Immune responses of oyster hemocyte subpopulations to in vitro and in vivo zinc exposure

Oysters are an excellent biomonitor of coastal pollution and the hyper-accumulator of toxic metals such as copper and zinc (Zn). One unique feature of molluscs is their hemocytes which are mainly involved in immune defenses. Different subpopulations of hemocytes have been identified, but their functions in metal transport and detoxification are not clear. In this study, we examined the immune responses of different subpopulations of oyster Crassostrea hongkongensis hemocytes under different periods of Zn exposure by using flow cytometer and confocal microscopy. In vitro exposure to Zn resulted in acute immune responses by increasing the reactive oxygen species (ROS) production and phagocytosis and decreased number of granulocytes and mitochondrial membrane potential (MMP) within 3 h. Granulocyte mortality and lysosomal pH increased whereas glutathione (GSH) decreased within 1 h of in vitro exposure, indicating the immune stimulation of granulocytes. Within the first 7 days of in vivo exposure, immunocompetence of granulocytes was inhibited with increasing granulocyte mortality but decreasing ROS production and phagocytosis. However, with a further extension of Zn exposure to 14 days, both phagocytosis and lysosomal content increased with an increasing number of granulocytes, indicating the increase of hemocyte-mediated immunity. Our study demonstrated that granulocytes played important roles in oyster immune defenses while other subpopulations may also participate in immune functions. The degranulation and granulation due to transition between semigranulocytes and granulocytes after Zn exposure were important in metal detoxification. The study contributed to our understanding of the immune phenomena and the adaptive capability of oysters in metal contaminated environments.