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Molecular Comparisons between Perkinsus marinus Isolates from the
Eastern Oyster, Crassostrea virginica,
and the Softshell Clam, Mya arenaria
Shaban. I.
Kotob
and M. Faisal Department of
Environmental Sciences, Virginia Institute of Marine Science, The College of
William and Mary, Gloucester Point, Virginia 23062, USA
Perkinisus
marinus
is a pathogenic protozoan of the eastern oyster that is associated with
significant losses. The P-1 clone of P. marinus has been cultured for over 8 years, yet retains virulence.
A major objective of this study
was to identify the complete sequence of P-1 small subunit (SSU) rRNA gene and
the internal transcribed spacer 1-5.8S rRNA gene-internal transcribed spacer 2
(ITS1-5.8S-ITS2) region of the rRNA operon and to compare between P-1 gene
sequence and those published for two other tissue-associated P. marinus. PCR-amplicons of the SSU rRNA gene and the ITS15.8S-ITS2 region
of P-1 isolate were 1805 and 842 base-pair long, respectively. A phylogenetic tree obtained from
Jukes-Cantor distances of the aligned rRNA gene sequences of 14 eukaryotic taxa
using the Neighbor-Joining method clustered P-1 within the genus Perkinsus. Recent, genetic, morphological and biochemical characterization of a Perkinsus spp. isolated from the softshell clam hemolymph
(designated as H-49) demonstrated its identity with P. marinus (McLaughlin & Faisal, 1998 and Kotob et al.,
1999). These results extend the host
range of P. marinus to include the softshell clam. Comparisons
of the SSU rRNA gene sequences of P-1
to H-49 and P. marinus from eastern
oyster (Fong et al., 1993) showed similarities of 99.9 and 99.6%,
respectively. Despite this high
sequence similarity, variations (0.1 to 0.4%) in this gene indicate that these three P. marinus isolates are not identical. Additional analyses using the ITS-5.8S regions demonstrated
sequence variations (6 to 7 nucleotides) between P-1, H-49 and that published
by Goggin (1994) for a tissue derived P.
marinus. These findings further confirm genetic variations among P.marinus isolates. Restriction Fragment
Length Polymorphism (RFLP) analysis of the SSU rRNA amplicons was used to
differentiate between P-1 from the eastern oyster and H-49 from the softshell
clam.
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