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.

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.

The invasion of non-native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non-native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA-based approach to quantitatively monitor the invasion of non-native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non-native DNA based on a single-nucleotide polymorphism using cycling probe technology in real-time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non-native DNA in the water was well correlated to the biomass ratio of native and non-native genotypes. This method provided quantitative estimates for the proportion of native and non-native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non-native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non-native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation.

Environmental DNA (eDNA)‐based assessments of macro‐organisms have now become an essential approach for biomonitoring. eDNA survey methods have a number of advantages over conventional survey methods. However, the value of the data that will accumulate would be greatly enhanced by standardizing the analysis methods, which would allow us to compare data from multiple monitoring sites at different points in time. The eDNA Society (http://ednasociety.org/en/about), whose founding members consist of Japanese researchers conducting eDNA studies on macro‐organisms, was established in 2018, with the aim of expanding eDNA technology and science. Here, we introduce our key publication, “Environmental DNA Sampling and Experiment Manual” (http://ednasociety.org/en/manual), which was published under the initiative of the eDNA Society. Detailed methods for the surveys and experiments are described in the manual, including the selection of sampling sites, sampling methods, filtration methods, DNA extraction, species‐specific detection by real‐time polymerase chain reaction, and fish eDNA metabarcoding. The manual assists users in conducting standardized surveys and quality experiments, and provides a basis for collecting comparable data. Given that the efficacy of methods can be context dependent and variable, and that procedures may sometimes conflict with standardization, it is difficult to ensure that all processes are equally effective. However, even in such cases, it is important to maintain sufficiently high data quality by setting the minimum standards to be followed. Implementation of such standardized methodologies will enable the systematic and frequent collection of flawless, comparable eDNA data from around the world; this will provide important fundamental information for biodiversity conservation, as well as the sustainable use of fisheries resources.

Cyprinid herpesvirus 3 (CyHV-3), which causes a lethal disease in common carp, Cyprinus carpio L., and koi, C. carpio koi, first occurred in Lake Biwa, Japan in 2004. To elucidate distribution of CyHV-3 in a wild common carp population, we conducted a PCR survey of CyHV-3 among such fish in Lake Biwa in 2006. Only 6% (1/18) of the common carp smaller than 300 mm were positive with PCR, whereas 31% (18/58) of fish larger than 300 mm were positive. To evaluate their past exposure to CyHV-3 infection based on the presence of antibodies, we also measured the levels of serum anti-CyHV-3 antibodies in the carp, using an enzyme-linked immunosorbent assay. None (0/26) of the fish smaller than 300 mm was positive for the antibodies, whereas 54% (33/61) of fish larger than 300 mm were positive. Of the antibody-positive individuals, 44% (14/32) were also positive by PCR strongly suggesting that wild common carp that survived infection become CyHV-3 carriers. Five individuals were positive by PCR but negative for antibodies indicating that their infection with CyHV-3 had occurred recently. These results suggest that transmission of CyHV-3 from carriers to naïve common carp is still occurring in Lake Biwa.

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the host's feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.

Emerging infectious diseases are major threats to wildlife populations. To enhance our understanding of the dynamics of these diseases, we investigated how host reproductive behavior and seasonal temperature variation drive transmission of infections among wild hosts, using the model system of cyprinid herpesvirus 3 (CyHV-3) disease in common carp. Our main findings were as follows: (1) a seroprevalence survey showed that CyHV-3 infection occurred mostly in adult hosts, (2) a quantitative assay for CyHV-3 in a host population demonstrated that CyHV-3 was most abundant in the spring when host reproduction occurred and water temperature increased simultaneously and (3) an analysis of the dynamics of CyHV-3 in water revealed that CyHV-3 concentration increased markedly in breeding habitats during host group mating. These results indicate that breeding habitats can become hot spots for transmission of infectious diseases if hosts aggregate for mating and the activation of pathogens occurs during the host breeding season.