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The group 1 carcinogen, the liver fluke Opisthorchis viverrini is the causative agent of opisthorchiasis and subsequent bile duct cancer (cholangiocarcinoma; CCA), which is an important public health problem in Southeast Asia. Bithynia snails are known to be the sole intermediate host of O. viverrini, and distributed throughout endemic areas of opisthorchiasis. Since 2001, the genetic variation investigation of O. viverrini has progressively been investigated. Comprehensive genetic variation studies of O. viverrini and Bithynia snails were undertaken and consecutively published in 2007 by Saijuntha and colleagues. These studies provided genetic evidence that O. viverrini and Bithynia snails are both species complex with evidence of co-evolution. Later, several studies have provided data in support of this finding, and have continuously to date reinforced that both O. viverrini and Bithynia are species complexes. Moreover, studies have shown that genetic variation of O. viverrini is related to geographical, temporal, fish host species including geographical genetic variation of its snail host, Bithynia siamensis sensu lato. This is significant and important in our understanding of the evolution and phylogenetic relationships between species within the O. viverrini and Bithynia species complexes. A comprehensive knowledge of the systematics and population genetics of O. viverrini and Bithynia snails provides a sound basis to instigate and develop effective prevention and control programs targeting opisthorchiasis and CCA in the endemic areas of Southeast Asia. Thus, this review examines the historical series of investigations of the systematics and population genetics of O. viverrini including Bithynia spp. in Southeast Asia since molecular genetic investigations commenced some 20 years ago., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Echinostomes are a diverse group of digenetic trematodes that are difficult to classify by predominantly traditional techniques and contain many cryptic species. Application of contemporary genetic/molecular markers can provide an alternative choice for comprehensive classification or systematic analysis. In this study, we successfully characterized the intron 5 of domain 1 of the taurocyamine kinase gene (TkD1Int5) of Artyfechinostomum malayanum and the other two species of the 37 collar-spined group, Echinostoma revolutum and Echinostoma miyagawai, whereas TkD1Int5 of Hypoderaeum conoideum cannot be amplified. High levels of nucleotide polymorphism were detected in TkD1Int5 within E. revolutum and E. miyagawai, but not in A. malayanum. Thus, TkD1Int5 can be potentially used as genetic marker for genetic investigation of E. miyagawai and E. revolutum. We therefore used TkD1Int5 to explore genetic variation within and genetic differentiation between 58 samples of E. miyagawai and five samples of E. revolutum. Heterozygosity was observed in 17 and two samples with 16 and three insertion/deletion (indel) patterns in E. miyagawai and E. revolutum, respectively. Heterozygous samples were then cloned and nucleotide sequence was performed revealing the combined haplotypes in a particular sample. Based on nucleotide variable sites (excluding indels), the 72 E. miyagawai and seven E. revolutum haplotypes were subsequently classified. The haplotype network revealed clear genetic differentiation between E. miyagawai and E. revolutum haplogroups, but no genetic structure correlated with geographical localities was detected. High polymorphism and heterogeneity of the TkD1Int5 sequence found in our study suggest that it can be used in subsequent studies as an alternate independent potential genetic marker to investigate the population genetics, genetic structure, and possible hybridization of the other echinostomes, especially the 37 collar-spined group distributed worldwide.

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F ST 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles.

Species and subspecies of the Oncomelania hupensis species complex are recognized as intermediate hosts of Schistosoma japonicum. Of these species and subspecies, O. quadrasi is distributed throughout the Philippines. This study used 12S ribosomal RNA sequences to explore the genetic structure of O. quadrasi populations in the Philippines. Three subspecies, O. h. hupensis, O. h. formosana, and O. h. chiui of this group were also examined. The phylogenetic tree and haplotypes network showed that O. quadrasi separated from the subspecies. Ten O. quadrasi haplotypes (Oq1-Oq10) clustered in relation to their geographic origin. Genetic differentiation (FST) and estimated gene flow (Nm) among populations showed significant differences, ranging from 0.556-1.000 to 0.00-0.74, respectively. Genetic differences among groups (FCT = 0.466), populations within a group (FSC = 0.727), and populations (FST = 0.854) were observed. These results indicate that the O. quadrasi populations in the Philippines have a substructure associated with their geographic origin., (© The American Society of Tropical Medicine and Hygiene.)

