Title

Immunomodulation of Crassostrea gigas and Crassostrea virginica cellular defense mechanisms by Perkinsus marinus

Date of Completion

January 2004

Keywords

Biology, Animal Physiology|Agriculture, Animal Pathology|Biology, Veterinary Science

Degree

Ph.D.

Abstract

The eastern oyster, Crassostrea virginica, is an economically and ecologically important species whose vitality is currently threatened by the protozoal parasite Perkinsus marinus. The roles of oyster cellular defense mechanisms, including phagocytosis, respiratory burst and apoptosis, are poorly understood in part due to suboptimal methods to precisely quantify them. To better understand if and how cellular defense mechanisms impart resistance to P. marinus infection, flow cytometric assays were developed to measure hemocyte phagocytosis, respiratory burst and apoptosis at the single cell level. The assays were used to quantify and compare host responses in resistant (Crassostrea gigas) and susceptible (Crassostrea virginica) oysters following in vivo and in vitro experimental infections with P. marinus compared to uninfected controls. Additionally, apoptosis was measured for C. virginica hemocytes and P. marinus trophozoites kept at different temperatures and salinities. Phagocytosis was measured by the ingestion of fluorescent beads. Reactive oxygen intermediate (ROI) production was quantified by the increase in dichlorofluorescin-associated fluorescence upon phorbol-12-myristate-13-acetate (PMA) stimulation. Apoptosis frequency was enumerated using the Annexin-V and TUNEL assays. ^ Phagocytosis and respiratory burst were greater in granulocytes while apoptosis was greater in hyalinocytes. The temperatures tested did not have a significant effect on apoptosis in host or parasite while high salinity increased host cell apoptosis but decreased parasite apoptosis. Following assay development and validation, in vivo and in vitro infection trials were performed. Phagocytosis was higher in the susceptible species and was similarly up-regulated in both species upon in vitro infection. ROI production did not significantly increase following in vitro and in vivo infections. Though in vitro infection suppressed apoptosis in both species, the resistant species overcame that suppression sooner than the susceptible one upon in vivo infection. Because neither host can effectively kill the parasite via ROI generation, the susceptible host's higher percentage of phagocytosis without effective intracellular destruction may be a marker of disease susceptibility rather than a resistance factor. If apoptosis is a viable mechanism to eliminate intracellular parasites after failure of ROI killing, the fact that the resistant species overcomes the parasite's suppression of apoptosis sooner than the susceptible species suggests that this may be part of C. gigas's resistance strategy. These studies have demonstrated the extent to which different oyster cellular defense mechanisms may be important to resistance to P. marinus infection, as well as the importance of environmental conditions on both host and parasite. Future studies will build upon and expand these initial findings with more detailed in vitro mechanistic studies and further comparisons of apoptosis between the species under different environmental and infectious conditions and variable time points. ^

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