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The protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo’s intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite’s life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

Marine diseases are a strong selective force that can have important economic and ecological consequences. Larval dispersal patterns, selective mortality and individual growth rates can modulate metapopulation responses to disease pressure. Here, we use a modeling framework that includes distinct populations, connected via larval transport, with varying disease selection pressure and connectivity to examine how these dynamics enhance or inhibit the evolu- tion of disease resistance in metapopulations. Our system, oysters and MSX disease, is one in which disease resistance is highly and demonstrably heritable. Simulations show that under con- ditions of population isolation (i.e. local retention of larvae) and strong disease selection, popula- tions rapidly evolve genetic disease resistance. Varying the patterns of larval dispersal alone dou- bles the time for evolution of disease resistance. Spatially varying disease in the absence of larval dispersal leaves some populations unable to respond to the disease, whereas adding larval disper- sal slows the response of populations under strong selection and speeds the response in popula- tions under low selection when fitness is based on relatively limited genetic structure ('juvenile fitness' in our simulations). Under spatially variable disease pressure, larval dispersal generates a fourfold greater variance in fitness outcomes across the dispersal patterns tested. In a meta - population, populations experiencing lower selection pressure will tend to slow the development of other, more heavily selected populations. This suggests that conservation efforts aimed at improving overall metapopulation resistance in the face of marine diseases should target those populations under modest or high disease pressure, rather than protecting those experiencing low selective pressure.

Humans rely on marine ecosystems for a variety of services but often impact these ecosystems directly or indirectly limiting their capacity to provide such services. One growing impact is the emergence of marine disease. We present results from a unique case study examining how oysters, a dominant organism in many coastal bays and estuaries that is often harvested for food, have responded to pathogens influenced by human activities, namely the introduction of novel pathogens. Climate change has enabled a northward spread and establishment of Dermo disease in oysters along the eastern seaboard of North America and human activities inadvertently introduced MSX disease along this same coast. Oysters in Delaware Bay have responded differently to each pathogen, and uniquely to MSX disease by developing a highly resistant baywide population not documented in any other bay. Offspring were produced using parents collected from low or high disease (MSX and Dermo) regions of Delaware Bay and exposed in a common garden experiment along with a naïve population from Maine. Results indicated widespread resistance to MSX disease, but not to Dermo disease, across Delaware Bay. One striking result was the demonstration of resilience in the population through its capacity to spread, presumably through larval transport, resistance to MSX disease into portions of the population that have experienced little to no MSX disease pressure themselves. Related studies indicated that larval transport mechanisms allowed widespread dispersal such that the entire metapopulation could acquire a high level of resistance over time if disease resistance is sufficiently heritable. The findings have implications for restoration, management and recovery of diseased populations. Namely, that if left to their own devices, natural selection may find a solution that enables populations to recover from introduced pathogens.

More and more infectious diseases affect marine molluscs. Some diseases have impacted commercial species including MSX and Dermo of the eastern oyster, QPX of hard clams, withering syndrome of abalone and ostreid herpesvirus 1 (OsHV-1) infections of many molluscs. Although the exact transmission mechanisms are not well understood, human activities and associated environmental changes often correlate with increased disease prevalence. For instance, hatcheries and large-scale aquaculture create high host densities, which, along with increasing ocean temperature, might have contributed to OsHV-1 epizootics in scallops and oysters. A key to understanding linkages between the environment and disease is to understand how the environment affects the host immune system. Although we might be tempted to downplay the role of immunity in invertebrates, recent advances in genomics have provided insights into host and parasite genomes and revealed surprisingly sophisticated innate immune systems in molluscs. All major innate immune pathways are found in molluscs with many immune receptors, regulators and effectors expanded. The expanded gene families provide great diversity and complexity in innate immune response, which may be key to mollusc's defence against diverse pathogens in the absence of adaptive immunity. Further advances in host and parasite genomics should improve our understanding of genetic variation in parasite virulence and host disease resistance.

