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

BackgroundHeterophyidiasis 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.ResultsThe 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.ConclusionsThe 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.

The life cycle of a zoonotic parasite reassessed: Experimental infection of Melanoides tuberculata (Mollusca: Thiaridae) with Centrocestus formosanus (Trematoda: Heterophyidae)

“…Recently, molecular studies have revealed new insights regarding the phylogeny of members of the superfamily Opisthorchioidea, including the paraphyly of Heterophyidae and an uncertain position of C . formosanus [ 55 , 58 – 60 ]. Future studies may suggest a new arrangement of the group.…”

“…For example, it is interesting to note that the grouping of the genera Euryhelmis , Cryptocotyle and Metagonimoides , which produce non-embryonated eggs, was recently verified [ 55 ]. The affinity between the two first genera was also reported using different markers [ 58 , 60 ]. On the other hand, the main clade containing species of Heterophyidae verified by these authors are formed by species that produce embryonated eggs (genera Haplorchis , Haplorchoides , Procerovum , Stellantchasmus and Metagonimus ) [ 58 , 60 ].…”

“…Regarding C . formosanus , its affinity with some members of Cryptogonomidae or a basal position in Opisthorchioidea has been demonstrated [ 60 ]. Interestingly, the partial 18S sequences obtained in our study were 98.6% similar to A .…”

Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam

“…Primer pairs OACOBF/OACOBR, OAND1F/OAND1R, OACO1F/OACO1R amplified complete cob , nad 1 and cox 1 genes, respectively. The primer pairs U18SF/U18SR were used for obtaining major fragments of ribosomal 18S and U28SF/U28SR for 28S, respectively [ 12 ]. Additional internal primers were designed and used as needed (Table 2 ).…”

“…Molecular phylogenetic/systematic studies are excellent aids for taxonomy [ 12 – 15 ]. Such studies require homologous sequences from as many taxa as possible within the group of interest.…”