Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two‐monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass‐fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long‐term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future.

During spawning activity, fish release large amounts of sperm and eggs into the water, which has been assumed to cause an increase in environmental DNA (eDNA) levels and nuclear DNA/mitochondrial DNA ratios. To test whether these assumptions are valid and whether nuclear and mitochondrial eDNA analysis can be used to monitor the spawning activity of freshwater fish, we conducted field eDNA surveys and traditional surveys using common carp (Cyprinus carpio), largemouth bass (Micropterus salmoides) and bluegill sunfish (Lepomis macrochirus) as model species. Fish spawning periods were estimated based on age, as estimated using the body lengths of juveniles collected in the Miharu reservoir in Fukushima, Japan. The results showed that the main spawning periods of largemouth bass and bluegill sunfish were from April to July and from July to August, respectively. Field eDNA surveys were conducted in the Hebisawagawa front reservoir, which is connected to the Miharu reservoir. From March to August 2019 and 2020, weekly eDNA sampling was conducted at three sites, and daily sampling was conducted at six sites from 23 June to 3 July 2020. The eDNA concentrations of the nuclear internal transcribed spacer 1 (ITS1) and mitochondrial cytochrome B (CytB), as well as the ITS1/CytB ratio, were measured for each of the three fish in each water sample. Water temperature had a statistically significant effect on eDNA concentration, probably reflecting the relationship between water temperature and spawning. We created generalised additive mixed models to estimate spawning activity periods based on weekly eDNA data. The estimated periods of spawning activity for common carp, largemouth bass and bluegill sunfish were March to May, May to July, and May to August, respectively. The estimated spawning periods coincided with known fish ecology or the results of traditional methods. This method also has been applied to daily eDNA samples, showing the feasibility of high‐resolution estimation of spawning activity. For common carp and bluegill sunfish, we were able to estimate the spawning period using this method. Although the method is affected by biomass and the diffusion and degradation of eDNA, it has the potential to accurately estimating spawning activities. These then can be estimated without conducting laborious traditional surveys, facilitating the monitoring of reproduction by rare, invasive or important fishery species. Further research on the diffusion distance and degradation time of the eDNA concentration peak caused by fish spawning activity may improve the accuracy of monitoring.

Dam-mediated biological invasions are a serious problem all over the world. Once established in reservoirs, the invasive species have catastrophic impacts on the river ecosystems downstream, and thus, rapid monitoring of invasive species is an urgent issue for the effective removal of them and the conservation management of native ecosystems. Here, we verified the utility of environmental DNA (eDNA) analysis as a tool to effectively monitor three invasive fish species (bluegill, largemouth bass, and smallmouth bass) in reservoirs using multiplex real-time PCR. First, to determine the optimal sampling location and season to detect eDNA from these species, we analyzed the eDNA in water samples from shore and offshore sites in three reservoirs all year around. We found that eDNA detection rates either did not differ between sampling locations or were higher for shore than offshore sites. In addition, eDNA detection rates were higher in spring (breeding season of target species) and/or summer than winter. Second, we extensively surveyed the distribution of the three species in 30 reservoirs in Japan using eDNA analysis. Consequently, a single eDNA-based surveillance in summer allowed to match approximately 90% of the presence/absence of the invasive fish species known from 27 yr of administrative capture-based surveillances. Given these results, we recommend collecting the replicated water samples from shore sites in summer or the breeding season for the effective detection of invasive fish eDNA in reservoirs. Our eDNA assays with multiplex real-time PCR enable the rapid and sensitive monitoring of invasive fish distribution in reservoirs.

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