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During routine histopathology of 180 juvenile hard clams, Mercenaria mercenaria, from a site in Virginia, USA, in 2007, we discovered a single individual heavily infected with a parasite resembling a haplosporidian, some members of which cause lethal bivalve diseases. Scanning electron microscopy of spores and sequencing of small subunit ribosomal DNA confirmed a new species: Minchinia mercenariae n. sp. Further sampling of clams at the site found prevalences up to 38% using polymerase chain reaction (PCR). No parasites were found in routine histological screening of the same individuals, but re-examination of clams judged positive by in situ hybridization (ISH) revealed very faintly staining plasmodia. No unusual mortalities have occurred among the sampled groups. Analysis of clams from Massachusetts to Florida by PCR failed to detect the parasite, but a haplosporidian found in a clam from New Jersey in 2001 was subsequently identified by ISH as M. mercenariae. No other haplosporidians have been reported in thousands of hard clams from the US east coast examined histologically since the mid-1980s. The discovery underscores critical questions about how to assess the risks associated with parasites in groups known to be lethal, but that themselves are not considered a problem.

We studied particle selection in the ribbed mussel Geukensia demissa, an important suspension-feeding inhabitant of estuaries and intertidal zones of salt marshes along the Atlantic coast of North America. Adult mussels were fed on several mixtures of microalgal cultures (1) in exponential or (2) in stationary phase of growth, and the proportional occurrence of algal species in pseudofeces was examined by flow cytometry. The Eastern oyster, Crassostrea virginica, was chosen as a reference. Results showed that both mussels and oysters were able to selectively ingest or reject our experimental microalgae. Moreover, the pre-ingestive particle selection was affected by microalgal growth phase, particularly in mussels. For instance, the sorting efficiency index increased significantly in mussels fed with a blend made of Nitzschia closterium, Isochrysis sp. and Tetraselmis suesica harvested in stationary growth phase, as compared to the same blend made with microalgae in exponential growth phase. Isochrysis sp. and T. suesica were preferentially ingested by both bivalves whereas N. closterium, was preferentially rejected in pseudofeces. These results demonstrate particle selection in ribbed mussel and underline the effect of algae growth phase on the sorting mechanisms.

The known range of the eastern oyster (Crassostrea virginica) parasite, Perkinsus marinus, expanded into the northeastern United States in the early 1990s. We used both in vitro and in vivo data to test the hypothesis that the northward expansion was associated with a low-temperature adapted strain of the parasite. In vitro proliferation of nine P. marinus isolates from three geographic sites, Massachusetts and New Jersey in the new range, and South Carolina in the historic southern range, was measured at seven temperatures (5 to 35 °C) using a tetrazolium blue dye assay. We wanted to determine if there were between- and within-geographic location differences in the P. marinus proliferation rate, and if so, whether they were associated with temperature. We found no evidence of low-temperature adaptation based on the fact that net proliferation rates for isolates from all three geographic locations were similar at temperatures from 5 to 20 °C. On the other hand, at temperatures of 25 to 35 °C, the South Carolina isolates exhibited higher proliferation rates than the northern isolates suggesting possible high-temperature adaptation of parasite strains that are routinely exposed to higher temperatures. Although there was significant within-location variation among isolates, the data tended to group together by geographic location supporting the hypothesis that there is an important regional component to the proliferation rate of P. marinus isolates. A survey of published data showed that the temperature at which in vivo proliferation was first observed in oysters at sites from the Gulf of Mexico to Massachusetts was typically between 20 and 23 °C with no evidence of a geographic cline. These results lend support to the hypothesis that the recent warming trend in the northeastern US is the most likely explanation for the P. marinus range extension.

Over a 2-year period in 1990 and 1991, coincident with a pronounced warming episode, Dermo disease outbreaks in the oyster, Crassostrea virginica, caused by the parasite Perkinsus marinus, occurred over a 500-km range from Delaware Bay to Cape Cod, in the northeastern United States. The parasite had not previously been recorded or known to cause mortalities in this region. To document infection patterns and levels in this region several years after the initial outbreaks, and to compare them with those in the parasite’s historic southern range, we deployed and sampled oysters from 1996 to 1998 at multiple sites spanning the expanded range. During this 2-year period, the parasite was documented to occur in oysters at high prevalences throughout the new range, in sites varying from small, enclosed embayments to large estuaries, and in both cultured and wild-set oysters. Infection and mortality patterns, and levels were similar to those in southern locations where the parasite has been enzootic for at least decades. The persistence of high P. marinus infection levels in the new range after the initial expansion is probably due to several factors: (1) winter temperatures continued to increase during the 1990s and early 2000s, albeit at a slower rate than in 1990–1991, facilitating overwinter survival of the parasite; (2) many oyster-growing sites in the northeast are in relatively shallow water in which summer temperatures offer ample time for the parasite to proliferate and spread; and (3) the combination of high parasite burdens and high host densities in oyster farms results in an abundance of parasites and high transmission rates. Colder winters and high rainfall after 2002 reduced prevalences in some regions, but P. marinus can survive low temperatures and low salinities, and epizootic conditions are likely to return if temperatures rise again, as predicted by climate-change models.

