Flow cytometric characterization of the hemocytes of blood cockles Anadara broughtonii (Schrenck, 1867), Anadara kagoshimensis (Lischke, 1869), and Tegillarca granosa (Linnaeus, 1758) as a biomarker for coastal environmental monitoring.

Marine bivalves are often used as a sentinel species in coastal environmental monitoring since changes in the environmental quality are often well preserved in their cells and tissues. Anadara and Tegillarca species of Arcidae, the blood cockles, are considered to be good sentinel species in monitoring coastal pollution and ecosystem health because they are distributed widely in the subsurface of intertidal mudflats. Internal cellular defense of the blood cockles to physical and biological stresses is mediated by the circulating hemocytes, while their hemocyte types and functions are poorly studied. In this study, we first characterized morphology and immune-related activities of hemocytes of three common blood cockles Anadara broughtonii, A. kagoshimensis, and Tegillarca granosa using flow cytometry. Based on cell morphology and immunological functions, we described five types of hemocytes identically in the three blood cockles: erythrocytes type-I (erythrocytes-I), erythrocytes type-II (erythrocytes-II), granulocytes, hyalinocytes, and blast-like cells. Erythrocytes were round cells containing hemoglobin with numerous granules in the cytoplasm and these cells consist of two central populations. Erythrocytes-I were the most abundant cells accounting for 80-89% of the total circulating hemocytes and exhibited a certain level of lysosome and oxidative capacity. Erythrocytes-II were the largest cells and displayed high lysosome content and the most active oxidative capacity. Both erythrocytes-I and erythrocytes-II did not show phagocytosis capacity. Granulocytes were intermediated-sized hemocytes characterized by granules in the cytoplasm and long pseudopodia on the cell surface, and these cells were mainly engaged in the cellular defense exhibiting the largest lysosome content, the most active phagocytosis, and high oxidative capacity. Contrary to granulocytes, hyalinocytes were comparatively small and round cells and exhibited no granules in the cytoplasm. Hyalinocytes displayed a certain level of lysosome and phagocytosis and oxidative capacities. Blast-like cells characterized by the smallest size and small quantity of cytoplasm and exhibited an absence of phagocytosis and extremely low oxidative capacity, suggesting that this population is not directly involved in the cell-mediated immune activities. In conclusion, flow cytometry indicated that three blood cockles had five types of hemocytes, and the erythrocytes and granulocytes were mainly involved in the immunological activities.