Fish environmental RNA sequencing sensitively captures accumulative stress responses through short-term aquarium sampling

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.