In microbial infections, the interaction between microorganisms and phagocytic cells is a crucial determinant in the outcome of the disease process. We used flow cytometry to study the in vitro interactions between Vibrio tapetis, the bacterium responsible for Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum, and haemocytes from three bivalve species: the Manila clam (susceptible to BRD), the hard clam Mercenaria mercenaria and the eastern oyster Crassostrea virginica (both non-susceptible to BRD). Results demonstrated that V. tapetis cells and extracellular products elicit major changes in the haemocytes of R. philippinarum, including decreased viability and phagocytic activity, and altered size and internal structure. V. tapetis was able to kill haemocytes from M. mercenaria and C. virginica but to a far lesser extent than those of R. philippinarum. These results suggest that disease resistance is not solely dependent on a host activity against the pathogen, but is also a function and magnitude of the injury to the host cell by a given pathogen.

Mass mortalities of cultured zhikong scallops (Chlamys farreri) have occurred each summer in most culture areas of northern China since 1996. Among the hypothesized causes are high culture density, infectious disease and genetic inbreeding. To investigate these potential agents, C. farreri were deployed at three densities (low, medium and high) at three sites (Jiaonan, Penglai and Yantai) in the summer of 2000. Scallops were sampled for survival, growth and histopathology before, during and after a mortality episode. Most of the mortality occurred in July and August, during and toward the later part of the spawning season, when water temperature reached 23–26 8C. Final cumulative mortalities reached 85% to 90% at all three sites. Scallops in the medium and high densities had higher initial death rates than did those at the low density. High densities also inhibited growth. Ciliates from the genus Trichodina, larvae of various organisms and anomalous secretions were observed in sections of the gill cavity, with highest prevalence during and at the end of the mortality period. Prokaryotic inclusion bodies were found in the soft tissues, but their prevalence was low and apparently without correlation with mortalities. Genetic analysis with random amplified polymorphic DNA markers showed slightly lower heterozygosity in the cultured stocks (0.301) than in the wild stocks (0.331). It is possible that the mortalities are caused by a combination of several factors such as stress associated with reproduction, high temperature, overcrowding and poor circulation in the growout cages, opportunistic invaders or pathogens, and possibly inbreeding. D 2005 Elsevier B.V. All rights reserved.

Quahog Parasite Unknown (QPX), a recently discovered pathogen of hard clams, Mercenaria mercenaria , appears to be most prevalent in clams held in hatchery or nursery tanks or grown to market in culture parks. The persistent link with cultured clams indicated that culture practices might increase the susceptibility of clams to an opportunistic or facultative parasite. We investigated two hypotheses: (1) that increased density would accelerate the development of QPX infections and (2) that seed originating from nonlocal (i.e. another state) sources would be more susceptible than seed from local sources. There was a significant trend toward higher QPX levels at higher planting densities, but considerable variability in the data made it difficult to determine the effect of density with a high degree of confidence. During 1995–1998, 3-year classes of clams imported as seed from a South Carolina hatchery and grown at several sites in New Jersey acquired heavy QPX infections and suffered mortalities of 26% to 92%. Clams of the same age from New Jersey hatcheries that had been planted in adjacent plots for the same length of time acquired few or no detectable infections and experienced no unusual deaths. QPX seems to be widespread in clam growing waters from Canada to at least Virginia, but causes disease and mortality only in certain groups that may be disadvantaged in some way, perhaps from an unfavorable genotype–environment interaction. The fact that no mortalities have been reported in New Jersey since the purchase of South Carolina seed was curtailed indicates that the problem was localized to these clams and has neither spread nor persisted to a noticeable extent. Nevertheless, the results underscore the potential dangers of using nonlocal stocks in molluscan aquaculture.

Petroleum seep mussels are often exposed to high hydrocarbon concentrations in their natural habitat and, thus, offer the opportunity to examine the relationship between parasitism, disease and contaminant exposure under natural conditions. This is the first report on the histopathology of cold-seep mussels. Seep mussels were collected by submersible from four primary sites in the Gulf of Mexico, lease blocks Green Canyon (GC) 184, GC-234, GC-233, and Garden Banks 425 in 550–650 m water depth. Five types of parasites were identified in section: (1) gill “rosettes” of unknown affinity associated with the gill bacteriocytes, (2) gill “inclusions” similar to chlamydia/rickettsia inclusions, (3) extracellular gill ciliates, (4) body “inclusions” that also resemble chlamydial/rickettsial inclusions, and (5) Bucephalus-like trematodes. Comparison to shallow-water mytilids demonstrates that: (1) both have similar parasite faunas; (2) seep mytilids are relatively heavily parasitized; and (3) infection intensities are extremely high in comparison to shallow-water mytilids for Bucephalus and chlamydia/rickettsia. In this study, the lowest prevalence for chlamydia/rickettsia was 67%. Prevalences of 100% were recorded from three populations. Bucephalus prevalence was ⩾70% in three of 10 populations. The parasite fauna was highly variable between populations. Some important parasites were not observed in some primary sites. Even within primary sites, some important parasites were not observed in some populations. Bucephalus may exert a significant influence on seep mussel population dynamics. Forty percent of the populations in this study are severely reproductively compromised by Bucephalus infection. Only a fraction of petroleum seep mussel populations are maintaining the entire beta-level population structure of this species. Variation in two parasites, gill ciliates and Bucephalus, explained most of the variation in PAH body burden between mussel populations. PAHs are known to be sequestered preferentially in gametic tissue. Bucephalus would be expected to reduce overall body burden, at high infection intensities, by replacing gametic tissue. PAH concentrations exceeded 1 ppm in 4 of 9 populations, a ratio significantly higher than the 8 of 30 mussel locales in the NOAA Mussel Watch Program. Only five Mussel Watch locales exceeded the highest value for a petroleum seep population. Digestive gland and gill tissue atrophy were not significantly correlated with PAH body burden, even though some populations were characterized by body burdens exceeding 1 ppm, suggesting that seep mussels may not be as sensitive to PAH exposure as are some shallow-water mytilid populations.

