Parasitology Research

, 100:351 | Cite as

Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences

Original Paper

Abstract

To clarify the genetic relationships between Clonorchis sinensis and Opisthorchis viverrini, patterns of inter-/intraspecific polymorphism were compared for four markers with nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) in liver flukes C. sinensis from Korea (Kimhae) and China (Shenyang and Nanning) and O. viverrini from Laos (Savannakhet). Intra- and interspecific variations in the 18S, ITS2, and 28S rDNA and mitochondrial cytochrome c oxidase subunit I (mtCOI) of mtDNA gene sequences were low and nearly identical. Three isolates of C. sinensis showed a high similarity (99–100%). No variation was detectable in the ITS2 sequence for the C. sinensis from Korea and China. ITS2 region sequences of O. viverrini vs C. sinensis showed 95% identity and differed at 28 nucleotide positions. Pairwise sequence divergence with three C. sinensis isolates and O. viverrini ranged from 0 to 3.94% in mtCOI gene. The mtCOI sequences were more highly conserved relative to the ITS2 sequences. These genetic data from different geographical areas showed that the liver flukes are not variable and are virtually identical almost despite belonging to entirely different genera.

Introduction

Clonorchis sinensis, the Chinese or oriental liver fluke, is an important human parasite and is widely distributed in southern parts of Korea, China, Taiwan, and in the northern part of Vietnam. Clonorchiasis is one of the most important endemic diseases in Korea. Although reports of this infection are infrequent in western countries, infection can be acquired by eating frozen, dried, or pickled freshwater fish imported from endemic areas (Rim 1990). People in endemic areas acquire infection by eating raw or uncooked freshwater fish. Substantial studies have been conducted on the biology, epidemiology, and pathology of C. sinensis and on the clinical symptoms and treatment of clonorchiasis (Park et al. 2000a,b). C.sinensis shows geographical differences in terms of host specificity and other biological features. The Korean isolates of C. sinensis utilize a snail, Parafossarulus manchouricus, as the first intermediate host and several species of freshwater fishes as second intermediate hosts, whereas the Chinese isolates utilize a snail, Alocinma longicornis, as the first intermediate host and freshwater fishes as second intermediate hosts (Choi 1984; Zou et al. 1994). Opisthorchis viverrini infection causes an endemic disease that creates a serious public health problem in Southeast Asia, especially in northeastern Thailand and Laos. Opisthorchis viverrini is the major parasitic trematode in Laos, with prevalence of over 50% in 2000; moreover, it has been implicated in the development of cholangiocarcinoma (Watana 1996; Wongratanacheewin et al. 2001). The closely related liver flukes, C. sinensis and O. viverrini, are bile tract parasites. In addition, the adult worms of C. sinensis and O. viverrini are difficult to distinguish because they are morphologically similar.

Molecular genetic characterization is increasingly being used to identify morphologically similar parasites. Recently, 18S, a second internal transcribed spacer region (ITS2), 28S of rDNA, and mitochondrial cytochrome c oxidase subunit I (mtCOI) of mitochondria have been used to analyze the genetic variations of several trematodes and cestodes (Bowles and McManus 1994; Iwagami et al. 2000). Mitochondrial sequences are likely to be of value in phylogenetic studies and in distinguishing among strains and species (Iwagami et al. 2000). Phylogenetic, or intraspecific, variations or both of geographical isolates have recently been studied using nuclear ribosomal DNA sequences in Paragonimus (Blair and Agatsuma 1997; Blair et al. 1997), Schistosoma japonicum (Bowles et al. 1995), and Echinostoma (Morgan and Blair 1998). ITS2 has been shown to be a sensitive marker at the species level in trematodes (Morgan and Blair 1995).

In the present study, the intra- and interspecies relationships of three C. sinensis isolates and of O. viverrini were investigated using DNA sequence data. For this purpose, 18S, ITS2, and 28S of nuclear DNA and mtCOI of mitochondrial DNA were selected for analysis.