Today, populations of eastern oysters, Crassostrea virginica, are commonly limited by disease mortality. Resistance to MSX disease has developed in a number of cases, but the development of resistance to Dermo disease would appear to be limited, despite the high mortality rates and frequency of epizootics. Can aspects of the host's genetics or population dynamics limit the response to the disease despite the apparent opportunity afforded by alleles conferring disease resistance or tolerance? To answer this question, we use a gene-based population dynamics model, configured for C. virginica, to simulate the development of disease resistance using mortality as the agent of selection. Simulated populations were exposed to 4 levels of mortality covering the range in mortality observed in Delaware Bay in the 1990s. In each case, disease resistance increased in the simulated population over time, normally proportional to the increase in mortality rate imposed by the disease. However, simulations show that the population responds even at its most rapid rate on multidecadal to half-century timescales. As the mortality rate declines with increasing disease resistance, the rate of further improvement in disease resistance likewise declines. Thus, disease resistance develops over decadal timescales at a 40%-per-year mortality rate, but, as mortality rate falls to 25% per year, the rate of further development of disease resistance extends to half-century timescales. The discouraging profundity is that a mortality rate of 25% per year, yielding a rate of selection profoundly slow, is still very high. In northern climes, significant decrements in oyster abundance will occur. Evidence from fisheries retrospectives suggests that oysters cannot withstand a constant removal at this scale without compromising population integrity noticeably. So, a mortality rate that grievously limits the development of disease resistance still sorely strains the species ability to maintain a vibrant population necessary to its long-term survival.

QPX (Quahog Parasite Unknown) a protistan pathogen of northern quahogs (=hard clams), Mercenaria mercenaria, has caused disease outbreaks in maritime Canada, and in Massachusetts, New York, New Jersey, and Virginia, USA. Although epizootics have occurred in wild hard clam populations, the parasite has most seriously affected cultured hard clams, suggesting that aquaculture practices may promote or predispose clams to the disease. In this investigation the influence of clam genetic origin and the geographic location at where they are grown on QPX disease susceptibility was examined in a common garden experiment. Aquaculture stocks were acquired from hatcheries in Massachusetts, New Jersey, Virginia, South Carolina, and Florida and spawned at a single hatchery in Virginia. All stocks were originally, although not exclusively, derived from wild hard clam populations from each state. The seed clams were deployed at two sites, New Jersey and Virginia, and evaluated during the subsequent 2.5 y for growth, survival, and QPX disease. At both sites, South Carolina- and Florida-derived clam stocks exhibited significantly higher QPX prevalence and lower survival than New Jersey and Massachusetts clam stocks. Levels in the Virginia stock were intermediate. In Virginia, mortality at the termination of the experiment was 78%, 52%, 36%, 33%, and 20% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey hard clam stocks, respectively. Mortality was significantly correlated with QPX prevalence. Maximum QPX prevalence in the South Carolina and Florida stocks ranged from 19% to 21% and 27% to 29%, respectively, whereas in the Virginia, New Jersey, and Massachusetts stocks prevalence was 10% or less. Similar trends were observed in New Jersey where mortality at the termination of the experiment was estimated to be 53%, 40%, 20%, 6%, and 4% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey clam stocks, respectively. QPX prevalence peaked at 18% in the Florida stock, 38% in the South Carolina, 18% in the Virginia, and 5% in the New Jersey and Massachusetts stocks. These results suggest that host genotype is an important determinant in susceptibility to QPX disease. As such, hard clam culturist should consider the genetic origin of clam seed stocks an important component of their QPX disease avoidance/management strategies.

To evaluate methods for calculating mortality in bivalve molluscs, we analyzed historical data from dredge surveys of both planted and natural oyster (Crassostrea virginica) grounds in Delaware Bay to compare total box-count mortality estimates with those made by accumulating short-term mortality rates obtained from fresh boxes identified by shell condition and degree of fouling. Box-count and cumulative-mortality patterns and values agreed best on grounds with planted oysters, where a cohort with very few dead oysters was broadcast on previously cleaned bottom and was followed over time. This situation is analogous to an artificially created reef with oysters either deployed or naturally set on it. When deaths predominated in late summer and early autumn, the two estimates were similar throughout the following year; when mortality was greatest in spring or early summer, the estimates were similar only through autumn of the same year. Correspondence was much weaker on natural beds, where new individuals constantly recruited to the population and variable numbers of boxes were always present. Nevertheless, total box-count mortality estimates made during autumn stock surveys were significantly correlated with cumulative mortalities calculated for the preceding year. We also estimated disarticulation rates of artificially created boxes by deploying them at three seasons and eight sites in Delaware Bay. Disarticulation time depended on the length of exposure at summer temperatures, with the average time to 50% disarticulation for boxes deployed in spring and in summer being 225 and 345 days, respectively. Disarticulation rates increased with decreasing size, and increasing salinity and temperature. Finally, we compared total box-count and cumulative-mortality estimates with those made using the disarticulation data. Annual averages for the three methods were within 5 percentage points of each other. Our data indicate that total box-count mortality estimates from fall stock surveys can provide a reliable index to total mortality for the previous year, although it cannot describe the seasonal patterns obtainable using the cumulative-mortality method.