We analyzed a data set collected over 15 yr, containing growth data from strains of eastern oysters, Crassostrea virginica (Gmelin, 1791), initiated from parent populations in Long Island Sound, Delaware Bay, and lower Chesapeake Bay. The long-term growth data proved to be a powerful tool for examining patterns of growth differentiation among separated populations of C. virginica. The oyster strains had been grown in a common environment in lower Delaware Bay for up to seven generations. We found that the oyster strains with origins in Long Island Sound were significantly larger over several generations than oyster strains from Delaware Bay and Chesapeake Bay. Chesapeake Bay oyster strains were larger than Delaware Bay oyster strains at 1.5 yr old, but Delaware Bay oysters were larger at 2.5 yr. Year-to-year variation in environmental conditions had a strong significant effect on absolute oyster size and the relative sizes of the oyster strains. Persistent differences between oyster strains from different origins over several generations support a hypothesis that these estuarine populations have experienced long-term genetically-based population differentiation. This result is consistent with hypotheses of population differentiation of oysters based on observations of local reproductive timing.

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.

Recently, PCR technology has been applied to search for marine microorganisms in environmental samples. Such sampling, however, has drawbacks, including the need to collect and filter large volumes of water, and the presence of substances in environmental samples that may destroy DNA or interfere with DNA isolation and amplification. We explored the possibility of using suspension-feeding bivalves in conjunction with PCR to investigate the environmental distribution of microparasites using the oyster pathogen Haplosporidium nelsoni (the MSX disease agent) with oysters and mussels in Delaware Bay, USA, as a model system. Delaware Bay oysters Crassostrea virginica have become very resistant to H. nelsoni infection development and rarely exhibit histologically detectable lesions. The ribbed mussel Geukensia demissa is also resistant. Infections were detected in only 6 % of the histologically examined oysters in the present study, although PCR-positive signals for H. nelsoni were found in feces, gill or heart samples of up to 100% of oysters. Positive signals were found in the feces and gill (but not heart) samples of up to 50% of mussels. The temporal evolution of PCR signals suggested a wave-like pattern of putative infective particles, which mimicked a pattern documented in early mortality studies. The present study demonstrated that H. nelsoni persists throughout Delaware Bay, although it is rarely detected using standard histology. We propose that the use of suspension-feeding bivalves as particle collectors in combination with PCR could be a method for detecting the presence of marine microparasites and might help answer questions about factors that prevent outbreaks of epizootics in certain regions.

Physiological rates of oysters Crassostrea virginica (Gmelin) selected and unselected for resistance to the endoparasite MSX Haplosporidium nelsoni (Haskin, Stauber & Mackin) were compared between June 1988 and May 1989 in lower Delaware Bay, USA. Prevalence of the parasite in the unselected (susceptible) group was 80% by July 1988, whereas no infections were detected in the selected (resistant) group until August 1988, and prevalence never exceeded 21%. In September 1988, cumulative mortality was 80% for the susceptible oysters compared to only 3% for the resistant oysters. Clearance rate was significantly (P < 0.001) greater in the resistant group that in the susceptible group in July and August 1988, but similar in June and September 1988 and May 1989. Oxygen consumption rate was significantly greater in the resistant group in June (P < 0.003) and August (P < 0.028) 1988, but similar in both groups in July and September 1988 and May 1989. During July and August 1988, when H. nelsoni was proliferating most rapidly and causing the greatest mortality of susceptible oysters, the difference between energy potentially taken in as food and energy respired was significantly (P < 0.002) greater in the resistant group than in the susceptible group. Although the greater degree of infection experienced by the susceptible oysters may have contributed to the differences in physiological rates, there was no consistent relationship between intensity of infection and physiological function. Alternatively, we suggest that “resistance” in the selected strain may be the result of a physiological response, perhaps induced by H. nelsoni, that inhibits development, as well as a basic metabolic adjustment to parasitism.