Materials and methods

Metacercariae of C. sinensis were harvested from the freshwater fish, Pseudorasbora parva, collected in Kimhae, Kyungsangnam-do, Korea and in Shenyang, Liaoning Province, and Nanning, Guangxi Province, China (Fig. 1). Metacercariae were orally fed to rabbits, which were killed 3–5 months after infection, and the adult flukes were recovered. Metacercariae of O. viverrini were taken from freshwater fish, Puntius orphoides, in Savannakhet, Laos, orally fed to six hamsters. The hamsters were killed 90–120 days later, and adult flukes were recovered.
Fig. 1

Collection sites of Clonorchis sinensis and Opisthorchis viverrini. 1 Kimhae, Korea, 2 Nanning, China, 3 Shenyang, China, 4 Savannakhet, Laos. Bar indicates 320 km

Adult worms were lyophilized and lysed with lysis buffer, proteinase K, and RNase. DNA was extracted in phenol/chloroform and precipitated by ethanol, as described by Sambrook et al. (1989). The three gene regions were amplified by the polymerase chain reaction (PCR) from 20 to 40 ng of worm DNA. For the 18S, the primers used, SB8 and PB, were as described by Barker and Blair (1996). For ITS2, the primers used were 3S and BD2 (Bowles et al. 1995), for 28S, the primers used were JB9 and JB10 (Bowles and McManus 1994), and for mtCOI, the primers used were JB3 and JB4.5 (Bowles et al. 1993). PCR amplification was conducted over 40 cycles using the following conditions: 1 min at 95°C, 1 min at 48–54°C, and 90 s at 72°C, with a final extension of 7 min at 72°C. The PCR products were purified using an Ultra Clean DNA purification kit (MO BIO Labs) and ligated into a TA cloning vector. DNA from positive recombinants was purified using the QIAprep spin plasmid kit. DNA sequencing was performed using the dideoxy chain termination method and an automated DNA sequencer. At least two clones were sequenced per isolate, and additional clones were sequenced as necessary to resolve ambiguous sites.

Nucleotide sequences were aligned using Clustal X (Thompson et al. 1997). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al. 2001). Gaps were considered as an additional character state in pairwise comparisons.

Results

All the sequences of the 18S, ITS2, 28S, and mtCOI genes obtained in this study have been deposited in the GenBank database under accession numbers AF408144 and AF408145 for 18S, AF217094 and AF408147 for ITS2, AF408149 and AF095330 for 28S, and AY055380 for mtCOI. Pairwise differences among 18S, ITS2, and 28S rDNA and mtCOI of mitochondrial gene sequences are shown in Table 1. The 1,077-bp 18S rDNA of the three C. sinensis isolates and O. viverrini was obtained. Intra- and interspecies variations were detected at a low level of 0.3–0.6% in the three C. sinensis isolates and at 1.0–1.2% in the O. viverrini as compared to C. sinensis. The phylogenetic tree shows the relationships among the intra- and interspecies based on the 18S sequences when Amphimerus ovalis of Opisthorchiidae was used as outgroup (Fig. 2). The distances between genera Amphimerus, Clonorchis, and Opisthorchis are significantly greater than some intra-/interspecies distances with the Clonorchis and Opisthorchis species. The alignment of the ITS2 sequence was 502 bp in length. Figure 3 shows the relationships among the intra- and interspecies used as inferred from their ITS2 sequences. The three isolates of C. sinensis from Korea and China had almost identical ITS2 sequences and differed at one or two sites. Interspecific variation of ITS2 sequence differed at 27 sites. The 28S rDNA sequences were 299 bp in length. The tree shown in Fig. 4 shows the relationships among species based on 28S rDNA sequences. The three isolates of C. sinensis from Korea and China had 100% identical sequences. The nucleotide sequences of the 28S gene of O. viverrini differed from those of C. sinensis at three sites. Interspecific variation was detected at comparatively low level of 1% in the 28S rDNA sequences of C. sinensis and O. viverrini. In contrast to the 18S and 28S rDNA data, the ITS2 sequences in C. sinensis and O. viverrini were found to be more variable. The alignment of mtCOI gene sequences was 457 bp in length. There were no insertions or deletions. The mtCOI sequences from Kimhae, Korea and Nanning, China of C. sinensis isolates were identical but differed from the ones from Shenyang, China at five sites. C.sinensis differed from that of O. viverrini at 18 of the 457 nucleotide positions examined in the mtCOI. Figure 5 presents these differences graphically. The tree shows that the C. sinensis group and O. viverrini form a tight cluster.
Table 1

Pairwise differences among 18S, ITS2, 28S, and mtCOI gene sequences of Clonorchis sinensis and Opisthorchis viverrini

Species

Origin

18S

ITS2

28S

mtCOI

 

 

2

3

4

2

3

4

2

3

4

2

3

4

C. sinensis

1. Kimhae, Korea

3

6

12

1

1

27

0

0

3

0

5

18

2. Nanning, China

 

