Characterization of the subpopulation and flow cytometric analysis of immune-related parameters of hemocytes in the green-lipped mussel Perna viridis

To provide a better understanding of the role of hemocytes in internal defence and to expand the knowledge of environmental immunotoxicology in P.viridis,it is essential to explore the fundamental knowledge of hemocytes.Three major types of hemocytes were identified in the hemolymph,including granulocyte,semi-granulocyte(small and large sizes)and hyalinocyte.Granulocytes were intermediate-sized cells with diameter of (7.49±1.32) μm and accounted for 40.4%±8.4%,containing numerous granules in the cytoplasm.Semi-granulocytes were the largest cells with diameter of (12.45±2.21) μm and percentage of 34.6%±9.4%,containing some granules.Small semi-granulocytes were only different from semi-granulocytes in the cell size with diameter of (6.64±1.05) μm.Hyalinocytes were the smallestdiameter,(4.69±1.01) μmand least abundant(15.6%±4.8%)with few granules.Flow cytometry has revealed that the dense-granulocytes are the most active in cell phagocytosis,whereas the dense-granulocytes and semi-granulocytes have similar activities in terms of esterase activity and reactive oxygen species(ROS)generation.In contrast,the hyalinocytes showed the lowest levels in phagocytosis,enzymatic activities and ROS production.Hyalinocyte mortality was higher compared with the other two hemocytes.The immune functions assessed by the flow cytometry indicated that the granulocytes are the main hemocytes involved in the cellular defence in the green-lipped mussel.