International audience; Brown ring disease (BRD) in the Manila clam is induced by the bacterium Vibrio tapetis. During the infection process, the pathogen enters the extrapallial compartment of the Manila clam and induces the formation of a characteristic brown deposit that gives the disease its name. Although post-infection processes have been widely described for this disease, the mechanisms of entry of the bacteria into the extrapallial compartment remains unclear. From relationships between clam size and BRD prevalence, and between grain-size distribution in natural habitats and prevalence, we propose a simple explanation for this step: V. tapetis benefits from mechanical disruptions of the periostracal lamina or valve margins to colonize the extrapallial compartment. Such disruptions may be induced by the presence of large sediment grains in natural habitats, which become lodged in the shell opening. This hypothesis suggests that limiting handling of clams may help to limit development of BRD in cultured clam beds.

International audience; The known negative effects of shellfish toxin producing dinoflagellates on feeding, burrowing and survival of some bivalve mollusks has prompted questions concerning whether they might also impair the internal defense system of affected bivalves and make them more susceptible to disease agents. The primary components of the cellular defense system are hemocytes. Many toxic dinoflagellates are too large to be ingested whole by hemocytes and would most likely be exposed to intracellular toxins only after the algae are consumed, broken down, and the water-soluble toxins, released. Therefore, we conducted a series of experiments in which hemocytes from two suspension-feeding bivalves - the Manila clam, Ruditapes philippinarum, and the softshell clam, Mya arenaria - were exposed in vitro to filtered extracts of one highly toxic paralytic shellfish toxin (PST)-producing and one nonPST-producing strain of Alexandrium tamarense (isolates PR18b, 76 ± 6 STXeq cell-1) and CCMP115, with undetectable PST, respectively). We measured adherence and phagocytosis, two hemocyte attributes known to be inhibited by bacterial pathogens and other stressors. We found no measurable effect of a cell-free extract from a highly concentrated suspension of the PSTproducing strain on hemocytes of either bivalve species. Instead, extract from the nonPST-producing strain had a consistent negative effect on both clams, resulting in significantly lower adherence and phagocytosis compared to strain PR18b and filtered seawater controls. The bioactive compound produced by strain CCMP115, which has yet to be characterized, may be similar to the PST-independent allelopathic compounds described for Alexandrium spp., which act on other plankters. These compounds and those produced by other harmful algae are known to cause immobilization, cellular deformation and lysis of co-occurring target organisms. Thus, nonPST producing Alexandrium spp., which do not cause paralysis and burrowing incapacitation of clams, may still produce a compound(s) that has negative effects not only on hemocytes, but on other molluscan cell types and their functions, as well.

The recent spread and intensification of Haplosporidium nelsoni (MSX) disease in oysters, Crassostrea virginica , along the Atlantic coast of the United States has heightened interest in the use of disease-resistant strains in aquaculture and increased the need for understanding all facets of how selective breeding affects oyster performance, including reproduction. Resistance does not prevent infection, but involves an ability to restrict parasite development and to tolerate infections. We examined selected and unselected oysters, from three geographical locations, all exposed to H. nelsoni infection, to determine the influence of selection, strain origin, and parasitism on gametogenesis. Gonadal development was most frequently correlated with geographic origin of oysters: northern strains (from Long Island Sound) ripened and spawned earlier than southern strains (from the James River), even after several generations of selection at a single location in Delaware Bay. Parasitism was associated with impaired gonadal development in May when gametogenesis was occurring, but not in August, when spawning was underway. Selection for disease resistance was associated with improved gametogenic development, but only in oysters with few or no patent infections. Both parasite burden and selection for resistance were correlated with gametogenesis only in strains from the two southern-most locations, the James River and Delaware Bay, and never in those from Long Island Sound. We suggest that our observations resulted from interference by H. nelsoni in energy acquisition, storage, and conversion cycles of the oyster, which, in turn, differed seasonally according to geographic origin. Gonad development appeared to be more sensitive to parasitism when gametes were in the formative stage rather than after they had matured. Thus, southern strains, which were still in the early stages of gametogenesis when they experienced typical high parasite loads in late May, showed significantly greater inhibition than northern strains, which already had well-developed gonads at the same time. In contrast to most reports that environmental stress correlates with an increased ratio of males to females, we found a three-fold increase in the proportion of females among H. nelsoni -infected oysters compared to uninfected oysters. There was no evidence of sex-associated differential infection or mortality rates and we suggest that parasitism inhibits the development of male gametes more than female gametes.

A bioenergetics model of the Manila clam (Ruditapes philippinarum) was built to simulate growth, reproduction and spawning in culture and fishery field sites in Marennes–Oleron Bay (French Atlantic coast). The model is driven by two environmental variables: temperature and food supply. Food supply and the clam's filtration rate determine soft tissue condition index, which in turn drives clam growth and reproduction. The model was calibrated and then validated using two independent data sets. This paper discusses the difficulty of comparing experimental data and individual model outputs when asynchronous spawning events occur in the studied population. In spite of this difficulty, the simulations reproduce the typical pattern of growth and reproduction of the Manila clam. Simulations showed that water column chlorophyll a concentration is not a perfect estimator of food resources for a near bottom suspension feeder such as the Manila clam and emphasize the lack of knowledge about Ruditapes philippinarum nutrition. The individual growth model presented in this paper will be integrated into a numerical population model describing the host– parasite–environment relationship in Brown Ring Disease, caused by the bacterium Vibrio tapetis. © 2007 Elsevier B.V. All rights reserved.

