Environmental RNA as a transformative tool for aquatic ecosystem health assessment: progress and challenges

• The feasibility exploration of eRNA technology is summarized. • General workflow of eRNA technology is proposed. • Applications of eRNA is wide and has a great potential. • Point out the “mists” of eRNA technology. The assessment of aquatic ecosystem health is undergoing a paradigm shift from traditional physicochemical monitoring to molecular biological monitoring. As an innovative biomonitoring tool, environmental RNA (eRNA) offers advantages such as a short persistence time and correlation with metabolic gene expression, which effectively reduce false-positive detection rates and identify metabolically active communities, providing real-time information on biological assemblages. Compared to environmental DNA (eDNA), eRNA possesses unique diagnostic capabilities for assessing living organisms and their functional states. The use of eRNA technology to obtain real-time transcriptional information can provide physiological insights into species. When combined with eDNA technology, it can achieve a relatively ideal effect while saving human and material resources compared to traditional techniques. Nevertheless, eRNA technology still faces challenges related to feasibility verification, improvement and optimization of detection methods, construction of application frameworks, and promotion of field experiments. Despite its promise, eRNA technology remains in its infancy and lacks comprehensive reviews synthesizing current research progress. To address this gap, we present a systematic analysis of eRNA studies to examine the technical characteristics and practical feasibility of this approach. We further outline standardized eRNA workflows and highlight its diverse applications, including (1) biodiversity monitoring, (2) physiological status assessment, (3) immune response evaluation and pathogen detection, and (4) assessment of environmental stress responses. We also identify unresolved questions (“mists”) around theory, methodology, and application. Finally, we discuss the impacts of these “mists” and propose possible solutions and future research directions. The potential of eRNA extends beyond species detection, revolutionizing aquatic ecosystem health monitoring by facilitating the acquisition of multidimensional biological data. In the future, eRNA technology is expected to be applied across diverse fields, including conservation biology, ecotoxicology, environmental health science, and ecosystem management. However, many obstacles must be overcome before it can be widely adopted in practical applications.