The Science of Nature

, 104:100 | Cite as

Identifying a breeding habitat of a critically endangered fish, Acheilognathus typus, in a natural river in Japan

  • Masayuki K. Sakata
  • Nobutaka Maki
  • Hideki Sugiyama
  • Toshifumi MinamotoEmail author
Original Paper


Freshwater biodiversity has been severely threatened in recent years, and to conserve endangered species, their distribution and breeding habitats need to be clarified. However, identifying breeding sites in a large area is generally difficult. Here, by combining the emerging environmental DNA (eDNA) analysis with subsequent traditional collection surveys, we successfully identified a breeding habitat for the critically endangered freshwater fish Acheilognathus typus in the mainstream of Omono River in Akita Prefecture, Japan, which is one of the original habitats of this species. Based on DNA cytochrome B sequences of A. typus and closely related species, we developed species-specific primers and a probe that were used in real-time PCR for detecting A. typus eDNA. After verifying the specificity and applicability of the primers and probe on water samples from known artificial habitats, eDNA analysis was applied to water samples collected at 99 sites along Omono River. Two of the samples were positive for A. typus eDNA, and thus, small fixed nets and bottle traps were set out to capture adult fish and verify egg deposition in bivalves (the preferred breeding substrate for A. typus) in the corresponding regions. Mature female and male individuals and bivalves containing laid eggs were collected at one of the eDNA-positive sites. This was the first record of adult A. typus in Omono River in 11 years. This study highlights the value of eDNA analysis to guide conventional monitoring surveys and shows that combining both methods can provide important information on breeding sites that is essential for species’ conservation.


Acheilognathus typus Breeding site Environmental DNA (eDNA) Omono River Real-time PCR Species-specific detection 


Funding information

This study was partly supported by JSPS KAKENHI Grant Number 17H03735 and by a donation from a private company to which one of the authors (NM) belongs. The funders had no role in designing and conducting this research.


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Graduate School of Human Development and EnvironmentKobe UniversityKobe CityJapan
  2. 2.Tohoku Branch, Pacific Consultants Co., LTDSendai CityJapan
  3. 3.Faculty of Bioresource SciencesAkita Prefectural UniversityAkita CityJapan

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