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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.

Attempts to decipher the life cycle of Haplosporidium nelsoni began almost immediately after it was identified as the pathogen causing MSX disease in eastern oysters, Crassostrea virginica. But transmission experiments failed and the spore stage, characteristic of haplosporidans, was extremely rare. Researchers concluded that another host was involved: an intermediate host in which part of the life cycle was produced, or—if the oyster was an accidental host—an alternate host that produces infective elements. A later finding that spores were found more often in spat (

Dermo disease caused by the protistan Perkinsus marinus in Eastern oysters Crassotrea virginica is an important source of mortality impacting oyster population dynamics resulting in substantial losses in fisheries and aquaculture. The rapid transmission and spread of the disease minimized the importance of transmission models and past models (proliferation-based models) assumed simple density-dependent transmission or rapid infection post-settlement. This approach is a good approximation only for low population densities. A transmission model was developed for P. marinus in Eastern oysters that accounts for the seasonal change in disease dynamics and density-dependent foraging (of suspended particles) interference among hosts. The model, verified and evaluated against field observations, incorporates parasite release to the water column from live and dead individuals, parasite consumption by living oysters, the diffusion of parasites in the water, body burden-based dose-dependent transmission, recruitment, and disease-caused mortality. The model returns a basic reproduction number R 0 for Dermo much greater than unity ( R 0 = 90) in accordance with the current persistence and pandemic nature of this disease in oysters. No population density is obtained that is low enough to suppress R 0 below 1 (i.e. disease extinction). R 0 is also estimated for high oyster densities (>300 individuals m −2 ) and particularly for relatively large oysters (∼90 mm), today rare but once common before generalized overfishing occurred on healthy oyster reefs. In this scenario, R 0 drops below 1, indicating that high oyster density can limit disease invasion through foraging interference and depletion of parasites in the water column. High intensity recruitment events allow the oyster population to attain such densities and limit the development of epizootics. These results provide insight into the transition from past populations, where Dermo is inferred to have been limited in its impact, to the current persistent and pandemic nature of this disease. Further coupling of this model into metapopulation and hydrodynamic models could be a promising tool to support management decision-making for bivalve populations impacted by Dermo disease.

During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, USA, a high prevalence and intensity of ciliate xenomas has been noted since sampling began in 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by intracellular ciliates, likely Sphenophrya sp. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high abundance. The objectives of this study were to characterize the xenomas, describe the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters. Upon gross examination, xenomas appeared as white nodules, up to 3mm in diameter, located in the gill tissue and occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes, which they often completely blocked. Higher magnification revealed dual nuclei, eight kineties, and conjugation of the ciliates. Transmission electron microscopy revealed dual nuclei that varied in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. These traits place the ciliates into the Order Rhynchodida, but insufficient molecular data exist to confirm classification of this ciliate to the Genus Sphenophrya. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained30 xenomas, but 2.1% contained100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters60mm. Infection intensity peaked in 70-90mm oysters and declined in larger oysters. Individual oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55-65% in November and dropping to10% by spring. The oyster population in Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation.

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.

The response of salinity in the Delaware Estuary to climatic variations is determined using statistical models and long-term (1950-present) records of salinity from the U.S. Geological Survey and the Haskin Shellfish Research Laboratory. The statistical models include non-parametric terms and are robust against autocorrelated and heteroscedastic errors. After using the models to adjust for the influence of streamflow and seasonal effects on salinity, several locations in the estuary show significant upward trends in salinity. Insignificant trends are found at locations that are normally upstream of the salt front. The models indicate a positive correlation between rising sea levels and increasing residual salinity, with salinity rising from 2.5 to 4.4 per meter of sea-level rise. These results are consistent with results from 1D and dynamical models. Wind stress also appears to play some role in driving salinity variations, consistent with its effect on vertical mixing and Ekman transport between the estuary and the ocean. The results suggest that continued sea-level rise in the future will cause salinity to increase regardless of any change in streamflow.

