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

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.

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

“…Further, it is known that a large fraction of functional proteins present in plasma are secreted by hemocytes [50]. In fact, previous work on bivalves has shown that the plasma proteome reflects some of the proteomic make-up of circulatory hemocytes [23,71]. We had hypothesized that there would be a correlation between the two; however, this was not true for EPF and only a slightly significant correlation was found for hemolymph.…”

“…In our study, there was a significant reduction in the phagocytic activity of agranulocytes in EPF and a reduction in cell viability for agranulocytes in both EPF and hemolymph, suggesting that there were immune costs to clam maintenance under OA. As hemocytes play dual roles in biomineralization and immunity [22,23], maintaining mineralization might come at a cost to immune function. While EPF is mostly associated with biomineralization, hemocytes in EPF are important components of the immune response [23,49,50].…”

“…As hemocytes play dual roles in biomineralization and immunity [22,23], maintaining mineralization might come at a cost to immune function. While EPF is mostly associated with biomineralization, hemocytes in EPF are important components of the immune response [23,49,50]. Under OA conditions, EPF may pivot mostly towards biomineralization processes, which is supported by transcriptomic and proteomic analyses showing upregulation of biomineralization genes and downregulation of immune genes in EPF from clams exposed to acidification (see below).…”

“…For instance, hemolymph pH, but not EPF pH, of M. galloprovincialis was significantly impacted by low pH [21]. EPF is the site where biomineralization takes place; however, hemolymph also plays a role in shell formation by mediating the transport of material needed for shell formation [22,23]. The acid-base balance of hemolymph is impacted by OA, which can have adverse effects if not corrected.…”

“…For bivalves, EPF is actually involved in immune response as well as calcification. Hemocytes, which populate EPF and hemolymph, contribute to both biomineralization and immunity [22,23,25]. Therefore, environmental stressors, such as OA, could represent significant challenges for organisms that must integrate biomineralization with maintenance of innate immune protection.…”