The eastern oyster is an economically and ecologically important species whose vitality is threatened by the protozoal parasite Perkinsus marinus. To better understand which cellular defense mechanisms impart resistance to P. marinus, resistant (Crassostrea gigas) and susceptible (Crassostrea virginica) oyster species were challenged by an experimental infection with P. marinus and their cellular responses were quantified and compared. Both in vivo and in vitro infection trials measured hemocyte phagocytosis, respiratory burst, apoptosis at 1, 3 and 7 days postinfection (in vivo) or 1-h postco-incubation (in vitro). Total parasite body burden concentrations were also measured at the end of in vivo infections. Infections were significantly more severe in C. virginica than C. gigas at 3 and 7 days postinfection confirming the resistance of C. gigas and validating the experimental model. There was more phagocytosis in infected C. virginica than infected C. gigas three days postinfection. In vitro, C. virginica granulocytes phagocytized significantly more parasites and fluorescent latex beads than C. gigas granulocytes, and infection increased bead phagocytosis in both species, equally in cells with or without intracellular parasites. Neither in vivo nor in vitro infections significantly increased respiratory burst activity. While in vitro infections suppressed hemocyte apoptosis in both species, in vivo infections increased hemocyte apoptosis frequency in C. gigas at 3 days postinfection. In vivo infection increased hemocyte apoptosis in C. virginica at 7 days postinfection but not at three days postinfection. From those experiments, we concluded that the increased phagocytosis without concomitant increase in respiratory burst activity seen in infected C. virginica might exacerbate infections. Also, while in vitro P. marinus infection suppresses hemocyte apoptosis in both species, C. gigas appeared to overcome that suppression faster than C. virginica upon in vivo infection, suggesting that hemocyte apoptosis may be an effective oyster defense response against P. marinus infection. The combination in vitro and in vivo infections in P. marinus disease resistant and susceptible oyster species with multiple time points and assays allowed the identification of apoptosis as the cellular defense mechanism most likely to play an important role in defense against P. marinus. This information may provide more accurate predictive criteria for disease resistance, allowing for the testing and selection of more disease resistant oysters.
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