International audience; Components of the haemolymph are understood to constitute the internal defense system of bivalve mollusks and their levels are often considered to be indicators of "health"; however, relatively little proof exists of the role that these elements play in the success or failure of defense against a pathogen. A change associated with infection may be the consequence of disease rather than a measure of the capacity to respond effectively to a pathogen. One way to assess whether haemocyte or serum-component concentrations are related to resistance to microbial infection is to sample individuals over time, both before and after they are experimentally or naturally infected. But sampling itself may alter the parameter being assessed. In addition, interindividual variation is large and the degree of intraindividual variation over time is largely unknown. To evaluate intra- vs interindividual variability measured over time and to assess the effects of repeated sampling, we subjected Manila clams, Ruditapes philippinarum, to multiple haemolymph samplings during both field and laboratory experiments, and measured four parameters: haemocyte density, protein concentration, and the activities of leucine amino peptidase and DOPA-oxidase. A repeated-measures ANOVA indicated that individuals with high or low levels at one sampling, tended to have high or low levels, respectively, at the other sampling times. Furthermore, the index of individuality, which is the ratio of intra- to interindividual variability, for these four parameters was comparable to that for human serum components. Repeated sampling had no measured effect on field-deployed clams, which were sampled at intervals of 1-3 months, but significantly depressed values in laboratory-held clams sampled at 1-month intervals. Results demonstrated relative intraindividual constancy in the measured variables and suggested that minimizing sample frequency and volume, and maintaining animals in a comparatively natural environment should all facilitate repeated sampling with minimum injury to experimental mollusks.

International audience; This work compared the effect of challenge with Vibrio tapetis, the etiologic agent of brown ring disease (BRD) in clams, and other bacterial strains on defence-related factors in four bivalve species: Ruditapes philippinarum (highly susceptible to BRD), R. decussatus (slightly susceptible to BRD), Mercenaria mercenaria and Crassostrea virginica (both non-susceptible to BRD). Results show that bacterial challenge modulated defence-related factors, namely total and differential haemocyte counts, percentage of viable haemocytes, and lysozyme activity, both in haemolymph and extrapallial fluid. Injection with bacteria induced a response that was dependent upon the bacterial and bivalve species investigated, and upon the site of inoculation: external (pallial cavity), pseudo-internal (extrapallial space), or internal compartment (adductor muscle). The most conspicuous changes were systematically measured in R. philippinarum injected with V. tapetis, indicating a bacterial pathogenicity particular to the host in which it causes a specific disease syndrome. Alterations of defence-related factors were maximal in haemolymph of clams injected with V. tapetis in the muscle, and in the extrapallial fluid when the bacteria were injected into the pallial or the extrapallial cavity. Resistance to the development of the BRD symptom was not related to the extent of the haemocyte reaction measured following in vivo challenge.

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally trans- mit parasites by 'natural' methods. In addition, the host's condition may affect a study's outcome. The experiments reported here examined the effect of dosing method and host metabolic condition on measures of virulence for the oyster parasite Perkinsus marinus. Oysters, Crassostrea virginica, were challenged with wild-type and cultured forms of P. marinus via feeding, shell-cavity injection, gut intubation and adductor-muscle injection. For both parasite types, adductor-muscle injections pro- duced the heaviest infections followed by shell-cavity injection, gut intubation, and feeding. There was no difference in parasite burdens between oysters fed cultured cells by acute vs chronic dosing, and parasite loads stabilized over time, suggesting a dynamic equilibrium between invasion and elimination. P. marinus distribution among tissues of challenged oysters indicated that parasites invaded the mantle and gill, as well as the gut, which has been considered the primary portal of entry. Frequency distributions of P. marinus in oysters challenged with 3 different culture phases indicated an aggregated distribution among hosts and suggested that stationary-phase parasites were easiest for the oyster to control or eliminate and log-phase parasites were the most difficult. Host metabolic condition also affected experimental outcomes, as indicated by increased infection levels in oysters undergoing spawning and/or exposed to low oxygen stress.

Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe. The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell. Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens. This study was designed to evaluate the pathogenicity of V. tapetis, in an attempt to help explain the mechanisms of mortality. We found high mortalities (up to 100%) for clams following the inoculation of V. tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R. philippinarum. Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation. In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption. Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle. In contrast, no mortalities were observed following injection of V. tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD. This clam rapidly eliminated the bacterium from hemolymph and soft tissues. Clam mortality associated with BRD in the field is likely to result from the penetration of V. tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium. Some bacteria also penetrate through the digestive epithelia. In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death.

