marinus infections (30 oysters/site) were diagnosed by Ray fluid thioglycollate medium (RFTM) assays (Ray 1954) from rectal-anal tissues. Because the State of Connecticut uses the Mackin infection intensity scale from 0 (uninfected) to 5 (heavy) (Mackin 1962) and the Maryland DNR uses a scale of 0-7, Maryland's data were transformed to the 0-5 Mackin scale by designating Maryland 7 as "5" (0 = 0, 1 = 0.7, 2 = 1.4, 3 = 2.1,4 = 2.8,5 = 3.6, 6 = 4.2 and 7 = 5). Infection prevalences and intensities were statistically compared by Kolmogorov-Smirnov tests for differences between sites, and by 1-way ANOVA for differences between years (Sokal & Rohlf 2000).

Comparison of Apoptotic Indices of Perkinsus marinus Infecting Long Island Sound and Chesapeake Bay Oysters

Long Island Sound samples were composed of 3, 30-oyster samples collected during spring, fall and summer at a salinity of 28 [per thousand]. Spring samples were collected between March 27 and April 10, summer samples on July 30, and fall samples between October 3 and November 29. Chesapeake Bay samples were composed of 90 oyster samples collected from different salinities in the fall of 2001. Oysters diagnosed as positive for P. marinus infections by RFTM assays were selected for this study. Archived hematoxylin-eosin-stained slides from the Bureau of Aquaculture, Connecticut and the Maryland DNR, Oxford Laboratory were screened, and 180 specimens having a high number of P. marinus cells in the stomach and intestinal epithelia were resectioned for hematoxylin-eosin staining and for in situ hybridization (ISH) apoptosis assays. Apoptotic indices were compared by Wilcoxon 2-sample tests for differences between sites, and by 1-way ANOVA for differences between seasons and salinities (Sokal & Rohlf 2000).

Detection of Apoptosis by In situ Hybridization

In situ hybridization was performed to detect apoptosis in P. marinus cells using Apoptag Plus Peroxidase kit S7101 (Intergen Company, Purchase, New York). Six-[micro]m sections of paraffin-embedded oyster tissues were cut, immobilized and dried onto positively charged slides. Tissue section deparaffinization and rehydration consisted of two 5-min washes each in 100% xylene and 100% ethanol, followed by single washes in 95% ethanol (5 min), 70% ethanol (3 min) and 1.0 M PBS (5 min). Davidson's fixed in vitro parasite cells were permeabilized for ISH assays by washing cells from experimental cultures in two changes of PBS, postfixing in ethanol: acetic acid 2:1 at -20[degrees]C, and then washing again in two changes of PBS.

Deparaffinized tissues and in vitro propagated cells were pretreated with proteinase K (15 min), 0.1% Triton X-100 (10 min), and quenched with 3.0% (w/v) [H.sub.2][O.sub.2] in PBS (10 min), after which an equilibration buffer was applied for up to 1 hour. Sample cells were incubated with terminal deoxynucleotidyl transferase enzyme (TdT) and digoxigenin-conjugated nucleotides in a saturated humidified chamber at 37[degrees]C for 1 h. Tissues were washed with Stop/Wash Buffer before an antidigoxigenin peroxidase antibody conjugate was applied for a 30-min incubation in a saturated humidified chamber at room temperature. Diaminobenzidine (DAB) Dilution Buffer containing 0.05% (w/v) DAB peroxidase substrate was applied and developed approximately 4.5 min for precipitation of brown, oxidized DAB chromophore. The staining reaction was terminated with a distilled water wash. Tissues were counterstained with 0.5% (w/v) methyl green in 0.1 M sodium acetate, pH 4.0, and dehydrated in 100% N-butanol.

Positive controls included postweaning rat mammary glands (Apoptag) and C. virginica tissues with apoptotic P. marinus cells from previous experiments. Negative controls included replacing TdT with water, and staining of rehydrated slides with peroxidase substrate without adding TdT or antibody conjugate, to detect possible endogenous peroxidase activity.

The apoptotic index was defined as the average percentage of positive staining P. marinus cells from five differential microscopic counts of 100 parasite cells.

Chesapeake Bay and Long Island Sound Perkinsus marinus Cell Cultures

In vitro P. marinus cultures from the Chesapeake Bay, Maryland (ATCC 50439) and Long Island Sound, Connecticut (ATCC 50508) were propagated in vitro at 27[degrees]C in 850 [mOsmkg.sup.-1] Dulbecco Modified Eagle (DME)/Ham's F-12 liquid growth medium, supplemented to 3% (v/v) fetal bovine serum (FBS) and 100 U:[micro]g [mL.sup.-1] of penicillin:streptomycin (DME/F12-3ps) (Dungan & Hamilton 1995). Parasite cell cultures were maintained in log-phase for 4 days prior to in vitro experiments.

