Utilizing ribosomal DNA gene marker regions to characterize the metacercariae (Trematoda: Digenea) parasitizing piscine intermediate hosts in Manipur, Northeast India

Abstract

Freshwater fishes in Manipur, Northeast India frequently harbour several types of metacercariae, which based on morphological criteria were identified as Clinostomoides brieni, Euclinostomum heterostomum (Clinostomidae) and Polylekithum sp. (Allocreadiidae). Molecular techniques utilizing PCR amplification of rDNA regions of larger subunit (LSU or 28S), smaller subunit (SSU or 18S) and inter transcribed spacers (ITS1, 2) were used for molecular characterization of these types. Sequences generated from the metacercariae were compared with their related sequences available in public databases; an analysis of the identity matrices and phylogenetic trees constructed was also carried out, which confirmed their identification. Similarly, the sequences generated from Polylekithum sp. were found to be highly similar to the species of the same genus. The rDNA ITS2 secondary structure provided additional confirmation of the robustness of the molecular marker as a tool for taxon-specific characterization.

Molecular data show Clinostomoides Dollfus, 1950 is a junior synonym of Clinostomum Leidy, 1856, with redescription of metacercariae of Clinostomum brieni n. comb.

“…Athokpam et al . (2014) provided rDNA sequences from C. brieni , but without supporting morphological information. Moreover, the identification of C. brieni by these authors was questioned by Briosio-Aguilar et al .…”

“…It is also relevant that the material sequenced by Athokpam et al . (2014) was from Heteropneustes in Northeastern India, rather than the region or hosts (central and southern Africa, Clarias , Ardea ) where the genus originated and is better known.…”

Species delimitation in trematodes using DNA sequences: Middle-American Clinostomum as a case study

“…In recent years, molecular methods have been used to obtain information that, in combination with morphology, has been very useful to distinguish among species of Clinostomum (e.g. Dzikowski et al 2004;Gustinelli et al 2010;Caffara et al 2011Caffara et al , 2013Caffara et al , 2014Sereno-Uribe et al 2013;Athokpam et al 2014;Senapin et al 2014;Locke et al 2015a;Pinto et al 2015). A large database of mitochondrial and nuclear DNA sequences of Clinostomum individuals is now available, improving our ability to produce a more reliable estimate of species diversity in the genus.…”

The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam

“…Although markers are often chosen from the mitochondrial genome, sequences from the nuclear ribosomal transcription unit (rTU) (including 18S, ITS1, ITS2 and 28S) are particularly useful and reliable for this purpose [10, 15–22]. A single rTU consists of three coding regions (the 18S, 5.8S and 28S rRNA genes) separated by two internal transcribed spacers (ITS1 and ITS2) [17].…”

“…Sequences from various portions of the rTU (18S, ITS1, ITS2 and 28S) have been widely used for inference of phylogenetic relationships and taxonomic clarification within and between many trematode families (e.g. [15, 18, 22, 25–31]). Sequences of complete or near-complete rTUs are only available for a few species of trematode [12, 16, 20, 32, 33].…”