Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenaria, by sequence analysis of two internal transcribed spacer regions and the 5·8S ribosomal RNA gene

Abstract

The internal transcribed spacer (ITS-1 and ITS-2) regions and the 5.8S ribosomal RNA gene of 2 Perkinsus spp. (G117 and H49) originating from the softshell clam, Mya arenaria, of the Chesapeake Bay were cloned and sequenced to obtain evidence for their genetic divergence. A high level of heterogeneity in both regions, probably resulting from deletions, insertions, and base substitutions, was evident from alignments of the sequences of the 2 isolates with published sequences of other Perkinsus spp. The isolate G117 and other Perkinsus spp. were highly divergent (13-26% and 19-20% sequence divergence in ITS-1 and ITS-2, respectively). These regions in the isolate H49 and Perkinsus marinus were similar (99.07% and 99% for ITS-1 and ITS-2, respectively). Evidence obtained from a phylogenetic analysis using the aligned sequences suggests that G117 and H49 belong to 2 distinct species of Perkinsus. The isolate G117 possibly belongs to an as yet undescribed species of Perkinsus, and H49 belongs to the species P. marinus. The conclusions drawn from the genetic analysis of H49 and G117 are supported by previously reported morphological characteristics (McLaughlin & Faisal, 1998b). Isolates H49 and G117 originated from the same molluscan species demonstrating that at least 2 different species of Perkinsus can co-exist in 1 host.

Phylogenetic relationship of Perkinsus olseni from the Ebro Delta, Spain, to other Perkinsus species, based on ribosomal DNA sequences

“…discrimination are the 5.8S sequence of the internal transcribed spacer (ITS) region and the nontranscribed spacer (NTS) region of ribosomal DNA (rDNA); the comparison of ribosomal sequences with reference sequences constitutes the only confirmatory method for the identification of Perkinsus spp. (Reece et al 1997, Kotob et al 1999, Robledo et al 1999, Coss et al 2001, Casas et al 2002, Murrell et al 2002, Burreson et al 2005, Park et al 2005.In the present study, we report the species identity of Perkinsus sp. found in Ruditapes philippinarum from Spanish Mediterranean waters based on an analysis of NTS and ITS (including ITS1, 5.8S and ITS2) sequences, and the phylogenetic relationship of P. olseni strains from this area to other Perkinsus spp.…”

“…discrimination are the 5.8S sequence of the internal transcribed spacer (ITS) region and the nontranscribed spacer (NTS) region of ribosomal DNA (rDNA); the comparison of ribosomal sequences with reference sequences constitutes the only confirmatory method for the identification of Perkinsus spp. (Reece et al 1997, Kotob et al 1999, Robledo et al 1999, Coss et al 2001, Casas et al 2002, Murrell et al 2002, Burreson et al 2005, Park et al 2005.…”

Comparison of diagnostic techniques to detect the clam pathogen Perkinsus olseni

“…A nested PCR was then performed. The first reaction was done according to Kotob et al (1999) with 10× buffer in a volume of 25 µl containing 10 mM of Tris, 50 mM KCl (pH 8.3), 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 µM of each primer, 1 µl (10 to 50 ng) of DNA, and 1.25 U of Taq polymerase. Buffer, MgCl 2 , and Taq were obtained from Roche, and primers and dNTPs from Invitrogen.…”

“…However, Perkinsus spp. can infect several alternate hosts in addition to the type host, and this is especially true for P. olseni (Kotob et al 1999, Dungan et al 2002, Bower & McGladdery 2003.…”

“…Internal transcribed spacers (ITS-1 and ITS-2) are non-coding regions of the RNA gene cluster that are less conserved than the ribosomal RNA coding genes and are suitable to distinguish among proximal species. They have been used in several studies to distinguish different species of the genus Perkinsus (Kotob et al 1999, Robledo et al 1999b, Casas et al 2002, Dungan et al 2002). Here we compared histopathology, RFTM, and PCR of the rDNA ITS region for the diagnosis of P. olseni in the principal clam species cultured in Galicia (NW Spain).…”