Genetic differentiation between two synonymous echinostomes species, Artyfechinostomum malayanum and Artyfechinostomum sufrartyfex was determined by using the first and second internal transcribed spacers (ITS1 and ITS2), the non-coding region of rDNA as genetic makers. Of the 699 bp of combined ITS1 and ITS2 sequences examined, 18 variable nucleotide positions (2.58 %) were observed. Of these, 17 positions could be used as diagnostic position between these two sibling species, whereas the other one variation was intraspecific variation of A. malayanum. A clade of A. malayanum was closely aligned with A. sufrartyfex and clearly distance from the cluster of other echinostomes. Our results may sufficiently suggest that the current synonymy of these species is not valid.

Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy-Weinberg equilibrium of 0.44-0.56. Assessment of population structure by pairwise F(ST) analysis showed inter-population differentiation (P<0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini.

A total of 314 individual Echinostoma revolutum were collected at different locations and times from domestic ducks from Khon Kaen Province, Thailand and Vientiane Province, the Lao People's Democratic Republic (PDR). Genetic variation of these parasites was analyzed using multilocus enzyme electrophoresis at three polymorphic loci namely, glucose-6-phosphate dehydrogenase (G6pd), malic enzyme (Me) and peptidase valine-leucine (PepA). High levels of genetic variability were found within and between populations. Significant heterozygote deficiencies compared with the predictions under Hardy-Weinberg equilibrium were detected in populations from Thailand and the Lao PDR for all loci except G6pd-1. Significant genetic differentiation was observed between spatially separated populations from Thailand and the Lao PDR. This as also true for some samples collected at different times in Thailand. The variability found may be consistent with a Wahlund effect, genetic drift and/or other factors such as the population structure of snail hosts. Our data provide further insight into the process of genetic divergence within and among geographically and temporally isolated populations of E. revolutum, and potentially other medically important echinostomes in Southeast Asia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Multilocus enzyme electrophoresis (MEE) and DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were used to genetically compare four species of echinostomes of human health importance. Fixed genetic differences among adults of Echinostoma revolutum, Echinostoma malayanum, Echinoparyphium recurvatum and Hypoderaeum conoideum were detected at 51-75% of the enzyme loci examined, while interspecific differences in CO1 sequence were detected at 16-32 (8-16%) of the 205 alignment positions. The results of the MEE analyses also revealed fixed genetic differences between E. revolutum from Thailand and Lao PDR at five (19%) of 27 loci, which could either represent genetic variation between geographically separated populations of a single species, or the existence of a cryptic (i.e. genetically distinct but morphologically similar) species. However, there was no support for the existence of cryptic species within E. revolutum based on the CO1 sequence between the two geographical areas sampled. Genetic variation in CO1 sequence was also detected among E. malayanum from three different species of snail intermediate host. Separate phylogenetic analyses of the MEE and DNA sequence data revealed that the two species of Echinostoma (E. revolutum and E. malayanum) did not form a monophyletic clade. These results, together with the large number of morphologically similar species with inadequate descriptions, poor specific diagnoses and extensive synonymy, suggest that the morphological characters used for species taxonomy of echinostomes in South-East Asia should be reconsidered according to the concordance of biology, morphology and molecular classification., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Genetic variation among 231 individuals of Opisthorchis viverrini from the Ban Phai District, Khon Kaen Province (Thailand) was examined at three polymorphic enzymes: enolase (ENOL), phosphoglucomutase (PGM), and triose phosphate isomerase (TPI). Four alleles were detected for TPI and PGM, whereas only two alleles were detected for ENOL. The inferred genotype frequencies for both TPI and ENOL were not significantly different from Hardy-Weinberg equilibrium. In contrast, the inferred genotype frequencies for PGM showed a significant departure from Hardy-Weinberg equilibrium, with a lack of heterozygous individuals. This heterozygote deficiency suggests non-random mating and/or potentially high self fertilization.

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People's Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.

