Enhanced fish detection sensitivity in aquatic systems using environmental RNA

Abstract Environmental RNA (eRNA) can potentially augment the biological monitoring ability of the established environmental DNA (eDNA) application in conservation. Despite faster degradation, environmental RNA could offer unique advantages: it may help reduce the legacy signal compared to eDNA, provide insights into physiological status through gene transcription and offer higher detection sensitivity due to the inherent amplification of certain genes in response to physiology and the environmental context. This study investigates the practical application and sensitivity of eRNA for detecting fish in the wild: an endangered freshwater fish in a seasonal stream throughout the year and a pelagic fish of commercial interest in the ocean. We developed attainable field sampling and laboratory protocols for eRNA preservation and extraction, addressing the challenges of its natural instability. Then, we compared the detection sensitivity of two mitochondrial genes using species‐specific digital PCR (dPCR) assays: the cytb gene (eDNA)/ cytb transcript (eRNA) and the 12S rRNA gene (eDNA)/12S rRNA product (eRNA), The results showed higher concentrations of 12S rRNA (eRNA), suggesting its superior utility for detecting low‐abundance organisms while providing empirical information regarding the amount of an eRNA transcript from the primary cellular pathway (cytochrome b). Importantly, in the freshwater system, eRNA and eDNA concentrations followed similar patterns reflecting the variation in hydrological conditions that affected the concentration of nucleic acids in the water. These findings highlight the importance of marker choice in eRNA studies, with a ribosomal gene offering enhanced sensitivity compared to its eDNA. This research provides insights into the temporal and spatial dynamics, sampling strategies, and molecular targets for eRNA‐based biomonitoring. It demonstrates the potential of eRNA to improve the detection of endangered species and inform conservation efforts by providing a more accurate snapshot of extant biological communities and paving the road for the full potential of eRNA to study the physiological status of target organisms.