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.

The field of environmental DNA (eDNA) analysis has rapidly developed over the past decade and the technique has become widely used for detecting aquatic macroorganisms in a variety of habitats. However, a variety of measurement protocols have been individually developed for different eDNA studies and this may lead to confusion for others who wish to incorporate eDNA analysis in their research. It is important therefore to synthesize the current status of—and future challenges to—the methodology of eDNA analysis. We here synthesized the protocols from total 438 published eDNA studies detecting aquatic macroorganisms were used to calculate the frequency of using each method in eDNA analysis steps. We found that the frequency of methods used converged to one or two methods for any analysis step. Furthermore, although the procedure with highest frequency is not always the best, it was shown that the eDNA collection by filtration and subsequent extraction/purification using a DNeasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) or PowerWater DNA Extraction Kit (Qiagen) is the most common procedure. An understanding of the characteristics of commonly used methods can help those newly engaged in eDNA studies to understand the basic outline of eDNA analysis. Our review will be useful for the future improvement and development of analytical eDNA techniques of eDNA by sharing the recognition of methodological characteristic including advantages and disadvantages in major analytical techniques.

Environmental DNA (eDNA) is DNA shed by organisms into surrounding environments such as soil and water. The new methods using eDNA as a marker for species detection are being rapidly developed. Here we explore basic knowledge regarding the dependence of the eDNA degradation rate on time and water temperature, and the relationship between eDNA degradation and bacterial abundance. This subject has not been well clarified, even though it is essential for improving the reliability of eDNA analysis. To determine the time- and water temperature-dependent degradation of eDNA, river water was sampled and eDNA concentrations were determined for ayu sweetfish (Plecoglossus altivelis altivelis) and common carp (Cyprinus carpio) at seven time points, over a 48-h period, and at three different water temperatures. The degradation of eDNA was modeled for each species using an existing exponential decay model with an extension to include water temperature effects. The degradation models were constructed for ayu sweetfish as Nt = 229,901.2 × exp [− (0.01062 × k − 0.07081) × t] and for common carp as Nt = 2,558.0 × exp [− (0.01075 × k − 0.07372) × t]. Nt is the DNA concentration at time t (elapsed time in hours) and k is the water temperature (°C). We also measured the concentration of eDNA derived from purified genomic DNA of the common carp, which was spiked into aquarium water without the target species, and we measured the bacterial abundance in the sample water after 12 and 24 h of incubation. Environmental DNA degradation was accelerated at higher water temperatures (generalized linear model, GLM; p < 0.001), but bacterial abundance did not have a significant effect on eDNA degradation (GLM, p = 0.097). These results suggest that the proper treatment of this temperature effect in data interpretations and adjustments would increase the reliability of eDNA analysis in future studies.

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) provide a noninvasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D‐loop region were contained in the sample and amplified by two‐step tailed PCR. The 15 PCR replicates indexed with different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes was compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR replicates, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes was further reduced. The approach proposed in this study successfully eliminated most of the false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

Environmental DNA (eDNA) analysis is a powerful tool within ecology for the study of the distribution or abundance of aquatic species, although the simplification of water sampling is required for enabling light and fast field sampling to expand further application of eDNA analysis. Here, certain candidate chemicals belonging to the group of cationic surfactants were examined for their effectiveness as preservatives for eDNA water samples by simply adding the chemicals to water samples to suppress the degradation of eDNA. The quaternary ammonium compound benzalkonium chloride (BAC) at a final concentration of 0.01% was effective to retain 92% of eDNA derived from the bluegill sunfish Lepomis macrochirus in an 8-h incubation test at ambient temperature, which assumed a transportation of water samples in 1-day field sampling during the daytime. Meanwhile, eDNA in water samples without BAC retained only 14% of the initial eDNA. Moreover, an additional long-term incubation test (up to 10 days) revealed BACtreated samples retained *70 and 50% of bluegill DNA compared to the initial amount after 1-and 10-day incubation at ambient temperature, respectively. Meanwhile, eDNA in naïve samples reduced to 20% after 1-day incubation and reached undetectable levels after 10 days. Up to now, many eDNA studies have adopted on-site filtration followed by filter fixation, which requires many pieces of equipment. Addition of BAC can protect eDNA in water samples with less effort and equipment resulting in an increase of measurement accuracy of the eDNA quantity and detection probability of rare species by preventing the disappearance of rare sequences in water samples.

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.

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization with N-methyl-N'-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

An understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual‐ and capture‐based monitoring generally require laborious, time‐consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient noninvasive approach for monitoring spawning events on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)‐based approach for monitoring and understanding spawning events by observing spikes in eDNA concentration after spawning events. We found in hybridization experiment using two medaka species (Oryzias latipes and Oryzias sakaizumii, 1:1 individual per tank) that a spike in eDNA concentration occurred in male species after spawning. Besides, the magnitude of the spike in eDNA concentration was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentrations after spawning were 3–25 times higher than before the expected time for spawning. Additionally, there was no spike in eDNA concentration during the non‐reproductive season. Therefore, our results demonstrated that spike in eDNA concentration is mainly caused by the released sperm during spawning events, and it can be used as evidence of spawning in the field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.