The Molecular Phylogeny Study On Paragonimus Heterotremus From Two Regions In Guangxi

Objective:To analyze the genetic variation between the two Paragonimus heterotremus isolates from two regions in Guangxi(Lingchuan,Napo)by compare their sequences of cytochrome c oxidase subunit I(CO1)and the ribosomal DNA internal transcribed spacer 2(ITS2)genes,then to reveal their genetic relationship and taxonomic status.Methods:Metacercariae collected from Lingchuan and Napo were inoculated into definitive host rat,The genomic DNA were extracted from the adult worms after it matured.CO1 and ITS2 gene were amplified by polymerase chain reaction(PCR)and sequenced.Their homologous sequences from other Paragonimus species were retrieved from Genebank.The gene sequences were aligned and analyzed by Megalign application to evaluate their similarity,then Mega and Phylip software were used to build phylogenetic tree based on the CO1 and ITS2 sequences by minimum evolution(ME),Neighbor joining(N-J), Maximum likelihood(ML)and Maximum parsimony(MP)methods.Kimura 2-parameter model were used to compute genetic distance in ME and N-J methods and bootstrap replicates were set to 1000.Bootstrap replicates used in ML and ME methods were 500.Results:The similarity between ITS2 gene sequences of isolates from Napo and Lingchuan was 100%.And the similarity between CO1 gene sequences was 97.5%,they were highly homologous.The similarity of CO1 gene between the isolate from Napo and one isolate from Thailand was 100% and that means a very high homology.In the ITS2 phylogenetic trees,all the isolates from Napo,Lingchuan, Vietnam,Thailand and India were in a clade of P.heterotremus,but isolate from India yielded a relatively independent branch,it has a far relationship with other P.heterotremus strains.In the CO1 phylogenetic trees,the isolate from Napo has a close relationship to isolates from Thailand and Vietnam,and they formed a monophyletic group with high value of bootstrap confidence level in trees built by different methods.The branch of isolate from Lingchuan was outside of this group.The relationship of isolates from India with them is still further.Conclusion:The homology of ITS2 and CO1 gene of isolates from Lingchuan and Napo is relatively high,and the gene homology from the isolates in Napo and Thailand are higher.Although the isolates form Napo and Lingchuan had some differences in morphology,but they were both in the clade of P.heterotremus of in the phylogenetic trees built by different methods.In the CO1 phylogenetic trees Napo isolate was close to Thailand and Vietnam isolates and they formed monophyletic group.The branch of Lingchuan isolate was always outside of this group.Lingchuan and Napo isolates may belong to different geographical strains.