Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences
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.
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
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.
Pairwise differences among 18S, ITS2, 28S, and mtCOI gene sequences of Clonorchis sinensis and Opisthorchis viverrini
1. Kimhae, Korea
2. Nanning, China
3. Shenyang, China
4. Savannakhet, Laos
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.
This work was supported by Kwandong University Research Fund 2002.
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