Species-specific detection of the endangered piscivorous cyprinid fish Opsariichthys uncirostris uncirostris, three-lips, using environmental DNA analysis

Opsariichthys uncirostris uncirostris is a subspecies of Amur three-lips Opsariichthys uncirostris (Temminck and Schlegel, 1846) and endemic to Japan. This only piscivorous fish among Cyprinidae in Japan, hereafter three-lips, is in a unique ecological situation, that is, the species is endangered in the native habitats such as Lake Biwa water system and Lake Mikata, but on the other hand, three-lips is threatening the other native species as an intra-nation invasive species in introduced habitats. In this study, a species-specific primer–probe set was developed for detecting three-lips using environmental DNA (eDNA) analysis for monitoring the species distribution. Ten out of 11 sites in Futatsugawa River system, Kyusyu, and 15 out of 28 sites in Lake Biwa tested positive. In the Futatsugawa, three-lips was confirmed to occupy both of the lotic sites in the main stem of the river and the lentic sites in the inner part of small ditches. In Lake Biwa, positives were biased to the sites in north basin of the lake than smaller south basin. The PCR amplicons in the positive samples were confirmed to have DNA sequences of the target species, suggesting the accuracy of this species-specific analysis. The eDNA detection system developed in this study will contribute to future follow-up monitoring by providing a cost-effective and less labor-intensive method for determining the distribution of three-lips with dual identities as a threatened and invasive species.