Brown ring disease (BRD) is a shell disease caused by Vibrio tapetis. This pathogen disturbs the periostracal lamina causing the appearance of a brown conchiolin deposit on the inner face of the shell, within the extrapallial space. Although differences in resistance to BRD have been documented, their relationship to possible defense functions has never been investigated. In this study, flow cytometry was used to analyze cellular parameters in asymptomatic and experimentally infected Ruditapes philippinarum from France and the west coast of the USA. Parallel analyses were made on Ruditapes decussatus, the native European clam, which is highly resistant to BRD. In the haemolymph and extrapallial fluid of animals without BRD, total haemocyte counts, the percentage of granulocytes, and the phagocytic activity against latex beads or V. tapetis by the haemocytes were significantly higher in American R. philippinarum than in French R. philippinarum. In most cases, levels in R. decussatus were the highest of all three groups. Four weeks following challenge with V. tapetis, BRD prevalence reached 52 in American clams and 100% in French specimens, but only 37% in R. decussatus. In symptomatic animals, phagocytosis of V. tapetis increased significantly in the resistant species of clam, R. decussatus, was unchanged in US clams, and decreased significantly in FR specimens when compared to asymptomatic individuals from each population. Ingestion of V. tapetis by haemocytes in the extrapallial fluid, which is in contact with the periostracal lamina, could be the main defense mechanism used to counter the pathogen. Our results suggest that resistance to BRD may well be related to the concentration of granular haemocytes and the phagocytic activity of haemocytes.

Eastern oysters (Crassostrea virginica) often undergo epizootics of MSX (Multinucleated Spore Unknown) disease, which is caused by the protozoan pathogen, Haplosporidium nelsoni. The disease has been present in oyster populations in the mid-Atlantic United States since the 1950s. During the 1980s and 1990s, it became established further north along the east coast of the United States. To investigate the factors underlying the northward progression of MSX disease, a model that simulates the host–parasite–environmental interactions was used. The model is physiologically-based and is structured around the transmission, proliferation and death rates of the parasite. Environmental conditions of temperature, salinity and oyster food supply provide the external forcing that results in variations in the biological rates. For this study, environmental data sets, both average and extreme, were obtained at a site in upper Chesapeake Bay for 1986 through 1995. This site is in the middle of one of the most productive oyster growing regions on the east coast of the United States; thus, both short- and long-term changes in the environment measured here realistically reflect conditions experienced by important oyster populations. The effect of short-term high-salinity (drought) or low-salinity (wet) conditions on MSX disease prevalence and intensity was relatively small because the average salinity regime already favors maximum parasite activity at this site. Even the extreme low salinity events were not low enough to significantly inhibit the parasite. Similarly, simulations using short-term high-temperature extremes for the same site showed only minor deviations from the average pattern because average temperatures were already high enough to support parasite development. In contrast, the measured low temperature conditions, applied for a single year, caused a dramatic reduction in parasite activity, which extended over a 2-year period. Additional simulations show that overall food supply to the host is of little consequence in determining the basic disease pattern; however, the timing of maximum food supply provided to the host, relative to specific times in the parasite life cycle, is important in determining whether or not the parasite attempts sporulation or undergoes density-independent growth. Simulations that test a sequence of changing environmental conditions show that when a year with cold winter temperatures (less than 3 °C) is followed by a year of low salinity (less than 15 ppt), prevalences and intensities of MSX disease are greatly reduced, with the disease becoming almost absent in the oyster populations; however, the disease returns when average environmental conditions return. Simulations using progressive cooling or warming conditions indicate that winter temperatures consistently lower than 3 °C limit the long-term development of MSX disease. These simulations support the suggestion that climate warming is a contributing factor to the northward spread of MSX disease.

Perkinsus marinus infection intensity was measured in eastern oysters Crassostrea virginica collected in October and December 1993, and March, May, and July 1994 from 3 U.S. sites: Apalachicola Bay (FL), Chesapeake Bay (VA), and Oyster Bay (NY). Gill, mantle, digestive gland, adductor muscle, hemolymph, and remaining tissue (including gonadal material and rectum) were dissected from 20 oysters from each site at each collection time. Samples were separately diagnosed for P. marinus infections by incubation in Ray's Fluid Thioglycollate Medium (RFTM) and subsequent microscopic quantification of purified enlarged hypnospores. At all sampling times and sites, average P. marinus infection intensity (g wet wt tissue-1 or ml hemolymph-1) was lowest in hemolymph samples, and generally highest in the digestive gland. Perkinsus marinus prevalence was 100% at both FL and NY sites for each of the 5 collection times, and, for the VA site, was less than 100% in only 1 month (May 1994). Seasonal intensity patterns and mean total body burdens differed among the sites. Average body burden was highest in VA during October and progressively declined to a minimum in May. This decline was probably due to mortality of heavily infected oysters and diminution of parasite activity associated with colder temperatures and reduced salinities. Intensities varied little during the months of October and December at both the FL and NY sites. Minimum average intensities were observed in March in FL oysters and May in NY oysters. Relatively high P. marinus infection levels that persisted throughout the winter in NY oysters compared with VA oysters could reflect constant high salinity in Long Island Sound which favors parasite activity, and also rapid decline in temperature in the fall that may have prevented epizootic oyster mortalities.

