Transcriptomics, proteomics, and physiological assays reveal immunosuppression in the eastern oyster Crassostrea virginica exposed to acidification stress.

Ocean acidification (OA) is recognized as a major stressor for a broad range of marine organisms, particularly shell-building invertebrates. OA can cause alterations in various physiological processes such as growth and metabolism, although its effect on host-pathogen interactions remains largely unexplored. In this study, we used transcriptomics, proteomics, and physiological assays to evaluate changes in immunity of the eastern oyster Crassostrea virginica exposed to OA conditions (pH = 7.5 vs pH = 7.9) at various life stages. The susceptibility of oyster larvae to Vibrio infection increased significantly (131 % increase in mortality) under OA conditions, and was associated with significant changes in their transcriptomes. The significantly higher mortality of larvae exposed to pathogens and acidification stress could be the outcome of an increased metabolic demand to cope with acidification stress (as seen by upregulation of metabolic genes) at the cost of immune function (downregulation of immune genes). While larvae were particularly vulnerable, juveniles appeared more robust to the stressors and there were no differences in mortality after pathogen (Aliiroseovarius crassostrea and Vibrio spp.) exposure. Proteomic investigations in adult oysters revealed that acidification stress resulted in a significant downregulation of mucosal immune proteins including those involved in pathogen recognition and microbe neutralization, suggesting weakened mucosal immunity. Hemocyte function in adults was also impaired by high pCO2, with a marked reduction in phagocytosis (67 % decrease in phagocytosis) in OA conditions. Together, results suggest that OA impairs immune function in the eastern oyster making them more susceptible to pathogen-induced mortality outbreaks. Understanding the effect of multiple stressors such as OA and disease is important for accurate predictions of how oysters will respond to future climate regimes.

Combination of RNAseq and RADseq to Identify Physiological and Adaptive Responses to Acidification in the Eastern Oyster (Crassostrea virginica).

Ocean acidification (OA) is a major stressor threatening marine calcifiers, including the eastern oyster (Crassostrea virginica). In this paper, we provide insight into the molecular mechanisms associated with resilience to OA, with the dual intentions of probing both acclimation and adaptation potential in this species. C. virginica were spawned, and larvae were reared in control or acidified conditions immediately after fertilization. RNA samples were collected from larvae and juveniles, and DNA samples were collected from juveniles after undergoing OA-induced mortality and used to contrast gene expression (RNAseq) and SNP (ddRADseq) profiles from animals reared under both conditions. Results showed convergence of evidence from both approaches, particularly in genes involved in biomineralization that displayed significant changes in variant frequencies and gene expression levels among juveniles that survived acidification as compared to controls. Downregulated genes were related to immune processes, supporting previous studies demonstrating a reduction in immunity from exposure to OA. Acclimation to OA via regulation of gene expression might confer short-term resilience to immediate threats; however, the costs may not be sustainable, underscoring the importance of selection of resilient genotypes. Here, we identified SNPs associated with survival under OA conditions, suggesting that this commercially and ecologically important species might have the genetic variation needed for adaptation to future acidification. The identification of genetic features associated with OA resilience is a highly-needed step for the development of marker-assisted selection of oyster stocks for aquaculture and restoration activities.

Using meta-analysis to explore the roles of global upwelling exposure and experimental design in bivalve responses to low pH.

