Genetic diversity of the Chinese liver fluke Clonorchis sinensis from Russia and Vietnam.

International Journal for Parasitology 44 (2014) 795–810

Contents lists available at ScienceDirect

International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Genetic diversity of the Chinese liver fluke Clonorchis sinensis from Russia and Vietnam Galina N. Chelomina a,⇑, Yulia V. Tatonova a, Nguyen Manh Hung b, Ha Duy Ngo b a b

Institute of Biology and Soil Science, Far Eastern Branch, Russian Academy of Sciences, 100-letiya Street, 159, Vladivostok 690022, Russia Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay Dist., Hanoi, Viet Nam

a r t i c l e

i n f o

Article history: Received 19 March 2014 Received in revised form 1 June 2014 Accepted 2 June 2014 Available online 12 August 2014 Keywords: Clonorchis sinensis cox1 gene COX1 Genetic diversity Intraspecific phylogeny

a b s t r a c t Clonorchiasis is a parasitic disease of high public health importance in many countries in southeastern Asia and is caused by the Chinese liver fluke Clonorchis sinensis. However, the genetic structure and demographic history of its populations has not been sufficiently studied throughout the geographic range of the species and available data are based mainly on partial gene sequencing. In this study, we explored the genetic diversity of the complete 1560 bp cytochrome c oxidase subunit 1 (cox1) gene sequence for geographically isolated C. sinensis populations in Russia and Vietnam, to our knowledge for the first time. The results demonstrated low nucleotide and high haplotype differentiation within and between the two compared regions and a clear geographical vector for the distribution of genetic diversity patterns among the studied populations. These results suggest a deep local adaptation of the parasite to its environment including intermediate hosts and the existence of gene flow across the species’ range. Additionally, we have predicted an amino acid substitution in the functional site of the COX1 protein among the Vietnamese populations, which were reported to be difficult to treat with praziquantel. The haplotype networks consisted of several region-specific phylogenetic lineages, the formation of which could have occurred during the most extensive penultimate glaciations in the Pleistocene Epoch. The patterns of genetic diversity and demographics are consistent with population growth of the liver fluke in the late Pleistocene following the Last Glacial Maximum, indicating the lack of a population bottleneck during the recent past in the species’ history. The data obtained have important implications for understanding the phylogeography of C. sinensis, its host-parasite interactions, the ability of this parasite to evolve drug resistance, and the epidemiology of clonorchiasis under global climate change. Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Clonorchis sinensis is a parasite from the class Trematoda that infects both people and some species of animals. This liver fluke is one of the most important fish-borne zoonotic parasites in east Asian countries such as China, Korea, Thailand and Vietnam, and the southern Far East of Russia (Mas-Coma and Bargues, 1997; Chai et al., 2005; Keiser and Utzinger, 2009; Besprozvannykh et al., 2012; Tatonova et al., 2012, 2013). More than 15 million people globally are infected with clonorchiasis, and more than 200 million are at risk of this infection (Fürst et al., 2012; Hong and Fang, 2012). Although a carcinogenic effect of C. sinensis was revealed in the early 1990s (Lee et al., 1993, 1994), this parasite was officially listed as a biological carcinogen to humans (Group 1) quite recently (Bouvard et al., 2009). As a tissue parasite, this ⇑ Corresponding author. Tel.: +7 (423) 231 0410; fax: +7 (423) 231 0193. E-mail address: [email protected] (G.N. Chelomina).

fluke contributes to human diseases, disability and death (Kruglyakova et al., 1987; Posokhov et al., 1987; Mas-Coma and Bargues, 1997) due to pathological changes in the liver and bile ducts. For all of these reasons, C. sinensis is of great scientific, medical and epidemiological significance. Until recently, it was believed that there were no true clonorchiasis nidi in the Primorye region of southern Far East Russia. This conclusion was based on an examination of more than 200 wild animals, belonging to different species of carnivores (Posokhov, 2004). However, over approximately the last 20 years, surveillance from the Russian Federal Service on Customers’ Rights Protection and Human Well-being has registered an increase in human clonorchiasis cases in Russia (Besprozvannykh et al., 2012). Clonorchiasis nidi were revealed in the native range of freshwater snails belonging to the genus Parafossarulus, and some nidi were estimated to be present due to the introduction of these snails. Thus, the number of clonorchiasis cases increased in recreational areas near Kronshtadtka as a result of the water storage formation

http://dx.doi.org/10.1016/j.ijpara.2014.06.009 0020-7519/Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

796

G.N. Chelomina et al. / International Journal for Parasitology 44 (2014) 795–810

in 1979. In this example, the first intermediate host of C. sinensis – the freshwater snail Parafossarulus manchouricus – was transferred from other places in the Primorye region with lotus rootstocks (Besprozvannykh et al., 2012). In addition, increased migration flows from endemic Asian countries and high human traffic from both local populations and people interested in exotic east Asian food can also lead to deterioration of the sanitary and epidemiological situation in the region. Unlike Russia, in Vietnam a high number of people and domestic animals are infected by C. sinensis, especially in the northern part of the country (e.g. Nam Dinh, Ninh Binh, Thai Binh provinces) and certain central provinces, e.g. Thanh Hoa and Nghe An (Nontasut et al., 2003; Dung et al., 2007; Hop et al., 2007; Ngo and Ermolenko, 2011; Clausen et al., 2012; Sithithaworn et al., 2012). The rapid development of aquaculture is suggested to be the major cause of the increase in clonorchiasis in southeast Asian countries, where fish are the main source of protein and people traditionally consume raw fish (Dung et al., 2007; Clausen et al., 2012; Hung et al., 2013a). In Vietnam, fish production has increased almost 10-fold (to 400,000 tons) over the past four decades (Dung et al., 2007), and a large part of this production occurs at integrated fish-livestock (VAC) systems. In these cases, ponds inhabited by both intermediate hosts (freshwater snails and farmed fish fry) become contaminated with parasite eggs through the faecal waste of definitive hosts (humans and animals), which increases the risk factors for clonorchiasis infection and transmission (Clausen et al., 2012). Despite its high epidemiological significance, the genetic diversity of C. sinensis has been insufficiently studied in different countries, including Russia and Vietnam, which represent the northernmost and southernmost parts of the C. sinensis distribution area, respectively. Genetic analysis of natural C. sinensis populations is usually conducted using nuclear (internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit 1, cox1) markers (Lee and Huh, 2004; Park, 2007; Liu et al., 2012; Tatonova et al., 2012, 2013; Sun et al., 2013). However, incomplete sequences for the cox1 gene are mainly used for genetic investigations of C. sinensis even though, in theory, the full-length gene sequences may reveal more complete information on species genetic diversity. COX is a small, highly conservative oligomeric enzymatic complex located on the inner membrane of the mitochondria and the terminal oxidase of cell respiration. COX1 is an important enzyme involved in the oxidation phosphorylation pathway and energy production. It was recently suggested (Abumourad, 2011) that COX1 may be included in the immune response against bacterial infection and that it may also play a specific role as an apoptotic inducer. Thus, information about the cox1 gene may be important for understanding the host-pathogen relationship. The cox1 gene is often used as molecular marker for phylogeographic studies in many taxa. The reconstruction of the historical biogeography of populations and the identification of the major genetic subdivisions within species are the main objectives of the phylogeographic analyses (Avise, 2000). Phylogeographic data can be also used for estimating the history of parasite colonisation and its migration routes, the potential source populations and time of invasion; and phylogeographic data can be used to evaluate drug resistance in the introduced locations (Criscione et al., 2005). In the present work, we used the complete 1560 bp sequence of the mitochondrial DNA (mtDNA) cox1 gene to gain insight into the genetic diversity of C. sinensis in both Russian and Vietnamese populations. We hypothesised that the gene characterisation may identify genetic information that is important for our understanding of the host immune response against trematode infection. Molecular data was also employed to address the question of historical demography and the phylogeography of C. sinensis across the large geographic range of this species.

2. Materials and methods 2.1. Sample collection and ethical statement Clonorchis sinensis parasitological material was collected in the summer of 2011 from three localities in southern Far East Russia and from two localities in different provinces in Vietnam. Each of these localities represents geographically isolated clonorchiasis nidus. In Russia, metacercariae were detected in the muscle of freshwater fish of the family Cyprinidae which were fed to laboratory rats (Sprague–Dawley strain) according to the standard method (Wang et al., 2009). We used two male rats for each locality, and each of these animals consumed 10–100 metacercariae (four to 17 fish according to their size and intensity of infection) at each feed for 1 week. Approximately 3 weeks after infection, the rats were examined for the presence of adult flukes in the liver. In Vietnam, adult flukes were extracted from naturally infected cats, and livers of these cats were bought at slaughterhouses. In total, 65 C. sinensis individuals (13 per each locality) were used in our analysis. The animal experiment was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Biology and Soil Science, Russia (Permit Number: 3 of 02.06.2011). Euthanasia was performed by decapitation, and all efforts were made to minimise suffering. 2.2. DNA extraction and PCR Liver flukes were stored in 96% ethanol. DNA was extracted from 65 flukes (13 worms from each locality) using the HotSHOT technique (Truett et al., 2000). The complete mitochondrial cox1 gene was amplified by PCR using the following primers: forward 5cF22 (50 -TAG-ACT-ATC-TGT-CTT-CAA-AAC-A-30 ), designed with the Lasergene PrimerSelect program (http://www.dnastar.com/ t-primerselect.aspx), and reverse CO1-Rv (50 -AAC-AAA-TCA-TGATGC-AAA-AGG-TA -30 ; Katokhin et al., 2008). PCR was performed in a total volume of 20 ll and contained 0.25 mM of each primer, 6 ll of DNA, 1x Taq buffer, 1.25 mM dNTP, 1.5 mM magnesium and 1.5 units of Taq polymerase (Medigen, Russia). Amplification of the cox1 sequence was performed on a GeneAmp 9700 (Applied Biosystems, USA) using the following cycling conditions: a 1 min initial denaturation step at 95 °C; 35 cycles of 30 s at 94 °C, 1 min at 52 °C, and 2 min at 72 °C; and a 7 min extension at 72 °C. Negative and positive controls with both primers were included. The PCR products of the cox1 genes for 65 C. sinensis specimens were purified by ethanol precipitation and sequenced directly on an ABI 3130 Genetic Analyzer (at the Institute of Biology and Soil Sciences, Far Eastern Branch, Russian Academy of Science) using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the following primers: CO1-Rv, CO10 3-Rv (50 -TGTTAA-TAT-TGC-CGG-GGT-TT-30 ; Katokhin et al., 2008) and newly designed primers 5cF22, 5cFS16 (50 -ATA-GGC-GGG-TGA-GGGAAC-GAC-A-30 ), 5cR4 (50 -GAT-CTC-ATA-GAC-GCT-CCC-GAG-T-30 ), and 5cRS5 (50 -TGT-CGT-TCC-CTC-ACC-CGC-CTA-T-30 ). 2.3. Analysis of genetic diversity The sequences were assembled manually and aligned using Clustal X (Thompson et al., 1997). We downloaded three cox1 gene sequences from GenBank for C. sinensis specimens from other regions: Khabarovsk, Russia (FJ381664); Guangdong, China (JF729303) and Korea (JF729304). Thus, complete sampling consisted of 68 complete cox1 gene sequences. Opisthorchis felineus