7

13

 

2

26

 

0

3

 

4

18

3. Shenyang, China

  

13

  

26

  

3

  

18

O. viverrini

4. Savannakhet, Laos

            
Fig. 2

Tree depicting relationships between Clonorchis sinensis isolates and Opisthorchis viverrini inferred from 18S rRNA gene sequence data using Amphimerus ovalis (GenBank accession number AY222121), Cryptocotyle lingua (AJ287492), Mitotrema anthostomatum (AJ287542), Siphodera vinaledwardsii (AY222122), Galactosomum lacteum (AY222120), and Haplorchoides sp. (AY222226) for the phylogenetic analysis. A distance matrix was calculated using the Kimura two-parameter model and the tree constructed using the neighbor-joining method

Fig. 3

Tree depicting relationships between Clonorchis sinensis isolates and Opisthorchis viverrini inferred from ITS2 rRNA gene sequence data using Plagiorchis muelleri (GenBank accession number AF151948), Plagiorchis elegans (AF151952), Plagiorchis vespertilionis (AF151951), Paryphostomum radiatum (AY240758), Isthmiophora melis (AY168932), Echinoparyphium recurvatum (AY168931), Echinostoma liei (U58099.2), and Echinostoma trivolvis (U58097.1) for the phylogenetic analysis. A distance matrix was calculated using the Kimura two-parameter model and the tree constructed using the neighbor-joining method

Fig. 4

Tree depicting relationships between Clonorchis sinensis isolates and Opisthorchis viverrini inferred from 28S rRNA gene sequence data using Fasciola hepatica (GenBank accession number AJ439738), Plagiorchis muris (AF096222), Pygidiopsis summa (AF181885), Stellantchasmus falcatus (AF181886), Metagonimus yokogawai (AF095331), and Metagonimus takahashii (AF095332) for the phylogenetic analysis. A distance matrix was calculated using the Kimura two-parameter model and the tree constructed using the neighbor-joining method

Fig. 5

Tree depicting relationships between Clonorchis sinensis isolates and Opisthorchis viverrini inferred from mtCOI gene sequence data using Neodiplostomum seoulense (GenBank accession number AF096233), Gymnophalloides seoi (AF096234), and Schistosoma edwardiense (AY197347) for the phylogenetic analysis. A distance matrix was calculated using the Kimura two-parameter model and the tree constructed using the neighbor-joining method

Discussion

Clonorchiasis, a disease caused by the Chinese liver fluke, and opisthorchiasis, caused by any of the several Opisthorchis spp., are bile duct infections and are acquired by the ingestion of uncooked fish or crustaceans containing metacercariae. In general, the morphology and the egg size of C. sinensis closely resemble that of O. viverrini.

Within the tandemly repeated rRNA gene complex, coding sequences for small (18S) and large (5.8S+28S) subunit rRNA components are flanked by nontranscribed and internal transcribed spacer regions. Because of functional constraints within the ribosome, coding regions are, in general, more conserved than the spacer regions (Raué et al. 1990). For organisms evolving over a short period, rRNA internal transcribed and terminal nontranscribed spacer sequences are known to be useful for taxonomy (Swofford et al. 1996). Therefore, it is possible to use the ribosomal cistron to develop phylogenies for both distantly and closely related organisms. rRNA molecules provide a good opportunity to examine the patterns of nucleotide sequence change (Wheeler and Honeycutt 1988). 18S rRNA genes evolve slower than 28S rRNA genes and are thus used to construct deeper phylogenies.

In the present study, the lack of intraspecific variability in the 18S rDNA sequence was confirmed by studying individuals from geographically isolated populations of C. sinensis. Sequences of 18S rDNA for the three C. sinensis isolates and O. viverrini were highly similar and differed at only 13 of the 1,077 base positions. It can be concluded that the 18S rDNA sequence is not a good marker for species determination and differentiation among these species, although 18S rRNA gene sequence data have proved useful as a marker for higher taxonomic levels within the platyhelminthes (Blair and Barker 1993).

