We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163–185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.

Environmental DNA (eDNA) from aquatic vertebrates has recently been used to estimate the presence of a species. We hypothesized that fish release DNA into the water at a rate commensurate with their biomass. Thus, the concentration of eDNA of a target species may be used to estimate the species biomass. We developed an eDNA method to estimate the biomass of common carp (Cyprinus carpio L.) using laboratory and field experiments. In the aquarium, the concentration of eDNA changed initially, but reached an equilibrium after 6 days. Temperature had no effect on eDNA concentrations in aquaria. The concentration of eDNA was positively correlated with carp biomass in both aquaria and experimental ponds. We used this method to estimate the biomass and distribution of carp in a natural freshwater lagoon. We demonstrated that the distribution of carp eDNA concentration was explained by water temperature. Our results suggest that biomass data estimated from eDNA concentration reflects the potential distribution of common carp in the natural environment. Measuring eDNA concentration offers a non-invasive, simple, and rapid method for estimating biomass. This method could inform management plans for the conservation of ecosystems.

Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to assess aquatic community structures. However, the method has hitherto lacked field tests that evaluate its effectiveness and practical properties as a biodiversity monitoring tool. Here, we evaluated the ability of eDNA metabarcoding to reveal fish community structures in species-rich coastal waters. High-performance fish-universal primers and systematic spatial water sampling at 47 stations covering ~11 km² revealed the fish community structure at a species resolution. The eDNA metabarcoding based on a 6-h collection of water samples detected 128 fish species, of which 62.5% (40 species) were also observed by underwater visual censuses conducted over a 14-year period. This method also detected other local fishes (≥23 species) that were not observed by the visual censuses. These eDNA metabarcoding features will enhance marine ecosystem-related research, and the method will potentially become a standard tool for surveying fish communities.

Knowledge of the presence of an invasive species is critical to monitoring the sustainability of communities and ecosystems. Environmental DNA (eDNA), DNA fragments that are likely to be bound to organic matters in the water or in shed cells, has been used to monitor the presence of aquatic animals. Using an eDNA-based method, we estimated the presence of the invasive bluegill sunfish, Lepomis macrochirus, in 70 ponds located in seven locales on the Japanese mainland and on surrounding islands. We quantified the concentration of DNA copies in a 1 L water sample using quantitative real-time polymerase chain reaction (qPCR) with a primer/probe set. In addition, we visually observed the bluegill presence in the ponds from the shoreline. We detected bluegill eDNA in all the ponds where bluegills were observed visually and some where bluegills were not observed. Bluegills were also less prevalent on the islands than the mainland, likely owing to limited dispersal and introduction by humans. Our eDNA method simply and rapidly detects the presence of this invasive fish species with less disturbance to the environment during field surveys than traditional methods.

The environmental DNA (eDNA) technique is expected to become a powerful, non-invasive tool for estimating the distribution and biomass of organisms. This technique was recently shown to be applicable to aquatic vertebrates by collecting extraorganismal DNA floating in the water or absorbed onto suspended particles. However, basic information on eDNA release rate is lacking, despite it being essential for practical applications. In this series of experiments with bluegill sunfish (Lepomis macrochirus), we examined the effect of fish developmental stage on eDNA release rate. eDNA concentration reached equilibrium 3 days after the individual fish were introduced into the separate containers, enabling calculation of the eDNA release rate (copies h⁻¹) from individual fish on the assumption that the number of eDNA released from the fish per unit time equals total degradation in the container (copies h⁻¹). The eDNA release rate was 3–4 times higher in the adult (body weight: 30–75 g) than in the juvenile group (0.5–2.0 g). Such positive relationship between fish size and eDNA release rate support the possibility of biomass rather than density estimation using eDNA techniques. However, the eDNA release rate per fish body weight (copies h⁻¹ g⁻¹) was slightly higher in the juvenile than the adult group, which is likely because of the ontogenetic reduction in metabolic activity. Therefore, quantitative eDNA data should be carefully interpreted to avoid overestimating biomass when the population is dominated by juveniles, because the age structure of the focal population is often variable and unseen in the field. eDNA degradation rates (copies l⁻¹ h⁻¹), calculated by curve fitting of time-dependent changes in eDNA concentrations after fish removal, were 5.1–15.9% per hour (half-life: 6.3 h). This suggests that quantitative eDNA data should be corrected using a degradation curve attained in the target field.