Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths

Abstract

The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9-25%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.

Evidence of new species ofHaematoloechus(Platyhelminthes: Digenea) using partialcox1sequences

“…The generation of primers that amplify the standard barcode region for a wide variety of platyhelminth groups has been a challenge (Moszczynska et al 2009), and is still at the experimental stage. Although the cox1 fragment traditionally used in platyhelminths is shorter than the standard barcodes, it has given valuable information about the taxonomy and phylogeny of different groups, most of the time supported by other genes and morphology (Iwagami et al 2000(Iwagami et al , 2003Morgan et al 2003;Razo-Mendivil et al 2004).…”

“…Smith et al 2006Smith et al , 2008, including some groups of helminths (Ferri et al 2009;Moszczynska et al 2009). The aim of this study is to test the performance of partial sequences of cox1 in the discrimination of Table I.…”

Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes

“…These were analysed with 1167 sequences generated by Moszczynska et al (2009), Locke et al (2010a, Désilets et al (2013), which are published herein, and 336 sequences published by Moszczynska et al (2009) Kuhn et al (2015), and Otachi et al (2015); details are in Supplementary Table S1. Alignment of these 2026 CO1 sequences from Diplostomum, Tylodelphys and Austrodiplostomum was unambiguous (Supplementary Data S1; GUIDANCE confidence scores of all sequences and all 621 positions P0.993) and 99.4% of the Kimura 2-parameter (K2P) distances were either less than 2.0% or greater than 9.0% (Fig.…”

“…Total DNA was extracted from individual parasites, except in a few cases where cercariae from individual snails were pooled. The barcode region of CO1 was amplified and sequenced at the Canadian Centre for DNA Barcoding in Guelph, Canada, as described by Moszczynska et al (2009) and in one specimen (Diplostomum ardeae) by Van Steenkiste et al (2015). In some samples from which CO1 sequences were obtained, ITS was amplified and sequenced as described by Galazzo et al (2002).…”

“…Diplostomids were collected during necropsies of fish (mainly), amphibians, birds and molluscs in Eurasia, North and South America, including collections described by Moszczynska et al (2009), Locke et al (2010a,b, 2011 and Désilets et al (2013) (Fig. 1).…”