Interspecies variation within Schistosoma spp. and Campulidae differed by 2.7–3.5% and 13–22.4% in terms of their 18S sequences, respectively (Johnston et al. 1993; Fernandez et al. 1998). Moreover, the 28S rRNA gene contains “conserved” core regions interspersed with more variable “expansion segments” or domains, which are designated as D1 to D18 (Raué et al. 1990). Sequence data from the 5′ end of the 28S rRNA gene, especially the D1 domain, have been successfully used for intrageneric resolution within the Digenea (Barker et al. 1993). Thus, it is believed that 28rRNA gene provided a more useful means of applying phylogenetic inferences within or among closely related families. In the present study, the genetic variation of the 28S gene of two species was very low, and, in fact, they were almost identical. In trematodes, intraspecific variation in ITS2 sequences is minimal or nonexistent (Hashimoto et al. 1997). Within the rDNA repeat, the transcribed spacers are expected to be significantly more variable than in the coding regions (Hillis and Dixon 1991).

In contrast to the 18S and 28S rDNA data, more variations between C. sinensis and O. viverrini were observed in the ITS2 data. No evidence of genetic variation was found among the three C. sinensis isolates examined in the current study. Intraspecific variation in ITS2 sequences among trematodes is virtually unknown. Morgan and Blair (1995) found no differences in ITS2 sequences between samples of Echinostoma revolutum from Germany and Indonesia. Similarly, Després et al. (1992) found no differences in ITS2 sequences among samples of Schistosoma mansoni from several sites in Africa and the Western Hemisphere.

Interspecies distances of mtCOI sequence for recognized two species range from four to 18 nucleotides, while intraspecies of C. sinensis differ at five positions. The two species utilize different snail hosts and a little differ in adult morphology. However, molecular phylogenetic studies using the relatively conserved ITS2 and mtCOI gene indicate that C. sinensis and O. viverrini are extremely closely related to each other. The mtCOI sequence divergence between the two species was 4% compared to 5.4% in the ITS2. Interspecies variation of mtCOI gene within the genus Fasciola reported differs by 1.2–7.2% (Agatsuma et al. 2000), 8–21% in Schistosoma (Bowles et al. 1995), and 0.5–20.6% in Paragonimus (Blair et al. 1997). Of the three genes sequenced, ITS2 and mtCOI appear to be the most informative for investigating relationships within the Opisthorchiidae.

The two Asian liver flukes, the C. sinensis and the O. viverrini, are very similar in adult morphology, although they have long been considered to belong to separate genera. Dawes (1946) suggested that Clonorchis had characteristics sufficiently similar to Opisthorchis to classify this species as C.sinensis. Based on the 18S, ITS2, 28S, and mtCOI sequence data in the present study, the genes of the two species appear to be highly conserved, and the differences are minor.

Notes

Acknowledgement

This work was supported by Kwandong University Research Fund 2002.