Low pH conditions, associated with ocean acidification, represent threats to many commercially and ecologically important organisms, including bivalves. However, there are knowledge gaps regarding factors explaining observed differences in biological responses to low pH in laboratory experiments. Specific sources of local adaptation such as upwelling exposure and the role of experimental design, such as carbonate chemistry parameter changes, should be considered. Linking upwelling exposure, as an individual oceanographic phenomenon, to responses measured in laboratory experiments may further our understanding of local adaptation to global change. Here, meta-analysis is used to test the hypotheses that upwelling exposure and experimental design affect outcomes of individual, laboratory-based studies that assess bivalve metabolic (clearance and respiration rate) responses to low pH. Results show that while bivalves generally decrease metabolic activity in response to low pH, upwelling exposure and experimental design can significantly impact outcomes. Bivalves from downwelling or weak upwelling areas decrease metabolic activity in response to low pH, but bivalves from strong upwelling areas increase or do not change metabolic activity in response to low pH. Furthermore, experimental temperature, exposure time and magnitude of the change in carbonate chemistry parameters all significantly affect outcomes. These results suggest that bivalves from strong upwelling areas may be less sensitive to low pH. This furthers our understanding of local adaptation to global change by demonstrating that upwelling alone can explain up to 49 % of the variability associated with bivalve metabolic responses to low pH. Furthermore, when interpreting outcomes of individual, laboratory experiments, scientists should be aware that higher temperatures, shorter exposure times and larger changes in carbonate chemistry parameters may increase the chance of suppressed metabolic activity.

Behavioral and physiological effects of ocean acidification and warming on larvae of a continental shelf bivalve.

The negative impacts of ocean warming and acidification on bivalve fisheries are well documented but few studies investigate parameters relevant to energy budgets and larval dispersal. This study used laboratory experiments to assess developmental, physiological and behavioral responses to projected climate change scenarios using larval Atlantic surfclams Spisula solidissima solidissima, found in northwest Atlantic Ocean continental shelf waters. Ocean warming increased feeding, scope for growth, and biomineralization, but decreased swimming speed and pelagic larval duration. Ocean acidification increased respiration but reduced immune performance and biomineralization. Growth increased under ocean warming only, but decreased under combined ocean warming and acidification. These results suggest that ocean warming increases metabolic activity and affects larval behavior, while ocean acidification negatively impacts development and physiology. Additionally, principal component analysis demonstrated that growth and biomineralization showed similar response profiles, but inverse response profiles to respiration and swimming speed, suggesting alterations in energy allocation under climate change.

Increased Food Resources Help Eastern Oyster Mitigate the Negative Impacts of Coastal Acidification.

Oceanic absorption of atmospheric CO2 results in alterations of carbonate chemistry, a process coined ocean acidification (OA). The economically and ecologically important eastern oyster (Crassostrea virginica) is vulnerable to these changes because low pH hampers CaCO3 precipitation needed for shell formation. Organisms have a range of physiological mechanisms to cope with altered carbonate chemistry; however, these processes can be energetically expensive and necessitate energy reallocation. Here, the hypothesis that resilience to low pH is related to energy resources was tested. In laboratory experiments, oysters were reared or maintained at ambient (400 ppm) and elevated (1300 ppm) pCO2 levels during larval and adult stages, respectively, before the effect of acidification on metabolism was evaluated. Results showed that oysters exposed to elevated pCO2 had significantly greater respiration. Subsequent experiments evaluated if food abundance influences oyster response to elevated pCO2. Under high food and elevated pCO2 conditions, oysters had less mortality and grew larger, suggesting that food can offset adverse impacts of elevated pCO2, while low food exacerbates the negative effects. Results also demonstrated that OA induced an increase in oyster ability to select their food particles, likely representing an adaptive strategy to enhance energy gains. While oysters appeared to have mechanisms conferring resilience to elevated pCO2, these came at the cost of depleting energy stores, which can limit the available energy for other physiological processes. Taken together, these results show that resilience to OA is at least partially dependent on energy availability, and oysters can enhance their tolerance to adverse conditions under optimal feeding regimes.

An apicomplexan parasite drives the collapse of the bay scallop population in New York.

