Targeted rDNA Sequence Determination from Geographically Isolated Populations of Proterometra macrostoma (Trematoda: Azygiidae)

Abstract

Proterometra macrostoma, a digenetic trematode, was described to have eight morphological variants from cercariae specimens collected from North Elkhorn Creek, Scott County, Kentucky. We have identified and characterized a new population of P. macrostoma collected from Clear Creek, Wayne County, Indiana, through examining shed cercariae. The objectives of this study were to conduct rDNA sequence comparisons among individual cercarial phenotypic variants from both populations to (1) determine the partial 28S and complete internal transcribed spacer 2 (ITS2) ribosomal DNA sequence for P. macrostoma, (2) demonstrate that Clear Creek samples are indeed P. macrostoma, and (3) phylogenetically place P. macrostoma among related trematodes. First, we identified and collected Pleurocera semicarinata livescens from Clear Creek and monitored these gastropod intermediate hosts for patent trematode infections. Of the four different species of trematodes observed, the characteristic furcocystocercous P. macrostoma cercariae were identified. Examination of the cercariae demonstrated phenotypic variation in spined and/or unspined papillae as established in the prior North Elkhorn Creek study. Genomic DNA was isolated and purified from individual cercariae, representing different morphological variants, in both populations. PCR was successful in amplifying across three rDNA loci (partial 5.8S, complete ITS2, and partial 28S) and multiple recombinant clones were independently sequenced. This study represents the first sequence determination for these rDNA regions in P. macrostoma. Subsequent rDNA sequence analysis confirmed 100% identity independent of population or phenotypic variant and phylogenetic analyses placed P. macrostoma within a monophyletic clade of the Proterometra genus. Ongoing specimen acquisition, sequence analysis, and phenetic studies should be conducted to further resolve Proterometra phylogeny.