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Paddy ecosystems are among the most biodiversity-rich ecosystems, but they can be degraded by field consolidation or the abandonment of rice cultivation. Snakes are the top predators in paddy ecosystems; therefore, understanding the distribution of snakes is important for the conservation of paddy biodiversity. Traditionally, surveys of snakes have been primarily conducted by sighting and trapping, but such methods require encountering snakes during surveys and require expertise in species identification. In this study, we developed environmental DNA (eDNA) detection assays for snakes as a method for surveying a wide area relatively easily. We designed species-specific assays for three common snake species in Japanese paddy ecosystems: Elaphe quadrivirgata, Rhabdophis tigrinus, and Gloydius blomhoffii. After confirming the detection of target species DNA from the rearing environment, the assays were applied to field samples. Eighty-seven and 51 samples were collected from paddy fields, reservoirs, and ditches in summer and autumn, respectively. We collected eDNA samples from eight traditional paddy areas that had not been consolidated, eight consolidated paddy areas, and two abandoned paddy areas in southeastern Hyogo Prefecture, Japan. We attempted to detect the DNA of the three target species in these samples using our eDNA assays and succeeded in identifying the DNA of R. tigrinus and G. blomhoffii in three areas and one area, respectively. Statistical analysis suggested that land-use type affected the detection of R. tigrinus eDNA. This study is the first attempt to survey snakes in paddy ecosystems using eDNA analysis and will contribute to the development of paddy ecosystem monitoring.

We have reported a case of a 44-year-old woman with anti-signal recognition particle (SRP) antibody-positive immune-mediated necrotizing myopathy triggered by human parvovirus B19 (PVB19) infection. She was admitted to the hospital because of lower leg edema and muscle weakness after erythema infectiosum. Magnetic resonance imaging of the lower extremities revealed high signals in the proximal muscles and subcutaneous edema on STIR. Muscle biopsy showed myofiber regenerative changes and variation in fiber size. A myositis-specific autoantibody profile indicated a positive result for anti-SRP antibodies. We diagnosed the patient with immune-mediated necrotizing myopathy (IMNM). Muscle strength and subcutaneous edema improved gradually in 3 months following immunotherapy. This is the first case report of an IMNM associated with PVB19 infection.

In the biomonitoring using environmental DNA (eDNA) analysis, the suppression of eDNA degradation has been an important issue to estimate accurately the presence of target species and their eDNA concentrations. A recently proposed eDNA preservation technique requires only the addition of benzalkonium chloride (BAC) to water samples and has been used for various studies as an alternative solution to on-site filtration. However, BAC remaining in filters may adsorb to and inhibit the function of the enzyme used to extract eDNA from filters. In this study, we tested whether eDNA yield is affected by BAC addition immediately before filtration and demonstrated that the BAC in water remaining in filters decreased the eDNA yield significantly. We found that simple pre-centrifugation is effective for removing the remaining water from the filters to prevent the eDNA yield reduction due to the use of BAC. This is the most important finding in the present study. Appropriate eDNA extraction techniques are needed when BAC is used to secure eDNA yields and expand the application of eDNA analysis for biomonitoring.

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionise phylogeography.

The combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

Estimating the presence and abundance of non-native species in the early stage of invasion is important to prevent further spread of non-native species in aquatic systems. Environmental DNA (eDNA) methods have been applied for aquatic plants; however, it is still questionable on the accuracy of the eDNA methods for the detection and abundance estimation of aquatic plants in lotic systems. Here, we studied the invasive aquatic plant Egeria densa in the tributaries of the Gonokawa River in Japan to compare the E. densa detection rate of the eDNA method with that of the three sampling methods and examine the relationships of the eDNA flux to the upstream plant coverage, plant stem fragment flux, and plant tissue DNA flux of E. densa in the study sites on each season. Our results indicated that the eDNA method better detected the presence of E. densa in our study sites than other methods. The eDNA flux was directly related to the coverage rank and the plant tissue DNA flux in the study sites in both months. These results suggest that the eDNA methods have a potential as monitoring tool for early detection and relative abundance estimation of invasive aquatic plants in lotic systems.

The global outbreak of the SARS-CoV-2 pandemic has increased the focus of Wastewater-based epidemiology (WBE) studies as a tool for understanding the epidemic and risk management. A highly sensitive and rapid method for the virus concentration from wastewater is needed to obtain the accurate information for early detection of SARS-CoV-2 outbreak and epidemic. In this study, we evaluated the efficiency of the direct capture method provided from Promega, based on column adsorption using the wastewater from actual infectious diseases ward. The efficiency of the nucleic acid extraction-purification process was also evaluated by Maxwell® RSC instrument (fully automated extraction) and QIAamp Viral RNA mini kit (manual extraction). The obtained SARS-CoV-2 data from wastewater were analyzed with the number of inpatients which is the consideration of the severity and the days of onset. The combination of direct capture and Maxwell’s method (DC-MW) was suggested to be a highly sensitive and simple method with better concentration efficiency and quantification than other methods. Moreover, the inpatient conditions (severity and days of after onset) should be considered to accurately understand the actual status of the correlation between the number of inpatients and SARS-CoV-2 concentration in wastewater. The highly sensitive method of DC-MW was suggested to assess more actual situation of SARS-CoV-2 shedding into the wastewater.

Medaka (comprising two species Oryzias latipes and Oryzias sakaizumii) is a small freshwater fish that is globally used as an experimental model organism. Wild Japanese medaka populations have been declining and their genetic diversity has been affected by extensive artificial introduction of non-native populations and ‘himedaka’, an orange-red variety derived from O. latipes, which is a commercially popular in Japan. To detect artificially caused genetic introgression in wild medaka populations, we developed a novel and effective nuclear markers. Sequencing analysis of a single-copy nuclear DNA (scnDNA) belonging to linkage group 21 (LG21b) was performed to identify specific haplotype in O. sakaizumii, nine genetically localized groups of O. latipes, and himedaka populations. A total of 28 haplotypes were identified from the 16 tested wild populations. Of the 28 haplotypes, 27 were considered specific to O. sakaizumii and to each genetically localized group of O. latipes; thus, these haplotypes could be used as diagnostic markers to detect artificially caused genetic introgression. Eight haplotypes were confirmed in himedaka, and four were considered specific to himedaka. Our tests involved direct sequencing of the 3′-sequence of the LG21b region as a diagnostic marker, which effectively detected artificially caused genetic introgression in wild populations. Analysis of the actual level of genetic introgression will be improved by incorporating the new scnDNA marker in combination with conventional DNA markers of medaka.

Palaemon paucidens has a large population and is an important food source for fish in Lake Biwa, Japan. They are abundant in shallow waters from spring to summer, after which most individuals migrate to offshore deep areas where they remain during autumn and winter. However, some individuals are nonmigratory, remaining in shallow waters over winter. It has been reported that P. paucidens individuals have declined in recent years; a better understanding of its seasonal distribution is needed to manage this species, and basic information on its seasonal distribution is indispensable. We tracked the environmental DNA (eDNA) distribution of P. paucidens in Lake Biwa over a year using a quantitative real‐time polymerase chain reaction method. We collected water samples from offshore (both from the surface and from the benthic) and from shallow shore sites adjacent to the shorelines of the main lake and connecting freshwater lagoons. Offshore sampling took place in summer and winter, and shallow shore and lagoon sampling in all four seasons. During summer, eDNA concentrations were significantly higher in shallow and lagoon areas than offshore bottom sites. Conversely, during winter, eDNA concentrations were higher in offshore bottom sites, and relatively high and low eDNA concentrations in lagoons and shallow shore, respectively. These results most likely reflect the spatial and temporal distribution of this species in Lake Biwa. The eDNA concentrations peaked in early August at shallow shore sites in the main lake, with a significant decline in mid‐October, while low eDNA concentrations were recorded at offshore bottom sites in late August. These results suggest that P. paucidens migrates from shallow waters to offshore bottom sites between early August and mid‐October. These results provide important information for the management of this species.

