Abstract
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.
Highlights
Bivalves have an open circulatory system populated by hemocytes, the quintessential component of bivalve immunity
The percentage of phagocytic agranulocytes was higher in extrapallial fluid (EPF) than hemolymph (p
This study used functional assays, transcriptomics, and proteomics to further reveal major functions of hemocytes from different fluids and found evidence to support the role of EPF hemocytes in both immunity and biomineralization
Summary
Bivalves have an open circulatory system populated by hemocytes, the quintessential component of bivalve immunity. The extrapallial space is an active site for microbial colonization and multiple infections affecting bivalves are initiated in this area This is the case for juvenile oyster disease (JOD) and brown ring disease (BRD), which are bacterial diseases that respectively affect Crassostrea virginica (eastern oyster) and Ruditapes philippinarum (Manila clam), and are often associated with hemocyte infiltration to mantle surfaces and the EPF, suggesting localized-immune response at the site of infection [8]. Despite these preliminary investigations, additional research is needed to allow the identification of molecular pathways that enable hemocytes in the EPF to contribute to biomineralization while still fulfilling their immune functions. This study used functional assays, transcriptomics, and proteomics to characterize hemolymph and EPF in M. mercenaria, and to determine potential specialization with regard to processes such as biomineralization, ion regulation, and immunity
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