In vitro Temperature Experiments

Thirty milliliters of DME/F12-3ps medium were added to log phase cells, and 10-mL aliquots were transferred to individual temperature treatment flasks. Flasks were incubated at 4, 10, 15, 20 and 25[degrees]C for 24 h. This preliminary experiment showed that the optimum incubation temperature (least apoptotic cells) was 20[degrees]C. This optimum temperature was later used for the salinity experiment. Temperature exposures were repeated by using 8 x [10.sup.7] cells of each isolate suspended in 80 mL of 28 [per thousand] DME/F12-3ps. Cell suspensions were dispensed into eight flasks, each containing 107 cells in 10 mL of culture medium, which were incubated at 0, 4, 10, 15, 20, 25, 30 or 35[degrees]C for 24 h.

In vitro Salinity Experiment

Cells (1 x [10.sup.7]) of each isolate were suspended in 10 mL of each salinity treatment medium, inoculated into T25 culture flasks, and incubated at 20[degrees]C. After 24 h, experimental cells were pelleted at x230g for 5 min. Cell pellets were resuspended in 5 mL of Davidson's fixative for 60 min at 20[degrees]C. Fixed cells were centrifuged again at x230g for 5 min, and pellets were resuspended in 10 mL of 70% ethanol for storage pending apoptosis assays. Osmolalities of experimental media were determined using a freezing point depression osmometer. Actual experimental salinities and osmolalities were as follows: 11.6 [per thousand] (330 [mOsmkg.sup.-1]), 17.2 [per thousand] (492 [mOsmkg.sup.-1]), 21.1 [per thousand] (603 [mOsmkg.sup.-1]), 27.9 [per thousand] (797 [mOsmkg.sup.-1]), 33.5 [per thousand] (957 [mOsmkg.sup.-1]), 37.6 [per thousand] (1075 [mOsmkg.sup.-1]) and 37.8 [per thousand] (1081 [mOsmkg.sup.-1]).

Cytospin slides were prepared by using a quantity of 1 x [10.sup.6] cells for a set of four positively charged slides, and centrifuging at x 100g for 3 min. Apoptotic indices of experimental cell cultures were compared after salinity and temperature exposures as paired observations by Signed-Ranks tests (Sokal & Rohlf 2000).

RESULTS

Comparison of Prevalence and Intensity of Perkinsus marinus Infections in the Chesapeake Bay and Long Island Sound Oysters

The 1997 to 2001 fall survey results of Perkinsus marinus infections were compared, with significantly higher infection prevalences estimated in Chesapeake Bay than in Long Island Sound oyster samples (Fig. 1A, P < 0.05). Both areas had individual samples with prevalences ranging from 0% to 100%. The comprehensive mean prevalence in Chesapeake Bay oysters was 78.6% (SD = 27.73), and in Long Island Sound oysters 72.9% (SD = 30.35). Mean infection intensities in Chesapeake Bay oysters were generally higher, with a comprehensive mean of 1.92 (SD = 0.96) and a range of 0-3.9 among individual samples, whereas the Long Island Sound oysters had a comprehensive mean of 1.12 (SD = 0.64) and a range from 0-2.5 among individual samples (Fig. 1B, P < 0.05). In Chesapeake Bay oysters, there was a significant increase in mean prevalence and intensity of P. marinus infections between year-groups 1997-1998 and 1999-2001 (groups A and B in Fig. 1A and 1B, P < 0.00001). The years 1999 to 2002 were consecutive drought years in Chesapeake Bay, during which actual salinities, and salinity-driven disease effects, consistently exceeded long-term averages in all Chesapeake Bay salinity zones. Such a trend was not seen in Long Island Sound oysters.

[FIGURE 1 OMITTED]

In Chesapeake Bay oysters, there was an increase in mean P. marinus infection prevalence and intensity in the lowest salinity Zone 1 (5 [per thousand] to 11 [per thousand]) between year-group (A) 1997-1998 and 1999-2001 (group B) (Fig. 2A and 2B, P < 0.0001). Mean infection intensity further increased from 2000 to 2001 (group C in Fig. 2B), reflecting continued drought-induced elevated salinity effects among Zone-1 oysters. Such a trend was not present in Chesapeake Bay medium- and high-salinity zone infection prevalences, but mean infection intensities increased in the medium-salinity zone (P < 0.00001) and in the high-salinity zone (P < 0.003) between 1997 and 2001. Because of the salinity-driven increase in P. marinus infection intensity between 1998 and 1999 in Chesapeake Bay salinity Zone 1, the difference between the Long Island Sound and Chesapeake Bay P. marinus mean infection intensities increased accordingly (Fig. 1B).

