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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Environmental RNA (eRNA) analysis is expected to infer species' physiological information (health status, developmental stage, and environmental stress response) and their distribution and composition more correctly than environmental DNA (eDNA) analysis. With the prospect of such eRNA applications, there is an increasing need for technological development for efficient eRNA detection because of its physicochemical instability. The present study conducted a series of aquarium experiments using zebrafish (Danio rerio) and validated the methodologies for capture, preservation, and extraction of eRNA in a water sample. In the eRNA extraction experiment, an approximately 1.5-fold increase in lysis buffer volume resulted in a more than sixfold increase in target eRNA concentration. In the eRNA capture experiment, although GF/F and GF/A filters yielded similar eRNA concentrations, a GF/A filter may be capable of passing through more volume of water samples and consequently collecting more eRNA particles, given the time required for water filtration. In the eRNA preservation experiment, the use of RNA stabilization reagent (RNAlater) allowed for stably preserving target eRNA on a filter sample at - 20 and even 4 °C for 6 days at least. Altogether, the findings enable the improvement of eRNA availability from the field and easily preserve eRNA samples without deep-freezing, which will contribute to the refinement of eRNA analysis for biological and physiological monitoring in aquatic ecosystems., (© 2023. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)