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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.

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.

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.

Lectins are a large and diverse group of sugar-binding proteins involved in nonself recognition and cell-to-cell interactions. Suspension-feeding bivalves, such as the oyster Crassostrea virginica, are capable of using these molecules to bind cell surface carbohydrates of food particles, allowing particle capture and selection. The aim of this project was to assess whether the expression of mucosal lectins in C. virginica is constant or changes with the season, and to determine whether lectin expression is linked to environmental parameters and/or internal biological factors (gametogenesis). A total of 130 oysters were placed in submerged cages at a tidal estuary and monitored for changes in lectin gene expression over a 1-year period. In parallel, environmental parameters prevailing at the field site, including seawater physicochemical characteristics (temperature, salinity and dissolved oxygen), particulate organic matter and chlorophyll contents, were also monitored. Throughout the study, oysters were dissected and the gills were collected and used for the assessment of the expression of three different lectin genes (CvML, CvML3914 and CvML3912). Remaining tissues were processed for histology and the classification of the gonad development stage. Results showed that when food is abundant, such as during the spring bloom, lectin gene expressions are low, and inversely lectin levels increase with lower food levels. These findings suggest that oysters increase lectin expression to enhance the capture and ingestion of scarce food, while during spring, enough food is already being ingested and lectins are not needed. Furthermore, results showed that as the energy demands of oysters increase (gonad maturation), lectin gene expressions also increase to enhance selective ingestion of nutritious food particles. This study, therefore, demonstrates the seasonality of lectin gene expression in C. virginica, and suggests that lectin regulation is related to the reproduction process and abundance of high-quality food.

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.

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.

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

The hard clam, Mercenaria mercenaria, is one of the most valuable commercial mollusk species along the eastern coast of the United States. Throughout the past 2 decades, the hard clam industry in the Northeast was significantly impacted by disease outbreaks caused by a lethal protistan parasite known as Quahog Parasite Unknown (QPX). QPX is an opportunistic pathogen and the infection has been shown to be a cold water disease, where warmer conditions (above 21 °C) lead to disease reduction and clam healing. In vitro studies also showed a sharp reduction in parasite growth and survivorship at temperatures exceeding 27 °C. In this study, we evaluated the effect of short-term exposures to high temperatures on QPX disease dynamic and clam recovery. Infected clams were collected from an enzootic site and subsequently submitted to one of ten “heat shock” treatments involving a gradient of temperatures and exposure times. QPX prevalence was compared before and 10 weeks after heat shock to assess the effect of each treatment on disease progress. Expression of several stress-related genes was measured 1 and 7 days after heat shock using qPCR to evaluate the effect of each treatment on clam physiology. Anti-QPX activity in clam plasma was also measured in an attempt to link changes in defense factors to thermal stress and disease progress. Our results suggest that brief exposures to moderate high temperatures promote the greatest remission while imposing the mildest stress to clams. These results are discussed with the aim of providing the industry with possible strategies to mitigate QPX disease.

Significant progress has been made in the understanding of cellular and molecular mediators of immunity in invertebrates in general and bivalve mollusks in particular. Despite this information, there is a lack of understanding of factors affecting animal resistance and specific responses to infections. This in part results from limited consideration of the spatial (and to some extent temporal) heterogeneity of immune responses and very limited information on host-pathogen (and microbes in general) interactions at initial encounter/colonization sites. Of great concern is the fact that most studies on molluscan immunity focus on the circulating hemocytes and the humoral defense factors in the plasma while most relevant host-microbe interactions occur at mucosal interfaces. This paper summarizes information available on the contrasting value of information available on focal and systemic immune responses in infected bivalves, and highlights the role of mucosal immune factors in host-pathogen interactions. Available information underlines the diversity of immune effectors at molluscan mucosal interfaces and highlights the tailored immune response to pathogen stimuli. This context raises fascinating basic research questions around host-microbe crosstalk and feedback controls of these interactions and may lead to novel disease mitigation strategies and improve the assessment of resistant crops or the screening of probiotic candidates.