Infectious diseases are common in marine environments, but the effects of a changing climate on marine pathogens are not well understood. Here we review current knowledge about how the climate drives host-pathogen interactions and infectious disease outbreaks. Climate-related impacts on marine diseases are being documented in corals, shellfish, finfish, and humans; these impacts are less clearly linked for other organisms. Oceans and people are inextricably linked, and marine diseases can both directly and indirectly affect human health, livelihoods, and well-being. We recommend an adaptive management approach to better increase the resilience of ocean systems vulnerable to marine diseases in a changing climate. Land-based management methods of quarantining, culling, and vaccinating are not successful in the ocean; therefore, forecasting conditions that lead to outbreaks and designing tools/approaches to influence these conditions may be the best way to manage marine disease.

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.

The Tikehau atoll (Tuamotu Archipelago, French Polynesia) is located at 14'5 148"W. Phytoplankton and sand microalgae are the most important primary producers of the lagoon. They were studied for 4 years. The relationship between light energy and lagoonal primary production was measured by using the 14C method for phytoplankton and O, method for phytobenthos. Incubations, carried out in situ, were made at different depths and light exposition times. Irradiance was high and 17% of the light energy measured at the surface reached 25 m (lagoon average depth). Maxima of phytoplankton and phytobenthos productions occurred at low depths; there was therefore no photoinhibition of photosynthesis. Correlations between light energy and primary production were strong, especially for phytobenthos. Multiplicative linear regression models (production VS light) associated with an exponential linear regression model (light vs depth), allowed planktonic and benthic primary production to be predicted from the depth and the light energy received at the surface. The benthic primary production exceeded the phytoplanktonic production in the upper 18 m. The total primary production (benthos + plankton) was constant with depth and depended only on light energy at the surface. One Einstein received at the lagoon surface allowed the growth production of 14 mg of carbon (water column + sediments).

In 1957–1959, the introduced protistan parasite, Haplosporidium nelsoni, killed 90–95% of the oysters (Crassostrea virginica) in lower Delaware Bay and about half of those in the upper bay. Shortly thereafter, H. nelsoni-caused mortality in the wild population of the lower bay declined, approximating that of first-generation selectively bred oysters. For nearly three decades thereafter no further change in survival of the wild population was evident, although steady improvement was achieved by continued selective breeding. Survival of the wild population is thought to have plateaued because the great majority of oysters inhabited the upper bay where they were protected from H. nelsoni infection and selective mortality by low salinity. Consequently, they contributed most of the offspring to the bay population. From 1957 through 1987, H. nelsoni prevalence was cyclic, but overall high (annual maxima of 60 to 85%) in the lower bay. Since 1988, however, prevalence in wild oysters has rarely exceeded 30% anywhere in the bay, even though unselected oysters continue to become heavily infected when exposed, and molecular evidence indicates that the parasite remains present throughout the bay. This apparent “second step” in the development of resistance in the wild oysters occurred after a drought-associated incursion of H. nelsoni into the upper bay in the mid-1980s. Mortalities were widespread, heavy and more extreme than during the 1957–59 epizootic. Resistant survivors of the second epizootic have apparently repopulated the bay. When compared to unselected stocks, common-garden exposure to H. nelsoni of oysters from both upbay and downbay sites indicates that a high degree of resistance to the development of MSX disease has become widespread in the wild oyster population of Delaware Bay after two major selection events separated by nearly 30 years.

Parasitic, disease-causing pathogens can exert strong control over marine populations yet few long-term studies exist that describe these relationships. Understanding the connections to longterm large-scale processes relative to local short-term processes should facilitate better planning for disease impacts in the management of marine resources. We describe a 21-yr dataset of dermo disease (Perkinsus marinus) in eastern oysters (Crassostrea virginica) in Delaware Bay, USA. Analyses indicated (1) a strong positive association between disease and mortality that was non-linear and defined by thresholds, (2) a clear spatial gradient of increasing disease and mortality with increasing salinity, (3) an apparent 7-year cycle in which peaks were associated with strong positive anomalies of the North Atlantic Oscillation (NAO), (4) an inverse relationship with freshwater inflow, and (5) no obvious response to natural selection from persistent disease pressure. These data quantify the impact of environmental variables on the disease in a wild population and provide new insight into how disease interacts with host populations by linking disease patterns with larger climate controlling processes. Understanding these connections will facilitate prediction of and response to disease outbreaks.