[FIGURE 2 OMITTED]

Comparison of Apoptotic Indices of Perkinsus marinus Infecting Long Island Sound and Chesapeake Bay Oysters

In the in vivo studies, P. marinus was observed and differentially counted in intestine and stomach epithelia after in situ hybridization for apoptosis detection. Apoptosis occurred in single and multiplying cells within or outside oyster hemocytes in the epithelia. Additionally, many apoptotic P. marinus cells phagocytosed by hemocytes were detected in mantle and connective tissues. Chesapeake Bay samples had more advanced stages of the disease, with higher frequencies of dividing P. marinus cells. Long Island Sound samples had fewer parasites, and reduced numbers of subdividing clusters of daughter cells. However, there was not a statistically significant difference in the apoptotic indices between the Long Island Sound fall samples (x = 34.0, SD = 34.4) and those pooled from Chesapeake Bay's three salinity zones (x = 40.1, SD = 21.4). Long Island Sound fall apoptotic indices had a range of 0% to 98.0%, whereas Chesapeake Bay samples had a range of 0% to 87.2%.

Comparison of Apoptotic Indices of Perkinsus marinus in Chesapeake Bay Oysters From Different Salinities

Figure 3 demonstrates the percentage of apoptotic P. marinus cells in Chesapeake Bay oysters from different salinities (10 [per thousand] to 20 [per thousand]). There was significantly more apoptosis of P. marinus in oysters collected from lower salinities 10 [per thousand] to 13 [per thousand] (group A) than in oysters collected higher salinities 14 [per thousand] to 20 [per thousand] (group B, P < 0.005). Low salinity indices ranged from 13.2% to 87.2% (n = 33, x = 52.8, SD = 21.2) and high salinity indices ranged from 0% to 71.0% (n = 57, x = 32.7, SD = 17.9). The threshold salinity for the increase in apoptosis was at 13 [per thousand], below which apoptotic indices increased significantly.

[FIGURE 3 OMITTED]

Comparison of Apoptotic Indices of Perkinsus marinus Infecting Long Island Sound in Spring, Summer and Fall Samples

The apoptotic indices of P. marinus in 30 Long Island Sound oysters were measured in the spring, summer and fall to detect possible seasonal differences (Fig. 4). There was more apoptosis of P. marinus in oysters collected during fall (x = 34.0, SD = 34.4) and spring (x = 29.2, SD = 30.9), than in oysters collected during the summer (x = 23.7, SD = 17.8). However, these differences were not statistically significant. There were large variations in the indices with significantly different standard deviations (Bartlett test 1.15, P = 0.002). Spring values varied from 0% to 95%, summer values from 0% to 75% and fall values from 0% to 98%.

[FIGURE 4 OMITTED]

In vitro Temperature Experiments

The preliminary experiment to optimize the temperature for salinity experiments using the Chesapeake Bay P. marinus isolate (ATCC 50439) revealed that the 20[degrees]C treatment had the lowest level of apoptosis (2%) with increasing apoptosis at higher and lower temperatures (Table 1). Expanded experiments testing Chesapeake Bay and Long Island Sound isolates with additional higher temperatures demonstrated similar patterns, with the lowest apoptosis indices in experimental treatments between 15[degrees]C and 25[degrees]C (Fig. 5). There was no significant difference between the Long Island Sound and Chesapeake Bay isolates under different temperatures (Signed Rank Test = 0.0, NS). Data points for Long Island Sound 0[degrees]C and 4[degrees]C and Chesapeake Bay 0[degrees]C treatments were not included, because of our inability to visualize apoptotic staining in the extremely small cells present at those temperatures. Microscopic examination of the cytospin preparations revealed the highest rates of parasite schizogony between 15[degrees]C and 25[degrees]C, and no schizogony between 30[degrees]C and 35[degrees]C, in both isolates.