The protozoan parasite Haplosporidium nelsoni (MSX) elicits an inflammatory-type response in oysters, Crassostrea virginica. We assayed circulating hemocytes of oysters exposed to H. nelsoni to quantify the effects of parasitism, selection for resistance, and season on total and differential counts. All factors had a significant (P0.02) effect on total counts, but explained relatively little of the overall variance [season (12%)selection (4%)infection (2%)]. Circulating hemocyte densities increased with infection intensity in resistant animals, but were depressed in susceptible oysters with light infections. Counts in both groups were lowest in August and highest in May, at which times densities in susceptible oysters were reduced (P0.02) compared to those in resistant animals. No differences existed in November. Susceptible oysters may be more debilitated by infections than resistant animals, resulting in impaired circulation and depressed cell counts. The number of tissue-infiltrating hemocytes increased with infection intensity (P0.0001), but showed no statistically significant (P0.05) association with numbers of circulating hemocytes in individual oysters. Size-frequency analysis with a Coulter counter indicated that the proportion of large cells (presumed to be granular hemocytes) was lower (P0.0001) in susceptible oysters, which were also heavily infected, compared to resistant oysters, which had very few infections. The loss of granular hemocytes may stem from degranulation associated with tissue damage and inflammation. Present evidence suggests that the principal role of the hemocyte response in MSX disease is to plug lesions, remove debris, and repair tissues and that these functions may help oysters survive infection.(ABSTRACT TRUNCATED AT 250 WORDS)

The spore stage of Haplosporidium nelsoni, the ascetosporan parasite causing multinucleated sphere unknown (MSX) disease in oysters, Crassostrea virginica, has been reported so rarely (less than 0.01% of infected oysters) that a second host has been postulated. However, recent intensive sampling of young (less than 1 year) oysters in Delaware Bay, U.S. suggests that spore formation occurs regularly in this group and that spores are produced in at least 75-85% of all infections reaching the advanced stage. Sporulation was seasonal, occurring over two to three weeks in late June/early July and again in late summer/early fall. Our data indicate that sporulation by H. nelsoni in oysters is more common than previously suspected, occurring in a segment of the host population that may not have been sufficiently sampled in the past, and that a direct life cycle should be reconsidered.

We used a total body parasite burden assay for the oyster pathogen Perkinsus marinus to investigate quantitative changes in microparasite burdens and frequency distributions. Heretofore, this type of study was limited mainly to macroparasites. The apparent in vivo growth pattern of P. marinus was characterized by a "lag" phase ( 10,000 cells/g wwt). Infections declined exponentially under conditions unfavorable to the parasite but with a lengthening half-life, suggesting that elimination of parasites became increasingly difficult at low densities. Oysters held for 11 wk at 15 C, a temperature considered more favorable for oyster hemocytes than for P. marinus, were not able to eliminate infections. Parasite distributions within host populations were aggregated; in each sample, 1 or 2 oysters contained more parasites than all other oysters in the sample combined and the variance-to-mean ratio exceeded 1:1. The few hosts with large parasite burdens may be the most important individuals for survival and transmission of parasites. They are likely to play a key role in initiating and maintaining epizootics either in situ or after introduction of infected animals into a naive population.

A number of stressors, including the endoparasiteHaplosporodium nelsoni (MSX), reduce metabolic condition of oysters; however, previous studies of the effect of the shell-boring polychaetePolydora sp. on condition have produced conflicting results. To assess the effects of both symbionts, singly and in combination, we examined 214 oysters for shell infestation byPolydora sp. mud blisters (by image analysis), intensity of infection byH. nelsoni (by histology), and nutritional state (estimated by soft tissue gravimetric condition index). Among all oysters tested, condition index was negatively (p

Resistance to disease caused by the oyster pathogen Haplosporidium nelsoni (MSX) has developed in nature and through selective breeding; however, examination of tissue slides shows no evidence that phagocytic cells are involved. To investigate this phenomenon further, we quantified in vitro phagocytosis of plasmodial stages of H. nelsoni by hemocytes from 2 oyster and 1 mussel species. Our results show that hemocytes from oysters Crassostrea virginica, whether selected or unselected for resistance to MSX disease, did not ingest plasmodia unless the parasites were damaged or killed. Time-lapse photography suggested that granular hemocytes retreated rapidly after initial contact with living plasmodia and thatnearby agranular hemocytes showed little movement

1. 1. Tolerance of plasmodial stages of the oyster parasite, Haplosporidium nelsoni (MSX), to acute change from high to low salinity was compared in vitro with that of hemocytes from the host, Crassostrea virginica , using exclusion of trypan blue dye as a measure of cell viability. 2. 2. At salinities of 15–20 ppt, equal proportions (4–8%) of parasites and hemocytes absorbed dye. 3. 3. The proportion of stained hemocytes from uninfected oysters remained the same at salinities down to 6 ppt, but rose to 14–17% at 9 and 6 ppt when hemocytes were from infected oysters. 4. 4. Between 15 and 9 ppt, the proportion of dyed parasites rose exponentially to about 75%, with little additional change at 6 ppt. 5. 5. Tolerance to salinity change in vitro parallels the distribution of host and parasite in nature and indicates that the reduced incidence of H. nelsoni in low salinity is most probably due to a physiological inability to tolerate reduced salinity, rather than to enhanced effectiveness of host defense mechanisms.

