Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 μm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 μm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 μm) and contained small basophilic granules (diameter, 0.3-0.4 μm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 μm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 105 cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor® 488-conjugated Escherichia coli bioparticle and a LysoTracker® lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system.