797


(Rivolta, 1884) (EU921260) and Opisthorchis viverrini (Poirier, 1886) (JF739555) were used as external controls. Sequence polymorphisms, amino acid variability and genetic distances among samples and haplotypes were estimated using MEGA version 5 (Tamura et al., 2011). Values for the number of haplotypes (H), polymorphic sites (S), haplotype (h) and nucleotide (p) diversities (Nei, 1987) and the average numbers of pairwise differences (k) (Tajima, 1983) were measured using DnaSP version 5.10 (Librado and Rozas, 2009) and ARLEQUIN version 3.11 (Excoffier et al., 2007). The protein structure of COX1 was analysed with using the three-dimensional structure of COX from bovine heart muscle (Tsukihara et al., 1996). Functional sites of the COX1 protein were obtained from GenBank (protein ID ACI95073).

The historical demographic parameters s (time since population expansion, expressed in units of mutational time), h0 and h1 (initial and current estimates of s, respectively) were calculated with ARLEQUIN. The time since expansion, s (with 95% confidence intervals (CIs)), was scaled with the equation s = 2lk, in which l is the mutation rate per nucleotide and k is the nucleotide number. The s values were converted to absolute time in millions of years (t). The molecular clocks for opisthorchids are unavailable due to a lack of fossils. Therefore, we used a nucleotide substitution rate of 2.5% per million years (Myr), as estimated from trematodes of the Schistosoma species, Schistosoma mekongi, (Attwood et al., 2008) to place the historical events on a relatively reliable time scale.

2.4. Intraspecific phylogenetic reconstructions

3.1. Sequence analysis

The genetic distance matrix was constructed using the Hasegawa–Kishino–Yano (HKY) model (Hasegawa et al., 1985) of evolution with the proportion of invariable sites and the gamma shape parameter (HKY + I + G). This model was selected by hierarchical Likelihood Ratio Tests (hLRTs) in Modeltest 3.07 (Posada and Crandall, 1998). Intraspecific phylogenies for the total dataset were reconstructed with the neighbour-joining (NJ) and maximum likelihood (ML) methods in the PAUP 4.0 b10 program (Swofford, 2002) and with the Bayesian Inference (BI) method in MrBayes 3.1.1. (Ronquist and Huelsenbeck, 2003). NJ and ML trees were constructed using 1000 and 100 bootstrap replicates, respectively. BI analyses were employed with two independent runs for 1,100,000 Markov chain Monte Carlo (MCMC) generations and sampling of the Markov chains at every 1000 generations (temp = 0.5). Consensus trees were constructed using the ‘‘sumt’’ command with the burnin = 250. Likewise, minimum spanning trees (MST) generated in ARLEQUIN version 3.11 and median-joining (MJ) networks generated in Network 4.6.1.0 (Bandelt et al., 1999) were used. Fst was calculated for estimation of the differentiation values among geographical populations with Analysis of Molecular Variance (AMOVA) statistics in ARLEQUIN. The null hypothesis of population panmixia was tested in two ways. First, the exact test was performed using 10,000 randomly permutated populations (r) with different haplotypes (k). Second, we used the Mantel test with 1000 randomisations (with Isolation by Distance (IBD) Web Service version 3.23) to estimate a correlation between the genetic and geographical distances among populations (Jensen et al., 2005). Additionally, gene flow (Nm) among populations was detected using DnaSP version 5.10.

A total of 65 complete sequences of the mtDNA cox1 gene (1560 bp) were determined for C. sinensis specimens from five isolated localities in Russia and Vietnam. We also used available GenBank data; thus, the total dataset included 68 sequences from seven geographic localities in Russia, Vietnam, China and Korea (Table 1; Fig. 1). The Chinese and Korean sequences were identical; therefore, were conventionally named the ‘‘Chinese-Korean haplotype’’. The cox1 gene was AT-rich; the AT/GC ratio was estimated to be 1.45 for both Russian (nucleotide frequencies were 13.46%, 42.24%, 16.92% and 27.38% for C, T, A and G, respectively) and Vietnamese (13.45%, 42.25%, 16.92% and 27.38%) samples, and 1.43 for the Chinese-Korean haplotype (13.53%, 42.18%, 16.73%, 27.56%). We detected 52 polymorphic sites in cox1 gene sequences from the total dataset, 29 of which were parsimony informative. The majority of mutations were transitions (transition/transversion (Ts/Tv) = 9.8). The mutation profile included 49 transitions (C M T and A M G), five transversions (T M G, T ? A, and C ? G) and no indels. No common transversions were detected for the studied populations and no differences in transition frequencies were estimated between the Russian and Vietnamese samples. However, the most northern populations in both countries showed a prevalence of C M T transitions, whereas southern populations demonstrated approximately an equal number of each transition type. It is worth noting that C ? T transitions were mainly at the 50 -end, and T ? C transitions were at the 30 -end of cox1 gene sequences in all studied populations. The distribution pattern of nucleotide diversity along the cox1 gene (Fig. 2) revealed an extended (575 bp) region with no substitutions in the central part of the sequences for samples from southern Vietnam. Interestingly, this region significantly decreased in size from southern to northern localities, with the shortest region (124 bp) found in an isolate from the northernmost locality

3. Results

2.5. Demographic analysis Mismatch distribution was applied for the demonstration of substitution differences between sequence pairs. Obtained distributions were compared with a fit to the Poisson model using DnaSP version 5.10. Tajima’s D (Tajima, 1989) and Fu’s Fs (Fu, 1997) statistics were used to test the mutation-drift equilibrium in the populations. Their significance was evaluated by 1000 random permutations. Significant negative D values can be due to demographic forces such as population expansion, bottlenecks and selection, while Fs is the most sensitive to demographic population expansion. A demographic model of population expansion was tested with the sum of squared deviations (SSD) and Harpending’s raggedness index (HRI) for observed and expected mismatch distributions, as implemented in ARLEQUIN version 3.11 (Rogers and Harpending, 1992).

Table 1 Complete mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences of Clonorchis sinensis analysed in the present study. Locality number

Geographic origin

Number of sequences

GenBank accession number

1 2

Magdikovoe, Russia Kronshtadtka, Russia Kondratenovka, Russia Thai Binh, Vietnam Danang, Vietnam Khabarovsk region, Russia Guangdong, China Korea

13 13

KJ204560-KJ204572 KJ204573-KJ204585

13

KJ204586-KJ204598

13 13 1

KJ204599-KJ204611 KJ204612-KJ204624 FJ381664

3 4 5 6 7 8

1 1

JF729303 JF729304

798


Fig. 1. Sampling locations for Clonorchis sinensis. Numbers correspond with those in Table 1.

in Russia. Additionally, the first 400 bp of the 50 -end in Vietnamese cox1 sequences demonstrated substitutions that were missing from the Russian samples. All transversions and 83.7% of transitions were located in the third codon position and all of these substitutions were synonymous. The first and second codon positions included eight (16.3% of total number) transitions, and six of them were non-synonymous substitutions: two in the Russian samples, one in the Vietnamese samples, and three in the Chinese-Korean haplotype. These nucleotide substitutions (at positions 112, 823, 1189, 1313, 1327 and 1399 bp) resulted in different amino acid substitutions: Val ? Met, Met ? Val, Thr ? Ala, Ser ? Leu, Phe ? Leu and Ile ? Val, respectively. Four of six non-synonymous nucleotide substitutions were singletons and specific for different countries. One parsimony informative non-synonymous nucleotide substitution (which resulted in Ile ? Val) was identified in the northern Russian populations (Magdikovoe and Kronshtadtka), while another (which resulted in Val ? Met) was identified in both Vietnamese populations. Most of the nucleotide substitutions (69%) were detected in a part of the gene that corresponds to a-helical regions of the COX1 protein (Fig. 3). Only 10 of 52 nucleotide substitutions were localised in the functional sites of the cox1 gene. Seven of the substitutions corresponding to the Val/Met38, Tyr 116, Arg208, Ser261, Ile313, Ala408 and Pro441 amino acid positions in the COX1 protein are at the site that is responsible for binding different subunit

interfaces; two nucleotide substitutions corresponding to Ser378 and Cys420 are at the site that enables chemical binding with heme a; and one nucleotide substitution corresponding to Asp360 is at the site involved in the putative water exit pathway. The single nonsynonymous substitution (Val/Met38) was identified in both Vietnam populations (see Fig. 3). The codon usage frequencies in different populations and geographical regions were very similar. However, the most frequent nucleotide substitution in the Russian samples was in the Ser codon, while in Vietnamese samples, the most frequent nucleotide substitution was in the Gly codon. The most frequently used codons in both geographical regions - UUU/Phe, GUU/Val, UUG/Leu, GGG/Gly, GGU/Gly and AUG/Met - comprised 32.7% of all codons. Only the CAA/Gln codon was not found in all populations within both geographical regions. A high level of haplotype diversity and a low level of nucleotide diversity were obtained for the total dataset (h = 0.976 ± 0.007; p = 0.00367 ± 0.00197), as well as within geographical regions and local populations (Table 2). The level of nucleotide diversity varied widely among local populations and was higher in the combined Russian samples (0.00343 ± 0.00188 versus 0.002 92 ± 0.00165). The level of haplotype diversity was somewhat higher in Vietnamese samples (0.969 ± 0.018 versus 0.955 ± 0.018). Both the genetic distances and AMOVA analysis did not reveal strong differences between regional groups (Table 3). The genetic


