Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis.

Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments, such as dissolved oxygen, hydrogen sulfide concentration, and hydrostatic pressure. In the view of phylogeny of veneroida clams including two vesicomyids, their hemocytes appear to be categorizable into three basic types, with the first containing ERCs and agranulocytes, the second including EGs, and the third comprised of BGs, small eosinophilic granulocytes, and other granulocytes. The present data showed no phagocytic activity of ERCs and a lack of agranulocytes in both vesicomyid species, and when combined with previous reports that other veneroid clams show low or no phagocytic activity, this suggests that ERCs have become evolutionarily differentiated from agranulocytes in the ancestral vesicomyid clam.