Molecular variation in the Paragonimus heterotremus complex in Thailand and Myanmar

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P heterotremus, and it is likely that P pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were FOR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P heterotremus formed a sister group to sequences from P pseudoheterotremus-type metacercariae. Sequences of P heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P heterotremus complex in Thailand and the form referred to as P pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region