799

Fig. 2. Nucleotide distribution for the cytochrome c oxidase subunit I (cox1) gene of Clonorchis sinensis in different isolates. Pi, nucleotide diversity. Grey arrows show the conservative region in the central part of cox1 gene sequences for the local populations: (A) Magdikovoe, northernmost Russia; (B) Kronshtadtka, Russia; (C) Kondratenovka, Russia; (D) Thai Binh, Vietnam; (E) Danang, southernmost Vietnam. The size of the conservative region increases with geographical southward direction.

distances among cox1 gene sequences ranged from 0% to 0.90%, with an average of 0.37% for the total dataset. The values for sequence differences within and among populations were slightly higher for the Russian samples (with an average of 0.30% and 0.36%, respectively) than for Vietnamese ones (with an average of 0.29% for both comparisons). The genetic distances between geographical regions were slightly higher: 0.33–0.44%, with an average of 0.38%. Fst statistics demonstrated low genetic

differentiation between the geographical regions (Fst = 0.15448, P < 0.0001) and among the local populations except for Vietnamese regions, for which no statistically supported differentiation was estimated. The exact test of population differentiation also showed no significant differentiation among the local populations or between the geographical regions (exact P value = 1.0). Additionally, genetic isolation by distance was not found among populations (Mantel test P value > 0.05 for all models of IBD analysis).

800


Fig. 3. The functional sites in nucleotide sequences of the cytochrome c oxidase subunit I (cox1) gene (A) and in predicted amino acid sequences of the COX1 protein (B) of Clonorchis sinensis. The protein structure for C. sinensis COX1 was based on the three-dimensional structure for beef (Tsukihara et al., 1996). Bolded residues are the functional sites of the protein which were taken from GenBank (ACI95073, AGB56952 are the protein sequences for C. sinesis and Bos taurus, respectively). Grey rectangles show a-helical regions; Roman and Arabic numerals denote the numbers of transmembrane helices and numbers of amino acid residues in helices, respectively; rectangles without numerals are the helices in the extramembrane regions (according to Tsukihara et al., 1996). Squares, triangles and CH denote the nucleotide substitutions for Russian, Vietnamese and Chinese/Korean samples.

The patterns of haplotype frequencies in Russia and Vietnam were estimated to be significantly different (Fig. 4). We detected 40 haplotypes in the total dataset and almost all of those (90%)

were unique (with one or two sequences). The major haplotype, with three to six copies, was found in each population except for Danang (Vietnam). The cox1 gene haplotypes demonstrated both

801

G.N. Chelomina et al. / International Journal for Parasitology 44 (2014) 795–810 Table 2 Descriptive statistics for genetic diversity in cytochrome c oxidase subunit 1 (cox1) sequences of Clonorchis sinensis. Region and site a

Magdikovoe Kronshtadtkaa Kondratenovkaa Thai Binhb Danangb Russiac Vietnam Total samplesd

n

H

S

h

p

k

D

13 13 13 13 13 40 26 68

8 9 8 9 9 24 18 40

15 17 13 18 16 31 27 52

0.808 ± 0.113 0.910 ± 0.068 0.859 ± 0.089 0.936 ± 0,051 0.936 ± 0.051 0.955 ± 0.018 0.969 ± 0.018 0.976 ± 0.007

0,00266 ± 0,00159 0.00370 ± 0.00213 0,00268 ± 0.00160 0.00332 ± 0.00193 0.00255 ± 0.00153 0.00340 ± 0.00187 0.00292 ± 0.00165 0.00367 ± 0.00197

4.15385 ± 2.20844 5.76923 ± 2.95257 4.17949 ± 2.22028 5.17949 ± 2.68131 3.97436 ± 2.12548 5.31026 ± 2.61841 4.54769 ± 2.30995 5.71905 ± 2.77343

0–0.0052 0–0.0065 0–0.0058 0–0.0058 0–0.0058 0–0.0065 0–0.0064 0–0.0090

(0.0027) (0.0037) (0.0027) (0.0033) (0.0026) (0.0034) (0.0029) (0.0037)

H, number of haplotypes; S, number of polymorphic (segregating) sites; h, haplotype diversity (±S.D.); p, nucleotide diversity (±S.D.); k, mean pair-wise difference (±S.D.); D, genetic distance with the average values in parentheses. a Located in Russia. b Located in Vietnam. c Authors’ own sequences and specimen from GenBank (FJ381664). d All Russian, Vietnamese and Chinese/Korean samples (JF729303, JF729304).

Table 3 Pairwise indices (D, above diagonal and Fst, below diagonal) of genetic differentiation between Clonorchis sinensis populations calculated from the nucleotide dataset for the cytochrome c oxidase subunit 1 (cox1) gene. Samples Russian populations Magdikovoe Kronshtadtka Kondratenovka Vietnamese populations Thai Bing Danang

Magdikovoe

Kronshtadtka

Kondratenovka

Thai Bing

Danang

– 0.1755 (P < 0.01) 0.2287 (P < 0.01)

0.00386 (0.00096) – 0.1038 (P < 0.05)

0.00346 (0.00090) 0.00356 (0.00091) –

0.00390 (0.00094) 0.00437 (0.00100) 0.00378 (0.00094)

0.00340 (0.00084) 0.00396 (0.00093) 0.00333 (0.00087)

0.2328 (P < 0.0001) 0.2334 (P < 0.0001)

0.1976 (P < 0.0001) 0.2106 (P < 0.0001)

– 0.2144 (P < 0.0001)

– 0.0124 (P > 0.5)

0.00290 (0.00072) –

Fst, co-efficient of gene fixation (ARLEQUIN); D, genetic distances (MEGA); P 6 0.05 was considered significant.

high local and regional specificity, with not more than one common haplotype identified in any pairwise comparison of populations, even within the same geographical region, and only three common haplotypes were revealed between the Russian and Vietnamese samples. 3.2. Intraspecific phylogeny The model test revealed that the HKY substitution model with invariable sites’ proportion (I = 0.6652) and gamma distribution shape parameter (G = 1.5251) provides the best fit for the complete cox1 gene data. Therefore, an HKY genetic distance for the estimation of intraspecific phylogenetic relationships was used in our study. The topologies of NJ, ML and Bayesian (Fig. 5) trees were very similar and only small differences in bootstrap support values were obtained among those. These trees demonstrated unresolved topology with low bootstrap support for most branches. Only a few clusters were statistically supported; nevertheless, none of those corresponded to the geographical localities. To better resolve the intraspecific phylogeny and establish the genealogical relationships between the haplotypes, we constructed both the MST and the MJ network. The MST was reconstructed based on complete gene sequences as well as on the three gene segments (1–500 bp, 501–1000 bp, and 1001–1560 bp) (Fig. 6). The phylogenetic signal increased from the first, second and last segment, with 32, 28 and 14 gene sequences, respectively, in the most common haplotype. These common haplotypes can be tentatively named as ‘Russian’, ‘Vietnamese’ or ‘Russian-Vietnamese’ because they principally include sequences from Russia, Vietnam or both equally, respectively. The MST based on complete gene sequences reflected genealogy more efficiently. There is a small, star-like structure in the centre of a genealogical tree with an ancestor from the Russian-Vietnamese haplotype (from the southern populations) and a number of secondary-order structures that

mainly comprise sequences from either Russia or Vietnam. The most common haplotypes for each Russian locality (especially the northernmost one) are well differentiated. The MJ network (Fig. 7) is quite similar to the MST based on complete gene sequences. In total, this reconstruction showed the central, star-like structure with several short rays around the common Russian–Vietnamese haplotype, and five branches that comprised different haplotypes with their hypothesised samples. These hypothesised samples of the MJ network clustered the haplotypes quite well according to their geographical distribution, with the exception of the single link between the mv5 and mv6 branches. Based on genetic distance data, the phylogenetic lineages diverged amongst themselves approximately 230–130 ka before present (BP; d = 0.332–0.577) while their split from a putative common ancestor occurred at 130–50 ka BP (d = 0.128–0.327). 3.3. Historical demography To test the hypothesis of C. sinensis population expansion, we have exploited the distribution of pairwise nucleotide differences among the haplotypes. The mismatch distributions of nucleotide substitutions were generated for each local population, geographical region and for the total dataset (Fig. 8). The patterns of mismatch distribution point to higher genetic heterogeneity of cox1 gene sequences in the Russian populations, especially in Magdikovoe, with statistically supported deviation from the unimodal distribution. The local populations from Vietnam are more homogeneous, with statistically supported unimodal distribution for Danang’s population. The mismatch distributions for both geographical regions and the total dataset were unimodal and matched the expected distribution according to the model, suggesting population expansion (Rogers, 1995). The peaks of these mismatch distributions were in the range of five to six, which excludes the possibility of a genetic bottleneck in the recent past.

802


Fig. 4. Haplotype frequencies for the cytochrome c oxidase subunit I (cox1) gene of Clonorchis sinensis samples. (Russia) This study and GenBank (FJ381664) sequences; (Total) All Russian, Vietnamese and Chinese/Korean (JF729303, JF729304) sequences.