▪ Abstract Many factors (climate warming, pollution, harvesting, introduced species) can contribute to disease outbreaks in marine life. Concomitant increases in each of these makes it difficult to attribute recent changes in disease occurrence or severity to any one factor. For example, the increase in disease of Caribbean coral is postulated to be a result of climate change and introduction of terrestrial pathogens. Indirect evidence exists that (a) warming increased disease in turtles; (b) protection, pollution, and terrestrial pathogens increased mammal disease; (c) aquaculture increased disease in mollusks; and (d) release from overfished predators increased sea urchin disease. In contrast, fishing and pollution may have reduced disease in fishes. In other taxa (e.g., sea grasses, crustaceans, sharks), there is little evidence that disease has changed over time. The diversity of patterns suggests there are many ways that environmental change can interact with disease in the ocean.

The current status of the Haplosporidia is reviewed as well as recent information on Haplosporidium nel- soni, the causative agent of MSX disease in oysters. Recent molecular phylogenetic analyses with greatly increased taxon sampling support monophyly of the Haplosporidia and hypothesize placement of the group as sister taxon to the phylum Cercozoa. Oyster pathogens in the genus Bonamia should be considered haplosporidians based on molecular sequence data. Thus, the group contains 4 genera: Uropsoridium, Haplosporidium, Bonamia and Minchinia. Molecular phylogenetic analyses support monophyly of Urosporidium, Bonamia and Minchinia ,b utHaplosporidium forms a pa- raphyletic clade. Reports of haplosporidia worldwide are reviewed. Molecular detection assays have greatly increased our ability to rapidly and specifically diagnose important pathogens in the phylum and have also improved our under- standing of the distribution and biology of H. nelsoni and H. costale. Much of the data available for H. nelsoni has been integrated into a mathematical model of host/parasite/environment interactions. Model simulations support hypotheses that recent H. nelsoni outbreaks in the NE United States are related to increased winter temperatures, and that a host other than oysters is involved in the life cycle. Evidence is presented that natural resistance to H. nelsoni has developed in oysters in Delaware Bay, USA. However, in Chesapeake Bay, USA H. nelsoni has intensified in historically low salinity areas where salinities have increased because of recent drought conditions. Efforts to mitigate the impact of H. nelsoni involve selective breeding programs for disease resistance and the evaluation of disease resistant non-native oysters.

The protozoan pathogen Perkinsus marinus is the causative agent of Dermo, a lethal disease of the eastern oyster Crassostrea virginica, but not the Pacific oyster Crassostrea gigas. To understand the response of these two oysters to parasite exposure, a suppression subtractive hybridization (SSH) method was employed to characterize genes up-regulated during parasite challenge in both hemocytes and gills. The number of differentially expressed gene sequences obtained was 107 for C. virginica and 69 for C. gigas, including 46 and 37 sequences, respectively, that matched known genes in GenBank. Most of the sequences have not been characterized in other molluscs. Nineteen genes involved in immune system and cell communication, protein regulation and transcription, cell cycle, respiratory chain and cytoskeleton were selected for expression analysis by semi-quantitative PCR. Although varying in magnitude and timing post exposure, all genes screened showed over-expression in challenged oysters in both species, validating the SSH method. Results of this study highlighted some differences in gene expression between the two oysters in response to P. marinus infection, providing candidate genes and pathways for further analysis.