References

  1. Agatsuma T, Arakawa Y, Iwagami M, Honzako Y, Cahyaningsih U, Kang SY, Hong SJ (2000) Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica. Parasitol Int 49:231–238PubMedCrossRefGoogle Scholar
  2. Barker SC, Blair D (1996) Molecular phylogeny of Schistosoma species supports traditional groupings within the genus. J Parasitol 82:292–298PubMedCrossRefGoogle Scholar
  3. Barker SC, Blair D, Garrett AR, Cribb TH (1993) Utility of the D1 domain of nuclear 28S rRNA for phylogenetic inference in the Digenea. Syst Parasitol 26:181–188CrossRefGoogle Scholar
  4. Blair D, Agatsuma T (1997) Molecular evidence for the synonymy of three species of Paragonimus, P. ohirai Miyazaki, 1939, P. iloktsuenensis Chen, 1940 and P. sadoensis Miyazaki. J Helminthol 71:305–310PubMedCrossRefGoogle Scholar
  5. Blair D, Barker SC (1993) Affinities of the Gyliauchenidae: utility of the 18S rRNA gene for phylogenetic inference in the Digenea (Platyhelminthes). Int J Parasitol 23:527–532PubMedCrossRefGoogle Scholar
  6. Blair D, Agatsuma TR, Watanabe T, Okamoto M, Ito A (1997) Geographical genetic structure within the human lung fluke, Paragonimus westermani, detected from DNA sequences. Parasitology 115:411–417PubMedCrossRefGoogle Scholar
  7. Bowles J, McManus DP (1994) Genetic characterization of the Asian Taenia, a newly described taeniid cestode of humans. Am J Trop Med Hyg 50:33–44PubMedGoogle Scholar
  8. Bowles J, Hope M, Tiu WC, Liu XS, McManus DP (1993) Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Schistosoma japonica. Acta Trop 55:217–229PubMedCrossRefGoogle Scholar
  9. Bowles J, Blair D, McManus DP (1995) A molecular phylogeny of the human Schistosomes. Mol Phylogenet Evol 4:103–109PubMedCrossRefGoogle Scholar
  10. Choi DW (1984) Clonorchis sinensis: life cycle, intermediate hosts, transmission to man and geographical distribution in Korea. Arzneimittelforschung 34:1145–1151PubMedGoogle Scholar
  11. Dawes B (1946) The trematode, with special reference to British and other European forms. Cambridge University Press, Cambridge, UK, pp 250Google Scholar
  12. Després L, Imbert-Establet M, Combes C, Bonhomme F (1992) Molecular evidence linking hominid evolution to recent radiation of Schistosomes (Platyhelminthes: Trematoda). Mol Phylogenet Evol 1:295–304PubMedCrossRefGoogle Scholar
  13. Fernandez M, Littlewood DT, Latorre A, Raga JA, Rollinson D (1998) Phylogenetic relationships of the family Campulidae (Trematoda) based on 18S rRNA sequences. Parasitology 117:383–391PubMedCrossRefGoogle Scholar
  14. Hashimoto K, Watanabe T, Liu CX, Init I, Blair D, Ohnishi T, Agatsuma T (1997) Mitochondrial DNA and nuclear DNA indicate that the Japanese Fasciola species is F. gigantica. Parasitol Res 83:220–225PubMedCrossRefGoogle Scholar
  15. Hillis DM, Dixon MT (1991) Ribosomal DNA: molecular evolution and phylogenetic inference. Q Rev Biol 66:411–453PubMedCrossRefGoogle Scholar
  16. Iwagami M, Ho LY, Su K, Lai PF, Fukushima M, Nakano M, Blair D, Kawashima K, Agasuma T (2000) Molecular phylogeographic studies on Paragonimus westermani in Asia. J Helminthol 74:315–322PubMedGoogle Scholar
  17. Johnston DA, Kane RA, Rollinson D (1993) Small subunit (18S) ribosomal RNA gene divergence in the genus Schistosoma. Parasitology 107:147–156PubMedGoogle Scholar
  18. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USAGoogle Scholar
  19. Morgan JAT, Blair D (1995) Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma: an aid to establishing relationships within the 37-collar-spine group. Parasitology 111:609–615PubMedCrossRefGoogle Scholar
  20. Morgan JAT, Blair D (1998) Relative merits of nuclear ribosomal internal transcribed spacers and mitochondrial COI and ND1 genes for distinguishing among Echinostoma species (Trematoda). Parasitology 116:289–297PubMedCrossRefGoogle Scholar
  21. Park GM, Yong TS, Im KI, Lee KJ (2000a) Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China. Korean J Parasitol 38:45–48PubMedCrossRefGoogle Scholar
  22. Park GM, Im KI, Huh S, Yong TS (2000b) Chromosomes of the liver fluke, Clonorchis sinensis. Korean J Parasitol 38:201–206PubMedGoogle Scholar
  23. Raué HA, Musters W, Rutgers CA, Vant’t Riet J, Planta RJ (1990) rRNA: from structure to function. In: Hill WE, Dahlberg A, Garrett RA, Moore PB, Schlessinger D, Warner JR (ed) The ribosome: structure, function and evolution. American Society of Microbiology, Washington, DC, pp 217–235Google Scholar
  24. Rim HJ (1990) Clonorchiasis in Korea. Korean J Parasitol 28:63–78 (Suppl)Google Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  26. Swofford DL, Olsen GJ, Waddell PJ, Hillis DM (1996) Phylogenetic inferrence. In: Hillis DM, Moritz C, Mable BK (eds) Molecular systematics. Sinauer Associates, Sunderland, Massachusetts, USA, pp 407–514Google Scholar
  27. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  28. Watana P (1996) Cholangiocarcinoma in patients with opisthorchiasis. Br J Surg 83:1062–1064CrossRefGoogle Scholar
  29. Wheeler WC, Honeycutt RL (1988) Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications. Mol Biol Evol 51:90–96Google Scholar
  30. Wongratanacheewin S, Pumidonming W, Sermswan RW, Maleewong W (2001) Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters. Parasitology 122:175–180PubMedCrossRefGoogle Scholar
  31. Zou H, Peng Y, Cai W, Lu S (1994) Studies on Clonorchis sinensis control in Sanshui City, Guangdong province. Chin J Parasitol Parasitic Dis 12:294–296Google Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of ParasitologyKwandong University College of MedicineGangneungSouth Korea

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