Molecular characterization of cultured Perkinsus marinus isolates

Perkinsus marinus is the causative agent of the oyster disease Dermo in the eastern oyster, Crassostrea virginica. In vitro propagation of the parasite has led to the establishment of multiple isolates by several investigators. Little work, however, has been done to characterize different isolates. In this study multiple isolates were examined for genetic and biochemical diversity. At two loci, the ITS region and the ATAN region, there was as much intra-isolate genetic variation among DNA sequences of some isolates as there was inter-isolate variation. Variation was also observed at a third loci, a subtilisin-like serine protease gene. This is the first report of a serine protease gene in P. marinus. In addition, a second, very similar subtilisin-like gene, may have also been isolated which was 95% similar in DNA sequence to the first and encoded amino acid changes in conserved regions. Examination of the extracellular proteins produced by eight P. marinus isolates from the Chesapeake Bay revealed differences in protein profiles, protease activity, protease profiles, growth rates, cell size and viability. The degree to which the differences observed were due to strain variation or to complex biochemical interactions of the parasite is unknown. However, isolates of similar viability, growth rates and cell size still had differences in protein and protease band profiles as well as in proteolytic activity, suggesting that there may be some genetic basis for the observed differences. The variation detected at both the genetic and molecular level suggests that multiple isolates should be examined when conducting biochemical and physiological studies on the parasite and for designing molecular diagnostic probes and PCR primers.