The soft body surface of marine invertebrates is covered by a layer of mucus, a slippery gel secreted by mucocytes lining epithelia. The functions of this gel are diverse including locomotion, cleansing, food particles processing and defense against physicochemical injuries and infectious agents. In oysters, mucus covering pallial organs has been demonstrated to have a major importance in the processing of food particles and in the interactions with waterborne pathogens. Given the limited information available on mucus in bivalves and the apparent wide spectra of activity of bioactive molecules present in this matrix, the characterization of these mucosal secretions has become a research priority. In this study, mucus was separately collected from the mantle, gills and labial palps of the eastern oyster (Crassostrea virginica) and analyzed by liquid chromatography and tandem mass spectrometry. Results showed the presence of a wide variety of molecules involved in host-microbe interactions, including putative adhesion molecules (e.g. c-type lectins) confirming that transcripts previously identified in epithelial cells are translated into proteins secreted in mucus. Mucus composition was different among samples collected from different organs. These results generate a reference map for C. virginica pallial mucus to better characterize the various physiological functions of mucosal secretions.

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.

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.

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.

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.

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.

Background The hard clam Mercenaria mercenaria is an important seafood species widely exploited along the eastern coasts of the United States and play a crucial role in coastal ecology and economy. Severe hard clam mortalities have been associated with the protistan parasite QPX (Quahog Parasite Unknown). QPX infection establishes in pallial organs with the lesions typically characterized as nodules, which represent inflammatory masses formed by hemocyte infiltration and encapsulation of parasites. QPX infection is known to induce host changes on both the whole-organism level and at specific lesion areas, which imply systemic and focal defense responses, respectively. However, little is known about the molecular mechanisms underlying these alterations. Results RNA-seq was performed using Illumina Hiseq 2000 (641 Million 100 bp reads) to characterize M. mercenaria focal and systemic immune responses to QPX. Transcripts were assembled and the expression levels were compared between nodule and healthy tissues from infected clams, and between these and tissues from healthy clams. De novo assembly reconstructed a consensus transcriptome of 62,980 sequences that was functionally-annotated. A total of 3,131 transcripts were identified as differentially expressed in different tissues. Results allowed the identification of host immune factors implicated in the systemic and focal responses against QPX and unraveled the pathways involved in parasite neutralization. Among transcripts significantly modulated upon host-pathogen interactions, those involved in non-self recognition, signal transduction and defense response were over-represented. Alterations in pathways regulating hemocyte focal adhesion, migration and apoptosis were also demonstrated. Conclusions Our study is the first attempt to thoroughly characterize M. mercenaria transcriptome and identify molecular features associated with QPX infection. It is also one of the first studies contrasting focal and systemic responses to infections in invertebrates using high-throughput sequencing. Results identified the molecular signatures of clam systemic and focal defense responses, to collectively mediate immune processes such as hemocyte recruitment and local inflammation. These investigations improve our understanding of bivalve immunity and provide molecular targets for probing the biological bases of clam resistance towards QPX. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2493-9) contains supplementary material, which is available to authorized users.

