Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England

PLOS ONE RESEARCH ARTICLE Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England Egie E. Enabulele1☯¤, Scott P. Lawton ID1,2☯, Anthony J. Walker1, Ruth S. Kirk ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Molecular Parasitology Laboratory, School of Life Sciences, Pharmacy and Chemistry, Kingston University, Kingston upon Thames, Surrey, United Kingdom, 2 Epidemiology Research Unit, Department of Veterinary and Animal Sciences, Northern Faculty, Scotland’s Rural College, Inverness, United Kingdom ☯ These authors contributed equally to this work. ¤ Current address: Disease Intervention and Prevention Program, Texas Biomedical Research Institute, San Antonio, Texas, United States of America * OPEN ACCESS Citation: Enabulele EE, Lawton SP, Walker AJ, Kirk RS (2023) Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England. PLoS ONE 18(2): e0270672. https://doi.org/10.1371/journal. pone.0270672 Editor: Hudson Alves Pinto, Universidade Federal de Minas Gerais, BRAZIL Received: December 21, 2021 Accepted: June 14, 2022 Published: February 6, 2023 Copyright: © 2023 Enabulele et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and Supporting Information files. The nucleotide sequences generated in this study are available in the GenBank database: nad1 gene for echinostomatid parasites (accession numbers ON653218-ON653298); cox1 gene for snail hosts (accession numbers ON653299-ON653379). Abstract Echinostoma revolutum (sensu stricto) is a widely distributed member of the Echinostomatidae, a cosmopolitan family of digenetic trematodes with complex life cycles involving a wide range of definitive hosts, particularly aquatic birds. Integrative taxonomic studies, notably those utilising nad1 barcoding, have been essential in discrimination of E. revolutum (s.s.) within the ‘Echinostoma revolutum’ species complex and investigation of its molecular diversity. No studies, however, have focussed on factors affecting population genetic structure and connectivity of E. revolutum (s.s.) in Eurasia. Here, we used morphology combined with nad1 and cox1 barcoding to determine the occurrence of E. revolutum (s.s.) and its lymnaeid hosts in England for the first time, in addition to other echinostomatid species Echinoparyphium aconiatum, Echinoparyphium recurvatum and Hypoderaeum conoideum. Analysis of genetic diversity in E. revolutum (s.s.) populations across Eurasia demonstrated haplotype sharing and gene flow, probably facilitated by migratory bird hosts. Neutrality and mismatch distribution analyses support possible recent demographic expansion of the Asian population of E. revolutum (s.s.) (nad1 sequences from Bangladesh and Thailand) and stability in European (nad1 sequences from this study, Iceland and continental Europe) and Eurasian (combined data sets from Europe and Asia) populations with evidence of subpopulation structure and selection processes. This study provides new molecular evidence for a panmictic population of E. revolutum (s.s.) in Eurasia and phylogeographically expands the nad1 database for identification of echinostomatids. Funding: E.E.E. received funding for this study from the Tertiary Education Trust Fund, Nigeria PLOS ONE | https://doi.org/10.1371/journal.pone.0270672 February 6, 2023 1 / 20 PLOS ONE (REG/SSA/P.10084/55) http://tetfundserver.com. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Molecular epidemiology of Eurasian Echinostoma revolutum (s.s.) Introduction The Echinostomatidae is a diverse, widely distributed family of hermaphrodite digenetic trematodes comprising 37 nominal genera, typically characterized by a circumoral collar of spines and a spiny tegument [1]. The adults commonly infect the intestine and bile ducts of aquatic or semi-aquatic birds and mammals [2, 3]. Some echinostomatid species cause disease in humans in specific foci in East and Southeast Asia as a result of ingestion of metacercariae in raw or undercooked molluscs, fish, crustaceans and amphibians [3]. Reports of such cases outside of Asia are limited, although accurate epidemiological mapping is undermined by misidentifications and diagnostic challenges [2, 3]. Furthermore, certain echinostomatid species are important pathogens of animals and have been associated with mortalities or severe pathology in amphibians [4, 5]. Host-parasite relationships of the Echinostomatidae have been extensively investigated as experimental models [6] and in ecological studies [7], particularly since echinostomes can castrate, reduce longevity and influence growth (stunting or gigantism) of first intermediate snail hosts [8]. There is a long history of controversy about the systematics of the Echinostomatidae due to inter-specific homogeneity of morphological characteristics, loss/unavailability of type material and incorrect identification of sequenced isolates resulting in extensive synonymies, nomenclatural issues and a requirement for systematic revisions [9]. Taxonomy of the cryptic 37-collar-spined ‘Echinostoma revolutum’ group has been particularly difficult to resolve [reviewed in 10]. Integrative taxonomic studies utilising morphological and molecular methods have discriminated genetically distinct echinostomatids worldwide [11–21]. Many studies have used the mitochondrial DNA barcoding marker NADH dehydrogenase subunit 1 (nad1) gene as it is the most informative locus for species discrimination of echinostomatids and inferences of phylogenetic relationships [14, 15, 22]. Numerous species were previously synonymised with E. revolutum [10]. The validity of E. revolutum and E. miyagawai as separate species was confirmed by morphometric [23], life cycle [24] and molecular differentiation [15, 17, 25]. Phylogenetic analyses of nad1 support the separation of E. revolutum into two cryptic species, Eurasian E. revolutum (sensu stricto) (as previously defined by Georgieva et al. [14] for E. revolutum from Europe) and North American E. revolutum [17, 18]. Isolates previously identified as E. revolutum (E. friedi from Valencia, Spain, Marcilla et al. Genbank; German isolate [22]) clustered with European [15] and Eurasian nad1 isolates of E. miyagawai [17, 21]. Australian isolates identified as E. revolutum [26] formed an Australasian lineage of E. miyagawai with an isolat (...truncated)