The Morphology And Molecular Genetics Study Of Paragonimus Bangkokensis In Yunnan Province

[Objective] Based on morphological classification and combined with DNA sequence analysis, this study aimed to identify the metacercaria of lung flukes from Mengla country in Yunnan Province and confirm its taxonomy in Paragonimus. Take the sequences of ITS2 and CO I gene from the metacercaria into GeneBank to compare homology and phylogenetic relationship with other species reported.[Methods] Crabs were collected from Mengla country in Yunnan Province. Metacercaria were separated and identified, part of which were fed to the experimental animals to obtain the adult worms and eggs. Metacercaria, excysted metacercaria, adult worms and eggs were observed and measured, part of which were used to analyse DNA sequences. Compare its homology with Blast program of GenBank, and construct its neighbor-joining trees with MEGA4.0 software.[Results] The living adult worm form cat's lung is small, deep fleshred and oval shape. The average size is 7.00×3.25 mm. The rate of width to length is 1:2.34. The size of ventral sucker is a bit larger than that of oral sucker, and the transverse diameter rate of oral sucker to ventral sucker is 1:1.14. The average size of ovary is 928.57X757.14μm. The ovary has 8-10 slender branches, while the testes have 2-4 short-clavate branches. The average size of left and right testes is 871.43×628.57μm and 892.86×592.86μm. The uterus is small. The vitelline gland is abundant. The adult worm form the thoracic cavity of rat is oval in shape, and its front is wider. The average size is 6.00×2.90 mm. The rate of width to length is 1:2.07. The transverse diameter rate of oral sucker to ventral sucker is 1:1.22. The size of ovary which has slender branches is 714.29×574.43μm. The testes have 3 stubbed branches, and the size of left and right testis is 600.00×342.86μm and 500.00×228.57μm. The uterus parallels arrangement with ovary. The vitelline gland is abundant. The end of excretory bladder is behind intestinal bifurcation.The metacercaria is approximate roundness, and have two layer cyst walls. There is a big space between the outer and inner wall. The average size of metacercaria which has the outer wall and which has inner only is (529.20±62.08)μm×(494.15±43.50)μm and (430.82±31.40)μm×(414.99±35.07)μm. The larval bodies crimp as the shape of "U"style. The eggs are golden yellow in color and oval in shape, most of which are asymmetric. The egg-shell's thickness is not the same, and the thickness in the opposite side of the operculum is thicker than that the other place. The operculum locates in the wide side and has a little slope.The acromion is clear. The average size of the eggs is (67.74±8.67)μm×(42.32±3.98)μm.According to the analysis of gene obtained from the metacercaria of Mengla country in Yunnan Province, it shows that homology of ITS2 and CO I gene between the specimen and P. bangkokensis form Vietanm is 100% and 99%, which between the specimen and P. bangkokensis form Thailand is 100% and 95%, and which betweem the specimen and P. harinasutai form Thailand is 99% and 94%. The phylogenetic tree of ITS2 gene shows that the specimen and P. harinasutai form Thailand cluster to one branch. The phylogenetic tree of CO I gene shows that the specimen and P. bankokensis form Vietanm cluster to one branch.[Conclusion] The morphology of the samples obtained from Mengla country in Yunnan Province is the same as that of P. bangkokensis in each stage of life cycle. Between the specimen in this study and P. bankokensis from Vietanm,homology of gene is highest, and the genetic relationship is closest in the phylogenetic tree of COⅠgene. Homology of ITS2 gene between the specimen and P. bangkokensis from Vietanm and Thailand is 100%. Based on morphological classification and combined with DNA sequence analysis, this study shows that the specimen obtain from Mengla country in Yunnan Province is the same specie with P. bangkokensis from Vietanm and Thailand. It is a new record of P. bangkokensis in Yunnan Province.