The Chemotaxonomy Study On Paragonimus From Three Geographical Areas In Yunnan Province

[Objective]This paper involved the distribution of Paragonimus in Lvchun country, Yunnan province;sequence analyses of ITS2 and CO1 genes of Paragonimus in Baoshan city,Jinping county and Lvchun county of Honghe state in Yunnan province to definite the genus relationship and genetic relationship with other Paragonimus.[Methods]Crabs were collected from Baoshan city,Jinping county and Lvchun county of Honghe state in Yunnan province.Metacercariae and excysted metacercariae were separated,identified and fed to rats and cats from which adult worms and eggs were obtained. Excysted metacercariae of Lvchun county were observed and measured.DNA was extracted from metacercariae,excysted metacercariae,adult worms and eggs,following standard methods,involving digestion with proteinase K and a phenol/chloroform extraction followed by precipitation of DNA using ethanol.Amplify ITS2 gene and CO1 gene then sequencing。The sequences of ITS2 and CO1 were aligned using DNASTAR software with default options.Then compare the gene identical with Blast Program of GenBank and DNASTAR software MagAlign Program;compare the gene variation with MAGE 4.0 software.For each data set,midpoint rooted neighbor-joining trees were constructed using PAUP 4.0 software.[Resultsl The excysted metacercariae were obtained from crabs of Niukong of Lvchun county.Metacercaria was not found.The positive infectious ratio is 27.59%.The excysted metacercariae were identified as Paragonimus proliferus.We successfully obtained 7 ITS2 and 5 CO1 sequences of Paragonimus of Baoshan city,7 ITS2 and 5 CO1 sequences of Paragonimus from Jinping county,9 ITS2 and 5 CO1 sequences of Paragonimus from Lvchun county.The ITS2 gene identities between the Paragonimus from Baoshan city,Jinping county and P.skrjabini from Xishuangbanna state in GenBank are 100%,the identities of CO1 gene are 99%.The ITS2 gene identities between the Paragonimus from Lvchun county and P.proliferus from Vietnam,P.hokuoensis from Xishuangbanna state in GenBank are 100%,the identities of CO1 gene are 99%.Above the two comparisons were used the Blast Program of Genbank.DNASTAR software MagAlign Program was used to get the identities of Paragonimus gene from Baoshan city and Jinping county,the identities of ITS2 gene are 100%,CO1 are 99%;the identities of ITS2 gene between Baoshan city,Jinping county and Lvchun county are 98.3%,the CO1 are 92.3%.As can be seen in both ITS2 and CO1 phylogenetic trees,the Paragonimus from Baoshan city and Jinping county were clustered into the same clade of P.skrjabini from Xishangbanna state.The same as the condition between the Paragonimus from Lvchun county,P.prolifers from Vietnam and P.hokuoensis from Xishuangbanna state.[Conclusionsl Distribution region of P.proliferus was evidenced in Lvchun county of Yunnan Province at first.The Paragonimus from Baoshan city and Jinping county are the same species which is P.cheni.The ITS2 and CO1 genes in Genbank of P.skrjabini from Xishuangbanna state are the same as P.cheni from Baoshan city and Jinping county,so they are all belong to P.cheni.In analyses of the DNA sequences of ITS2 and CO1 genes and morphology of P.proliferus obtained in Lvchun county with those of the Vietnamese isolates,they are found to be identical with each other.According to analyses of ITS2 and CO1 gene sequences,P.hokuoensis from Xishuangbanna state should be taken as the same species of P.proliferus from Lvchun county.