Oysters inhabiting areas enzootic for the parasite Haplosporidium nelsoni (MSX) are exposed to infective particles each summer, and it is often difficult to distinguish newly acquired lesions from older infections. To study long-term parasitism without the complication of new infections, MSX-infected oysters were moved from Delaware Bay to a disease-free area on the New Jersey coast. Because infections seen after the transfer were acquired in Delaware Bay during a known infective period, it was possible to determine how long oysters remained infected and how they were affected by chronic parasitism. Chronic infections displayed the same seasonal cycle (high levels in winter and late spring, low levels in summer and early spring) that occurs in enzootic areas with annual reinfection. Within individual oysters, chronic MSX became localized, relapsed into general infections, and then became localized again in a sequence that was probably controlled by temperature. Some experimental oysters survived with MSX for at least 4 years. Hemocytosis persisted in these oysters, and their poor condition suggested that chronically infected individuals would have lowered resistance to additional stress.

1. 1. A method for repeated sampling of hemolymph from bivalve molluscs was evaluated by bleeding oysters, Crassostrea virginica, from the adductor muscle through a notch in the shell, at 2-week and 1-month intervals over a 4-month period. 2. 2. Hemolymph protein concentrations in oysters bled monthly did not change significantly from initial or final control levels; however, levels in oysters sampled bi-weekly were consistently lower than those of the 1-month group during the entire experiment. 3. 3. Increased bleeding frequency had no measurable effect on growth or final mortality, but did increase within-oyster variability in hemolymph protein concentrations and was associated with increased parasitism by Haplosporidium nelsoni (MSX).

Serum protein constituents in laboratory-reared stocks of oysters, Crassostrea virginica , susceptible to mortality caused by the parasite Haplosporidium nelsoni (MSX) were compared with constituents in resistant oysters exposed to the same level of infection activity. Virtually all of the susceptible oysters became infected and 71% died by the end of the 9-month study, while only half of the resistant group had patent H. nelsoni infections and only 7% died. Total serum protein concentrations fell sharply and in approximate proportion to disease intensity in oysters with systemic H. nelsoni infections. Oysters with heavy parasitism had about one-third the protein levels of uninfected individuals. When infections were confined to the gill, however, as was the case with mortality-resistant oysters, there was no depression of protein levels. In all oysters examined, SDS electrophoresis showed three rapidly migrating proteins, with molecular weights between 25,000 and 75,000, that comprised nearly all the total protein. Up to 12 larger proteins, with molecular weights up to 500,000, were also present, but with considerable individual variability in number and concentration. There was no obvious correlation of electrophoresis patterns with H. nelsoni disease or with genetic background. Results of the study suggest that depletion of certain metabolic substrates such as circulating protein and/or interruption of biosynthetic pathways by the parasite may be a primary cause of death in infected oysters.

1. 1. Tissue biochemical composition of oysters (Crassostrea virginica) from Delaware Bay (USA) was examined from May to November 1985 as a function of infection intensity by the endoparasite, Haplosporidium nelsoni (MSX). 2. 2. The lipid, glycogen, protein and ash content (mgDW) of a standard 100mm (shell height) oyster was determined for uninfected, epithelial and systemic infection categories. 3. 3. All biochemical components generally decreased in content with increasing MSX infection intensity (and duration). Overall reductions were significant in glycogen [epithelial (P < 0.05) and systemic (P = < 0.001)], protein [systemic (P < 0.05)] and ash [systemic (P < 0.02)] categories. 4. 4. Glycogen was the substrate most readily catabolized to meet the energetic burden posed by MSX. The reduction by MSX of nutrient reserves affects other metabolic functions such that the ecological fitness of surviving oysters is reduced.

Strains of oysters Crassostrea virginica resistant to mortality caused by the parasite Haplosporidium nelsoni (MSX) were developed and tested through 6 generations. In addition, strains in each generation were followed for up to 6 yr of continuous exposure to the parasite in nature. Selected strains responded to challenge by the parasite with gradually improved survival in successive generations. They were slower to develop patent infections than were unselected groups and were able to delay mortality after infections did develop, but under repeated exposure most oysters eventually died with H. nelsoni parasitism. Many selected strains, however, reached market size before significant mortalities occurred. The data suggest that resistance to H. nelsoni mortality is under the influence of many genes. No clear defense mechanism has been described and we hypothesize that resistance to H. nelsoni may, in part, involve a physiological state in which selected oysters temporarily fail to provide a suitable habitat for the parasite. Temporary insusceptibility would, in this view, be followed by an increased ability to tolerate the parasite when conditions for its development are present. Selection would then favor individuals that are able to prolong periods of insusceptibility and/or to carry out basic life processes while parasitized.