Both the Tajima’s D (1.57, P = 0.03) and Fu’s Fs (25.477, P = 0.00) indices were negative and significant for the total dataset, while only Fu’s Fs was significant (50% is shown for maximum likelihood/neighbour-joining/Bayesian Inference analyses (as indicated). Opisthorchis felineus and Opisthorchis viverrini were used for comparison. Samples 18.1– 18.13, 1.1–1.17, 2.1–2.17, 26.1–26.14, 24.1–24.13 are the samples from Magdikovoe, Kronshtadtka and Kondratenovka in Russia and Thai Binh and Danang in Vietnam, respectively; RusKha, Russia, Khabarovsk; ChGng, China, Guangdong; Kor, Korea.

mtDNA gene sequences (Park and Yong, 2001; Lee and Huh, 2004; Park, 2007; Liu et al., 2012; Tatonova et al., 2013) including multilocus analysis (Sun et al., 2013), and only genetic research has been conducted based on complete ITS1-5.8S-ITS2 region (Tatonova et al., 2012). To our knowledge the present study is the first population genetic analysis of C. sinensis based on complete cox1 gene sequences and has provided new data about the genetic diversity of the Chinese liver fluke. Additionally, we compared the most northern (Russian) populations with the most southern (Vietnamese) populations of C. sinensis for the first known time. The chosen marker has proved to be effective for studying the

phylogeography, population structure and the dynamics of the genetic processes within populations of different organisms (Avise, 2000). On the whole, the patterns of nucleotide substitutions, GC-content, and codon usage for the C. sinensis cox1 gene were similar to those of other trematode species such as Fasciola hepatica, Schistosoma mansoni and O. felineus (Le et al., 2002a, 2004; Shekhovtsov et al., 2010). These results are also consistent with our previously reported data, which compared Russian and Chinese C. sinensis samples (Tatonova et al., 2013). The absence of statistically supported population subdivisions over the area studied was observed

804


Fig. 6. Minimum spanning trees for the cytochrome c oxidase subunit I (cox1) haplotypes of Clonorchis sinesis. n, number of haplotypes; lengths of branches correspond to substitution numbers. (A) Tree for complete cox1 gene (1560 bp); (B, C, D) Trees for partial gene sequences (1–500 bp, 501–1000 and 1001–1560 bp, respectively). RusKha, Russia, Khabarovsk; ChGng, China, Guangdong; Kor, Korea.

with a number of approaches based on the nucleotide diversity: the Fs statistics, the exact test and Mantel tests. Additionally, the data obtained clearly showed no significant genetic differentiation between regional C. sinensis populations despite the significant geographical distances between Russia and Vietnam (more than 4000 km). The lack of genetic differentiation may be due to high gene flow among parasite populations that should counteract local adaptation (Slatkin, 1987; Lively, 1999). However, significant differentiation among cox1 gene haplotypes was revealed in this study and was also observed previously when we compared the Russian and Chinese C. sinensis samples (Tatonova et al., 2013). Most likely, these data are partially due to a restricted sample set for each locality, resulting in the identification of only the most frequent haplotypes. Nevertheless, this explanation is not sufficient for the extraordinarily high haplotype differences that we observed in both cases. Perhaps the use of other molecular markers will be more efficient in addressing this issue. For example, multilocus Random Amplified Polymorphic DNA (RAPD) (Sire et al., 2001; Semyenova et al., 2003, 2007; Theron et al., 2004; Korsunenko et al., 2009) and microsatellite (Blair et al., 2001; Curtis et al., 2002; Agola et al., 2006, 2009; Rudge et al., 2008; Gower et al., 2013) markers were demonstrated to be highly successful in estimating genetic diversity among different trematode species.

The patterns of genetic diversity and the population structure of parasites, especially those with a complex life cycle, depend on the interplay of many factors. In contrast to free-living organisms, the parasites are associated with two habitats i.e. the host’s external environment and the internal environment of the host and both have a significant impact on parasites. The abiotic factors affecting the free-living stage have a direct impact on parasites and an indirect impact via parasite hosts (Harvell et al., 2002; van Dijk et al., 2010; Sherrard-Smith et al., 2013). Trematodes, for example, are known to infect up to five hosts during their life cycle. The consecutive change in hosts during their life cycle is associated with an alternation between sexual and asexual (clonal) reproduction, where the mating system includes selfing, biparental inbreeding or outcrossing (Criscione et al., 2005; Prugnolle et al., 2005a; Sherrard-Smith et al., 2013). One of hypotheses regarding trematode evolution states that molluscs have been primarily included in the trematode life cycle as their hosts (Ginetsinskaya, 1968). Therefore, to date, parasites have shared and expressed equivalent molecules with their hosts that contribute greatly to high specificity of host-parasite interactions (Wide et al., 2006; Shalaby et al., 2010). Approximately 10 species of freshwater snails, belonging to different families, are the first intermediate hosts of C. sinensis (Hung et al., 2013a;


805

Fig. 7. Median-joining network for the cytochrome c oxidase subunit I (cox1) haplotypes of Clonorchis sinensis. Pale grey and dark grey circles denote the Russian and Vietnamese haplotypes, respectively; mv1-mv6 are hypothetical haplotypes generated by Network ver. 4.6.1.0.

Petney et al., 2013). However, in the southern part of Far East Russia, P. manchouricus and Parafossarulus spiridonovi (Bithyniidae) are the first intermediate hosts for C. sinensis, while in Vietnam the first intermediate hosts are more southern species of molluscs: Parafossarulus striatulus (Bithyniidae) and Melanoides tuberculata (Thiaridae) and, perhaps, Bithynia fuchsiana (Bithyniidae) and Alocinma longicornis (Hydrobiidae) (De, 2004; Posokhov, 2004; Ngo and Ermolenko, 2011; Besprozvannykh et al., 2012; Yoshida, 2012; Hung et al., 2013a). Fish belonging to the family Cyprinidae are the major secondary intermediate hosts for Chinese liver fluke. More than 100 species of freshwater fish have been shown to be infected with C. sinensis and, generally, the prevalence of infection among them is significantly higher than in the first intermediate hosts (Hung et al., 2013a,b). In Vietnam, the secondary intermediate hosts are mainly fish slide Anabus testudineus, goldfish Carassius auratus, mud carp Cirrhina molitorella, spotted snakehead Channa maculata, and silver carp Hypophthalmichthys molitrix (De, 2004; Le et al., 2013). In southern Far East Russia, the main secondary intermediate hosts of C. sinensis are Amur chebachok Pseudorasbora parva, Amur sleeper Perccottus glenii, minnow Manchu Phoxinus perenurus manthschuricus, Amur carp Cyprinus carpio haematopterus (in the Amur River), silver Prussian carp Carassius gibelio, and Ray-finned sungarik Chanodichthys erythropterus (Posokhov, 2004; Besprozvannykh et al., 2012; Yoshida, 2012). Clonorchis sinensis is known to infect humans and various wild and domestic fish-eating mammals, e.g. dogs, cats, pigs, rats and so on (Posokhov, 2004; Lun et al., 2005). The host’s agility should be a major determinant of gene flow in parasites, and for species with a complex life cycle the gene flow will be controlled by the most mobile host (Jarne and Theron, 2001; Criscione et al., 2005; Prugnolle et al., 2005a,b). Often, the definitive host is the most mobile hosts species, which can influence the genetic structure

of the parasites because these hosts provide a high gene flow among local populations that leads to decreased genetic differentiation (Criscione and Blouin, 2004; Louhi et al., 2010). Notably, it has been reported that trematode species have less structured populations and lower genetic diversity compared with snails, their freshwater intermediate hosts (Dybdahl and Lively, 1996; Criscione and Blouin, 2004; Keeney et al., 2007). The molluscan dispersal and mating system was indicated to be important for genetic differentiation in the schistosome-transmitting freshwater snails (Mavárez et al., 2002) and therefore may be associated with the genetic diversity of their trematode parasites. Previously, it was believed that parthenogenetic progeny of trematodes (cercariae) in snails were a set of genetically similar individuals (clones). Today, a high heterogeneity of cercariae from the same sporocyst as well as between daughter sporocysts is known for numerous trematode species (Bayne and Grevelding, 2003; Semyenova et al., 2005, 2007; Korsunenko et al., 2009). Nevertheless, intrahost genetic diversity of trematodes in the larval stages in snails was estimated to be much lower than that of adult flukes in definitive hosts, and a single definitive host can maintain approximately half of the total genetic diversity detected within the whole parasite population (Theron et al., 2004). Based on both the data obtained previously (Liu et al., 2012; Tatonova et al., 2012; Sun et al., 2013) and in this study, there are no species-specific phylogenetic lineages for definitive hosts of C. sinensis, presumably due to the substantially smaller evolutionary interaction times compared with those for the first and second hosts. Therefore, low genetic differentiation was found among C. sinensis isolates from fish, cats and dogs, while each of those demonstrated high and almost the same level of nucleotide diversity (Sun et al., 2013). Meanwhile, host-specific haplotypes that only belong to the cat isolates were found for C. sinensis, although it is not clear whether there are internal host-parasite associations

806


Fig. 8. Mismatch distribution of the cytochrome c oxidase subunit I (cox1) haplotypes of Clonorchis sinensis from different localities, geographical regions, and for the total dataset. Exp, expected frequency; Obs, observed frequency. Magdikovoe, Kronshtadtka and Kondratenovka are in Russia and Thai Binh and Danang are in Vietnam. Russia, this study and GenBank (FJ381664) sequences; Total, all Russian, Vietnamese and Chinese/Korean (JF729303, JF729304) sequences.

