Molecular Phylogeny On Intermediate Host Snails Of Opisthorchis Sinensis In China

Opisthorchis sinensis of family Opisthorchiidae is the member of Clonorchis. It is parasitic on human liver system and a foodborne parasitic disease that is serious harmful for human health. It is the causative agent of cholelithiasis, cholecystitis, angiocholitis, cancer of biliary duct, etc. Clonorchiosis is prevalent and one of the most important parasitic diseases in China. Opisthorchis sinensis has a complex life cycle. It requires two intermediate hosts and one definitive host to complete the whole life history. Researches on the first intermediate host snails are still not adequate. Differentiation of the first intermediate host snails has important value on the study of snail control in the parasitic disease prevention and control and biological research on snails. At present, it’s not easy to distinguish species only by morphological taxonomy for the sake of the subtle morphological characters. A new approach with higher universality is to be explored to assist the taxonomic study of species.DNA barcoding is a new method for taxon differentiation which uses a short sequence as a mark for the rapid identification on species. It established a one to one correspondence between sequences and species. This technology is extensively used among organisms and microorganisms. In this study, we first tested the feasibility of the method and then used this technique to make a preliminary phylogenetic study. Furthermore, we discussed if there is a phenomenon of synonym between two species in Heng county, Guangxi. Finding are as follows:1. The mitochondrial cytochrome oxidase (COI) gene from 136 specimens of 9 species (there are another three unknown species) which collected from the extensive districts of the first intermediate host snails in China perform Polymerase Chain Reaction (PCR) amplification and bi-directional sequencing. At last, we have amplified partial sequences of COI gene from 85 specimens, the success rate is 62.5%. The average intraspecific distance is 1.31% among species in the family of Bithyniidae (ranged from 0.2-14.5%) and the average interspecific distance is 14.8% which has a clear gap after analyzing COI sequences. In other families of snails, such as Melanoides tuberculate in the family of Thiaridae, Semisulcospira cancellata and Semisulcospira libertina in the family of Pleuroseridae, Assimineidae sp., the average intraspecific distance is less than 1%. The interspecific distance is ten times than the intraspecific distance which has a clear gap. This section confirms the efficacy of COI barcoding for identifying species in this study.2. In this section, we used 4 genes (COI,16S rDNA, ITS-1,28S rDNA) to make the phylogenetic relationship of snails in this study. It’s not easy to amplify 16S rDNA gene by using general primer while easier to amplify ITS-1 gene and 28S rDNA gene to get DNA barcoding. In the phylogenetic tree based on COI gene, most of species come into being monophyletic group and get higher supports. Genus and families come into being own branches. Unfortunately, the phylogenetic trees based on 16S rDNA gene, ITS-1 gene and 28S rDNA gene didn’t so match the result of morphology.3. By using morphological and DNA barcoding methods to distinguish two bithynia species, Bithynia fuchsiana and Bithynia robusta collected from Heng county, Guangxi Province, which are morphologically similar and share the same ecological habitat. Adult B. fuchsiana and B. robusta were collected from Heng county of Guang Xi province, China. The two species specimen were compared by measuring shell morphological parameters, comparing the characters of male reproductive system, using COI gene barcoding technique and building phylogenetic tree. The results indicate that B. fuchsiana and B. robusta are similar morphologically in shell appearance and and male reproductive structure, and COI gene has low sequence divergence with 11 variation sites among 22 sequences. The length of COI gene segment is 517bp and no insertion sites and deletion loci after sequence edited. All individuals of the two species gathered to one clade in phylogenetic tree based on COI gene. According to the evidence of morphology and COI gene coding sequence, B. fuchsiana and B. robusta from Heng county, Guangxi Province are likely infer the same species.