Teruhiko Takahara

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.

Critical considerations for the application of environmental DNA methods to detect aquatic species

Summary Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed. Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms. Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA. We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

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.

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.

Trophic Position and Metabolic Rate Predict the Long-Term Decay Process of Radioactive Cesium in Fish: A Meta-Analysis

Understanding the long-term behavior of radionuclides in organisms is important for estimating possible associated risks to human beings and ecosystems. As radioactive cesium (137Cs) can be accumulated in organisms and has a long physical half-life, it is very important to understand its long-term decay in organisms; however, the underlying mechanisms determining the decay process are little known. We performed a meta-analysis to collect published data on the long-term 137Cs decay process in fish species to estimate biological (metabolic rate) and ecological (trophic position, habitat, and diet type) influences on this process. From the linear mixed models, we found that 1) trophic position could predict the day of maximum 137Cs activity concentration in fish; and 2) the metabolic rate of the fish species and environmental water temperature could predict ecological half-lives and decay rates for fish species. These findings revealed that ecological and biological traits are important to predict the long-term decay process of 137Cs activity concentration in fish.

Stress response to daily temperature fluctuations in common carp, Cyprinus carpio L.

The littoral zone of lakes and lagoons is often used by fish for feeding or reproduction. However, the large changes in temperature that are typical of natural environments, including the littoral zone, represent a potential stressor for fish. Despite the importance of this habitat, little is known about the effect of daily temperature fluctuations on the stress responses of fish. We monitored daily temperature changes in the near-shore and offshore regions of a natural lagoon between May and July 2008–2010. We observed large temperature fluctuations more frequently in the near-shore zone than the offshore zone. We then exposed common carp (Cyprinus carpio) to a temperature regime similar to that observed in the near-shore zone and measured the levels of cortisol released into the water. The rate of cortisol release increased when carp were exposed to an increase in temperature of ~0.6°C/h over a 5-h period. Conversely, there was no change in the rate of release when temperatures decreased. Our results highlight the importance of maintaining high temporal resolution when evaluating the stress response to daily fluctuations temperature.

Benefit of suites of defensive behavior induced by predator chemical cues on anuran tadpoles, Hyla japonica

When predator chemical cues are present, low activity of prey is a commonly seen defensive behavior. However, few studies have explored the functional implications of the defensive behaviors and, thus, elucidated the possible linkages between behavioral responses and its consequences. In this study, we experimentally investigated how behavioral responses of Hyla japonica tadpoles to predator chemical cues affect vulnerability to a dragonfly nymph Anax parthenope julius. The frequency of tadpoles attacked by dragonfly nymphs was lower with chemical cues of predator was present than without chemical cues, and most of attacks occurred when tadpoles were mobile. When tadpoles were exposed to chemical cues, on the other hand, their swimming speed was quicker and swimming distance was longer, respectively, and the rates of being approached of the swimming tadpoles by dragonfly nymph was lower than those not exposed to chemical cues. We found that the tadpoles are induced by predator chemical cues not only to generally lower activity but also to swim in bursts as additional behavior and that the suite of their behavioral responses reduce the vulnerability against dragonfly nymph. Tadpoles can receive information about the predation risks by chemical cues and adjust their defensive behavior accordingly.

Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio

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.

Temporal changes in vertical distribution of 137Cs in litter and soils in mixed deciduous forests in Fukushima, Japan

ABSTRACT Downward migration of 137Cs in soils was studied in three mixed deciduous forests c.a. 40 km northwest of the Fukushima Daiichi Nuclear power plant (FDNPP), Japan. We selected three different types of forest regarding to environmental condition such as slope inclinations and snow coverage conditions at the time of deposition. We examined temporal changes in the vertical distribution of 137Cs from litter layers to 10 cm soil depth for two years (2.3 to 4.3 years after the FDNPP accident in 2011). At all three study sites, the 137Cs in the litter layer had largely migrated to surface soil by 2013. After 2014, about 80% of the 137Cs in forest soils (litter layer to 10 cm soil depth) remained within 0–5 cm soil layer. The vertical distribution had not changed substantially since 2014, suggesting that changes to the downward migration rates of 137Cs in soils drastically decreased with time. In addition, small amounts of migrating 137Cs could not be detected by the present method because there was a large spatial variation in the distribution of soil 137Cs. The results showed similar patterns of soil 137Cs distribution among the three study sites although there were differences in the environmental conditions.