Polyester wool-based environmental RNA collection enables detection of diel per2 expression in the Malabar grouper...

Environmental RNA (eRNA) analysis has attracted increasing attention as a non-invasive approach for monitoring aquatic organisms because it can provide information on actively expressed genes. However, many existing eRNA sampling methods rely on filtration-based procedures that require specialized materials and substantial sample processing, limiting their practical application in aquaculture settings. In this study, we developed a simple polyester wool-based method for collecting eRNA directly from fish rearing tanks. Using the Malabar grouper (Epinephelus malabaricus) as a model species, we conducted three experiments to evaluate the detectability, persistence, and temporal expression patterns of eRNA collected using this approach. eRNA obtained from rearing water contained detectable transcripts of housekeeping genes and the circadian clock gene per2. In a closed recirculating system, the target transcript remained detectable for up to 8 h after fish removal. Furthermore, diel expression patterns of per2 detected in rearing-water eRNA were broadly similar to those observed in muscle, pituitary, and liver tissues. Heatmap visualization, cross-correlation analysis, and cosinor analysis suggested temporal similarity between aqueous eRNA signals and tissue expression profiles. These findings demonstrate the feasibility of detecting diel clock gene expression using polyester wool-based eRNA collection. The proposed approach provides a simple and non-invasive method for eRNA sampling in aquaculture systems, although further validation across additional genes, species, and culture conditions is required.