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

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  • Environmental DNA as a Practical Tool for Aquatic Conservation and Restoration
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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.

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All the qPCR results used in this study are shown in Supplementary Table 1.


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We thank Mr. Satoshi Furumoto, Mr. Hidenori Yoshigou and Dr. Shigefumi Kanao for providing information on the previous distribution of Rhodeus atremius suigensis in the Ashida River basin. We also thank Mr. Kazuya Takeuchi (Okayama University) and the staff of the Environment Conservation Division, City of Fukuyama for their help in our field investigations. We are grateful to Dr. Risa Monna (Rizo Inc.) for her advice on this research. We thank Dr. Steven D. Aird for editing the manuscript. This study was financially supported by the Chugoku Regional Development Bureau, Ministry of Land, Infrastructure, Transport and Tourism of Japan and by the Suma Aqualife Park, Kobe City. This study complies with current Japanese law.

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Correspondence to Kazuyoshi Nakata.

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Supplementary file1 (XLSX 14 kb)


Supplementary Fig. 1. Rhodeus atremius suigensis eDNA concentration is not correlated with water depth or velocity. Water depth (A)/water velocity (B) (horizontal axis) and eDNA concentration (vertical axis) at each sampling site are plotted. There was no significant rank correlation between water depth or water velocity and eDNA concentration (Kendall rank correlation coefficient, water depth: τ = 0.05, P > 0.05, flow velocity: τ = 0.03, P > 0.05) Supplementary file2 (PPTX 517 kb)

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Otsuki, K., Hamada, M., Koizumi, N. et al. Quantitative PCR method to detect an extremely endangered bitterling fish (Rhodeus atremius suigensis) using environmental DNA. Landscape Ecol Eng 19, 79–86 (2023).

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