[FIGURE 5 OMITTED]

In vitro Salinity Experiment

Apoptotic indices of Long Island Sound and Chesapeake Bay P. marinus isolates were calculated after exposure to different salinities (Fig. 6). There was a significant difference between apoptotic indices of the two isolates (Signed Rank Test 2.01, P < 0.5), with lower rates of apoptosis in Chesapeake Bay isolate cell cultures when compared with Long Island Sound isolate cultures. The Chesapeake Bay isolate exhibited low apoptotic indices, minimized at salinities above 21.1 [per thousand], over a wide range of salinities. Apoptosis in the Chesapeake Bay isolate was elevated at 11.6 [per thousand]. The Long Island Sound isolate had relatively high apoptotic indices at all salinities tested, except at 27.9 [per thousand]. At that salinity, the apoptotic index did not differ from that of the Chesapeake Bay isolate. Microscopic examination of cytospin preparations suggested morphologic differences between the strains after exposure to different salinities. The Chesapeake Bay isolate exhibited schizogony at all salinities with highest rates occurring at salinities from 21.1 [per thousand] to 33.5 [per thousand]. The Long Island Sound isolate showed no schizogony between 37.6 [per thousand] and 37.8 [per thousand], and many predivision hypnospores at salinities between 11.6 [per thousand] and 17.2 [per thousand].

[FIGURE 6 OMITTED]

DISCUSSION

The prevalences and intensities of Perkinsus marinus infections in Long Island Sound and Chesapeake Bay oysters have not been compared until this study. This comparison may reveal valuable information concerning differences in the pathogenesis of this parasite between the two regions. Both mean prevalences and intensities of P. marinus infections were higher in Chesapeake Bay than in Long Island Sound oysters during the 5-year study period. Apoptosis of P. marinus was first observed in oysters collected from Long Island Sound by Sunila and LaBanca (2003). This article describes apoptotic P. marinus also in infected Chesapeake Bay oysters.

P. marinus in Chesapeake Bay oysters sampled from lower salinities, 10 [per thousand] to 13 [per thousand], had significantly more apoptosis than P. marinus in oysters sampled from higher salinities, 14 [per thousand] to 20 [per thousand], (see Fig. 3) with a threshold value at 13 [per thousand]. These results suggest that low salinity controls infection intensity via the induction of P. marinus apoptosis, which allows oysters to better survive with lower parasite body burdens. The in vitro experiments of the present work (see Fig. 6), and of another study (Goedken et al. 2005b) demonstrate higher parasite apoptosis at low salinity, which supports the results observed in vivo in this study.

Apoptosis of P. marinus was inducible in cell cultures in this study (see Fig. 5 and 6), as in another related study (Goedken et al. 2005b). These in vitro studies demonstrate that P. marinus variably undergoes apoptosis without the influence of the host. Whereas our parasite isolates didn't significantly differ in their responses to temperature exposures, the isolates had different apoptotic responses to variations in medium salinities. The viability and proliferation of P. marinus cultures of different Chesapeake Bay isolates has been studied in several previous works at different salinities and temperatures (Burreson et al. 1994, Dungan & Hamilton 1995, Gauthier & Vasta 1995, O'Farrell et al. 2000, La Peyre 1996). La Peyre (1996) reviewed articles about different Chesapeake Bay isolates of P. marinus in different culture conditions as well as different temperature and salinity ranges. Results differed depending on culture media and method for measuring proliferation. Optimum temperatures varied between 28[degrees]C to 32[degrees]C and optimum salinities between 24 [per thousand] to 36 [per thousand]. According to Dungan and Hamilton (1995), proliferation of the ATCC 50439 isolate used in this study occurred between 10[degrees]C to 40[degrees]C and 11.9 [per thousand] to 68 [per thousand]. Culture medium and exposure conditions used in these experiments were selected according to those results to ensure the presence of proliferating cells throughout the exposure ranges.

O'Farrell et al. (2000) studied mortality based on neutral red supra-vital stain and volume regulation of P. marinus culture after exposure to different salinities. They reported a volume-regulatory acclimation to a hypoosmotic shock at 2.5 [per thousand]. Also, cells cultured at the low osmolality (168 [mOsmkg.sup.-1], 6.5 [per thousand]) were significantly larger than cells cultured at the higher osmolalities (341 and 737 [mOsmkg.sup.-1], 12.7 [per thousand] and 27 [per thousand]). The cells cultured at the osmolalities of 341 and 737 [mOsmkg.sup.-1], however, were not significantly different from each other in size. Based on these results acclimation did not play a role within the salinity range studied here.

