Molecular Analysis of Echinostome Metacercariae from Their Second Intermediate Host Found in a Localised Geographic Region Reveals Genetic Heterogeneity and Possible Cryptic Speciation

Abstract

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km2 in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.

Echinostoma ` revolutum ¿ (Digenea: Echinostomatidae) species complex revisited: species delimitation based on novel molecular and morphological data gathered in Europe

“…Several notes of caution are required before considering the recent papers on "Echinostoma" spp. reported recently from Asian locations (Saijuntha et al [62][63][64]; Noikong et al [65]). First, the authors should be aware that annotations in GenBank solely reflect the identification (in most cases not supported by voucher material and/or morphological data) of the authors submitting the sequences.…”

“…Echinoparyphium ellisi (AF026791, isolate PMeta3 of Morgan & Blair [14,26]) and Echinoparyphium hydromyos (AF026290, isolate Rat-Ad of Morgan & Blair [14]) re-identified by Kostadinova et al [28] based on examination of the available voucher material, are still being referred to as "Echinostoma sp." (see Noikong et al [65]).…”

“…The examination of the experimental material of E. hortense used for obtaining the sequence data of Morgan & Blair [14,25,26] confirmed its affiliation to Isthmiophora (see Kostadinova et al [28]). However, this species is still referred to as E. hortense by Saijuntha et al [62] and Noikong et al [65].…”

Phylogenetic relationships of some species of the family Echinostomatidae Odner, 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis

“…The use of molecular approaches to determine phylogenetic relationships of digeneans has grown very rapidly since 1990s and molecular-based studies on echinostomes have been carried out to date (Morgan and Blair 1995, 1998a, 1998b, 2000, Petrie et al 1996, Grabda-Kazubska et al 1998, Kostadinova et al 2003, Saijuntha et al 2011, Georgieva et al 2013, 2014, Noikong et al 2014, Selbach et al 2014, Kudlai et al 2015). The genus Echinochasmus Dietz, 1909 (as well as Echinostoma and Echinopharyphium ) is one of the most species–rich genera in Echinostomatidae (Kostadinova and Gibson 2000); however, no one species of this genus was involved in molecular phylogenetic studies of the Digenea (Cribb et al 2001, Olson et al 2003, Olson and Tkach 2005).…”

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

“…The 16S sequences of Custuteria metacercariae was matched with C. australis with >0.5 % divergence while Acanthoparyphium metacercariae sequences showed >0.6 % divergence. Noikong et al (2014) studied the identifi cation and intra-specifi c genetic diversity of metacercariae from snails from Thailand using ITS-2 rRNA and mitochondrial NDI region. The fi ndings pointed that major prevalent species were most closely asoociated to Echinostoma revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis.…”