Three selected aquaculture strains of the commonly used hard clam, Mercenaria mercenaria (Linnaeus, 1758)—Massachusetts (MA), New Jersey (NJ), and South Carolina (SC)—were reared in a single hatchery. They were planted in replicate plots in Barnstable Harbor, MA, and Dry Bay, NJ, in spring 2008 and grown to market size. Growth, survival, and QPX (quahog parasite unknown) prevalence and severity were measured in fall 2008, spring 2009, and fall 2009. Growth was similar at both sites for the first summer, but during the second year growth was better in Massachusetts. Overall survival was better for all strains in New Jersey when compared with Massachusetts. Survival of strains in New Jersey was 53.4% and 34.8% for NJ and MA, respectively, closely followed by those in Massachusetts (41.8% and 26.2% for NJ and MA, respectively). Strains from SC seed had the highest prevalence and severity of QPX and the lowest survival (36% and 6.6% in New Jersey and Massachusetts, respectively). Infections with QPX were low at both sites, but this study confirms earlier work indicating that QPX infection rates appear to be strain specific, with strains of southern origin being more susceptible than those of northern origin. Our study also supported anecdotal reports that QPX disease is more severe in Massachusetts than in more southern sites where it has been found. As in most previous field studies, we found that although mortality was correlated with QPX levels, it was considerably higher than infection prevalence would indicate, which suggests that strain interactions with stressful environmental conditions or unidentified factors may be also involved in mortality.

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.

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 evaluate a 54-y survey time series for the Delaware Bay oyster beds in New Jersey waters to identify the characteristics of regime shifts in oyster populations and the influence of MSX and Dermo diseases on population stability. Oyster abundance was high during the 1970s through 1985. Oyster abundance was low at the inception of the time series in 1953, remained low through 1969, and has been low since 1985 and very low since 2000. Natural mortality was low in most years prior to the appearance of MSX in 1957. From 1957 through 1966, natural mortality generally remained above 10% annually and twice exceeded 20%. Natural mortality remained well below 15% during the 1970s and into the early 1980s when oyster abundance was continuously high. The largest mortality event in the time series, an MSX epizootic that resulted in the death of 47% of the stock, occurred in 1985. Mortality rose again with the incursion of Dermo in 1990 and has remained above 15% for most years since that time and frequently has exceeded 20%. The primary impact of MSX and Dermo diseases has been to raise natural mortality and ultimately to cause a dispersed stock to retreat into its habitat of refuge in the moderately low salinity reach of the bay. The time series of oyster abundance on the New Jersey oyster beds of Delaware Bay is dominated by two regime shifts, the 1970 abundance increase that was maintained for about 15 y thereafter, and the 1985 abundance decrease that continues through today. These two regime shifts ushered in long-term periods of apparent constancy in population dynamics. The 1985 regime shift was induced by the largest MSX epizootic on record that produced high mortalities throughout a population distributed broadly throughout its habitat range after 15 y of high abundance. A putative new regime commenced circa 2000 as a consequence of a series of Dermo epizootics. Mortalities routinely exceeded 20% of the population annually during this period, with the consequence of a greater degree of stock consolidation than any previous time in the 54-y record. Extreme consolidation of the stock would appear to be a characteristic of the population's response to Dermo disease. The 1970-1984 and post-1985 regimes each were ushered in by a confluence of events unique in the 54-y time series. Each was characterized by a period of relative stability in population abundance. However, the stability in total population abundance belies a more dynamic process of stock redistribution during both time intervals, demonstrating that the appearance of constancy in stock abundance is not necessarily a result of invariant stock dynamics. Rather, the Delaware Bay oyster time series suggests that regime shifts delimit periods during which differential, often offsetting, local trends impart similar abundance levels, and thus constancy at the level of the stock masks substantive changes in local population dynamics potentially fostering future catastrophic changes in population-level attributes. Understanding such regime shifts will likely determine the success of decadal management goals more so than measures designed to influence population abundance.

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 increase in disease related mortality has made managing oyster resources increasingly difficult. In Delaware Bay sustaining harvest requires specific information on growth rates of adult oysters on the different beds. We reviewed the literature and found such information, particularly that depicting oyster growth directly on subtidal oyster beds, lacking. We used three methods to determine growth of oysters on Delaware Bay seed beds: measuring the new growth at the lip of oysters retrieved from the bottom, tethering individually identifiable oysters on specially designed frames, and size-at-age information based on ages developed from check marks in growth lines in the oyster hinge. Measuring new growth on the lip of the oyster was not accurate. Use of the frames was exact but very labor intensive and only provided data for the one season. Use of the check marks in the hinge to estimate age provided a growth history, and was relatively accurate. Intensive harvesting of oysters appeared to sig...

