The combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

No abstract

The Ayu Plecoglossus altivelis altivelis is an amphidromous fish that is not only the most important commercial fishery species in Japanese rivers but also has a high economic value in recreational fishing. However, the degradation of its spawning grounds has caused a decrease in its abundance. In this study, we used environmental DNA (eDNA) to monitor the Ayu in the Takatsu River in Japan to (1) identify the spawning season in three known spawning grounds, (2) clarify changes in the main spawning grounds during the spawning season, and (3) discover unknown spawning grounds. We collected 1 L of the surface river water at three known spawning grounds nine times in 2018 and seven times in 2019 in the lower reaches of the Takatsu River. We also collected samples from seven unknown sites in 2018. The water samples were filtered through glass fiber filters. Total eDNA was extracted from each filtered sample and a Real-time quantitative PCR was performed with the specific primers and probe for Ayu. The results of the eDNA analyses showed that (1) the spawning season was in November in 2018 and in September in 2019. (2) One site was used as a spawning ground in both the early and the late spawning season, depending on the year. At the second site, the frequency of use changed year by year. The third site was the main spawning ground in the middle to late spawning season every year. From these results, we elucidated that some spawning grounds are used regularly every year, while the use of others varies year by year. (3) In five of the seven unknown sites, the nighttime eDNA concentrations were high at least once during the four surveys, suggesting that these sites may have functioned as spawning grounds. In particular, one site could be an important new spawning ground.

Genetic disturbance in wild populations of medaka (Oryzias latipes complex) has been mainly caused by the introduction of the orange-red commercial variety medaka (himedaka) in Japan. To examine whether survival, reproduction, and species recognition would be influenced by this difference in body coloration, we conducted three laboratory experiments (predatory pressure, mate choice, schooling behavior) using wild type medaka and himedaka. In the predation experiment using dark chub (Candidia temminckii) as a predator, himedaka were predated upon more often than wild type medaka. However, individuals did not choose mates or select schooling groups based on himedaka or wild type medaka phenotypes. The results indicate that himedaka receive higher predation pressure but are able to easily mate with wild type medaka in a natural environment. To conserve the genetic diversity of wild medaka populations, we need to control the risk of genetic disturbance caused by himedaka.