Table 4 Tajima’s D, Fu’s Fs statistics, their P values and mismatch distribution parameter estimates for Clonorchis sinensis in different localities, geographical regions, and for the total dataset. Tajima’s D

Magdikovoea Kronshtadtkaa Kondratenovkaa Thai Binhb Danangb Russiac Vietnam Totald

Fu’s Fs

Mismatch distribution

Goodness-of-fit tests

D

P

Fs

P

s

h0

h1

SSD

P

HRI

P

0.587 0.224 0.010 0.454 0.963 0.937 1.315 1.575

0.295 0.632 0.547 0.373 0.177 0.195 0.081 0.031

10.603 8.542 10.562 9.187 10.902 25.448 25.698 25.329

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

7.434 7.043 3.801 6.314 5.086 6.174 5.354 6.172

0.000 0.000 1.062 0.007 0.000 0.004 0.012 0.007

9.326 50.453 15.352 37.773 14.160 41.094 23.323 63.086

0.046 0.022 0.035 0.013 0.008 0.004 0.003 0.001

0.250 0.250 0.200 0.410 0.810 0.450 0.830 0.630

0.143 0.059 0.084 0.035 0.027 0.015 0.013 0.010

0.210 0.280 0.230 0.510 0.860 0.640 0.960 0.630

s, time co-efficient of population expansion; h0, h1, mutation parameters before and after expansion, respectively; SSD, sum-of-squared deviations; HRI, HarDQ6798’s Raggedness index. a Located in Russia. b Located in Vietnam. c This paper and GenBank (FJ381664) sequences. d All Russian, Vietnamese and Chinese/Korean sequences (JF729303, JF729304).


between haplotypes or other factors related to geography (Sun et al., 2013). The genetic subdivision of parasitic populations between their host species or between the races of species have been reported for other examples, although the differences were often associated with geography or habitat type (McCoy et al., 2001; Johnson et al., 2002; Le et al., 2002b; Wang et al., 2006; Rudge et al., 2009). In our study, a clear geographical vector for the distribution of genetic diversity patterns among the studied populations was revealed, and these results are strongly supported by previously reported data on nDNA markers (Lai et al., 2008; Tatonova et al., 2012). Thus, the geographical origin of the local C. sinensis populations may contribute to an increase (when there are large geographical distances) or decrease (when there are closely spaced localities) in its genetic differentiation. Notably, Vietnamese populations (with no common haplotypes) belong to two different zoogeographical subregions: the northern population belonging to the Chinese subregion and the southern population belonging to the Indo-Malayan subregion (Starobogatov, 1970). In contrast, all Russian populations (which maintain the common haplotypes) belong to the same Amur-Japanese subregion (Starobogatov, 1970). Nevertheless, all local populations under comparison were well differentiated in terms of haplotype frequency, which could be a consequence of a deep local adaptation of the parasite to their particular environment, including water and temperature mode, water saltiness, dark/light conditions and intermediate hosts (see above). This finding is consistent with the Red Queen hypothesis, which states that interactions among species such as hosts and parasites lead to constant natural selection for adaptation and counter-adaptation and contribute to the spatial mosaic (Van Valen, 1973; Ebert, 1994; Gandon et al., 1996; Gandon and Michalakis, 2002; Lively, 1999; Peters and Lively, 1999). This antagonistic co-evolution is predicted to have complex evolutionary consequences, often within a short time period. It resulted, particularly, in greater genetic divergence between replicate populations (Lively and Dybdahl, 2000; Paterson et al., 2010). Local adaptation by parasites to their hosts is facilitated by higher parasite migration (Davies et al., 1999). In this study, we have attempted, to our knowledge for the first time, to analyse both the amino acid and nucleotide sequence variations in sites with different functional importance in the COX1 protein and cox1 gene of C. sinensis, respectively. We revealed only one parsimony informative substitution, Val ? Met, in COX1 of Vietnamese populations, and this substitution not only changes the primary protein structure but also can affect the protein function due to its location in the functional region. Actually, the Val ? Met substitution should be of considerable importance because in mammals, even the substitution of a single sulphurcontaining methionine in proteins can result in irreversible effects such as hereditary diseases (Murray et al., 1992; RodriguezMartinez et al., 2010). Additionally, there is evidence that nucleotide substitutions in the third codon position (which do not generally influence the primary protein sequence) can change the angle at which the next amino acid is incorporated into the growing polypeptide chain during protein synthesis (Kushelev, A.Y., Sokolik, V.V., 2012. Pikotechnology – a new approach to modelling the spatial protein structure. Materials of International scientific and practical conference ‘‘Modern Science: tendencies of development’’. SIC Apriori, Krasnodar. pp. 203–207 (in Russian).). Thus in C. sinensis, nucleotide substitutions in the third codon positions of the cox1 gene can also influence the protein spatial structure and hence its function. For example, they can change the operational efficiency of functional sites in the first subunit of the COX1 protein. Hypothetically, the particularities of molecular organisation revealed for Vietnamese cox1 genes and those proposed for the COX1 protein can be associated with the high resistance of human

807

populations in that country to praziquantel (Tinga et al., 1999) which is shown to be highly effective in different countries (Choi et al., 2006). There are a number of examples that certain molecular characteristics of parasites are closely correlated with their adaptive ability and infectivity (e.g. Schönian et al., 2001; Rooney, 2004; Subbotin et al., 2011). We came to the same conclusion based on data of the nuclear rDNA sequences of C. sinensis (Tatonova et al., 2012). Additionally, there is evidence that genes associated with infection or resistance to infection have particularly high rates of molecular evolution (Blanc et al., 2005; Obbard et al., 2006; Barrett et al., 2009). At the very least, it may be important for C. sinensis populations that the ability of parasites to evolve drug resistance will be affected both by gene flow among populations and genetic drift within them (Criscione et al., 2005). In this study, we demonstrated that using complete cox1 gene sequences is more efficient for reconstructing the intraspecific phylogeny of C. sinensis in comparison with a gene fragment, including the most variable and usable 30 -end of the gene. Particularly, the short gene fragments (due to a limited set of the parsimony informative sites) can generate a simple, star-like structure, implying a false bottleneck for the species in its recent past. The star-like phylogeny is associated with a rapid expansion from the species refuge, which would generate little genetic differentiation in the colonised area (Hewitt, 1996), and it is not strictly consistent with our results. Global climatic fluctuations in the Pleistocene Epoch are known to have had a deep impact on species distribution and the genetic structure of the current species worldwide (Avise, 2000; Hewitt, 2000). The Chinese liver fluke is an Asian species and the historical records and archaeological evidence of C. sinensis infection in human populations indicate an ancient origin of this species in China (e.g. Sun et al., 2013). Due to monsoons, the climatic fluctuations in Asia during the Pleistocene Epoch were not as extensive as those in Europe or America, although cold and arid weather fatefully affected the evolution and distribution of plants and animals, resulting in the disappearance of many species (Li et al., 1979; Qiu et al., 2011; Wang et al., 2013). Based on the divergent time estimated for mtDNA lineages, the population history of C. sinensis could be traced back to the extensive Saalian glaciations (230– 100 ka BP) in Europe, which correspond to the penultimate Pleistocene glaciations in China (Liu et al., 2000; Zheng et al., 2002; You et al., 2010). The divergent time estimated for the split of phylogenetic lineages with their putative common ancestor may be an indication of the spatial expansion of the Chinese liver fluke during the last, and one of the warmest, interglacial periods (130–75 ka BP), followed by a decrease in the population size during the last glaciation in China (74–11.5 ka BP) (Liu et al., 2001; You et al., 2010). The possibility of a population expansion event during the last interglacial period was previously suggested based on data obtained for samples from Russia, China and other countries of southeastern Asia (Tatonova et al., 2013). The presence of different phylogenetic lineages within the studied species suggests the isolation and differentiation of populations in multiple refuges and may be linked to the presence of biogeographic barriers such as mountains, rivers and deserts in the diversification and isolation of new genetic lineages within species (Avise, 2000). In eastern Asia, the Little Khingnan Mountains and Manchuria Korean Mountains are known to be efficient barriers to animal migration (Driscoll et al., 2009), and the uplift of the Qinghai-Tibet Plateau has induced changes in the distribution of many plant and animal populations in China (You et al., 2010). In southern Far East Russia, there were significant changes in the river systems, at least in part due to volcanic activity during the middle Pleistocene Epoch (Korotky et al., 2011). In theory (Excoffier and Schneider, 1999), the patterns of mismatched distribution, with peaks in the range of one or more,

808


assume a general population growth and the lack of a population bottleneck in the recent past in C. sinensis evolutionary history. Significantly negative values in the tests for neutrality obtained in this study are typical for populations that experienced a recent demographic expansion (Tajima, 1989; Fu, 1997). The extensive spatial expansion often provides the same signal in the panmictic populations but only when the adjacent populations have a large number of migrants (Calvo et al., 2009). Considering that the error associated with the mutation rates is large and the resulting time estimates based on molecular data are therefore approximate (Arbogast et al., 2002), the population expansion event of C. sinensis most likely occurred in the Late Pleistocene after the Last Glacial Maximum (23–15 ka BP). These estimates are lower than those predicted for the dataset with samples from China (Tatonova et al., 2013). Clearly, the extended history of the species is closely related to the migration of ancient human tribes and the high activity and national traditions of modern human populations. Comparison of the nucleotide and gene diversity parameters can also provide insight into the demographic history of the populations and allow for speculation concerning past demographic events (Avise, 2000; Painter et al., 2007). Thus, high haplotype and relatively low nucleotide diversities, which were observed in our study for the Russian and Vietnamese populations, may be a result of the long-term isolation of their small ancestral populations. In the east Asian region, southern China, which is one of the most species-rich regions in the world, may have served as a refuge during the Pleistocene glaciations or as the centre of speciation (Wang, 1992; Lei et al., 2003; Dai et al., 2011). One of the current phylogeographic scenarios also implies long-term isolation and species survival in the multiple refuges of China during the Quaternary climate change (Qiu et al., 2011). The Pleistocene refuges have been reported in eastern China and in the south-eastern plateau, and areas such as the Yunnan and Sichuan Basin are known to be large-scale relict refuges for many species (You et al., 2010). The Korean peninsula is also of great interest as a potential Pleistocene refuge for C. sinensis. This region, with a southern temperate mountain climate, may have allowed a number of species to survive during the coldest periods in the Pleistocene Epoch (Sakka et al., 2010). The species refuges should be characterised by not only a comparatively stable environment during climate fluctuations but also by the ability to exhibit an increased level of genetic diversity (Hewitt, 1996; Avise, 2000). Unlike Russia, Vietnam was not deeply affected by climatic changes and its environment was relatively stable during the global climate changes during the Pleistocene Epoch. However, the genetic diversity of Vietnamese and Russian populations was estimated to be almost identical. At the same time, the populations inhabiting central China demonstrated an increase in genetic diversity and exhibited direct linkages with ancient samples (Sun et al., 2013). Thus, we report novel data on genetic diversity in C. sinensis populations within and between two geographical regions, Russia and Vietnam, and we believe that these data can have important epidemiological, evolutionary and medical implications. The fullsize cox1 gene and predicted structure of the COX1 protein have proved to be promising molecular markers for genetic research on trematodes. Further samples and other molecular markers are necessary to provide additional information on the population genetic structure of this liver fluke in future studies. A large-scale genetic analysis is needed to better understand the Pleistocene history of the Chinese liver fluke and to shed light on its migratory routes in the post-glaciation epoch. In answering these questions, it is important to take into account the biological peculiarities of this parasitic species which may have influenced the microevolutionary processes in these populations.

