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The study of Cytochrome B (CYTB): species-specific detection and phylogenetic relationship of Echinostoma revolutum, (Froelich, 1802).

ABSTRACT: Echinostoma revolutum is known as a significant intestinal trematode in various species of animals and humans. It presents complexities in terms of both the morphological and molecular biological data. This is the first study of the application of Cytochrome B gene (CYTB) as a target for studying the phylogeny and designing species-specific primer of E. revolutum. Adult trematodes were harvested from experimentally infected hamsters at 18 days of post-infection. Each worm was identified based on their morphological appearance. The novel CYTB primers were designed from other Echinostoma species to initially amplify CYTB region in E. revolutum. All sequence data of E. revolutum in five provinces of Central Thailand were used as the target for designing the species-specific primer for E. revolutum. The results revealed that CYTB gene can separate E. revolutum into two sister groups by geographical distribution, comprising the eastern and western area groups. Moreover, it also separates E. revolutum from other Echinostoma species, including two sibling species; E. caproni and E. paraensei. In addition, we developed the high performance species-specific primer of E. revolutum. It can detect DNA from a single egg, as well as cercaria, metacercaria and adult stages of this trematode with no cross-reactions to other trematodes and their hosts. Therefore, this research is a positive initial step for the future study of E. revolutum CYTB. The future studies based on this gene should be continued with all species in revolutum complex to overcome the problems of systemic classification that arise in this complex group.

SUBMITTER: Anucherngchai S

PROVIDER: S-EPMC6423160 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The study of Cytochrome B (CYTB): species-specific detection and phylogenetic relationship of Echinostoma revolutum, (Froelich, 1802).

Anucherngchai Sothorn S Chontananarth Thapana T Tejangkura Thanawan T Chai Jong-Yil JY

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20181120 1

Echinostoma revolutum is known as a significant intestinal trematode in various species of animals and humans. It presents complexities in terms of both the morphological and molecular biological data. This is the first study of the application of Cytochrome B gene (CYTB) as a target for studying the phylogeny and designing species-specific primer of E. revolutum. Adult trematodes were harvested from experimentally infected hamsters at 18 days of post-infection. Each ...[more]

PMID: 30956448

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Project description:BackgroundThe systematics of echinostomes within the so-called 'revolutum' group of the genus Echinostoma, which encompasses the type-species E. revolutum and a number of morphologically similar species, has long been controversial. Recent molecular studies indicate the existence of more species than previously considered valid, thus stressing the need for wider taxon sampling from natural host populations. This is especially true for Europe where morphological evidence indicates higher species diversity than previously thought, but where molecular data are virtually lacking. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in the investigation of a dataset with larger taxonomic and geographical coverage.MethodsMore than 20,000 freshwater snails belonging to 16 species were collected during 1998-2012 from various localities in eight countries in Europe. Snail screening provided representative larval isolates for five species of the 'revolutum' group, identified by their morphology. Adult isolates for four species recovered from natural and experimental infections were also identified. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for 74 and 16 isolates, respectively; these were analysed together with the sequences of Echinostoma spp. available on GenBank.ResultsDelineation of the European Echinostoma spp. was carried out based on molecular, morphological and ecological data. The large-scale screening revealed infections with five Echinostoma spp., including one new species: E. revolutum (sensu stricto), E. miyagawai, E. paraulum, E. bolschewense and Echinostoma n. sp. The newly-generated nad1 sequences from Europe fall into six distinct, well-supported, reciprocally monophyletic lineages corresponding to the species identifications based on morphology; this was corroborated by the 28S rDNA sequences. The analyses of the total nad1 dataset provided evidence for 12 monophyletic groups and five singletons, which represent seven described/named species and ten cryptic species-level lineages of Echinostoma.ConclusionWe conclude that nad1 should be the first choice for large-scale barcode-based identification of the species of the 'revolutum' group. Our study provides a comprehensive reference library for precisely identified isolates of the European species and highlights the importance of an integrative approach for species identification linking molecular, morphological and biological data.

Project description:BACKGROUND: The digenean species of Echinostoma (Echinostomatidae) with 37 collar spines that comprise the so-called 'revolutum' species complex, qualify as cryptic due to the interspecific homogeneity of characters used to differentiate species. Only five species were considered valid in the most recent revision of the group but recent molecular studies have demonstrated a higher diversity within the group. In a study of the digeneans parasitising molluscs in central and northern Europe we found that Radix auricularia, R. peregra and Stagnicola palustris were infected with larval stages of two cryptic species of the 'revolutum' complex, one resembling E. revolutum and one undescribed species, Echinostoma sp. IG. This paper provides morphological and molecular evidence for their delimitation. METHODS: Totals of 2,030 R. auricularia, 357 R. peregra and 577 S. palustris were collected in seven reservoirs of the River Ruhr catchment area in Germany and a total of 573 R. peregra was collected in five lakes in Iceland. Cercariae were examined and identified live and fixed in molecular grade ethanol for DNA isolation and in hot/cold 4% formaldehyde solution for obtaining measurements from fixed materials. Partial fragments of the mitochondrial gene nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) were amplified for 14 isolates. RESULTS: Detailed examination of cercarial morphology allowed us to differentiate the cercariae of the two Echinostoma spp. of the 'revolutum' species complex. A total of 14 partial nad1 sequences was generated and aligned with selected published sequences for eight species of the 'revolutum' species complex. Both NJ and BI analyses resulted in consensus trees with similar topologies in which the isolates from Europe formed strongly supported reciprocally monophyletic lineages. The analyses also provided evidence that North American isolates identified as E. revolutum represent another cryptic species of the 'revolutum' species complex. CONCLUSION: Our findings highlight the need for further analyses of patterns of interspecific variation based on molecular and morphological evidence to enhance the re-evaluation of the species and advance our understanding of the relationships within the 'revolutum' group of Echinostoma.

Dataset Information

The study of Cytochrome B (CYTB): species-specific detection and phylogenetic relationship of Echinostoma revolutum, (Froelich, 1802).

Publications

The study of <i>Cytochrome B</i> (<i>CYTB</i>): species-specific detection and phylogenetic relationship of <i>Echinostoma revolutum</i>, (Froelich, 1802).

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