Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria).

BACKGROUND: The hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability. RESULTS: Whole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies. CONCLUSIONS: The 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species' genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

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.

Warming and hypoxia reduce the performance and survival of northern bay scallops (Argopecten irradians irradians) amid a fishery collapse.

Warming temperatures and diminishing dissolved oxygen (DO) concentrations are among the most pervasive drivers of global coastal change. While regions of the Northwest Atlantic Ocean are experiencing greater than average warming, the combined effects of thermal and hypoxic stress on marine life in this region are poorly understood. Populations of the northern bay scallop, Argopecten irradians irradians across the northeast United States have experienced severe declines in recent decades. This study used a combination of high-resolution (~1 km) satellite-based temperature records, long-term temperature and DO records, field and laboratory experiments, and high-frequency measures of scallop cardiac activity in an ecosystem setting to quantify decadal summer warming and assess the vulnerability of northern bay scallops to thermal and hypoxic stress across their geographic distribution. From 2003 to 2020, significant summer warming (up to ~0.2°C year-1 ) occurred across most of the bay scallop range. At a New York field site in 2020, all individuals perished during an 8-day estuarine heatwave that coincided with severe diel-cycling hypoxia. Yet at a Massachusetts site with comparable DO levels but lower daily mean temperatures, mortality was not observed. A 96-h laboratory experiment recreating observed daily temperatures of 25 or 29°C, and normoxia or hypoxia (22.2% air saturation), revealed a 120-fold increased likelihood of mortality in the 29°C-hypoxic treatment compared with control conditions, with scallop clearance rates also reduced by 97%. Cardiac activity measurements during a field deployment indicated that low DO and elevated daily temperatures modulate oxygen consumption rates and likely impact aerobic scope. Collectively, these findings suggest that concomitant thermal and hypoxic stress can have detrimental effects on scallop physiology and survival and potentially disrupt entire fisheries. Recovery of hypoxic systems may benefit vulnerable fisheries under continued warming.

Triploid animals, a potential model for ETosis research: Influence of polyploidy on the formation and efficacy of extracellular traps in the eastern oyster.

Decondensation and the subsequent release of chromatin from specific immune cells in response to inflammatory stimuli is a highly conserved aspect of the innate immune system and leads to the formation of extracellular traps, observable in nearly all forms of multicellular life. This process is known as ETosis, with the release of DNA and its associated antimicrobial proteins physically capturing and neutralizing pathogens following an infection or tissue damage. Despite the universality of this response, data concerning extracellular traps in non-model organisms is limited, with most invertebrate studies doing little more than proving their existence due to difficulties in stimulation and high interindividual variability in trap production. This study provides a novel, simple, and inexpensive method for the consistent stimulation of extracellular traps in eastern oyster (Crassostrea virginica) hemocytes. Using the methods described in this study, we compared how ploidy impacts the rate, size, and efficacy of extracellular traps. Findings demonstrated that hemocyte extracellular traps were potent antimicrobials against both Gram-positive and Gram-negative bacteria. Furthermore, we provide evidence to suggest that agranulocytes may be the primary ETosis effector cells in C. virginica. This study is the first to describe extracellular traps in C. virginica and highlights the possible benefits of using triploid animals to gain a further understanding of ETosis and the factors that regulate its induction and efficacy.

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.

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.

Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

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.

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.

Quantification of the inflammatory responses to pro-and anti-inflammatory agents in Manila clam, Ruditapes philippinarum.

Inflammation is a form of innate immune response of living organisms to harmful stimuli. In marine bivalves, inflammation is a common defense mechanism. Several studies have investigated the morphological features of inflammation in bivalves, such as hemocyte infiltration. However, the molecular and biochemical responses associated with inflammation in marine bivalves remain unexplored. Here, we investigated changes in nitric oxide (NO) levels, cyclooxygenase 2 (COX-2) activity, and allograft inflammatory factor-1 (AIF-1) gene expression levels in hemolymph samples collected from Manila clam (Ruditapes philippinarum) exposed to pro- and anti-inflammatory substances. These included the pro-inflammatory agent lipopolysaccharide (LPS), and the nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac, all widely used in vertebrates. Our study showed that NO levels, COX-2 activity, and AIF-1 expression increased in response to the treatments with LPS and decreased in response to the treatments with NSAIDs in a concentration-dependent manner. These results suggest that the mechanism of inflammatory responses in bivalves is very similar to that of vertebrates, and we propose that inflammatory responses can be quantified using these techniques and used to determine the physiological status of marine bivalves exposed to biotic or abiotic stresses.

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.

First report of signs of infection by Coccomyxa-like algae in wild blue mussels, Mytilus spp., in the Gulf of Maine (USA, Maine).

In August 2019, visual inspection of intertidal zones of the Gulf of Maine (ME, USA) revealed young and adult wild blue mussels, Mytilus spp., in Alley Bay (Jonesport area) with the distinctive L-shaped shell deformity (LSSD) and green spots (GS) in the mantle and adductor muscle. LSSD is a characteristic sign of current or previous mussel infection by photosynthetic unicellular alga from the group Coccomyxa, while GS are algal colonies. Based on these findings, this study represents the first report of infection signs by pathogenic Coccomyxa-like algae in mussels from the coastal waters of the Northeastern United States, providing a base for future large scale monitoring of the alga in the region.