Acknowledgements We thank Dr. V.V. Besprozvannykh for providing the biological samples from the Russian Far East. This research was partially supported by a grant from the Far Eastern Branch of the Russian Academy of Science (project No. 11-III-D-06-014).

References Abumourad, I.M.K., 2011. Cytochrome c oxidase subunit-1 (cox1) gene in Tilapia (Oreochromis niloticus): its cloning and characterization. Int. J. Genet. Eng. 1, 1–5. Agola, L.E., Mburu, D.N., DeJong, R.J., Mungai, B.N., Muluvi, G.M., Njagi, E.N.M., Loker, E.S., Mkoji, G.M., 2006. Microsatellite typing reveals strong genetic structure of Schistosoma mansoni from localities in Kenya. Infect. Genet. Evol. 6, 484–490. Agola, L.E., Steinauer, M.L., Mburu, D.N., Mungai, B.N., Mwangi, I.N., Magoma, G.N., Loker, E.S., Mkoji, G.M., 2009. Genetic diversity and population structure of Schistosoma mansoni within human infrapopulations in Mwea, central Kenya assessed by microsatellite markers. Acta Trop. 111, 219–225. Arbogast, B.S., Edwards, S.V., Wakeley, J., Beerli, P., Slowinski, J.B., 2002. Estimating divergence times from molecular data on phylogenetic and population genetic timescales. Annu. Rev. Ecol. Syst. 33, 707–740. Attwood, S.W., Fatih, F.A., Upatham, E.S., 2008. DNA-sequence variation among Schistosoma mekongi populations and related taxa; phylogeography and the current distribution of Asian schistosomiasis. PLoS Negl. Trop. Dis. 2 (3), e200. http://dx.doi.org/10.1371/journal.pntd.0000200. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard Univ. Press, Cambridge. Bandelt, H.J., Forster, P., Röhl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Barrett, L.G., Thrall, P.H., Dodds, P.N., van der Merwe, M., Linde, C.C., Lawrence, G.J., Burdon, J.J., 2009. Diversity and evolution of effector loci in natural populations of the plant pathogen Melampsora lini. Mol. Biol. Evol. 26, 2499–2513. Bayne, C.J., Grevelding, C.G., 2003. Cloning of Schistosoma mansoni sporocysts in vitro and detection of genetic heterogeneity among individuals within clones. J. Parasitol. 89, 1056–1060. Besprozvannykh, V.V., Ermolenko, A.V., Rumiantseva, E.E., Maslov, D.V., Voroniuk, V.M., Tatonova, Y.V., 2012. Nosoareas of clonorchiasis in the Primorye Territory. Med. Parazitol. (Mosk) 2, 7–14 (in Russian). Blair, L., Webster, J.P., Barker, G.C., 2001. Isolation and characterization of polymorphic microsatellite markers in Schistosoma mansoni from Africa. Mol. Ecol. 1, 93–95. Blanc, G., Ngwamidiba, M., Ogata, H., Fournier, P.-E., Claverie, J.-M., Raoult, D., 2005. Molecular evolution of Rickettsia surface antigens: evidence of positive selection. Mol. Biol. Evol. 22, 2073–2083. Bouvard, V., Baan, R., Straif, K., Grosse, Y., Secretan, B., Ghissassi, F.E.I., BenbrahimTallaa, L., Guha, N., Freeman, C., Galichey, L., Cogliano, V., 2009. A review of human carcinogens - part B: biological agents. The Lancet Oncol. 10, 321–322. Calvo, J.H., Calvete, C., Martinez-Royo, A., Estrada, R., Miranda, M.A., Borras, D., Monteys, V., Sarto, I., Pages, N., Delago, J.A., Collantes, F., Lucientes, J., 2009. Variations in the mitochondrial cytochrome c oxidase subunit I gene indicate northward expanding populations of Culicoides imicola in Spain. Bull. Entomol. Res. 99, 583–591. Chai, J.Y., Murrell, K.D., Lymbery, A.J., 2005. Fish-borne parasitic zoonoses: status and issues. Int. J. Parasitol. 35, 1233–1254. Choi, D., Lim, J.H., Lee, K.T., Lee, J.K., Choi, S.H., Heo, J.S., Jang, K.-T., Lee, N.Y., Kim, S., Hong, S.-T., 2006. Cholangiocarcinoma and Clonorchis sinensis infection: a casecontrol study in Korea. J. Hepatol. 44, 1066–1073. Clausen, J.H., Madsen, H., Murrell, K.D., Van, P.H., Thu, H.N., Do, D.T., Thi, L.A.N., Mahn, H.N., Dalsgaard, A., 2012. Prevention and control of fish-borne zoonotic trematodes in fish nurseries, Vietnam. Emerg. Infect. Dis. 18, 1438–1444. Criscione, C.D., Blouin, M.S., 2004. Life cycle shape parasite evolution: comparative population genetics of salmon trematodes. Evolution 58, 198–202. Criscione, C.D., Poulin, R., Blouin, M.S., 2005. Molecular ecology of parasites: elucidating ecological and microevolutionary processes. Mol. Ecol. 14, 2247– 2257. Curtis, J., Sorensen, R.E., Minchella, D.J., 2002. Schistosome genetic diversity: the implications of population structure as detected with microsatellite markers. Parasitology 125, S51–S59. Dai, C., Zhao, N., Wang, W., Lin, C., Gao, B., Yang, X., Zhang, Z., Lei, F., 2011. Profound climatic effects on two East Asian Black-Throated tits (Ave: Aegithalidae), revealed by ecological niche models and phylogeographic analysis. PLoS ONE 6, e29329. http://dx.doi.org/10.1371/journal.pone.0029329. Davies, C.M., Webster, J.P., Krüger, O., Munatsi, A., Ndamba, J., Woolhousw, M.E.J., 1999. Host-parasite population genetics: a cross sectional comparison of Bulinus globosus and Schistosoma haematobium. Parasitology 119, 295–302. De, N.V., 2004. Fish-borne trematodes in Vietnam. Southeast Asian. J. Trop. Med. Public Health 35, 229–231. Driscoll, C.A., Yamaguchi, N., Bar-Gal, G.K., Roca, A.L., Luo, S., Mcdonald, D.W., O’Brien, S.J., 2009. Mitochondrial phylogeography illuminates the origin of the extinct Caspian Tiger and its relationship to the Amur Tiger. PLoS One 4, e4125. http://dx.doi.org/10.1371/journal.pone.0004125.

G.N. Chelomina et al. / International Journal for Parasitology 44 (2014) 795–810 Dung, D.T., De, N.V., Waikagul, J., Dalsgaard, A., Chai, J.-Y., Sohn, W.-M., Murrell, K.D., 2007. Fishborne zoonotic intestinal Trematodes, Vietnam. Emerg. Infect. Dis. 13, 1828–1833. Dybdahl, M.F., Lively, C.M., 1996. The geography of coevolution: comparative population structures for a snail and its trematode parasite. Evolution 50, 2264– 2275. Ebert, D., 1994. Virulence and local adaptation of a horizontally transmitted parasite. Science 256, 1084–1086. Excoffier, L., Laval, G., Schneider, S., 2007. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinf. 1, 47–50. Excoffier, L., Schneider, S., 1999. Why hunter-gatherer populations do not show signs of pleistocene demographic expansions. Proc. Natl. Acad. Sci. 96, 10597– 10602. Fu, Y.X., 1997. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147, 915–925. Fürst, T., Keiser, J., Utzinger, J., 2012. Global burden of human food-borne trematodiasis: a systematic review and meta-analysis. Lancet Infect. Dis. 12, 210–221. Gandon, S., Capoweiz, Y., Dubois, Y., Michalakis, Y., Olivieri, I., 1996. Local adaptation and gene-for-gene coevolution in a metapopulation model. Proc. R. Soc. B 263, 1003–1009. Gandon, S., Michalakis, Y., 2002. Local adaptation, evolutionary potential and hostparasite coevolution: interactions between migration, mutation, population size and generation time. J. Evol. Biol. 15, 451–462. Ginetsinskaya, T.A., 1968. Trematodes, their Life Cycles, Biology and Evolution. Nauka, Leningrad (in Russian). Gower, C.M., Gouvras, A.N., Lamberton, P.H.L., Deol, A., Shrivastava, J., Mutombo, P.N., Mbuh, J.V., Norton, A.J., Webster, B.L., Stothard, J.R., Garba, A., Lamine, M.S., Kariuki, C., Lange, C.N., Mkoji, G.M., Kabatereine, N.B., Gabrielli, A.F., Rudge, J.W., Fenwick, A., Sacko, M., Dembelé, R., Lwambo, N.J.S., Tchuem Tchuenté, L.-A., Rollinson, D., Webster, J.P., 2013. Population genetic structure of Schistosoma mansoni and Schistosoma haematobium from a cross six sub-Saharan African countries: implications for epidemiology, evolution and control. Acta Trop. 28, 261–274. Harvell, C.D., Mitchel, C.E., Ward, J.R., Altizer, S., Dobson, A.P., Ostfeld, R.S., Samuel, M.D., 2002. Climate warming and disease risks for terrestrial and marine biota. Science 296, 2158–2162. Hasegawa, M., Kishino, H., Yano, T., 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174. Hewitt, G.M., 1996. Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc. 58, 247–276. Hewitt, G.M., 2000. The genetic legacy of the Quaternary ice ages. Nature 405, 907– 913. Hong, S.-T., Fang, Y., 2012. C. sinensis and clonorchiasis, an update. Parasitol. Int. 61, 17–24. Hop, N.T., De, N.V., Murrell, D., Dalsgaard, A., 2007. Occurrence and species distribution of fishborne zoonotic trematodes in wastewater-fed aquaculture in northern Vietnam. Trop. Med. Int. Health 2, 66–72. Hung, N.M., Madsen, H., Fried, B., 2013a. Global status of fish-borne zoonotic trematodiasis in humans. Acta Parasitol. 58, 231–258. Hung, N.M., Stauffer, J.R., Madsen, H., 2013b. Prey species and size choice of the molluscivorous fish, black carp (Mylopharyngodon piceus). J. Freshwater Ecol.. http://dx.doi.org/10.1080/02705060.2013.800826. Jarne, P., Theron, A., 2001. Genetic structure in natural populations of flukes and snails: a practical approach and review. Parasitology 123, S27–S40. Jensen, J.L., Bohonak, A.J., Kelley, S.T., 2005. Isolation by distance, web service. BMC Genet. 6, 13. Johnson, K.P., Williams, B.L., Drown, D.M., Adams, R.J., Clayton, D.H., 2002. The population genetics of host specificity: genetic differentiation in dove lice (Insecta: Phthiraptera). Mol. Ecol. 11, 25–38. Katokhin, A.V., Shekhovtsov, S.V., Konkow, S., Yurlova, N.I., Serbina, E.A., Vodianitskai, S.N., Fedorov, K.P., Loktev, V.B., Muratov, I.V., Ohyama, F., Makhnev, T.V., Pel’tek, S.E., Mordvinov, V.A., 2008. Assessment of the genetic distinctions of Opisthorchis felineus from O. viverrini and Clonorchis sinensis by ITS2 and CO1 sequences. Dokl. Biochem. Biophys. 421, 214–217. Keeney, D.B., Waters, J.M., Poulin, R., 2007. Diversity of trematode genetic clones within amphipods and the timing of same-clone infections. Int. J. Parasitol. 37, 351–357. Keiser, J., Utzinger, J., 2009. Food-borne trematodiases. Clin. Microbiol. Rev. 22, 466– 483. Korotky, A.M., Korobov, V.V., Skrylnik, G.P., 2011. Anomalous natural processes and their influence on state of geosystems of south of Russian Far East. Dalnauka Press, Vladivostok (in Russian). Korsunenko, A.V., Tyutin, A.V., Semyenova, S.K., 2009. Clonal and population RAPD variation of cercariae obtained from Bucephalus plymorphus sporocysts (Trematoda: Bucephalidae). Russ. J. Genet. 45, 63–69. Kruglyakova, L.V., Grigorenko, A.A., Chertov, A.D., 1987. Clonorchiasis human event with high intensity and lethal outcome. In: Kikot, V.I. (Ed.), Helminthiases of Far East. Research Institute of Epidemiology and Microbiology Press, Khabarovsk, pp. 55–59. Lai, D.-H., Wang, Q.-P., Chen, W., Cai, L.-S., Wu, Z.-D., Zhu, X.-Q., Lun, Z.-R., 2008. Molecular genetic profiles among individual Clonorchis sinensis adults collected from cats in two geographic regions of China revealed by RAPD and MGE-PCR methods. Acta Trop. 107, 213–216. Le, T.H., Blair, D., McManus, D.P., 2002a. Mitochondrial genomes of parasitic flatworms. Trends Parasitol. 18, 206–213.

