Quantitative PCR method to detect an extremely endangered bitterling fish (Rhodeus atremius suigensis) using environmental DNA

Rhodeus atremius suigensis is an extremely endangered bitterling fish, designated as one of the Nationally Endangered Species of Wild Fauna and Flora by the Ministry of Environment of Japan. To prevent its extinction, effective ecological survey methods are necessary in order to guide habitat management. Recently, environmental DNA (eDNA) analysis has proven useful for detection and censusing of endangered aquatic species that are difficult to locate and capture. In this study, we developed and evaluated a semi-quantitative system to track R. a. suigensis using eDNA. We designed primers and a fluorescent probe specific to the mitochondrial gene for NADH dehydrogenase subunit 1 (ND-1) of R. a. suigensis for quantitative real-time PCR. We first confirmed the utility of this analysis in aquarium experiments, before performing field surveys. We sampled water for eDNA analysis and set fish traps at 48 points in an agricultural channel in the Ashida River basin, inhabited by R. a. suigensis, and examined the relationship between fish occurrence and eDNA concentration. The concentration was correlated with the distance downstream from the point where specimens of R. a. suigensis were captured, indicating that the eDNA concentration reflects distribution and abundance of R. a. suigensis. These results not only demonstrate the reliability of this method, but also suggest its feasibility for conservation measures.