Genetic markers for studies on the systematics and population genetics of snails, Bithynia spp., the first intermediate hosts of Opisthorchis viverrini in Thailand

Snails are the critical amplifying hosts of the liver fluke Opisthorchis viverrini, the causative agent of hepatobiliary disease and cholangiocarcinoma in the Mekong area of Southeast Asia. Bithynia funiculata, B. siamensis goniomphalos and B. s. siamensis are the first intermediate hosts of O. viverrini in Thailand. Morphological similarity between Bithynia species and subspecies creates problems for their taxonomic identification and an understanding of Bithynia systematics. In this study, multilocus enzyme electrophoresis (MEE) was applied to define genetic markers that could prove useful for investigating the systematics and population genetics of this genus in Thailand. Of the 34 enzymes examined, 20 encoding a presumptive 24 loci showed sufficient staining intensity and resolution for genetic interpretation. Of these, three loci were monomorphic and eight loci were diagnostic among the three Bithynia taxa. The remaining 13 loci were diagnostic between combinations of the three taxa. Fixed genetic differences were detected at 67–73% of loci among these taxa which in turn differed from a closely related species, Hydrobioides nassa, at 88% of loci. Seventy three percent fixed genetic differences were detected between B. funiculata and the two sub-species B. s. siamensis and B. s goniomphalos. Our data reveals similarly large genetic divergence, 67% fixed genetic differences, between B. s. siamensis and B. s. goniomphalos, which may well represent different species rather than subspecies as currently defined. The genetic markers detected will form the basis for subsequent comprehensive studies on the systematics and population genetics of Bithynia snails as well as for their role in the transmission of O. viverrini and opisthorchiasis.