Simple Summary: The zoonotic liver flukes Opisthorchis viverrini and Clonorchis sinensis infect small mammals, such as cats, dogs, pigs, rodents, and rabbits, as well as humans. Human infection subsequently develops into bile duct malignancy, also referred to as cholangiocarcinoma (CCA). Understanding the molecular systematics and population genetics of these liver flukes has an important role in prevention and control, and is important in comprehending their roles in zoonotic transmission. Different molecular markers have varying evolution rates and contain different genetic information. Polymorphic genetic markers are necessary and more suitable for such investigations. Therefore, we screened seven intron regions of the taurocyamine kinase gene (TK) to determine their potential as genetic markers for population genetic investigations of the liver flukes O. viverrini and C. sinensis which were collected from a range of geographical isolates and animal hosts. We identified a suitable intron region of TK, i.e., intron 5 of domain 1 (TkD1Int5) as having the most potential as a polymorphic marker. Results showed that TkD1Int5 is effective in examining the genetic variation and heterozygosity of O. viverrini and C. sinensis, but further studies are required to better understand the role of different species of animals as reservoir hosts of these zoonotic liver flukes. Opisthorchiasis and clonorchiasis are prevalent in Southeast and Far-East Asia, which are caused by the group 1 carcinogenic liver flukes Opisthorchis viverrini sensu lato and Clonorchis sinensis infection. There have been comprehensive investigations of systematics and genetic variation of these liver flukes. Previous studies have shown that O. viverrini is a species complex, called "O. viverrini sensu lato". More comprehensive investigations of molecular systematics and population genetics of each of the species that make up the species complex are required. Thus, other polymorphic genetic markers need to be developed. Therefore, this study aimed to characterize the intron regions of taurocyamine kinase gene (TK) to examine the genetic variation and population genetics of O. viverrini and C. sinensis collected from different geographical isolates and from a range of animal hosts. We screened seven intron regions embedded in TK. Of these, we selected an intron 5 of domain 1 (TkD1Int5) region to investigate the genetic variation and population genetics of theses liver flukes. The high nucleotide and haplotype diversity of TkD1Int5 was detected in O. viverrine. Heterozygosity with several insertion/deletion (indel) regions were detected in TkD1Int5 of the O. viverrine samples, whereas only an indel nucleotide was detected in one C. sinensis sample. Several O. viverrine samples contained three different haplotypes within a particular heterozygous sample. There were no genetic differences between C. sinensis isolated from various animal host. Heterozygous patterns specifically detected in humans was observed in C. sinensis. Thus, TkD1Int5 is a high polymorphic genetic marker, which could be an alternative marker for further population genetic investigations of these carcinogenic liver flukes and other related species from a wide geographical distribution and variety of animal hosts. [ABSTRACT FROM AUTHOR]

Abstract: Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host. [Copyright &y& Elsevier]

Abstract: The liver fluke, Opisthorchis viverrini, is one of the major food borne trematodes in Southeast Asia, where infection causes hepatobiliary disease and subsequent development of cholangiocarcinoma. In Thailand, O. viverrini is most prevalent in the northeast where there is marked regional variation in the rate of infection in humans at provincial, district and village levels. To date, the roles of genetic variation of O. viverrini on this observed variability in infection, transmission and associated disease are not known. We have applied multilocus enzyme electrophoresis (MEE), specifically allozyme electrophoresis, to isolates of O. viverrini from Thailand and Laos to establish genetic markers to examine its systematics and population structure. Forty-six enzymes commonly found useful for genetic characterisation in parasitic helminths were screened, and of these, 33 enzymes gave sufficient staining and resolution to act as potential genetic markers. Sixteen enzymes were monomorphic and 17 enzymes were polymorphic in the pools of worms examined. Whether they are indicative of different enzyme loci, heterozygosity or unique genotypes within the pools of worms examined remains to be determined. Preliminary investigations examining five individual worms at enzyme loci where pools of worms showed multiple bands have confirmed the diagnostic value of the enzyme loci established as well as providing evidence of potential population sub structuring and heterozygosity. For the first time, we have established at least 17 enzymes that provide the basis to undertake comprehensive genetic analyses of the systematics and population structure of O. viverrini, a medically important food borne trematode in Southeast Asia. [Copyright &y& Elsevier]

