Co-existence of Paragonimus harinasutai and Paragonimus bangkokensis metacercariae in fresh water crab hosts in central Viet Nam with special emphasis on their close phylogenetic relationship.

During our epidemiological surveys for Paragonimus species in central Viet Nam, we found four morphologically different Paragonimus metacercariae in mountainous crabs. They were identified as metacercariae of Paragonimus westermani, P. bangkokensis, P. proliferus, and P. harinasutai in the order of their prevalence in crab hosts. This is the first discovery of P. harinasutai in Viet Nam, co-inhabiting with P. bangkokensis and other species. Metacercariae of P. harinasutai were given orally to a cat to obtain adult worms. Then, ITS2 and CO1 sequences of metacercariae and adults of P. harinasutai, and metacercariae of P. bangkokensis collected from the same place were determined for analyses of phylogenetic relationships to other P. harinasutai and P. bangkokensis populations as well as related species. The results of molecular analyses showed that P. harinasutai from Quang Binh province of central Viet Nam was almost completely identical with those from Vientiane, Lao PDR; P. bangkokensis from Quang Binh, Viet Nam was also almost completely identical with those from Lao PDR and from Quang Ninh province, Viet Nam. Except for one P. harinasutai isolate from China, all populations of P. harinasutai and P. bangkokensis from Thailand, Lao and Viet Nam make a single clade in both ITS2 and CO1 trees. In ITS2 sequences, AT deletion and ATC insertion were observed in some isolates of both species, indicating recent gene flow between P. harinasutai and P. bangkokensis. Moreover, because of their extremely high genetic similarities and their co-inhabitation in the same crab hosts found in Thailand, Lao PDR and Viet Nam, they should be considered as the sister species at the early stage of divergence. In addition, P. microrchis previously described from Yunnan, China should be placed as the synonym of P. harinasutai, because of their morphological and molecular similarities.