Environmental <scp>RNA</scp> ‐Based Metatranscriptomics as a Novel Biomonitoring Tool: A Case Study of Glyphosate‐Based Herbicide Effects on Freshwater Eukaryotic Communities

Traditional morphology- and molecular-based biodiversity surveys provide essential information on species composition and diversity, but they rarely provide information about the physiological states of organisms, which are key indicators of ecosystem health. Environmental RNA (eRNA) has the potential to significantly enhance biomonitoring by providing insights beyond species detection. Recent studies suggest that extra-organismal RNA released into the environment could help identify differentially expressed genes of single species. However, the feasibility of eRNA-based metatranscriptomics on complex environmental samples, containing both extra-organismal and organismal eukaryotic RNA, remains untested due to numerous experimental and analytical challenges. In this study, we explored the potential of eRNA-based metatranscriptomics, enriched for eukaryotes, as a tool to monitor environmental stress. We used outdoor mesocosms to examine the acute effects of a glyphosate-based herbicide (GBH) on gene transcription across diverse freshwater eukaryotic taxa. Our metatranscriptomics data revealed diverse eukaryotic taxa spanning multiple trophic levels, including phytoplankton, zooplankton, ciliates, and aquatic insects. GBH treatment significantly altered the relative transcript abundances of most eukaryotic classes, with longer-lived taxa demonstrating greater tolerance compared to shorter-lived taxa. Differential expression analysis showed more gene downregulation than upregulation in response to GBH, likely due to its acute toxicity. Many differentially expressed genes were involved in molecular pathways associated with responses to GBH exposure, such as oxidative stress response and detoxification. Our results demonstrate that eRNA-based metatranscriptomics captures transcriptional signals from diverse aquatic eukaryotic taxa, providing insights into functional gene expression. As such, its application to support environmental monitoring of aquatic ecosystems warrants further exploration.