Genetic variation among Clonorchis sinensis isolates from different geographic regions in China revealed by sequence analyses of four mitochondrial genes | Journal of Helminthology | Cambridge Core (original) (raw)

Abstract

The present study examined sequence variation in four mitochondrial (mt) genes, namely cytochrome c oxidase subunits 1 (cox1) and 2 (cox2), and NADH dehydrogenase subunits 1 and 2 (nad1 and nad2) among Clonorchis sinensis isolates from different endemic regions in China, and their phylogenetic relationships with other zoonotic trematodes were reconstructed. A portion of the cox1 and cox2 genes (pcox1 and pcox2), and nad1 and nad2 genes (pnad1 and pnad2) were amplified separately from individual liver flukes by polymerase chain reaction (PCR) and the amplicons were subjected to sequencing from both directions. The intra-specific sequence variations within C. sinensis were 0–1.6% for pcox1, 0–1.4% for pcox2, 0–0.9% for pnad1 and 0–1.0% for pnad2. Phylogenetic analyses based on the combined sequences of pcox1, pcox2, pnad1 and pnad2 revealed that all the C. sinensis isolates grouped together and were closely related to Opisthorchis felineus. These findings revealed the existence of intra-specific variation in mitochondrial DNA (mtDNA) sequences among C. sinensis isolates from different geographic regions, and demonstrated that mtDNA sequences provide reliable genetic markers for phylogenetic studies of zoonotic trematodes.

References

Ai, L., Chen, M.X., Alasaad, S., Elsheikha, H.M., Li, J., Li, H.L., Lin, R.Q., Zou, F.C., Zhu, X.Q. & Chen, J.X. (2011) Genetic characterization, species differentiation and detection of Fasciola spp. by molecular approaches. Parasites and Vectors 4, 101.CrossRefGoogle ScholarPubMed

Cai, X.Q., Liu, G.H., Song, H.Q., Wu, C.Y., Zou, F.C., Yan, H.K., Yuan, Z.G., Lin, R.Q. & Zhu, X.Q. (2012) Sequences and gene organization of the mitochondrial genomes of the liver flukes Opisthorchis viverrini and Clonorchis sinensis (Trematoda). Parasitology Research .CrossRefGoogle ScholarPubMed

Cerutti, M.C., Citterio, C.V., Bazzocchi, C., Epis, S., D'Amelio, S., Ferrari, N. & Lanfranchi, P. (2010) Genetic variability of Haemonchus contortus (Nematoda: Trichostrongyloidea) in alpine ruminant host species. Journal of Helminthology 84, 276–283.CrossRefGoogle ScholarPubMed

Chilton, N.B., Gasser, R.B. & Beveridge, I. (1995) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). International Journal for Parasitology 25, 647–651.CrossRefGoogle Scholar

Dai, R.S., Liu, G.H., Song, H.Q., Lin, R.Q., Yuan, Z.G., Li, M.W., Huang, S.Y., Liu, W. & Zhu, X.Q. (2012) Sequence variability in two mitochondrial DNA regions and internal transcribed spacer among three cestodes infecting animals and humans from China. Journal of Helminthology 86, 245–251.CrossRefGoogle ScholarPubMed

Gasser, R.B. & Newton, S.E. (2000) Genomic and genetic research on bursate nematodes: significance, implications and prospects. International Journal for Parasitology 30, 509–534.CrossRefGoogle ScholarPubMed

Guindon, S. & Gascuel, O. (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52, 696–704.CrossRefGoogle ScholarPubMed

Kang, S., Sultana, T., Loktev, V.B., Wongratanacheewin, S., Sohn, W.M., Eom, K.S. & Park, J.K. (2008) Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda). Parasitology International 57, 191–197.CrossRefGoogle ScholarPubMed

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. Doklady Biochemistry and Biophysics 421, 214–217.CrossRefGoogle ScholarPubMed

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 Tropical 107, 213–216.CrossRefGoogle ScholarPubMed

Li, J., Zhao, G.H., Zou, F.C., Mo, X.H., Yuan, Z.G., Ai, L., Li, H.L., Weng, Y.B., Lin, R.Q. & Zhu, X.Q. (2010) Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes. Parasitology Research 107, 561–569.CrossRefGoogle ScholarPubMed

Li, M.W., Lin, R.Q., Song, H.Q., Sani, R.A., Wu, X.Y. & Zhu, X.Q. (2008) Electrophoretic analysis of sequence variability in three mitochondrial DNA regions for ascaridoid parasites of human and animal health significance. Electrophoresis 29, 2912–2917.CrossRefGoogle ScholarPubMed

Lin, R.Q., Qiu, L.L., Liu, G.H., Wu, X.Y., Weng, Y.B., Xie, W.Q., Hou, J., Pan, H., Yuan, Z.G., Zou, F.C., Hu, M. & Zhu, X.Q. (2011) Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens. Gene 480, 28–33.CrossRefGoogle ScholarPubMed

Liu, G.H., Lin, R.Q., Li, M.W., Liu, W., Liu, Y., Yuan, Z.G., Song, H.Q., Zhao, G.H., Zhang, K.X. & Zhu, X.Q. (2011) The complete mitochondrial genomes of three cestode species of Taenia infecting animals and humans. Molecular Biology Reports 38, 2249–2256.CrossRefGoogle ScholarPubMed

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 Infectious Diseases 5, 31–41.CrossRefGoogle ScholarPubMed

Page, R.D. (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358.Google ScholarPubMed

Park, G.M. (2007) Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences. Parasitology Research 100, 351–357.CrossRefGoogle ScholarPubMed

Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.CrossRefGoogle ScholarPubMed

Saijuntha, W., Sithithaworn, P., Wongkham, S., Laha, T., Chilton, N.B., Petney, T.N., Barton, M. & Andrews, R.H. (2008) Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes. Parasitology 135, 1479–1486.CrossRefGoogle ScholarPubMed

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). Parasitology International 59, 100–103.CrossRefGoogle ScholarPubMed

Shin, H.R., Oh, J.K., Masuyer, E., Curado, M.P., Bouvard, V., Fang, Y.Y., Wiangnon, S., Sripa, B. & Hong, S.T. (2010) Epidemiology of cholangiocarcinoma: an update focusing on risk factors. Cancer Sciences 101, 579–585.CrossRefGoogle ScholarPubMed

Swofford, D.L. (2002) PAUP*: Phylogenetic analysis using parsimony (and other methods). Sunderland, Massachusetts, Sinauer Associates.Google Scholar

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882.CrossRefGoogle Scholar

Zhao, G.H., Mo, X.H., Zou, F.C., Weng, Y.B., Lin, R.Q., Xia, C.M. & Zhu, X.Q. (2009) Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes. Veterinary Parasitology 162, 67–74.CrossRefGoogle Scholar