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Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future. [ABSTRACT FROM AUTHOR]

Environmental DNA and RNA (eDNA and eRNA; collectively eNA) analyses have the potential for non-invasive and cost-efficient biomonitoring compared with traditional capture-based surveys. Although various types of eNA particles, including not only mitochondrial eDNA but also nuclear eDNA and their transcripts, are present in the water, performances of eNA detection and quantification have not yet been evaluated sufficiently across multiple mitochondrial and nuclear genes. We conducted a tank experiment with ayu (Plecoglossus altivelis) to compare the detection sensitivity, yields per water sample, and quantification variability between replicates of each type of eNAs. The assay targeting the multi-copy nuclear gene exhibited a higher sensitivity than the assay targeting the mitochondrial gene, and both the target eDNA and eRNA concentrations per water sample were higher for the nuclear gene. On the contrary, variation in eRNA quantifications per sample does not necessarily correspond to that in eDNA, and the intra-sample quantification variability (represented as the CVs between PCR replicates) tended to be larger for eRNA than eDNA. Our results suggested that, even if suitable to the sensitive detection of species occurrence, the use of eRNA particularly derived from multi-copy nuclear gene may not be necessarily appropriate for the reliable assessment of species abundance. The findings in this study would help optimize eNA analyses for making biomonitoring and stock assessment in aquatic environments more efficient and reliable. [ABSTRACT FROM AUTHOR]

Japanese giant salamander (Andrias japonicus) is one of the largest amphibian species in the world and an iconic species in Japan. However, as its distribution has recently declined across the country, rapid and extensive monitoring of the distribution is urgently needed for its efficient conservation. Here, we used environmental DNA (eDNA) analysis to assess the Japanese giant salamander's distribution in western Japan and, for that purpose, we collected 410 water samples from 12 rivers. We then developed a new eDNA assay for multi-copy nuclear DNA (nuDNA) of the giant salamander and compared the eDNA detectability of the nuDNA marker with that of a previous mitochondrial DNA (mtDNA) marker. Throughout the survey, we detected target eDNA from 162 water samples using either of the markers, which generally corresponded to the known natural distribution of the species. Additionally, the use of the nuDNA marker allowed for higher detection rate of target eDNA than the mtDNA marker. Moreover, the detection rate of target eDNA decreased substantially in water samples with higher conductivity and also partly in those with higher pH, suggesting their negative impacts on the salamander's ecology. Our results demonstrated that eDNA analysis with multi-copy nuDNA marker is highly useful for efficient and sensitive surveillance of Japanese giant salamander's distribution. Our study provided the methodology for efficiently monitoring the Japanese giant salamander's distribution via eDNA analysis and facilitating conservation activities for them. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis can facilitate surveillance of invasive aquatic plant's distribution. However, it is still questionable how accurately eDNA concentration reflects and predicts aquatic plant abundance. Although previous studies investigated relationships between aquatic plant eDNA concentration and their abundance in freshwater environments, the conclusions were not consistent between the studies. Here, a systematic literature search was conducted to summarize previous findings and meta-analyze the relationships between eDNA concentration and aquatic plant abundance. Hierarchical random-effect meta-analyses showed significant positive, but weak, relationships between them (r = 0.45 in average), meaning that eDNA concentration accounted for approximately 20% of the observed variation in plant abundance. Additionally, the relationships tended to be rather stronger in the field than in the laboratory environments, which may be attributed to limited quantifiability and species specificity of plant coverage as abundance metrics, heterogeneous dispersion of plant eDNA particles, and phenology-dependent eDNA production from plant bodies. The finding indicates that aquatic plant eDNA particles could reflect more regional (but not local) abundance, and thus, eDNA-based abundance estimation might be more appropriate at a broader scale (i.e., lower spatial resolution). Further studies focusing on various environmental parameters and plant ecotypes are needed to clarify the relationship. [ABSTRACT FROM AUTHOR]

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (β) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future. [ABSTRACT FROM AUTHOR]