Quahog Parasite Unknown (QPX) is a fatal protistan parasite that causes severe losses in the hard clam (Mercenaria mercenaria) fisheries along the northeastern coast of the US. Field and laboratory studies of QPX disease have demonstrated a major role for water temperature and M. mercenaria genetic origin in disease development. Infections are more likely to occur at cold temperatures, with clam stocks originating from southern states being more susceptible than clams from northern origin where disease is enzootic. Even though the influence of temperature on QPX infection have been examined in susceptible and resistant M. mercenaria at physiological and cellular scales, the underlying molecular mechanisms associated with host-pathogen interactions remain largely unknown. This study was carried out to explore the molecular changes in M. mercenaria in response to temperature and QPX infection on the transcriptomic level, and also to compare molecular responses between susceptible and resistant clam stocks. A M. mercenaria oligoarray (15 K Agilent) platform was produced based on our previously generated transcriptomic data and was used to compare gene expression profiles in naive and QPX-infected susceptible (Florida stock) and resistant (Massachusetts) clams maintained at temperatures favoring disease development (13 °C) or clam healing (21 °C). In addition, transcriptomic changes reflecting focal (the site of infection, mantle) and systemic (circulating hemocytes) responses were also assessed using the oligoarray platform. Results revealed significant regulation of multiple biological pathways by temperature and QPX infection, mainly associated with immune recognition, microbial killing, protein synthesis, oxidative protection and metabolism. Alterations were widely systemic with most changes in gene expression revealed in hemocytes, highlighting the role of circulating hemocytes as the first line of defense against pathogenic stress. A large number of complement-related recognition molecules with fibrinogen or C1q domains were shown to be specially induced following QPX challenge, and the expression of these molecules was significantly higher in resistant clams as compared to susceptible ones. These highly variable immune proteins may be potent candidate molecular markers for future study of M. mercenaria resistance against QPX. Beyond the specific case of clam response to QPX, this study also provides insights into the primitive complement-like system in the hard clam.

International audience; Loripes lacteus inhabits reduced sediments in shallow waters and harbors chemotrophic sulfur-oxidizing bacteria in its gills. In this study, a combination of molecular (16S rRNA analysis, real time PCR and fluorescence in-situ hybridization) and microscopy approaches (confocal and transmission electron microscopy) was used to characterize this symbiotic association and evaluate factors potentially involved in endosymbiont acquisition and regulation. 16S rRNA sequencing revealed the presence of two phylotypes of endobacteria specifically associated with clam gills. The more abundant phylotype is a close relative to thiotrophic symbionts found in several symbiotic Lucinidae, while the second phylotype clustered with bacteria not known to be involved in symbiotic association. Endobacteria were not detected in other organs including gonads strongly suggesting a horizontal mode of transmission of symbionts to aposymbiotic juveniles. Clams were exposed to 3 weeks of starvation, which caused a significant decrease in the relative abundance of both phylotypes as compared to fresh animals suggesting a lytic degradation of endobacteria within clam gills. In addition, endosymbiont-depleted clams were not able to re-acquire their symbionts once moved back to ``optimal'' conditions. These results highlight the need for a better understanding of the molecular mechanisms that mediate acquisition, regulation and control of endosymbiotic bacteria by L lacteus. (C) 2013 Elsevier B.V. All rights reserved.

The body surface of marine invertebrates is often covered by a layer of mucus, a slippery gel secreted by mucocytes (or goblet cells) lining epithelia. The functions of this gel are diverse and include locomotion, cleansing, food particles processing and defense against physicochemical injuries and infectious agents. In oysters, mucus covering pallial organs (mantle, gills and labial palps) has been demonstrated to have a major importance in particles (microalgae, pathogens) processing. Given the limited information available on pallial mucus in bivalves and the apparent wide spectra of activity of bioactive molecules present in this gel, the characterization of these mucosal secretions has become a research priority. In this study, mucus was collected from the pallial organs of eastern oysters (Crassostrea virginica) and analyzed by liquid chromatography and tandem mass spectrometry. Additionally, in order to investigate glycoconjugate molecules, preliminary glycosyl composition was also performed on mucus using gas chromatography/mass spectrometry (GC/MS) methods. Results showed the presence in pallial mucus of a wide range of molecules involved in non-self recognition, defense mechanisms and interactions with microbes. Mucus composition was different among samples collected from different organs. These results generate a reference map for C. virginica pallial mucus to better characterize the various physiological functions of the mucus. Research is ongoing to assess changes in mucosal immune proteins in response to pathological or environmental alterations.