The Ayu Plecoglossus altivelis altivelis is an amphidromous fish that is not only the most important commercial fishery species in Japanese rivers but also has a high economic value in recreational fishing. However, the degradation of its spawning grounds has caused a decrease in its abundance. In this study, we used environmental DNA (eDNA) to monitor the Ayu in the Takatsu River in Japan to (1) identify the spawning season in three known spawning grounds, (2) clarify changes in the main spawning grounds during the spawning season, and (3) discover unknown spawning grounds. We collected 1 L of the surface river water at three known spawning grounds nine times in 2018 and seven times in 2019 in the lower reaches of the Takatsu River. We also collected samples from seven unknown sites in 2018. The water samples were filtered through glass fiber filters. Total eDNA was extracted from each filtered sample and a Real-time quantitative PCR was performed with the specific primers and probe for Ayu. The results of the eDNA analyses showed that (1) the spawning season was in November in 2018 and in September in 2019. (2) One site was used as a spawning ground in both the early and the late spawning season, depending on the year. At the second site, the frequency of use changed year by year. The third site was the main spawning ground in the middle to late spawning season every year. From these results, we elucidated that some spawning grounds are used regularly every year, while the use of others varies year by year. (3) In five of the seven unknown sites, the nighttime eDNA concentrations were high at least once during the four surveys, suggesting that these sites may have functioned as spawning grounds. In particular, one site could be an important new spawning ground.

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

A mitochondrial cytochrome b genotype of Oryzias latipes, “mitotype B15”, is widely and discontinuously distributed in various localities in Japan. To re-examine the origin of mitotype B15 detected in various localities, we conducted a sequence analysis of the NADH dehydrogenase subunit 2 (ND2) gene region. Twenty-eight haplotypes were defined among mitotype B15 fish from 23 localities throughout Japan. Most of the haplotypes detected in each area are unique to the location and genetically closely related to each other, indicating that they are native haplotypes. Our results suggest that ND2 analysis is important for identifying native populations of O. latipes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In order to maximize the performance of lithium ion batteries, we have developed a new method for detecting the status of hybrid electric vehicle (HEV) batteries. The method obtains an accurate state of charge (SOC) by combining the SOC based on open circuit voltage (OCV) and the SOC based on current integration. In addition to that, our method has an auto-tuning function, which is realized by relatively simple processing, for the characteristic parameter of batteries. Our method can also obtain the state of health (SOH) by watching the tuned characteristic parameter. We perform simulation evaluations of our method with a battery model, and we confirm that the SOC error is less than 5% even if SOH set in the battery model and in our method are under mismatched condition. We also confirm that the SOH gradually converges to the true value and SOC error is improved by performing simulation evaluation repeatedly.

Genetic disturbance in wild populations of medaka (Oryzias latipes complex) has been mainly caused by the introduction of the orange–red commercial variety medaka (himedaka) in Japan. To examine whether survival, reproduction, and species recognition would be influenced by this difference in body coloration, we conducted three laboratory experiments (predatory pressure, mate choice, schooling behavior) using wild type medaka and himedaka. In the predation experiment using dark chub (Candidia temminckii) as a predator, himedaka were predated upon more often than wild type medaka. However, individuals did not choose mates or select schooling groups based on himedaka or wild type medaka phenotypes. The results indicate that himedaka receive higher predation pressure but are able to easily mate with wild type medaka in a natural environment. To conserve the genetic diversity of wild medaka populations, we need to control the risk of genetic disturbance caused by himedaka. J. Exp. Zool. 323A: 349–358, 2015. © 2015 Wiley Periodicals, Inc.

Genetic disturbance in wild populations of medaka (Oryzias latipes complex) has been mainly caused by the introduction of the orange-red commercial variety medaka (himedaka) in Japan. To examine whether survival, reproduction, and species recognition would be influenced by this difference in body coloration, we conducted three laboratory experiments (predatory pressure, mate choice, schooling behavior) using wild type medaka and himedaka. In the predation experiment using dark chub (Candidia temminckii) as a predator, himedaka were predated upon more often than wild type medaka. However, individuals did not choose mates or select schooling groups based on himedaka or wild type medaka phenotypes. The results indicate that himedaka receive higher predation pressure but are able to easily mate with wild type medaka in a natural environment. To conserve the genetic diversity of wild medaka populations, we need to control the risk of genetic disturbance caused by himedaka.