The bay scallop, Argopecten irradians, represents a commercially, culturally and ecologically important species found along the United States' Atlantic and Gulf coasts. Since 2019, scallop populations in New York have been suffering large-scale summer mortalities resulting in 90-99% reduction in biomass of adult scallops. Preliminary investigations of these mortality events showed 100% prevalence of an apicomplexan parasite infecting kidney tissues. This study was designed to provide histological, ultrastructural and molecular characteristics of a non-described parasite, member of the newly established Marosporida clade (Apicomplexa) and provisionally named BSM (Bay Scallop Marosporida). Molecular diagnostics tools (quantitative PCR, in situ hybridization) were developed and used to monitor disease development. Results showed that BSM disrupts multiple scallop tissues including kidney, adductor muscle, gill, and gonad. Microscopy observations allowed the identification of both intracellular and extracellular stages of the parasite. Field surveys demonstrated a strong seasonal signature in disease prevalence and intensity, as severe cases and mortality increase as summer progresses. These results strongly suggest that BSM infection plays a major role in the collapse of bay scallop populations in New York. In this framework, BSM may synergistically interact with stressful environmental conditions to impair the host and lead to mortality.

RNAi Silencing of the Biomineralization Gene Perlucin Impairs Oyster Ability to Cope with Ocean Acidification.

Calcifying marine organisms, including the eastern oyster (Crassostrea virginica), are vulnerable to ocean acidification (OA) because it is more difficult to precipitate calcium carbonate (CaCO3). Previous investigations of the molecular mechanisms associated with resilience to OA in C. virginica demonstrated significant differences in single nucleotide polymorphism and gene expression profiles among oysters reared under ambient and OA conditions. Converged evidence generated by both of these approaches highlighted the role of genes related to biomineralization, including perlucins. Here, gene silencing via RNA interference (RNAi) was used to evaluate the protective role of a perlucin gene under OA stress. Larvae were exposed to short dicer-substrate small interfering RNA (DsiRNA-perlucin) to silence the target gene or to one of two control treatments (control DsiRNA or seawater) before cultivation under OA (pH ~7.3) or ambient (pH ~8.2) conditions. Two transfection experiments were performed in parallel, one during fertilization and one during early larval development (6 h post-fertilization), before larval viability, size, development, and shell mineralization were monitored. Silenced oysters under acidification stress were the smallest, had shell abnormalities, and had significantly reduced shell mineralization, thereby suggesting that perlucin significantly helps larvae mitigate the effects of OA.

Molecular Features Associated with Resilience to Ocean Acidification in the Northern Quahog, Mercenaria mercenaria.

The increasing concentration of CO2 in the atmosphere and resulting flux into the oceans will further exacerbate acidification already threatening coastal marine ecosystems. The subsequent alterations in carbonate chemistry can have deleterious impacts on many economically and ecologically important species including the northern quahog (Mercenaria mercenaria). The accelerated pace of these changes requires an understanding of how or if species and populations will be able to acclimate or adapt to such swift environmental alterations. Thus far, studies have primarily focused on the physiological effects of ocean acidification (OA) on M. mercenaria, including reductions in growth and survival. However, the molecular mechanisms of resilience to OA in this species remains unclear. Clam gametes were fertilized under normal pCO2 and reared under acidified (pH ~ 7.5, pCO2 ~ 1200 ppm) or control (pH ~ 7.9, pCO2 ~ 600 ppm) conditions before sampled at 2 days (larvae), 32 days (postsets), 5 and 10 months (juveniles) and submitted to RNA and DNA sequencing to evaluate alterations in gene expression and genetic variations. Results showed significant shift in gene expression profiles among clams reared in acidified conditions as compared to their respective controls. At 10 months of exposure, significant shifts in allele frequency of single nucleotide polymorphisms (SNPs) were identified. Both approaches highlighted genes coding for proteins related to shell formation, bicarbonate transport, cytoskeleton, immunity/stress, and metabolism, illustrating the role these pathways play in resilience to OA.

Proteomic and Transcriptomic Responses Enable Clams to Correct the pH of Calcifying Fluids and Sustain Biomineralization in Acidified Environments.

