| [Objective] Based on morphological classification and combined with DNA sequence analysis, This paper aimed to further study the independence of Paragonimus heterotremus, taxonomic status in paragonimus and reconstruction neighbor-joining trees of P. heterotremus in order to gradually clarify the controversy of classification in Paragonimus, investigate more objective methods for taxonomy and identification, and make a foundation for studying the biologic characteristics of Paragonimus, geographical distribution, pathogenicity to human, prevention and treatment of paragonimiasis, which can offer an important scientific information to research the paragonimus and prevent paragonimiasis in Yunnan province.[Methods] Crabs were collected from endemic areas of Paragonimus heterotremus. Metacercaria were separated, identified and fed to SD rats, from which adult worms and eggs were obtained. Metacercaria, adult worms and eggs were observed and measured. Morphological characteristic spines of adult worms were observed with scanning electron microscope (SEM). With the help of reagent kits, DNA was extracted from metacercaria and adult worms, then ITS2 and CO1 gene were amplified with gene primer. Analysis of gene identity was finished with Blast program of GenBank and DNASTAR software MagAlign program and neighbor-joining trees were constructed with MEGA4.0 software.[Results] Two kinds of crabs, both of which belong to potamodiea, potamidea and potamon, were collected in this research. These kind of crabs were identified with Potamon chinghungense and Potamon hispidum. Morphological characteristic of Potamon chinghungense shows swelling carapace, dark red color, clear region, about 10 pieces of serration in antero-lateral border, and asymmetric cheliped leg which indicates that right one was bigger than the left. The first pleopod of male shows sturdy and the length of Penultimate section is 3.6 times bigger than the final section. The end edge of the first pleopod which seems like protruding peaks points to outside. The last section of male's abdomen shows triangle, but female shows oval. Morphological characteristic of Potamon hispidum shows flat carapace, dark brown color, enjoying short bristle hair, about 13-15 pieces of serration in antero-lateral border, and asymmetric cheliped leg. Exopod of the third maxilliped has short whips. The first pleopod of male shows flat and the length of penultimate section is 3.0 times bigger than the final section. The male's abdomen shows triangle and the last section indicates long triangle. The female's abdomen shows oval and the last section indicates semicircular in shape.Living adult worms of P. heterotremus shows pink in color, leech-like movement. After low-pressing and fixing, it shows elliptic in shape. To the whole adult worms, the average rate of width to length is 1:1.56-2.12. The average rate of oral sucker to ventral sucker is 1.62-2.09:1, so oral suckers are apparently bigger than ventral suckers. In this species, ovary is mostly located at the left of ventral suckers, and the front edge line is located at the level of ventral suckers, and also shows more thin branches, Uterus convolute into a group, parallels arrangement with ovary, but the size of which indicates bigger than the size of ovary. Front edge line of uterus mostly exceed to the level of ventral sucker, even partly or completely overlap. Testis are located below uterus and ovary and the central part shows small. The end of the branches swell apparently. The living metacercaria of P. heterotremus show elliptic in shape and have a thin outer cyst wall and thick inner cyst wall. The inner cyst wall thicks apparently in the bipolar and the gap between inner and outer cyst wall indicates clear. Eggs of P. heterotremus are always oval in shape, golden yellow in color, most of which is symmetry. The thickness of egg shows uneven with apparently thicker end edge. The eggs' cover always locates to the relative width end with a clear acromion. With scanning electron microscope observation, the surface of adult worms covers single spines which show rule and intensive at the central part, but irregular and thin at the end part. The shape of spines is dagger-like or triangle. The oral sucker has longitudinal folds and the ventral sucker has circular folds.The result of Blast homology analysis indicates that P. heterotremus homology of ITS2 gene between Yunnan, China and Arunacha,Manipur, India is 98%, but identity between Yunnan, China and Saraburi, Thailand is 99%. Homology of ITS2 gene between P. heterotremus and P. proliferus, all of which come from Yunnan, China is 95%, but comparison between P. heterotremus from Yunnan and P. westermani from India shows 93%. P. heterotremus homology of CO1 gene between Yunnan, China,Thailand,Vietnam and Guangxi, China is 98%. But to India, this data changes to 91%. The neighbor-joining trees of ITS2 and CO1 gene indicates that all species of P. heterotremus cluster to one branch and separate apart from other species.[Conclusion] Potamon chinghungense and Potamon hispidum collected from Yunnan are the second intermediate host of Paragonimus heterotremus. Cat's susceptibility to P. heterotremus is better than SD rat's. Cat is the appropriate experimental definitive host, but SD rat is not. Paragonimus heterotremus collected from Yunnan indicates no apparently different from the same species from other regions. DNA sequence analysis shows highly identity of P. heterotremus from different areas, clusters to one branch in neighbor-joining trees and separates apart from other species.
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