Development of an in vitro assay to examine intracellular survival of Perkinsus marinus trophozoites upon phagocytosis by oyster (Crassostrea virginica and Crassostrea ariakensis) hemocytes.

Perkinsus marinus is a facultative intracellular parasite that causes "Dermo" disease in the eastern oyster Crassostrea virginica. Although hemocytes from healthy oysters rapidly phagocytize P. marinus trophozoites, they fail to efficiently kill them. Instead, trophozoites survive and proliferate, eventually overwhelming the host. Because Chesapeake Bay oyster populations have been reduced to unprecedented levels, the introduction of the Suminoe oyster, Crassostrea ariakensis (synonymous C. rivularis), has recently been proposed. Although this species is refractory to developing Dermo disease, it can be infected by Perkinsus spp. and, thus, the mechanistic basis of its disease resistance remains intriguing. To examine whether the resistance to develop Dermo is due to a high capacity of C ariakensis hemocytes to kill internalized P. marinus, we developed an in vitro assay to compare intracellular survival and proliferation of P. marinus in C. virginica and C ariakensis hemocytes. Our results revealed that P. marinus cultured trophozoites have a similar capacity for in vitro survival within hemocytes from both oyster species, suggesting that the resistance of C. ariakensis to develop Dermo disease is most likely due to reduced parasite pathogenicity for the latter oyster species, rather than to infectivity. Together with the currently available P. marinus genome, EST sequences, and the transfection methodology we recently developed, this assay should significantly contribute to a rigorous identification of the P. marinus genes responsible for its intrahemocytic survival.