First intermediate hosts of Paragonimus spp. in Vietnam and identification of intramolluscan stages of different Paragonimus species

Pham Ngoc Doanh, Luu Anh Tu, Hoang Van Hien, Nguyen Van Duc, Yoichiro Horii, David Blair, Yukifumi Nawa

Members of the genus Paragonimus require at least three hosts in their life-cycles. The obligatory first intermediate hosts are freshwater snails. In Vietnam, although seven Paragonimus species have been recorded, the natural first intermediate hosts of almost all species have not been confirmed. The aim of this study was, therefore, to investigate snail hosts of Paragonimus species in Vietnam, and to identify Paragonimus species at intramolluscan stages. Freshwater snails were collected from streams in Yen Bai and Quang Tri Provinces, where high prevalences of Paragonimus metacercariae in crab hosts have been reported. Snails were morphologically identified and then examined individually for Paragonimus cercariae using shedding and crushing methods. Chaetomicrocercous cercariae, the morphological class to which Paragonimus cercariae belong, were collected for morphological description and molecular species identification by analyses of ITS2 sequences. The infected snail species were identified based on analyses of nucleotide sequences of the cox1 gene. Three snail species were found to be infected with Paragonimus cercariae at low infection rates, ranging between 0.07-1.0%. The molecular analyses identified them as Sulcospira quangtriensis and 2 species of subfamily Triculinae. In a phylogenetic tree, these two triculine snails were related to the genera Gammatricula and Tricula with low posterior probabilities. Thus we named them as Triculinae sp. 1 and Triculinae sp. 2. Cercariae from the three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, were molecularly identified as Paragonimus westermani, P. heterotremus and P. proliferus, respectively. The cercariae of the three species are morphologically similar to each other, but their daughter rediae can be distinguished by the length of the intestine and the number of cercariae per redia. The rediae of P. westermani have a long intestine and each contain 6-8 cercariae. In contrast, those of P. heterotremus and P. proliferus have a short intestine and each redia contain 10-12 and 5-6 cercariae, respectively. Three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, serve as the first intermediate hosts of P. westermani, P. heterotremus and P. proliferus, respectively, in Vietnam. The length of the intestine of rediae and the number of cercariae per redia are valuable characteristics for distinguishing between larvae of these Paragonimus species.