Haemonchus contortus is one of the most economically important parasitic nematodes affecting small ruminant livestock worldwide. This study was conducted to elucidate the genetic diversity and population structure of this nematode in Thailand based on mitochondrial DNA markers, the nicotinamide dehydrogenase subunit 4 (nad4) and the cytochrome c oxidase subunit 1 (cox1) genes. One hundred and thirty-six adult worms were obtained from 86 abomasa of slaughtered goats from 13 different localities in 5 regions of Thailand. Identification to the genus Haemonchus was done using morphology. DNA sequences of the nuclear ribosomal second internal transcribed spacer (ITS2) identified each specimen to species: three fixed nucleotide (SNP) differences distinguished H. contortus from H. placei. Genetic analysis defined 118 and 122 unique haplotypes in partial sequences of nad4 (alignment length 723 bp) and cox1 (645 bp) genes, respectively. Nucleotide diversities were 0.031 and 0.043 for nad4 and cox1 genes, respectively. Low genetic differentiation was observed among H. contortus samples from various provinces in Thailand. This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. This study has provided insights into the transmission dynamics of this parasitic nematode, information which is essential for farm management and parasite control. • This is the first study on the genetic diversity and population structure of H. contortus of goats in Thailand. • Our cox1 and nad4 sequence data revealed little sub-structuring among H. contortus samples from 13 provinces in Thailand. • High level of gene flow throughout the country, with the exception of the western region. • High within-population variation and low population genetic differentiation seems to be a global pattern for this species. [ABSTRACT FROM AUTHOR]

• COI sequence was used to identify species and sub-species of Bithynia. • Bithynia siamensis siamensis and B. s. goniomphalos were found in central Thailand. • Bithynia s. siamensis, B. s. goniomphalos and B. funiculata were found in northern Thailand. • Genetic structure of B. s. goniomphalos was related to catchment systems. • Bithynia s. siamensis and B. s. goniomphalos should be classified as " B. siamensis sensu lato" The freshwater snails, Bithynia are the first intermediate hosts of the liver fluke, Opisthorchis viverrini , the causative agent of cholangiocarcinoma (CCA) in Southeast Asia. In Thailand, there are three traditionally recognized taxa of Bithynia: Bithynia funiculata; B. siamensis siamensis; B. s. goniomphalos. This study examines the geographical distribution and genetic structure of Bithynia species from five previously reported water catchments and six new catchments in Thailand. Of these, three new catchments Kok, Wang, and Nan are from the north and the remaining three new catchments are Phetchaburi, Prachuap Khiri Khan Coast, Mae Klong from the west of Thailand. We sampled 291 Bithynia snails from 52 localities in 11 catchment systems in the northern, western and central regions of Thailand. Mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal DNA (16S rDNA) sequences were used to examine genetic diversity of Bithynia snails which revealed 200 and 27 haplotypes of COI and 16S rDNA, respectively. However, as 16S rDNA is a conserved gene, it is not suitable to distinguish Bithynia at the species and sub-species levels in our study. The phylogenetic tree and haplotype network analyses included sequences of COI from GenBank. B. funiculata was found only in the north of Thailand and the genetic structure did not differ among populations. Genetic differentiation (Φ ST) analyses showed that B. s. goniomphalos contained three distinct lineages. Lineage I contained B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR. Lineage II contained all B. s. goniomphalos from the Prachin Buri and Bang Pakong catchment systems in eastern and central Thailand, including samples from all catchment systems in Cambodia. While lineage III contained B. s. goniomphalos from the Songkram and Nam Kam catchment systems in Thailand and the Nam Ngum and Huai Som Pak catchment systems in Lao PDR. Furthermore, results showed that all samples of B. s. siamensis were classified into one lineage and placed phylogenetically between B. s. goniomphalos lineages I and II. Thus, the taxonomic status of B. s. goniomphalos and B. s. siamensis requires reassessment, and they should be reclassified as belonging to the species complex " Bithynia siamensis sensu lato". [Display omitted] [ABSTRACT FROM AUTHOR]