Host preference of Perkinsus species: epizootiological, environmental, and molecular aspects

Abstract

Perkinsus species are protistan parasites of mollusks. In Chesapeake Bay, P. marinus, P. chesapeaki, P. andrewsi, and a Perkinsus isolate from the hard clam Mercenaria mercenaria [Perkinsus sp. (M. mercenaria)] are sympatric.

In vitro experiments by others suggest a preference of P. marinus for the eastern oyster Crassostrea virginica. Studies conducted in this dissertation on the distribution of Perkinsus species in C. virginica and M. mercenaria from Virginia to Maine using PCR-based detection assays provided further evidence for a host preference of P. marinus. While P. marinus was the most prevalent species in C. virginica, its prevalence was significantly lower in M. mercenaria.

Interestingly, the assay designed to be specific for Perkinsus sp. (M. mercenaria) also amplified P. andrewsi. Characterization of the rRNA gene loci of both Perkinsus species revealed the presence of a second rRNA gene unit in P. andrewsi with high percent sequence identity to the unit of Perkinsus sp. (M. mercenaria), explaining the cross-amplification. Furthermore, DNA samples of M. mercenaria inhibited PCR amplification, which was overcome by adding bovine serum albumin and dimethyl sulfoxide to the PCR reaction mixture.

P. marinus resides in oyster hemocytes scavenging and/or inhibiting the production of reactive oxygen species (ROS) usually generated by oyster hemocytes. ROS scavenging enzymes include superoxide dismutases (SODs) and peroxidases such as catalases. SODs have been characterized in P. marinus, but peroxidases have not been detected. Results from the present study suggest that, while lacking catalase, P. marinus has ascorbate dependent peroxidases usually found in plants. Alternatively, P. marinus may suppress the production of ROSs by enzymes such as phosphatases. A secreted acid phosphatase activity reported earlier in P. marinus was further characterized in the present study, and its purification attempted. Furthermore, a search of a genome database yielded several phosphatase-like genes, including a putative protein phosphatase 2C predicted to be secreted. Further analysis could neither confirm its secretion nor its involvement in host preference.

However, the approaches implemented throughout this research represent a strategy for processing additional phosphatase and other gene sequences identified in genome databases that will further the understanding of the biology of Perkinsus species.