SNP hot-spots in the clam parasite QPX.

BACKGROUND: Quahog Parasite Unknown (QPX) is an opportunistic protistan pathogen of the clam Mercenaria mercenaria. Infections with QPX have caused significant economic losses in the Northeastern United States. Previous research demonstrated a geographic gradient for disease prevalence and intensity, but little information is available on the genetic diversity of the parasite throughout its distribution range. Also, QPX virulence factors are not well understood. This study addresses the occurrence of QPX genetic variants with a particular focus on functions involved in virulence and adaptation to environmental conditions. RESULTS: Analyses were performed using transcriptome-wide single-nucleotide polymorphism (SNP) of four QPX isolates cultured from infected clams collected from disparate locations along the Northeastern United States. For contig assembly and mapping, two different genome builds and four transcriptomes of the parasite were examined. Genomic variants appeared at a differential rate relative to sequenced transcripts at 20.18 and 22.55% occurrence under 1000 base pairs upstream and downstream protein domains respectively and at 57.26% rate in protein domain coding sequences. QPX strains shared 30.50% of the mutations and exhibited a preferential nucleotide substitution towards thymine. Sequence identity suggested relatedness between different QPX strains, with the parasite being possibly introduced to Virginia from the Massachusetts region during clam trading, while QPX could have been naturally present in New York. Diversity in virulence, temperature, and salinity domains suggested a common variability between strains, but with a preferential higher variation in local adaptation genes. This could explain differences in disease prevalence noted in different regions. Overall, the results supported views that this opportunistic parasite might be able to adapt to varying environmental conditions. CONCLUSION: Relatedness and mutations between the four QPX strains suggested that variability in environmental-related functions favors parasite survival, potentially promoting resilience against stressful conditions. These findings are in agreement with the widespread presence of QPX in the environment. Although QPX levels are enzootic in most areas, an increase in disease outbreaks were often associated with seasonal changes in environmental conditions. A selection mediated by the parasitic life of QPX remains possible, but the effect of the environment on the biology of the parasite appears more obvious.

Reverse genetics demonstrate the role of mucosal C-type lectins in food particle selection in the oyster Crassostrea virginica.

Prey selection governs species interactions and regulates physiological energetics of individuals and populations. Suspension-feeding bivalves represent key species in coastal and estuarine systems for their ecological and economic value. These animals are able to sort and selectively ingest nutritious microalgae from dilute and composite mixtures of particulate matter. This aptitude was suggested to be mediated by interactions between carbohydrates associated with the surface of microalgae and C-type lectins present in mucus covering the feeding organs, although a direct, unequivocal, role of lectins in food sorting in bivalves remains elusive. This study was designed to identify and characterize mucosal C-type lectins from oysters and manipulate the expression of these proteins in order to obtain decisive information regarding their involvement in food choice. Thus, two mucosal C-type lectins (CvML3912 and CvML3914) were identified based on transcriptomic and proteomic information. Transcripts of these lectins were detected in the feeding organs and their expression was upregulated following starvation. Recombinant lectin (rCvML3912) competitively inhibited the binding of commercial mannose/glucose-specific lectins to microalgae. Short Dicer-substrate small interfering RNA (DsiRNA) targeting these two lectins were designed and used to evaluate the effect of gene silencing on food particle sorting. As a result, the abundance of the two cognate transcripts significantly decreased and food sorting ability was significantly reduced among silenced oysters as compared with control animals. Overall, these findings propose a novel concept establishing the role of carbohydrate-protein interactions to provide efficient food particle sorting, and establish a new dimension for the role of evolutionarily conserved mannose/glucose-binding proteins in metazoans.

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.

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.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

The influence of temperature stress on the physiology of the Atlantic surfclam, Spisula solidissima.

Atlantic surfclam populations have significantly declined in state and federal waters from the south shore of Long Island, New York to the Delmarva Peninsula since the early 2000s. Previous studies have demonstrated that surfclams in this geographic range show signs of physiological stress, suggested to be a result of increasing ocean temperatures. In this study, we examined the effect of 2 temperature regimes (19 °C and 23 °C) on surfclam physiology. These temperatures were chosen because they represent maximal (23 °C) and minimal (19 °C) temperatures prevailing in New York clamming areas during summer. Results demonstrated enhanced energy metabolism and significant reductions in filtration rate, scope for growth, and immune functions in clams exposed to the warmer temperature treatment. Although net energy gains remained positive in both treatments under our experimental conditions, the findings suggest that temperature stress is involved in the recent observations of surfclams in poor condition. The impact of elevated temperatures on phytoplankton quantity/quality and other environmental variables in combination with the direct impact on surfclam filtration and metabolic rates could lead to a negative energy balance. While some uncertainties remain about population-scale impacts of overall warming trends, we fear that future increases in temperature may lead to the collapse of the Atlantic surfclam between New York and Virginia, especially within inshore regions.

Differential Gene Expression in Five Isolates of the Clam Pathogen, Quahog Parasite Unknown (QPX).

Quahog parasite unknown (QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed (DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay (RB, NY) and from Provincetown Harbor (MA) were more similar to each other and diverged from QPX isolates from Peconic Bay (PB, NY) and Old Plantation Creek (VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern (MA) and deeper (RB, NY) locations as compared to southern (VA) and shallower (PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions.