Environmental DNA (eDNA) metabarcoding is widely used for species analysis, while the use of environmental RNA (eRNA) metabarcoding is more limited. We conducted comparative eDNA/eRNA metabarcoding of the algae and arthropods (aquatic insects) in water samples from Naka River, Japan, to evaluate their potential for biological monitoring and water quality assessment. Both methods detected various algae and arthropod species; however, their compositions were remarkably different from those in traditional field surveys (TFSs), indicating low sensitivity. For algae, the species composition derived from eDNA and eRNA metabarcoding was equivalent. While TFSs focus on attached algae, metabarcoding analysis theoretically detects both planktonic and attached algae. A recently expanded genomic database for aquatic insects significantly contributed to the sensitivity and positive predictivity for arthropods. While the sensitivity of eRNA was lower than that of eDNA, the positive predictivity of eRNA was higher. The eRNA of terrestrial arthropods indicated extremely high or low read numbers when compared with eDNA, suggesting that eRNA could be an effective indicator of false positives. Arthropod and algae eDNA/eRNA metabarcoding analysis enabled water quality estimates from TFSs. The eRNA of algae and arthropods could thus be used to evaluate biodiversity and water quality and provide insights from ecological surveys.

The utility of environmental RNA (eRNA) in capturing biological responses to stresses has been discussed previously; however, the limited number of genes detected remains a significant hindrance to its widespread implementation. Here, we investigated the potential of eRNA to assess the health status of Japanese medaka fish exposed to linear alkylbenzene sulfonate. Analyzing eRNA and organismal RNA (oRNA) in aquarium water within 12 h, we achieved high mapping rates and 10 times more differentially expressed genes than previously reported. This advancement has facilitated the previously unattainable capability of gene ontology (GO) analysis. The GO analysis revealed that eRNA can detect nuclear genes associated with cellular components and reflect cumulative gene expression signatures over time, while oRNA provided short-term gene expression signatures in biological process. Moreover, eRNA exhibited high sensitivity in responding to genes associated with sphingolipid and ceramide biosynthesis, which are involved in inflammatory responses possibly originating from impaired cells. This finding aligns with the observations made in oRNA. In conclusion, eRNA-sequencing (eRNA-seq) using aquarium water emerges as a valuable high sensitivity tool for analyzing physiological stress. The findings of this study lay the foundation for further development of eRNA-seq technologies.

Metabarcoding analysis using fish environmental RNA (eRNA) has demonstrated higher performance compared to that using fish environmental DNA (eDNA) in the Naka River in Japan. However, the results from representative sampling sites were insufficient to estimate the diversity of organisms in the entire river. Therefore, we validated the evaluation performance using additional traditional field survey (TFS) data. Data from seven traditional field surveys on fish, covering the area near the sampling points where eDNA/eRNA metabarcoding analyses were conducted, were obtained. Ten fish species were newly identified, with nine determined using the eDNA and eRNA metabarcoding analyses. Performance was evaluated in terms of both positive predictive value (PPV) and sensitivity. Except for species specifically detected only near the estuary, the additional use of field survey data increased the PPV from 26.5% to 36.3% for eDNA and from 43.6% to 57.7% for eRNA. After combining the results of multiple surveys, the sensitivities for eDNA and eRNA increased from 52.4% to 67.4% and from 57.1% to 69.8%, respectively. The increase in PPV was more pronounced for eRNA than for eDNA, indicating that eRNA does not decrease PPV when multiple datasets are merged. The superior performance of eRNA compared to eDNA suggests that eRNA metabarcoding analysis is valuable for accurately conducting ecological surveys and biodiversity assessments and may reduce the labor required for monitoring the entire river by optimizing sampling points for metabarcoding analysis.

To achieve the current Nature Positive goals, it is important to identify the effects of chemicals on ecosystems and strategically reduce their risks. We therefore estimated the safe concentration (SC) of linear alkylbenzene sulfonate (LAS) by investigating the relationship between benthic communities detected via environmental RNA (eRNA) and environmental DNA (eDNA) metabarcoding and the water quality in a river contaminated with LAS. Non-metric multidimensional scaling of benthic communities detected via eRNA indicated significant differences between contaminated and non-contaminated sites, and redundancy analysis showed significant correlation between the benthic communities and LAS concentration. These results demonstrate that eRNA is more sensitive than eDNA for capturing the community changes that result from LAS contamination. No significant differences were observed in biodiversity indices between the highest LAS concentration site and non-contaminated site; thus, the SC was estimated at >47 µg/L. The results suggest that, although hazard assessment using the assessment factor (AF) leads to conservative estimation of the effects of chemicals because the predicted no-effect concentration in previous studies is lower than the SC, hazard assessment based on the response of biological communities using eRNA metabarcoding can refine such ecological assessments, developing the environmental management standards to achieve nature-positive goals.

The use of environmental DNA (eDNA) for molecular ecological surveys has become widespread in ecological assessments of different water environments, including river, coastal, and marine areas. Considering potential contamination by environmental nucleic acids is crucial when interpreting these results. Research on fish ghosts, i.e., false positives generated by eDNA present in fish feed, remains limited, we examined the fish species present in the feed that may influence surveys near aquaculture facilities and fish processing plants using metabarcoding analysis of eDNA/RNA. Additionally, we simultaneously assessed whether the fish species present in the feed used for aquaculture in a heavily cultivated bay could be detected via metabarcoding analysis. Thus, we evaluated whether fish species present in the feed could be detected in ecological surveys in the marine environment. Overall, 51 fish species (DNA: 46, RNA: 31) were detected in 3 fish feed types. The detected species in DNA were more than those in RNA. Furthermore, the common species that were detected in feed and water samples in the bay were remarkably higher in eDNA than in eRNA. Moreover, the number of species detected in the eDNA tended to be maintained at a greater distance from the fishpond where they originated than those detected in the eRNA. Thus, eRNA is useful for fish ecosystem surveys in bays contaminated by fish feed owing to the lower RNA content in the samples than DNA and because it is not easily detected and diffused in the environment.

The symposium “The New Era Shaped by Environmental Genome Monitoring,” held in December 2024 by the Japanese Environmental Mutagen and Genome Society (JEMS), aimed to explore the interdisciplinary collaborations that are essential for the development of new scopes in environmental genome monitoring. This event highlighted the necessity of integrating mutagenicity research with ecological assessments to enhance public health and biodiversity conservation. Presentations focused on the evolving landscape of environmental genomics, including metagenomic analyses for antibiotic resistance, viral genomic surveillance in wastewater, and innovations in noninvasive biodiversity and stress monitoring through environmental DNA and RNA. This report summarizes the key discussions and presentations from the symposium, underscoring the critical role of environmental genome monitoring in shaping future safety research.