Toshifumi Minamoto

Estimation of Fish Biomass Using Environmental DNA

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.

Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds

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.

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

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.

The Release Rate of Environmental DNA from Juvenile and Adult Fish

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.

Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.

A basin-scale application of environmental DNA assessment for rare endemic species and closely related exotic species in rivers: a case study of giant salamanders in Japan.

Summary To prevent the invasion of exotic species causing a decline in an endangered endemic species, it is important to determine the distribution of both species at an early stage, when the density of the exotic species is still low, and to manage the invasion immediately. However, distinguishing between closely related species is difficult because they share similar characteristics. The identification of DNA fragments sampled from a body of water (environmental DNA) has become a popular technique for rapidly determining the distribution of a target species. In this study, we analysed environmental DNA in water samples from 37 sites across the Katsura River basin in Japan. We used TaqMan real-time PCR to distinguish the Japanese giant salamander Andrias japonicus from the closely related Chinese giant salamander Andrias davidianus, which is known to invade Japanese rivers and hybridize with the Japanese species. In environmental samples, we detected mtDNA of the endemic species at 25 sites and mtDNA of the exotic species at nine sites. The DNA detection sites were concentrated in the upstream region. The exotic species DNA was found beyond the limits of an earlier capturing survey. Synthesis and applications. Using environmental DNA to monitor the two salamander species requires less time and effort than traditional surveys, so a wide-ranging survey can be conducted rapidly. Our results showed that performing three environmental DNA surveys for each site between autumn and winter is desirable for giant salamanders. Further collection of environmental DNA, in combination with conventional population surveys, will provide valuable information that can help protect rare endemic species in a variety of aquatic ecosystems and can help monitor the invasion of exotic species.

Droplet Digital Polymerase Chain Reaction (PCR) Outperforms Real-Time PCR in the Detection of Environmental DNA from an Invasive Fish Species

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

Environmental DNA as a 'Snapshot' of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay, Sea of Japan.

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R2 value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.

Detection of cyprinid herpesvirus 3 DNA in river water during and after an outbreak.

The disease caused by cyprinid herpesvirus 3 (CyHV-3) brings catastrophic damages to cultivated carp and koi and to natural carp populations; however, the dynamics of the virus in environmental waters are unclear. In July 2007, CyHV-3 DNA was detected in a dead common carp collected from the Yura River in Kyoto Prefecture, Japan, and this was followed by mass mortality. We collected water samples at eight sites along the Yura River for 3 months immediately after confirmation of the disease outbreak and attempted to detect and quantify CyHV-3 DNA in the water samples using molecular biological methods. The virus concentration was carried out by the cation-coated filter method, while the purification of DNA from the samples was achieved using phenol-chloroform extraction and a commercial DNA extraction kit. CyHV-3 was detected by PCR using six sets of conditions, three sets of primers (SphI-5, AP, and B22Rh exon 1), and two volumes of template DNA, and was quantified using real-time PCR. Our results indicate broader distribution of CyHV-3, even though dead fish were found only in a limited area; moreover, the virus was present at high levels in the river not only during the mass mortality caused by the disease but also for at least 3 months after the end of mass mortality. Our results suggest the possibility of infection by CyHV-3 via environmental water. The sequences of CyHV-3 collected from the Yura River matched perfectly with that of the CyHV-3 Japanese strain, suggesting that they share the same origin.

Molecular cloning of lysozyme-encoding cDNAs expressed in the salivary gland of a wood-feeding termite, Reticulitermes speratus

Two kinds of PCR-product cDNAs that encode premature lysozyme peptides (Rs-Lys1 and Rs-Lys2) were cloned from workers of a Japanese damp-wood termite, Reticulitermes speratus. The Rs-Lys1 and Rs-Lys2 cDNAs encoded deduced sequences of 170 and 164 amino acids, respectively. Alignment of these sequences with those of other insect lysozymes showed that the cDNAs encode lysozyme homologues with putative signal peptides, insertions eight amino acids long, and a relatively long C-terminus (13-17 amino acids). A maximum likelihood tree, constructed using the cDNA sequences, indicated that the termite lysozymes are related to those of mosquitoes and lepidopterans. Southern-blotting analysis identified single copies of these lysozyme genes in the termite. Reverse transcript (RT)-PCR and in situ hybridization experiments showed that Rs-Lys1 and Rs-Lys2 are expressed in the salivary glands of worker termites. Here, we discuss the possible digestive function of these lysozymes.