Seawater pH and carbonate saturation are predicted to decrease dramatically by the end of the century. This process, designated ocean acidification (OA), threatens economically and ecologically important marine calcifiers, including the northern quahog (Mercenaria mercenaria). While many studies have demonstrated the adverse impacts of OA on bivalves, much less is known about mechanisms of resilience and adaptive strategies. Here, we examined clam responses to OA by evaluating cellular (hemocyte activities) and molecular (high-throughput proteomics, RNASeq) changes in hemolymph and extrapallial fluid (EPF-the site of biomineralization located between the mantle and the shell) in M. mercenaria continuously exposed to acidified (pH ~7.3; pCO2 ~2700 ppm) and normal conditions (pH ~8.1; pCO2 ~600 ppm) for one year. The extracellular pH of EPF and hemolymph (~7.5) was significantly higher than that of the external acidified seawater (~7.3). Under OA conditions, granulocytes (a sub-population of hemocytes important for biomineralization) were able to increase intracellular pH (by 54% in EPF and 79% in hemolymph) and calcium content (by 56% in hemolymph). The increased pH of EPF and hemolymph from clams exposed to high pCO2 was associated with the overexpression of genes (at both the mRNA and protein levels) related to biomineralization, acid-base balance, and calcium homeostasis, suggesting that clams can use corrective mechanisms to mitigate the negative impact of OA.

Chromosomal assembly of the flat oyster (Ostrea edulis L.) genome as a new genetic resource for aquaculture.

The European flat oyster (Ostrea edulis L.) is a native bivalve of the European coasts. Harvest of this species has declined during the last decades because of the appearance of two parasites that have led to the collapse of the stocks and the loss of the natural oyster beds. O. edulis has been the subject of numerous studies in population genetics and on the detection of the parasites Bonamia ostreae and Marteilia refringens. These studies investigated immune responses to these parasites at the molecular and cellular levels. Several genetic improvement programs have been initiated especially for parasite resistance. Within the framework of a European project (PERLE 2) that aims to produce genetic lines of O. edulis with hardiness traits (growth, survival, resistance) for the purpose of repopulating natural oyster beds in Brittany and reviving the culture of this species in the foreshore, obtaining a reference genome becomes essential as done recently in many bivalve species of aquaculture interest. Here, we present a chromosome-level genome assembly and annotation for the European flat oyster, generated by combining PacBio, Illumina, 10X linked, and Hi-C sequencing. The finished assembly is 887.2 Mb with a scaffold-N50 of 97.1 Mb scaffolded on the expected 10 pseudochromosomes. Annotation of the genome revealed the presence of 35,962 protein-coding genes. We analyzed in detail the transposable element (TE) diversity in the flat oyster genome, highlighted some specificities in tRNA and miRNA composition, and provided the first insight into the molecular response of O. edulis to M. refringens. This genome provides a reference for genomic studies on O. edulis to better understand its basic physiology and as a useful resource for genetic breeding in support of aquaculture and natural reef restoration.

A Transcriptomic Analysis of Phenotypic Plasticity in Crassostrea virginica Larvae under Experimental Acidification.

Ocean acidification (OA) is a major threat to marine calcifiers, and little is known regarding acclimation to OA in bivalves. This study combined physiological assays with next-generation sequencing to assess the potential for recovery from and acclimation to OA in the eastern oyster (Crassostrea virginica) and identify molecular mechanisms associated with resilience. In a reciprocal transplant experiment, larvae transplanted from elevated pCO2 (~1400 ppm) to ambient pCO2 (~350 ppm) demonstrated significantly lower mortality and larger size post-transplant than oysters remaining under elevated pCO2 and had similar mortality compared to those remaining in ambient conditions. The recovery after transplantation to ambient conditions demonstrates the ability for larvae to rebound and suggests phenotypic plasticity and acclimation. Transcriptomic analysis supported this hypothesis as genes were differentially regulated under OA stress. Transcriptomic profiles of transplanted and non-transplanted larvae terminating in the same final pCO2 converged, further supporting the idea that acclimation underlies resilience. The functions of differentially expressed genes included cell differentiation, development, biomineralization, ion exchange, and immunity. Results suggest acclimation as a mode of resilience to OA. In addition, the identification of genes associated with resilience can serve as a valuable resource for the aquaculture industry, as these could enable marker-assisted selection of OA-resilient stocks.