809

Le, N.T., Ngo, H.D., Ermolenko, A.V., 2013. Trematodes of Terrestrial Vertebrates in Vietnam. Dalnauka Press, Vladivostok (in Russian). Le, T.H., Pearson, M.S., Blair, D., Dai, N., Zhang, L.H., McManus, D.P., 2002b. Complete mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of Echinococcus granulosus. Parasitology 124, 97–112. Le, T.H., McManus, D.P., Blair, D., 2004. Codon usage and bias in mitochondrial genomes of parasitic platyhelminthes. Korean J. Parasitol. 42, 159–167. Lee, J.H., Rim, H.J., Bak, U.B., 1993. Effect of Clonorchis sinensis infection and dimethylnitrosamine administration on the introduction of cholangiocarcinoma in Syrian golden hamsters. Korean J. Parasitol. 31, 21–30. Lee, J.H., Yang, H.M., Buk, U.B., Rim, H.J., 1994. Promotion role of Clonorchis sinensis infection on introduction of cholangiocarcinoma during two-step carcinogenesis. Korean J. Parasitol. 32, 15–18. Lee, S.U., Huh, S., 2004. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J. Parasitol. 42, 145–148. Lei, F., Qu, Y., Lu, J., Liu, Y., Yin, Z., 2003. Conservation on diversity and distribution patterns of endemic birds in China. Biodivers. Conserv. 12, 239–254. Li, J.J., Wen, S.X., Zhang, Q.S., Wang, F.B., Zheng, B.X., Li, B.Y., 1979. A discussion on the period, amplitude and type of the uplift of the Qinghai-Xizang Plateau. Sci. Sin. 22, 1314–1328 (in Chinese with English abstract). Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452. Liu, D., Shi, Y., Wang, R., Zhao, Q., Jian, Z., Cheng, X., Wang, P., Wang, S., Yuan, B., Wu, X., Zhangxiang, Q., Qinq, X., Wanbo, H., Weiwen, H., Zhisheng, A., Huayu, L., 2000. Table of the Chinese stratigraphic correlation remarked with climate change. Quat. Sci. 20, 108–128. Liu, G.H., Li, B., Li, J.Y., Song, H.Q., Lin, R.Q., Cai, X.Q., Zou, F.C., Yan, H.K., Yuan, Z.G., Zhou, D.H., Zhu, X.Q., 2012. Genetic variation among Clonorchis sinensis isolates from different geographic regions in China revealed by sequence analyses of four mitochondrial genes. J. Helminthol. 86, 479–484. Liu, J.Q., Ni, Y.Y., Chu, G.Q., 2001. Main palaeoclimatic events in the Quaternary. Quat. Sci. 21, 239–248. Lively, C.M., 1999. Migration, virulence, and the geographic mosaic of adaptation by parasites. Am. Nat. 153, S34–S47. Lively, C.M., Dybdahl, M.F., 2000. Parasite adaptation to locally common host genotypes. Nature 405, 679–681. Louhi, K.-R., Karvonen, A., Rellstab, C., Jokela, J., 2010. Is the population genetic structure of complex life cycle parasites determined by the geographic range of the most motile host? Infect. Genet. Evol. 10, 1271–1277. Lun, Z.R., Gasser, R.B., Lai, D.-H., Li, A.-X., Zhu, X.-Q., Yu, X.-B., Fang, Y.-Y., 2005. Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect. Dis. 5, 31–41. Mas-Coma, S., Bargues, M.D., 1997. Human liver flukes: a review. Res. Rev. Parasitol. 57, 145–218. Mavárez, J., Pointier, J.-P., David, P., Delay, B., Jarne, P., 2002. Genetic differentiation, dispersal and mating system in the schistosome-transmitting freshwater snail Biomphalaria glabrata. Heredity 89, 258–265. McCoy, K.D., Boulinier, T., Tirard, C., Michalakis, Y., 2001. Host specificity of a generalist parasite: genetic evidence of sympatric host races in the seabird tick Ixodes uriae. J. Evol. Biol. 14, 395–405. Murray, E.W., Giles, A.R., Lillicrap, D., 1992. Germ-line mosaicism for a valine-tomethionine substitution at residue 553 in the glycoprotein lb-binding domain of von Willebrand factor, causing type IIB von Willebrand disease. Am. J. Hum. Genet. 50, 199–207. Nei, M., 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. Ngo, K.Z., Ermolenko, A.V., 2011. The spread of the pathogen of human and animal clonorchiasis in North Vietnam. Med. Parazitol. 4, 39–41 (in Russian). Nontasut, P., Thong, T.V., Waikagul, J., Anantaphruti, M.T., Fungladda, W., Imamee, N., De, N.V., 2003. Social and behavioral factors associated with Clonorchis infection in one commune located in the Red river delta of Vietnam. Southeast Asian. J. Trop. Med. Public Health. 34, 269–273. Obbard, D.J., Jiggins, F.M., Halligan, D.L., Little, T.J., 2006. Natural selection drives extremely rapid evolution in antiviral RNAi genes. Curr. Biol. 16, 580–585. Painter, J.N., Siitonen, J., Hanski, I., 2007. Phylogeographical patterns and genetic diversity in three species of Eurasian boreal forest beetles. Biol. J. Linn. Soc. 91, 267–279. Park, G.M., 2007. Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences. Parasitol. Res. 100, 351–357. Park, G.M., Yong, T.S., 2001. Geographical variation of the liver fluke, Clonorchis sinensis, from Korea and China based on the karyotypes, zymodeme and DNA sequences. Southeast Asian. J. Trop. Med. Publ. Health 32, 12–16. Paterson, S., Vogwill1, T., Buckling, A., Benmayor, R., Spiers, A.J., Thomson, N.R., Quail, M., Smith, F., Walker, D., Libberton, B., Fenton, A., Hall1, N., Brockhurst, M.A., 2010. Antagonistic coevolution accelerates molecular evolution. Nature 464, 275–278. Peters, A.D., Lively, C.M., 1999. The Red Queen and actuating epistasis: a population genetic analysis of antagonistic coevolution. Am. Nat. 154, 393–405. Petney, T.N., Andrews, R.H., Saijuntha, W., Wenz-Mücke, A., Sithithaworn, P., 2013. The zoonotic, fish-borne liver flukes Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini. Int. J. Parasitol. 43, 1031–1046. Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Posokhov, P.S., 2004. Clonorchiasis in Amur River region. Far Eastern State Medicine University Press, Khabarovsk (in Russian).

