MOLECULAR AND MORPHOLOGICAL DISCRIMINATION OF DICROCOELIID LARVAE (TREMATODA: DIGENEA) FROM TERRESTRIAL MOLLUSKS IN JAPAN.

Trematodes of the family Dicrocoeliidae commonly use terrestrial mollusks as the first intermediate host. Despite the abundant studies on the adult worms in birds and mammals, few reports exist on their larval stage in snail intermediate hosts. A present survey of mollusks in Japan led us to the discovery of dicrocoeliid sporocysts with cercariae in 16 out of 303 individuals, encompassing eight snail species and one slug species. A DNA barcoding based on sequencing of mitochondrial cytochrome c oxidase subunit 1 showed that the larvae consisted of five species. Phylogenetic trees of nuclear 18S and 28S ribosomal DNAs confirmed the five species to be members of the Dicrocoeliidae. These were temporarily termed dicrocoeliid spp. 1 to 5, since conclusive identification was impossible without adult worms. These unknown species were phylogenetically related to each other, except sp. 5. The phylogenetic trees demonstrated close genetic relationships between sp. 3 and the genus Lutztrema, and between sp. 5 and the genus Lyperosomum. The phylogenetic analysis also suggested that a division into the subfamilies Dicrocoeliinae and Leipertrematinae is a wrong classification due to the paraphyly of the Dicrocoeliinae. Morphological characterization of the cercariae and their DNA barcodes provide a primary platform for differentiating dicrocoeliids from various mollusks in Japan. The DNA barcodes, in particular, will enable tracing the parasite life cycles, in case of finding metacercariae and adults from presently unknown hosts.