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Abstract 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. 

The adhesion between food particles and mucus is a fundamental process in particle sorting in suspension-feeding bivalves that requires specific recognition. Interactions between carbohydrate-binding proteins (lectins) expressed on the feeding organs and carbohydrates present on microbial cell surface can provide this specificity. Microalga cell surface carbohydrates (MCSC) represent unique patterns that can be considered as species-specific fingerprints. In this study, sorting efficiencies in blue mussels Mytilus edulis fed with microalgae having modified MCSC and engineered microspheres coated with target carbohydrates was measured. The nature and quantities of surface carbohydrates required to trigger sorting in mussels was evaluated and the relationship between ligand quantities and sorting efficiency (SE) was determined. Mussels fed with Chlamydomonas which MCSC were blocked with ConA or PEA lectins (affinity to mannose and glucose) led to a significant decrease of the sorting efficiencies, not observed when the lectin UEA (affinity to fucose) was used. The ability of commercial lectins to inhibit sorting was not linear and a threshold was noted between 30 and 45 ug lectins per million algae cells. Further, mussels were fed with microspheres coated with neoglycoproteins. Results showed that glucose-BSA, but not fucose-BSA, has an effect on particle sorting in mussels, and 1.08 x 10 9 molecules of glucose per microspheres, corresponding to a density of 6.99 x 10 6 molecules of glucose per µm 2 , triggers particle selection. These findings support that selection of food particles by mussels rely on the strength of the bond between suspended particle and the mucosal layer that mediate sorting, and that these bonds depend on the quantity of compatible ligands on each particle. 

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 Ca 2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca 2+ 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. 

ABSTRACT 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. 

ABSTRACT 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. 

Numerous studies have classified the effects of ocean acidification on development and shell growth, but less studied are the effects of acidification on oyster physiology and the potential mechanisms that may enable vulnerable larvae to survive and adapt. Research described in this article was designed to understand the impacts of ocean acidification on the eastern oyster. Specifically, the study assessed the effects of acidification on survival, growth and metabolic rates of oyster larvae. In addition, the study began to identify the processes that may enable survival by first assessing whether food availability enhances resilience of these early life stages. These questions are particularly important because a decrease in survival and size of larvae can significantly impact the commercial industry as a result of decreased yield. Studying metabolic rates is vital to understanding the health of an organism as it can indicate whether an animal is under stress or has high energetic demands that must be met with high food availability.