810


Posokhov, P.S., Dovgalev, A.S., Bryunetkina, N.M., 1987. Intravitally nonrecognized event of clonorchiasis invasion. Med. Parazitol. 5, 45–46 (in Russian). Prugnolle, F., Liu, H., de Meeus, T., Balloux, F., 2005a. Population genetics of complex life-cycle parasites: an illustration with trematodes. Int. J. Parasitol. 35, 255– 263. Prugnolle, F., Theron, A., Pointier, J.P., Jabbourn-Zahab, R., Jarne, P., Durand, P., de Meeu, T., 2005b. Dispersal in a parasitic worm and its two hosts and its consequence for local adaptation. Evolution 50, 296–303. Qiu, Y.-X., Fu, C.-X., Comes, H.P., 2011. Plant molecular phylogeography in China and adjacent regions: tracing the genetic imprints of quaternary climate and environmental change in the world’s most diverse temperate flora. Mol. Phylogen. Evol. 59, 225–244. Rodriguez-Martinez, A.B., Garrido, J.M., Zarranz, J.J., Arteagoitia, J.M., de Pancorbo, M.M., Atares, B., Bilbao, M.J., Ferrer, I., Juste, R.A., 2010. A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/ valine at codon 129: case report. BMC Neurol. 10, 99. Rogers, A.R., 1995. Genetic evidence for a Pleistocene population explosion. Evolution 49, 608–615. Rogers, A.R., Harpending, H., 1992. Population growth makes waves in the distribution of pairwise genetic differences. Mol. Biol. Evol. 9, 552–569. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rooney, A.P., 2004. Mechanisms underlying the evolution and maintenance of functionally heterogeneous 18S rRNA genes in Apicomplexans. Mol. Biol. Evol. 21, 1704–1711. Rudge, J.W., Carabin, H., Balolong, E., Tallo, V., Shrivastava, J., Lu, D.-B., Basánez, M.G., Olveda, R., McGarvey, S.T., Webster, J.P., 2008. Population genetics of Schistosoma japonicum with the Philippines suggest high levels of transmission between human and dogs. PLoS Negl. Trop. Dis. 2, e340. http://dx.doi.org/ 10.1371/journal.pntd.0000340. Rudge, J.W., Lu, D.-B., Fang, G.-R., Wang, T.-P., Basánez, M.-G., Webster, J.P., 2009. Parasite genetic differentiation by habitat type and host species: molecular epidemiology of Schistosoma japonicum in hilly and marshland areas of Anhui province. China Mol. Ecol. 18, 2134–2147. Sakka, H., Quere, J.-P., Kartavtseva, I.V., Pavlenko, M.V., Chelomina, G.N., Atopkin, D.M., Bogdanov, A.S., Michaux, J., 2010. Comparative phylogeography of four Apodemus species (Mammalia: Rodentia) in the Asian Far East: evidence of Quaternary climatic changes in their genetic structure. Biol. J. Linn. Soc., 797– 821. Schönian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, D., Pesber, W., Jaffe, C.L., 2001. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn. Microbiol. Infect. Dis. 47, 349–358. Semyenova, S.K., Khrisanfova, G.G., Filippova, E.K., Beer, S.V., Voronin, M.V., Ryskov, A.P., 2005. Individual and population variation in cercariae of bird schistosomes of the Trichobilharzia ocellata species group as revealed with the polymerase chain reaction. Russ. J. Genet. 41, 17–22. Semyenova, S.K., Khrisanfova, G.G., Korsunenko, A.V., Voronin, M.V., Beer, S.V., Vodyanitskaya, S.V., Serbina, E.A., Yurlova, N.I., Ryskov, A.P., 2007. Multilocus variation in cercariae, parthenogenetic progeny of different species of the class Trematoda. Doklady Biol. Sci. 414, 235–238. Semyenova, S.K., Morozova, E.V., Khrisanfova, G.G., Asatrian, A.M., Movsessian, S.O., Ryskov, A.P., 2003. RAPD variability and genetic diversity in two populations of liver fluke, Fasciola hepatica. Acta Parasitol. 48, 125–130. Shalaby, H.A., El-Moghazy, F.M., Khalil, F.A.M., 2010. Specificity of snail derived antigens in diagnosis of their trematode parasites. Res. J. Parasitol. 5, 224–230. Shekhovtsov, S.V., Katokhin, A.V., Kolchanov, N.A., Mordvinov, V.A., 2010. The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda). Parasitol. Int. 59, 100–103. Sherrard-Smith, E., Chadwick, E.A., Cable, J., 2013. Climate variables are associated with the prevalence of trematodes in otter. Int. J. Parasitol. 43, 729–737. Sire, C., Durand, P., Pointier, J.-P., Theron, A., 2001. Genetic diversity of Schistosoma mansoni within and among individual hosts (Rattus rattus): infrapopulation differentiation at microspatial scale. Int. J. Parasitol. 31, 1609–1616. Sithithaworn, P., Andrews, R.H., De, N.V., Wongsaroj, T., Sinuon, M., Odermatt, P., Nawa, Y., Liang, S., Brindley, P.J., Sripa, B., 2012. The current status of opisthorchiasis and clonorchiasis in the Mekong Basin. Parasitol. Int. 61, 10–16. Slatkin, M., 1987. Gene flow and the geographic structure of natural populations. Science 236, 787–792. Starobogatov, Y.I., 1970. Fauna of mollusks and zoogeographical zonation of the continental waters. Nauka, Leningrad (in Russian).

Subbotin, S., Deimi, A.M., Zheng, J., Chizhov, V.N., 2011. Length variation and repetitive sequences of internal transcribed spacer of ribosomal RNA gene, diagnostics and relationships of populations of potato rot nematode, Ditylenchus destructor Thorne, 1945 (Tylenchida: Anguinidae). Nematology 13, 773–785. Sun J., Huang Y., Huang H., Liang P., Wang X., Mao Q., Men J., Chen W., Deng C., Zhou C., Lv X., Zhou J., Zhang F., Li R., Tian Y., Lei H., Liang C., Hu X., Xu J., Li X., Yu X. 2013. Low divergence of Clonorchis sinensis in China based on multilocus analysis. PLoS One B, e67006. doi: 10.1371/journal.pone.0067006. Swofford, D.L., 2002. PAUP⁄. Phylogenetic analysis using parsimony (⁄and other methods). Sunderland, Massachusetts, Sinauer Associates. Tajima, F., 1983. Evolutionary relationships of DNA sequences in finite populations. Genetics 105, 437–467. Tajima, F., 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585–595. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetic analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tatonova, Y.V., Chelomina, G.N., Besprosvannykh, V.V., 2012. Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East. Parasitol. Int. 61, 664– 674. Tatonova, Y.V., Chelomina, G.N., Besprosvannykh, V.V., 2013. Genetic diversity of Clonorchis sinensis (Trematoda: Opisthorchiidae) in the Russian southern Far East based on mtDNA cox1 sequence variation. Folia Parasitol. 60, 155–162. Theron, A., Sire, C., Rognon, A., Prugnolle, F., Durand, P., 2004. Molecular ecology of Schistosoma mansoni transmission inferred from the genetic composition of larval and adult infrapopulations within intermediate and definitive hosts. Parasitology 129, 571–585. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougi, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface. flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25, 4876–4882. Tinga, N., De, N., Vien., H.V., Chau, L., Toan, N.D., Kager, P.A., Vries, P.J., 1999. Little effect of praziquantel or artemisinin on clonorchiasis in northern Vietnam. A pilot study. Trop. Med. Int. Health. 4, 814–818. Truett, G.E., Heeger, P., Mynatt, R.L., Truett, A.A., Walker, J.A., Warman, M.L., 2000. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29, 52–54. Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yoshikawa, S., 1996. The whole structure of the 13subunit oxidized cytochrome c oxidase at 2.8 A. Science 272, 1136–1144. Van Dijk, J., Sargison, N.D., Kenyon, F., Skuce, P.G., 2010. Climate change and infectious disease: helminthological challenges to farmed ruminants in temperate regions. Animal 4, 377–392. Van Valen, L., 1973. A new evolutionary law. Evol. Theor. 1, 1–30. Wang, B., Jiang, J., Xie, F., Li, C., 2013. Phylogeographic patterns of mtDNA variation revealed multiple glacial refugia for the frog species Feirana taihangnica endemic to the Quinling Mountains. J. Mol. Evol. 76, 112–128. Wang, T.P., Shrivastava, J., Johansen, M.V., Zhang, S.Q., Wang, F.F., Webster, J.P., 2006. Does multiple hosts mean multiple parasites? Population genetic structure of Schistosoma japonicum between definitive host species. Int. J. Parasitol. 36, 1317–1325. Wang, W.T., 1992. On some distribution patterns and some migration routes found in the Eastern Asiatic region (cont.). Acta Phytotaxonomica Sin. 30, 97–117. Wang, X., Liang, C., Chen, W., Fan, Y., Hu, X., Xu, J., Yu, X., 2009. Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically. Parasitol. Res. 106, 15–21. Wide, A., Capaldo, J., Zerpa, N., Pabon, R., Noda, A., Noya, B., González, J., Noya, O., 2006. Sharing of antigens between Plasmodium falciparum and Anopheles albimanus. Rev. Inst. Med. Trop. S. Paulo. 48, 327–332. Yoshida, Y., 2012. Clonorchiasis – a historical review of contributions of Japanese parasitologists. Parasitol. Int. 61, 5–9. You, Y., Sun, K., Xu, L., Wang, L., Jiang, T., Liu, S., Lu, G., Berquist, S.W., Feng, J., 2010. Pleistocene glacial cycle effects on the phylogeography of the Chinese endemic bat species, Myotis davidii. BMC Evol. Biol. 10, 208–219. Zheng, B., Xu, Q., Shen, Y., 2002. The relationship between climate change and Quaternary glacial cycles on the Qinghai-Tibetan Plateau: review and speculation. Quat. Int. 97–98, 93–101.

Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis.

Clonorchis sinensis, an oriental liver fluke, as a human biological agent of cholangiocarcinoma: a brief review.

Acute cholecystitis associated with Clonorchis sinensis infection.

Identification and molecular characterization of Parkin in Clonorchis sinensis.

Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

Oxidative stress-mediated mouse liver lesions caused by Clonorchis sinensis infection.

Characterization of a gut-associated asparaginyl endopeptidase of Clonorchis sinensis.

Identification, immunolocalization, and immunological characterization of nitric oxide synthase-interacting protein from Clonorchis sinensis.

threonine protein kinase 17A from Clonorchis sinensis.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production.

Pathological Lesions and Inducible Nitric Oxide Synthase Expressions in the Liver of Mice Experimentally Infected with Clonorchis sinensis.

Pancreatic mucinous cystadenoma of borderline malignancy associated with Clonorchis sinensis.

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

Painless Jaundice Caused by Clonorchis sinensis Infection: A Case Report.

Prevalence of Clonorchis sinensis Metacercariae in Fish from Water Systems of Seomjin-gang (River).

ClonorESTdb: a comprehensive database for Clonorchis sinensis EST sequences.

Letter: Bithionol therapy in Clonorchis sinensis infestation in Canada.

Th17 in Clonorchis sinensis-Infected Mice.

Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis.

Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma.

Structural insights into a 20.8-kDa tegumental-allergen-like (TAL) protein from Clonorchis sinensis.