Use of ITS rDNA for discriminating of larval stages of two microphallid (Digenea) species using Hydrobia ulvae (Pennant, 1777) and Corophium volutator (Pallas, 1766) as intermediate hosts

Abstract

Digenean trematodes encompass several species with little morphological differentiation in the larval stages and, as a result, uncertainty prevails regarding species identification. The microphallid trematode Maritrema subdolum occurs widespread geographically in mud snail and crustacean hosts in European marine shallow-water ecosystems. Larval stages of this and other congeneric species are, however, difficult to separate morphologically. To verify the species status and to secure identification of two co-occurring microphallids, M. subdolum and microphallid sp. no. 15 (Deblock, 1980), we examined the nucleotide sequences of the internal transcribed spacer regions (ITS1, ITS2). From fragments consisting of both ITS regions and the 5.8S gene (nearly 1,200 bp), a sequence divergence of 2.9% between the two types was recorded. In accordance with the morphological traits of the cercariae (stylet shape, length), the results support the view that the two types actually represent different species. Species-specific primers were prepared for each species. They proved to be efficient diagnostic tools for identifying single larvalstage individuals. Using these primers, infections in host organisms were also verified without performing a dissection of the host individuals.

Description of Maritrema portucalensis sp. nov. (Digenea, Microphallidae) parasite of Carcinus maenas (Crustacea, Decapoda) from Aveiro estuary, Northern Portugal

“…15 by Deblock (1980). The cercariae of this species are morphologically similar to those of Maritrema subdolum Jäger-skiöld, 1909, and both exploit H. ulvae as first intermediate host (Mouritsen 2001, Hust et al 2004. There have been records of double infections in this host comprising Microphallidae sp.…”

“…no. 15 by Hust et al (2004). The query-ITS1 sequence showed differences in 28 nucleotide positions (3.8% divergence) with the same ITS1 region of M. subdolum, and was found to be equal to the sequence of Microphallidae sp.…”

“…In fact, complete ITS1 sequences usually allow quick and accurate identification of genetically distinct but morphologically similar species (Nolan and Cribb 2005). Alignments between the query-ITS1 with the corresponding sequences of two species of microphallid cercariae published by Hust et al (2004) showed 3.8% divergence with the sequence of M. subdolum, and a complete match with the sequence of Microphallidae sp. no.…”

“…The mud snail Hydrobia ulvae (Pennant, 1777), which is a widespread and ecologically important gastropod in the Atlantic shorelines of Europe, is intermediate host to numerous digenean species (Hust et al 2004). One of these, whose biology is largely unknown, was named Microphallidae sp.…”

A review on the molecular characterization of digenean parasites using molecular markers with special reference to ITS region

“…Hust et al (2004) identifi ed and characterized the two congeneric microphallid species (Maritrema subdolum and microphallid sp. No.…”