This study demonstrated the use of environmental DNA (eDNA) to determine habitat connectivity for migration of fishes between the sea and river. Environmental DNA is DNA fragments released by fishes ...in water, which can be used as a species-specific marker of the presence/absence of the target species. A year-round water sampling regime at 15 sites on the Yodo River, Japan, was conducted to determine whether three major man-made barriers on the river inhibited the migration of fishes using species-specific detection of DNA fragments from three target migrant species, temperate seabass, Lateolabrax japonicus, flathead grey mullet, Mugil cephalus, and ayu, Plecoglossus altivelis altivelis. The presence/absence of eDNA from target species was consistent with known patterns of species’ seasonal migration. The detection of the DNA of temperate seabass and flathead grey mullet at sites upstream of the dam closest to the river mouth indicated successful upstream migration of these species via a fish ladder bypassing the dam. On the other hand, DNA of these two species was not detected from the upstream side of the two remaining dams, which are not equipped with fish ladders. Ayu is the only species among the three target species with a land-locked population in Lake Biwa located at the headwater of Yodo River. Ayu DNA was detected at most of the sites in the freshwater area during the warm months; however, in the coldest month of February, eDNA was only detected in the uppermost site of Yodo River at the southern tip of Lake Biwa. The eDNA we detected at this site suggests that it was derived from juvenile ayu spending their winter months in the lake. These results suggest that the eDNA analysis presented here can accurately track the seasonal migration of fishes in a river, demonstrating its application as an indicator of habitat connectivity for fishes in association with man-made barriers in a river. The sampling of eDNA involves merely scooping a tank full of water; therefore, it is a simple, rapid, and cost-effective method for long-term monitoring of habitat connectivity associated with the construction of barriers in a river.
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.
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.
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.
Summary
Environmental DNA (eDNA) analysis for detecting the presence of aquatic and terrestrial organisms is an established method, and the eDNA concentration of a species can reflect its ...abundance/biomass at a site. However, attempts to estimate the abundance/biomass of aquatic species using eDNA concentrations in large stream and river ecosystems have received little attention.
We determined the eDNA concentration and abundance/biomass of a stream fish, Plecoglossus altivelis, by conducting a snorkelling survey in the Saba River, Japan. Furthermore, we evaluated the relationship between eDNA concentrations and the estimated abundance/biomass of P. altivelis, and determined its spatial distribution within the river.
Across the three seasons from spring to autumn, we found significant correlations between the eDNA concentration of P. altivelis and its abundance/biomass at study sites within the river. We detected the eDNA at the sites where we found only feeding traces on stones (where P. altivelis was not directly observed), but not at sites without feeding traces. Additionally, we tested the optimal number of qPCR replicates needed for the eDNA evaluation of P. altivelis abundance and biomass; only a small number of replicates was required when the eDNA concentration was high.
Our findings suggest that eDNA analysis is a useful tool to estimate fish abundance/biomass as well as their spatial distribution in rivers.
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.
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.
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.
Aim
Although at least five Tylototriton species were recorded in Thailand, only Tylototriton verrucosus is registered as a protected species under the Wildlife Preservation and Protection Act in ...Thailand. Populations of T. uyenoi are now severely declining, caused by anthropogenic activities. As intense human pressure is having profound effects on the diminishment in T. uyenoi populations, a conservation plan is needed. Information such as the abundance and distribution of a species is necessary. Yet, current established survey methods are either time‐consuming or labour‐intensive. Here, eDNA‐based detection was developed for tracking the presence of the T. uyenoi.
Location
Thailand.
Methods
We target the surveillance of T. uyenoi using eDNA. Primers and a probe specific to T. uyenoi were designed and tested for their specificity and sensitivity. Water samples were collected once a month from August to January at three sites in Doi Suthep and at three extra sites within the range of the species. Three hundred ml samples of water were collected and filtered. Environmental DNA was extracted and then subjected to qPCR assay in an attempt to detect T. uyenoi.
Results
The qPCR assay was found to be species‐specific to T. uyenoi. Both PCR and qPCR did not result in any positive detection of three congeners or other non‐target species. The LOD and the LOQ of the assay were determined by an analysis of the standard curve, and it was found that the LOD and the LOQ were 7.99 and 9.0 copies per reaction, respectively. Environmental DNA was detected in water samples from all sites where T. uyenoi has been known to occur, but detection varied among sites and sampling times. In addition, low amounts of eDNA were detected in three sites with unknown occupancy of newts, but within the species’ range.
Conclusions
Our findings suggest that eDNA survey is a powerful tool for tracking T. uyenoi throughout the year regardless of the sampling site conditions. Similar to other amphibians, T. uyenoi is severely declining due to anthropogenic factors. In order to have an effective conservation plan, knowledge of a species’ distribution is needed. To our knowledge, this was the first study that used eDNA to track the crocodile newt in Thailand.
A lack of reliable tools for determining the presence and distribution of fish species can impede understanding of predator-prey interactions and fishery management. Conventional fish survey methods ...are invasive, and can be size or species selective. Combining netting and electrofishing is a current method used to monitor fish species in Phayao Lake (Kwan Phayao), Thailand. However, the methods are inefficient and time-consuming. Recently, locals who rely on inland fisheries in Kwan Phayao expressed their deep concerns about the giant snakehead, Channa micropeltes (Cuvier, 1831) destroying other fish there. The giant snakehead prey on many commercially important fish species, as the prey species is reduced, negative effects on both biodiversity and the fishery sector could follow. Here, an eDNA-based survey was developed to detect the presence of the giant snakehead. Water samples were collected from six sites within Kwan Phayao and 17 sites in Ing River where water flowed into and out of Kwan Payao. The eDNA of the giant snakehead was detected in water samples from all collection sites using the developed qPCR assay with various concentrations. The eDNA was shown here to be a sensitive and reliable tool for fish surveillance so there will be a better chance for developing an effective management strategy.
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