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.

International audience; Viruses are highly abundant in the oceans, and how filter-feeding molluscs without adaptive immunity defend themselves against viruses is not well understood. We studied the response of a mollusc Crassostrea gigas to Ostreid herpesvirus 1 µVar (OsHV-1μVar) infections using transcriptome sequencing. OsHV-1μVar can replicate extremely rapidly after challenge of C. gigas as evidenced by explosive viral transcription and DNA synthesis, which peaked at 24 and 48 h post-inoculation, respectively, accompanied by heavy oyster mortalities. At 120 h post-injection, however, viral gene transcription and DNA load, and oyster mortality, were greatly reduced indicating an end of active infections and effective control of viral replication in surviving oysters. Transcriptome analysis of the host revealed strong and complex responses involving the activation of all major innate immune pathways that are equipped with expanded and often novel receptors and adaptors. Novel Toll-like receptor (TLR) and MyD88-like genes lacking essential domains were highly up-regulated in the oyster, possibly interfering with TLR signal transduction. RIG-1/MDA5 receptors for viral RNA, interferon-regulatory factors, tissue necrosis factors and interleukin-17 were highly activated and likely central to the oyster's antiviral response. Genes related to anti-apoptosis, oxidation, RNA and protein destruction were also highly up-regulated, while genes related to anti-oxidation were down-regulated. The oxidative burst induced by the up-regulation of oxidases and severe down-regulation of anti-oxidant genes may be important for the destruction of viral components, but may also exacerbate oyster mortality. This study provides unprecedented insights into antiviral response in a mollusc. The mobilization and complex regulation of expanded innate immune-gene families highlights the oyster genome's adaptation to a virus-rich marine environment.

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.

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.

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.

▪ 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.

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.

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.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60–90% in oysters

Means to control infection by pathogenic organisms are needed to help ensure that aquaculture is not a source for the spread of infectious diseases in wild and cultured stocks. Questions often arise as to whether larval or juvenile stages become infected in the hatchery or nursery phase of production, and if so, how they might be protected. To help answer these questions, we utilized both traditional and molecular diagnostic methods to detect two eastern oyster, Crassostrea ˝irginica, pathogens Haplosporidium nelsoni, cause of MSX disease and Perkinsus marinus, . cause of Dermo disease in larval and juvenile oysters reared at a hatcheryron-shore nursery receiving water in which both parasites are enzootic. Our study indicated that filtration of water to y1 y2 . 1 mm using a cartridge filter followed by exposure to 30,000 mW s cm ultraviolet UV irradiation was an effective means of preventing infections of the larvae and post-set. Once the juveniles were moved from the highly treated hatchery water to an upweller nursery receiving .

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.

A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), was developed for detection of oyster MSX disease. The technique included using Haplosporidium nelsoni pathogen-specific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA plasmid DNA (for use as a capture probe). Digoxigenin was incorporated into the pathogen-specific PCR products, which were captured by the coated probe in a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stained agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.

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.

To avoid phagocytosis, parasites may mask themselves with host-like molecules that prevent recognition as nonself or they may produce substances that interfere with host cellular defenses. The protozoan parasiteHaplosporidium nelsoni,which causes MSX disease in the eastern oysterCrassostrea virginica,is not ingested by host hemocytes. To assess potential avoidance mechanisms, oyster hemocytes were incubated with plasmodial stages of the parasite that had been pretreated with one of a variety of enzymes (proteases and carbohydrases) to alter surface molecules or with metabolic inhibitors to prevent the synthesis or active uptake of “masking” molecules, as well as the production and discharge of inhibitory substances. The maximum increase in phagocytosis resulting from treatment with carbohydrases was 12.5% (β-galactosidase) and with proteases was 18% (Proteinase K). Inhibitors of aerobic metabolism resulted in a similar level of enhancement. In contrast, treatment of parasites with the glycolysis inhibitor iodoacetate enhanced phagocytosis by up to 66%. Thus, the process that obstructs phagocytosis involves aerobic and, especially, anaerobic pathways. The greater effect of a metabolic inhibitor compared to enzymes suggests that the mechanism involves more than just surface modification and may include the production of interference molecules.

Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated ( r 2 = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22°C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.

From its initial discovery in the Gulf of Mexico in the late 1940s until 1990,Perkinsus marinus, the parasite responsible for Dermo disease in the eastern oyster,Crassostrea virginica, was rarely found north of Chesapeake Bay. In 1990–92, an apparent range extension of the parasite led to epizootic outbreaks of the disease over a 500 km range north of Chesapeake Bay. One of the hypotheses for the range extension argues that small, undetected numbers of parasites were already present in northern oysters as the result of repeated historical introductions, and that a sharp warming trend in 1990–92 stimulated the disease outbreak. This argument was based on trends in air temperature. The present study examined this hypothesis by analysing water temperatures, rather than air temperatures, for five stations located in areas affected by the recent epizootics. At all five stations, there was a strong increasing trend in winter sea-surface temperature (SST) between 1986 and 1991. At four of the five stations, there was a smaller increasing trend in winter temperatures after 1960. There were no consistent or obvious trends in summer (August) temperatures. In Delaware Bay, which has a 40 year history of monitoring for oyster diseases, occasional findings ofP. marinusin oysters were correlated with warming episodes that were especially notable in the winter (February) record. Empirical orthogonal function (EOF) analysis showed that winter temperatures varied consistently at the stations examined and were associated with variations inP. marinusprevalence. Associations using EOF analysis with August temperatures were much weaker. The SST record is consistent with the hypothesis that increasing winter water temperatures have been important in the recent outbreak ofP. marinusepizootics in the north-eastern U.S.A.

Automated techniques for counting molluscan hemocytes and measuring certain of their characteristics are becoming more common, but there has not been a concerted attempt to compare results with more traditional microscopy. We compared electrical resistance (Coulter counter) and light-scatter (flow-cytometry) measurements of eastern oyster, Crassostrea virginica, hemocytes with those from phase-contrast light microscopy. Our findings showed that the clarity of results from the two automated measurements was inversely related to the complexity, in type and size, of the cell population being analyzed. Microscopy produced the most detailed information, but flow-cytometric measurements, even when relying on light scatter alone, clearly surpassed the discriminatory ability of the Coulter counter. Microscopic measurements documented considerable size overlap among morphologically different types of hemocytes. Because it measures size only, the Coulter counter is relatively insensitive to these differences. The flow cytometer consistently described three subpopulations: two granular and one agranular. Because of the wide range and overlap in hemocyte size and intracellular contents (the two criteria measured by light scatter) within individuals, however, we found only a loose correlation between subpopulation counts from microscopy and from flow cytometry. By combining light scatter with the flow cytometer's ability to detect specific fluorescent dyes and its capacity to physically sort cells according to defined parameters, however, we expect to greatly increase its usefulness for studying molluscan hemocytes.

Accurate detection and quantification of parasite body burden are critical for understanding many aspects of host-parasite interactions. The standard assay for diagnosing Perkinsus marinus infections in Crassostrea virginica involves incubation of oyster tissue in Ray's fluid thioglycollate medium (RFTM), followed by iodine staining and microscopic examination for parasites. The original RFTM tissue assay is destructive and provides only a ranked level of infection intensity. A recent modification provides a technique to enumerate P. marinus after incubation in RFTM and determines total body burden. Application of this technique to hemolymph samples has provided a nondestructive assay. We provide a critical evaluation of these three assays. Data from previous studies were not adequate to critically evaluate performance of the tissue and hemolymph assays. Sensitivity and accuracy of these assays were therefore compared against weight-standardized body burden in Delaware Bay oysters over the course of a year. Determination of total body burden was significantly more sensitive than the other assays. Neither tissue nor hemolymph assays provided accurate estimates of individual infection intensities and were insensitive at low infection levels. The tissue assay was easier to use and slightly more accurate than the hemolymph assay, and both provided reasonable estimates of average infection level in the population. In summary, total body burden assessment is recommended when highly accurate measures of infection intensity or prevalence are necessary, the tissue assay is recommended for monitoring epizootics because of its simplicity and accuracy at the population level, and the hemolymph assay is only recommended when oysters must be kept alive.

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.