Transcriptomic, Proteomic, and Functional Assays Underline the Dual Role of Extrapallial Hemocytes in Immunity and Biomineralization in the Hard Clam Mercenaria mercenaria.

Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the "biomineralization toolkit" and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca2+ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

High spatial resolution mapping of the mucosal proteome of the gills of Crassostrea virginica: implication in particle processing.

In the oyster Crassostrea virginica, the organization of the gill allows bidirectional particle transport where a dorsal gill tract directs particles meant to be ingested while a ventral tract collects particles intended to be rejected as pseudofeces. Previous studies showed that the transport of particles in both tracts is mediated by mucus. Consequently, we hypothesized that the nature and/or the quantity of mucosal proteins present in each tract is likely to be different. Using endoscopy-aided micro-sampling of mucus from each tract followed by multidimensional protein identification technologies, and in situ hybridization, a high spatial resolution mapping of the oyster gill proteome was generated. Results showed the presence in gill mucus of a wide range of molecules involved in non-self recognition and interactions with microbes. Mucus composition was different between the two tracts, with mucus from the ventral tract shown to be rich in mucin-like proteins, providing an explanation of its high viscosity, while mucus from the dorsal tract was found to be enriched in mannose-binding proteins, known to be involved in food particle binding and selection. Overall, this study generated high-resolution proteomes for C. virginica gill mucus and demonstrated that the contrasting functions of the two pathways present on oyster gills are associated with significant differences in their protein makeup.

Identification of variants associated with hard clam, Mercenaria mercenaria, resistance to Quahog Parasite Unknown disease.

Severe losses in aquacultured and wild hard clam (Mercenaria mercenaria) stocks have been previously reported in the northeastern United States due to a protistan parasite called QPX (Quahog Parasite Unknown). Previous work demonstrated that clam resistance to QPX is under genetic control. This study identifies single nucleotide polymorphism (SNP) associated with clam survivorship from two geographically segregated populations, both deployed in an enzootic site. The analysis contrasted samples collected before and after undergoing QPX-related mortalities and relied on a robust draft clam genome assembly. ~200 genes displayed significant variant enrichment at each sampling point in both populations, including 18 genes shared between both populations. Markers from both populations were identified in genes related to apoptosis pathways, protein-protein interaction, receptors, and signaling. This research begins to identify genetic markers associated with clam resistance to QPX disease, leading the way for the development of resistant clam stocks through marker-assisted selection.

Experimental acidification increases susceptibility of Mercenaria mercenaria to infection by Vibrio species.

Ocean acidification alters seawater carbonate chemistry, which can have detrimental impacts for calcifying organisms such as bivalves. This study investigated the physiological cost of resilience to acidification in Mercenaria mercenaria, with a focus on overall immune performance following exposure to Vibrio spp. Larval and juvenile clams reared in seawater with high pCO2 (~1200 ppm) displayed an enhanced susceptibility to bacterial pathogens. Higher susceptibility to infection in clams grown under acidified conditions was derived from a lower immunity to infection more so than an increase in growth of bacteria under high pCO2. A reciprocal transplant of juvenile clams demonstrated the highest mortality amongst animals transplanted from low pCO2/high pH to high pCO2/low pH conditions and then exposed to bacterial pathogens. Collectively, these results suggest that increased pCO2 will result in immunocompromised larvae and juveniles, which could have complex and pernicious effects on hard clam populations.