O'Farrell et al. (2000) stated that P. marinus was more tolerant of hyper- than hypoosmotic shock. This result, based on a Chesapeake Bay isolate, is in accordance with the Chesapeake Bay isolate of this study (see Fig. 6), but not the Long Island Sound isolate. The Long Island Sound isolate had high apoptotic rates at all salinities, except those prevalent in its native Long Island Sound (~28 [per thousand]). The conclusions about osmotic tolerance of P. marinus by O'Farrell et al. (2000) as well as those by Burreson et al. (1994) and Gauthier and Vasta (1995) are based on studies on individual Chesapeake Bay isolates, and may not describe general characteristics of the parasite throughout its entire range.

The Chesapeake Bay isolate in this study had low apoptotic indices at a wide range of salinities, which may reflect adaptation to the more variable estuarine salinity characteristics of the Chesapeake Bay. The higher P. marinus apoptotic indices observed in oysters sampled from lower salinities (<13 [per thousand]) areas in the field (see group A in Fig. 3) are in accordance with higher apoptotic indices during in vitro exposures of the Chesapeake Bay isolate to low salinities (see Fig. 6).

Prevalences and intensities of P. marinus infections have recently increased in Chesapeake Bay's low-salinity areas (Krantz & Jordan 1996, Burreson & Calvo 1996, Tarnowski 2003, Ragone Calvo & Burreson 2003). In this study, the mean prevalence and intensity of P. marinus infections were significantly higher in the Chesapeake Bay, because of the significant activity increase by the parasite between 1998 and 1999, particularly in Chesapeake Bay's low-salinity areas (see Fig. 1 and 2). These disease measures during 1997 and 1998 did not differ significantly from each other, nor did 1999 to 2001 values. The general intensification of P. marinus infections reflected the effects of a 5-y period of drought-elevated Chesapeake Bay salinities that began in 1999 (Ragone Calvo & Burreson 2003, Tarnowski 2003). Mean annual adult oyster disease-related mortality rates in Maryland's portion of Chesapeake Bay from 1997 to 2001 were estimated at 18%, 19%, 31%, 35% and 38%, respectively (Tarnowski 2003).

Since the 1980s, the Chesapeake Bay oyster industry has been restricted to low-salinity areas, after diseases decimated oyster populations in high-salinity areas (Krantz & Jordan 1996). This may have resulted in an increase in apoptosis-resistant pathogens in low salinity, and therefore more severe infections in low-salinity oysters. Long Island Sound does not have a salinity gradient, which leaves little impetus for parasites of osmoconforming oyster hosts to adapt to variable salinities. The differences in apoptosis between Chesapeake Bay and Long Island Sound P. marinus isolates could be explained by differences that are either genetic, adaptive, or both. Reece et al. (2001) analyzed 12 different composite genotypes and found no genetic difference between the Long Island Sound and Chesapeake Bay P. marinus isolates. However, differences in genes regulating apoptosis have not yet been specifically studied. It is likely that these two parasite strains share common ancestry from which they have independently adapted to different host habitats.

This study investigated apoptosis of P. marinus in oysters collected from Chesapeake Bay and Long Island Sound and in cell cultures established from parasites collected from these areas. The combination of natural infections and in vitro propagation of P. marinus cultures allowed the determination of the effects of temperature and salinity on different strains of the parasite. We suggest that the coevolution of the host and the parasite drove the Chesapeake Bay P. marinus strain to modify its apoptosis regulating genes to secure its survival in oysters from a wider, estuarine salinity range.

ACKNOWLEDGMENTS

Alison Yee thanks Dr. Robert Pavlica for his inspiration and support and Inke Sunila acknowledges Dr. Gary Wikfors for his help in statistical analysis.

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ALISON YEE, (1,2) CHRISTOPHER DUNGAN, (3) ROSALEE HAMILTON, (3) MICHAEL GOEDKEN, (4) SYLVAIN DE GUISE (4) AND INKE SUNILA (1) *

(1) State of Connecticut, Department of Agriculture, Bureau of Aquaculture, P.O. Box 97, Milford, Connecticut 06460; (2) Mount Holyoke College, 3090 Blanchard Student Center, South Hadley, Massachusetts 01075; (3) Maryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, Maryland 21654; (4) Department of Pathobiology and Veterinary Sciences, University of Connecticut, 61 N. Eagleville Road, Storrs, Connecticut 06260

* Corresponding author. E-mail: [e-mail]

TABLE 1.

A preliminary experiment for exposing a in vitro Chesapeake Bay
isolate of Perkinsus marinus to different temperatures. Apoptosis
was detected on cytospin preparations with in situ hybridization
using Apoptag

Temperatures     Percentage of
([degrees]C)     Apoptotic Cells (%)

 4                   37
10                    8
15                   12
20                    2
25                   31