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

• Main Author: Goedken, Michael Jacob
• Format: Text
• Language: English
• Published: OpenCommons@UConn 2004
• Subjects: Biology; Animal Physiology|Agriculture; Animal Pathology|Biology; Veterinary Science; Crassostrea gigas
• Online Access: https://opencommons.uconn.edu/dissertations/AAI3165993; http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3165993

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 ...