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis can promote efficient ecosystem monitoring and resource management. However, limited knowledge of the factors affecting the relationship between eDNA concentration and organism abundance causes uncertainty in relative abundance estimates based on eDNA concentration. Pooling of data points obtained from multiple locations within a site has been used to mitigate intra-site variation in eDNA and abundance estimates, but decreases the sample size used for estimating the relationship. I here assessed how the pooling of intra-site measurements of eDNA concentration and organism abundance impacted the reliability of the correlative relationship between eDNA concentration and organism abundance. Mathematical models were developed to simulate measurements of eDNA concentrations and organism abundances from multiple locations in a given survey site, and the CVs (coefficient of variability) of the correlations were compared depending on whether data points from different locations were individually treated or pooled. Although the mean and median values of the correlation coefficients were similar between the scenarios, the CVs of the simulated correlations were substantially higher under the pooled scenario than the individual scenario. Additionally, I re-analyzed two empirical studies conducted in lakes, both showing higher CVs of the correlations by pooling intra-site measurements. This study suggests that it would make eDNA-based abundance estimation more reliable and reproducible to individually analyze target eDNA concentrations and organism abundance estimates. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis allows cost-effective and non-destructive biomonitoring with a high detection sensitivity in terrestrial and aquatic environments. However, the eDNA results can sometimes include false-positive inferences of target organisms owing to the detection of aged eDNA that has long since been released from the individual and is more likely to be detected at a site further away from its source. In order to address the issue, this manuscript focuses on the state of eDNA, proposing new methodologies to estimate the age of eDNA: (1) DNA damage rate, (2) eDNA particle size distribution, and (3) viable cell-derived eDNA. In addition, the manuscript also focuses on the shorter persistence of environmental RNA (eRNA) compared with eDNA, highlighting the application of eRNA and environmental nucleic acid ratio for assessing the age of the genetic materials in water. Although substantial further research is essential to support the feasibility of these methodologies, incorporating time-scale information into eDNA analysis would update current eDNA analysis, improve the accuracy and reliability of eDNA-based monitoring, and further refine eDNA analysis as a useful monitoring tool in ecology, fisheries and various environmental sciences. [ABSTRACT FROM AUTHOR]

Precise abundance estimation via environmental DNA (eDNA) analysis is challenging because of myriad effects of biotic/abiotic factors on eDNA persistence in water. However, it remains unclarified how the relationship between eDNA concentration and species abundance is influenced by environmental conditions. Warm temperature can accelerate eDNA degradation and its turnover in water, which may make eDNA signals fresher and strengthen the relationship. Here, mathematical models were present to simulate the effect of temperature-dependent eDNA degradation, modeled based on the previous meta-analysis, on the relationship between eDNA concentration and species abundance under different temperature and eDNA production scenarios. The R2 values tended to be higher at warmer temperatures regardless of eDNA production scenarios, although differences in R2 between temperatures were relatively small. Additionally, the R2 values were higher for a smaller variance of eDNA production rate. Furthermore, previous studies regarding the relationship between fish eDNA concentration and their abundance were meta-analyzed and correlation coefficients of the relationship were significantly higher for warmer temperatures. The findings indicated the importance of taking water temperatures into account for better abundance estimation via eDNA, although further technical consideration in the modelling framework was needed. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) analysis is a promising tool for the sensitive and effective monitoring of species distribution and abundance. Traditional eDNA analysis has targeted mitochondrial DNA (mtDNA) fragments due to their abundance in cells; however, the quantification may vary depending on cell type and physiology. Conversely, some recent eDNA studies have targeted multi-copy nuclear DNA (nuDNA) fragments, such as ribosomal RNA genes, in water, and reported a higher detectability and more rapid degradation than mitochondrial eDNA (mt-eDNA). These properties suggest that nuclear eDNA (nu-eDNA) may be useful for the accurate estimation of species abundance relative to mt-eDNA, but which remains unclear. In this study, we compiled previous studies and re-analyzed the relationships between mt- and nu-eDNA concentration and species abundance by comparing the R2 values of the linear regression. We then performed an aquarium experiment using zebrafish (Danio rerio) to compare the relationships across genetic regions, including single-copy nuDNA. We found more accurate relationships between multi-copy nu-eDNA and species abundance than mt-eDNA in these datasets, although the difference was not significant upon weighted-averaging the R2 values. Moreover, we compared the decay rate constants of zebrafish eDNA across genetic regions and found that multi-copy nu-eDNA degraded faster than mt-eDNA under pH 7, implying a quick turnover of multi-copy nu-eDNA in the field. Although further empirical studies of nu-eDNA applications are necessary to support our findings, this study provides the groundwork for improving the estimation accuracy of species abundance via eDNA analysis. [ABSTRACT FROM AUTHOR]