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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.

ABSTRACT: Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis.The near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies.The 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon (Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The phylogenetic tree inferred from these sequences again confirmed anomalies in molecular placement of some members of the family Heterophyidae and demonstrates the need for reappraisal of the entire superfamily Opisthorchioidea. The new sequences provided here supplement those already available in public databases and add to the array of molecular tools that can be used for the diagnosis of heterophyid species in human and animal infections.

SUBMITTER: Le TH

PROVIDER: S-EPMC5223338 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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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.

Le Thanh Hoa TH Nguyen Khue Thi KT Nguyen Nga Thi Bich NT Doan Huong Thi Thanh HT Dung Do Trung DT Blair David D

Parasites & vectors 20170109 1

<h4>Background</h4>Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units ...[more]

PMID: 28069063

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Project description:BackgroundMetagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity.Methodology/principal findingsConserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes.Conclusions/significanceThe identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets for metazoan metagenetic analyses, are discussed.

Project description:To facilitate the process of spiral nematode species delineation, populations of Helicotylenchus canadensis, H. pseudorobustus, and H. varicaudatus deriving from various locations and diverse natural and anthropogenic environments from Poland were investigated and characterized. For the first time, 28S rDNA sequences are reported for H. canadensis and H. varicaudatus, whereas new mtCOI sequences were acquired for all three analyzed species. A Bayesian phylogenetic analysis of the 28S rDNA fragments revealed that H. canadensis and H. varicaudatus are members of a clade that is a sister group to all other Helicotylenchus species; however, the closest known sister group to H. canadensis is H. vulgaris type A. Both 28S rDNA- and mtCOI-based phylogenetic results suggest that this clade excludes H. pseudorobustus, whose most recent common ancestor with the former species was inferred to be the ancestor of all Helicotylenchus species. Moreover, within the mtCOI sequences obtained from H. pseudorobustus, unlike from the other two, a simultaneous presence of TAG and TAA codons was identified. This may indicate mitochondrial genetic code alterations or other genomic rearrangements in H. pseudorobustus.To facilitate the process of spiral nematode species delineation, populations of Helicotylenchus canadensis, H. pseudorobustus, and H. varicaudatus deriving from various locations and diverse natural and anthropogenic environments from Poland were investigated and characterized. For the first time, 28S rDNA sequences are reported for H. canadensis and H. varicaudatus, whereas new mtCOI sequences were acquired for all three analyzed species. A Bayesian phylogenetic analysis of the 28S rDNA fragments revealed that H. canadensis and H. varicaudatus are members of a clade that is a sister group to all other Helicotylenchus species; however, the closest known sister group to H. canadensis is H. vulgaris type A. Both 28S rDNA- and mtCOI-based phylogenetic results suggest that this clade excludes H. pseudorobustus, whose most recent common ancestor with the former species was inferred to be the ancestor of all Helicotylenchus species. Moreover, within the mtCOI sequences obtained from H. pseudorobustus, unlike from the other two, a simultaneous presence of TAG and TAA codons was identified. This may indicate mitochondrial genetic code alterations or other genomic rearrangements in H. pseudorobustus.

Dataset Information

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.

Publications

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.

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