Development of primer–probe sets for environmental DNA-based monitoring of pond smelt Hypomesus nipponensis and Japanese icefish Salangichthys microdon

The pond smelt Hypomesus nipponensis and Japanese icefish Salangichthys microdon are economically important fishery resources in Japan. The catching of these fish species has decreased owing to environmental pollution and habitat loss caused by human impacts. We developed species-specific primer–probe sets to detect and quantify environmental DNA (eDNA) in these fish species. The primer–probe sets were specific to each target species with the detection limits of two (H. nipponensis) and one (S. microdon) copies per reaction, sufficient for eDNA analysis in the field. Using these assays, the eDNA was detected with a high probability (100% and 86% for H. nipponensis and S. microdon, respectively) at the sites where each fish species was captured by a cast net survey in the Hirata–Funagawa River (one of the inflowing rivers of Lake Shinji, Shimane Prefecture, Japan), where it was frequently detected without cast net capture. The eDNA was detected in S. microdon a little earlier than in H. nipponensis, and the eDNA concentrations of both species gradually decreased after peaking in July and reached negligible levels by October. The utility of the assays for estimating the seasonal appearances/disappearances of both species in this river was demonstrated. The species-specific primer–probe sets of the eDNA-based method represent a potentially useful monitoring tool that requires minimal labor and time and could be useful for managing H. nipponensis and S. microdon fisheries in the future.