Comparative Decay Dynamics and Detectability of eDNA and eRNA in Connected and Isolated Freshwater Mesocosms Using Digital PCR.

Efficient use of environmental nucleic acids (eNAs) in freshwater biodiversity monitoring requires understanding their degradation and detectability in interconnected ecosystems. We employed a novel field-scale assay to compare environmental DNA (eDNA) and environmental RNA (eRNA) decay rates and detectability across four genetic markers (16S, 18S, COI and LDHA) in connected and isolated 1000-L mesocosms containing natural planktonic assemblages. This design provides ecologically relevant and complex settings to assess how connectivity influences the detectability of eNA over time. Isolated and head mesocosms were spiked with eNAs from cultured Daphnia pulex, absent from the water source, while downstream mesocosms received eNAs via unidirectional water transfers. Using digital PCR (dPCR), we captured fine-scale temporal patterns across mitochondrial and nuclear markers and transcript types (mRNA and rRNA), an approach rarely combined in previous research. eRNA degraded significantly faster than eDNA across markers and mesocosm types. Among RNA types, mRNA (COI, LDHA) degraded faster than rRNA (16S, 18S). eRNA followed a uniform monophasic decay pattern, whereas eDNA displayed biphasic decay for nuclear markers and monophasic decay for mitochondrial markers. eNA decay rates in this field-relevant mesocosm network exceeded those from laboratory scale. While decay rates remained consistent across networks, detectability declined with dilution. Even after a 10,000-fold dilution, both eNAs were detected in terminal mesocosms, demonstrating effective transport across the network. Although RNA degrades rapidly, high detectability was achieved across diverse dilutions using dPCR, highlighting eRNA's potential for detecting active biological communities in freshwater systems.