From the raw bar to the bench: Bivalves as models for human health.

Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell differentiation, the ability to fight opportunistic and specific pathogens in the absence of adaptive immunity, as source of alternative drugs, mucosal immunity and, microbiome turnover, toxicology, and cancer research. There is still a long way to go; however, the next time you order a dozen oysters at your favorite raw bar, think about a tasty model organism that will not only please your palate but also help unlock multiple aspects of molluscan biology and improve human health.

Regulation of apoptosis-related genes during interactions between oyster hemocytes and the alveolate parasite Perkinsus marinus.

The alveolate Perkinsus marinus is the most devastating parasite of the eastern oyster Crassostrea virginica. The parasite is readily phagocytosed by oyster hemocytes, but instead of intracellular killing and digestion, P. marinus can survive phagocytosis and divide in host cells. This intracellular parasitism is accompanied by a regulation of host cell apoptosis. This study was designed to gain a better understanding of the molecular mechanisms of apoptosis regulation in oyster hemocytes following exposure to P. marinus. Regulation of apoptosis-related genes in C. virginica, and apoptosis-regulatory genes in P. marinus, were investigated via qPCR to assess the possible pathways involved during these interactions. In vitro experiments were also carried out to evaluate the effect of chemical inhibitors of P. marinus antioxidant processes on hemocyte apoptosis. Results indicate the involvement of the mitochondrial pathway (Bcl-2, anamorsin) of apoptosis in C. virginica exposed to P. marinus. In parallel, the antioxidants peroxiredoxin and superoxide dismutase were regulated in P. marinus exposed to C. virginica hemocytes suggesting that apoptosis regulation in infected oysters may be mediated by anti-oxidative processes. Chemical inhibition of P. marinus superoxide dismutase resulted in a marked increase of reactive oxygen species production and apoptosis in infected hemocytes. The implication of oxygen-dependent apoptosis during P. marinus infection and disease development in C. virginica is discussed.

Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks.

Regulation of oyster (Crassostrea virginica) hemocyte motility by the intracellular parasite Perkinsus marinus: A possible mechanism for host infection.

Hemocytes associated with the mucus lining of pallial (mantle, gill) surfaces of the oyster Crassostrea virginica have been recently suggested to facilitate infection by the Alveolate parasite Perkinsus marinus by mediating the uptake and dispersion of parasite cells. These "pallial hemocytes", which are directly exposed to microbes present in surrounding seawater, are able to migrate bi-directionally between mucosal surfaces and the circulatory system, potentially playing a sentinel role. Interestingly, P. marinus was shown to increase trans-epithelial migration of hemocytes suggesting it may regulate cell motility to favor infection establishment. The purpose of this study was to investigate the effect of P. marinus on hemocyte motility and identify specific molecular mechanisms potentially used by the parasite to regulate hemocyte migration. In a first series of experiments, various components of P. marinus (live P. marinus cells, extracellular products, fragments of P. marinus cell membrane, membrane-modified live P. marinus cells, heat-killed P. marinus) along with components of the opportunistic bacterial pathogen Vibrio alginolyticus (bacterial cells and extracellular products) were investigated for their effects on hemocyte motility. In a second series of experiments, inhibitors of specific molecular pathways involved in motility regulation (Y-27632: inhibitor of Rho-associated protein kinase, RGDS: integrin inhibitor, CK-666: Arp2/3 inhibitor) were used in conjunction with qPCR gene expression experiments to identify pathways regulated by P. marinus exposure. Results showed a specific increase in hemocyte motility following exposure to live P. marinus cells. The increase in motility induced by P. marinus was suppressed by RGDS and CK-666 implicating the involvement of integrins and Arp2/3 in cell activation. Gene expression data suggest that Arp2/3 is possibly regulated directly by an effector produced by P. marinus. The implications of increased hemocyte motility prompted by P. marinus during the early stage of the infection process are discussed.