International audience; An individual-based mathematical model was developed to investigate the biological and environmental interactions that influence the prevalence and intensity of Brown Ring Disease (BRD), a disease, caused by the bacterial pathogen, Vibrio tapetis, in the Manila clam (Venerupis (=Tapes, =Ruditapes) philippinarum). V. tapetis acts as an external microparasite, adhering at the surface of the mantle edge and its secretion, the periostracal lamina, causing the symptomatic brown deposit. Brown Ring Disease is atypical in that it leaves a shell scar that provides a unique tool for diagnosis of either live or dead clams. The model was formulated using laboratory and field measurements of BRD development in Manila clams, physiological responses of the clam to the pathogen, and the physiology of V. tapetis, as well as theoretical understanding of bacterial disease progression in marine shellfish. The simulation results obtained for an individual Manila clam were expanded to cohorts and populations using a probability distribution that prescribed a range of variability for parameters in a three dimensional framework; assimilation rate, clam hemocyte activity rate (the number of bacteria ingested per hemocyte per day), and clam calcification rate (a measure of the ability to recover by covering over the symptomatic brown ring deposit), which sensitivity studies indicated to be processes important in determining BRD prevalence and intensity. This approach allows concurrent simulation of individuals with a variety of different physiological capabilities (phenotypes) and hence by implication differing genotypic composition. Different combinations of the three variables provide robust estimates for the fate of individuals with particular characteristics in a population that consists of mixtures of all possible combinations. The BRD model was implemented using environmental observations from sites in Brittany, France, where Manila clams routinely exhibit BRD signs. The simulated annual cycle of BRD prevalence and intensity agrees with observed disease cycles in cultured clam populations from this region, with maximum disease prevalence and intensity occurring from December to April. Sensitivity analyses of modeled physiological processes showed that the level of hemocyte activity is the primary intrinsic determinant of recovery of infected clams. Simulations designed to investigate environmental effects on BRD suggested that the outcome of the host-parasite interaction is dependent on food supply (high values being favorable for the host) and temperature. Results of simulations illustrate the complex interaction of temperature effects on propagation and viability of the bacterium, on the phagocytic activity of the hemocytes, and on other physiological processes of the host clam. Simulations using 1 degrees C and 2 degrees C increases in temperature generally favored disease development, indicating that climate warming might favor the spread of BRD.

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.

International audience; In this study of short-term exposure of Ruditapes philippinarum from Brittany, France, to an Alexandrium tamarense isolate that produces high concentrations of paralytic shellfish toxins (PSTs), the effects of the isolate on clam fitness, toxin uptake, and the response of hemocytes (responsible for internal defense in bivalves) were examined. Adults exhibited individual variability in resistance to the effects of PSTs, measured by their burrowing capacity. If genetically based, this may (1) indicate that resistance is under natural selection and (2) provide a new model to advance our understanding of the molecular basis for PST resistance in bivalves, so far determined only in Mya arenaria. Juvenile clams were more vulnerable to PSTs than adults, experiencing 100% burrowing incapacitation within 1 d of exposure to A. tamarense. They also experienced growth suppression and high mortalities following 4 d of toxification. Juveniles challenged with Vibrio tapetis, the cause of brown ring disease (BRD), and exposed to A. tamarense, developed significantly fewer BRD symptoms relative to controls fed non-toxic algae, but suffered higher mortality. Adult clams exposed to A. tamarense showed a significant increase in hemocyte concentrations and a small, but significant, decrease in phagocytic activity, and no effect on hemocyte viability or other functional parameters. We speculate that the inhibitory effects on BRD progression may be attributable to toxicity of PSTs to V. tapetis, inability of juveniles to activate the shell conchiolin-deposition response, and/or an overall increase in phagocytic cells induced by A. tamarense. Harmful algae and pathogens may thus interact and modulate the effects of disease in bivalve populations.

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.

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.

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.

The decline of the eastern oyster Crassostrea virginica in Chesapeake Bay from overfishing, disease, and habitat degradation is well documented ([1][1]–[3][2]). Oysters provided important ecological and economic benefits to the Chesapeake Bay states of Maryland and Virginia, which have poured

Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea vir- ginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cul- tured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites. In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) viru- lence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential defi- ciency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more viru- lent than those obtained from lag- or stationary-phase cultures.

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.

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.

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

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.