Can carcasses cause environmental nucleic acid pollution? Implications for interpreting environmental RNA/DNA survey results

Environmental nucleic acid (eRNA and eDNA) metabarcoding is a promising tool for biodiversity assessment as it supports low-cost, noninvasive biological surveys. A critical challenge for eRNA/eDNA analyses is accounting for nucleic acid contamination that can compromise the accuracy of biomonitoring results. The primary aim of the current study was to examine carcasses as potential sources of eRNA/eDNA contamination, a contribution that remains poorly understood. Using Japanese medaka (Oryzias latipes), we quantified the detectability and persistence of carcass-derived eRNA/eDNA in water through two complementary experiments: (i) a shedding study comparing eRNA/eDNA release from live and dead individuals and (ii) a decay experiment measuring temperature-dependent degradation from 4 to 34 {degrees}C. Dead fish released detectable amounts of both eDNA and eRNA, indicating that eRNA-based surveys cannot fully eliminate carcass-derived signals. Arrhenius-based analysis showed that eRNA degradation was more temperature-dependent than eDNA degradation. These results suggest that eRNA may improve temporal resolution for recent organismal presence under warm conditions, whereas carcass-derived eRNA may persist under cold conditions and complicate interpretation of positive detections. Overall, eRNA may help reduce carcass-related false positives, particularly in warm conditions, but should be paired with temperature-aware sampling strategies and temporal replication during colder periods.