Wastewater and warming effects on aquatic invertebrates: Experimental insights into multi-level biodiversity consequences

•Species-specific responses to single and combined stressors were observed.•Metabolome and lipidome of taxa changed in response to warming and pollution.•The lowest total lipid content was observed in multiple stressor treatments.•Phylogenetic diversity of communities decreased in response to stressors.•Stable ecosystem health indicators; swift molecular responses to pressures. Wastewater effluents and global warming affect freshwater ecosystems and impact their crucial biodiversity. Our study aimed at characterizing individual and combined impacts of wastewater effluent and increased water temperature (as one aspect of climate change) on model freshwater communities. We tested the effect of experimental treatments on genetic diversity, survival, body weight, total lipid content, lipidome and metabolome of individual species as well as community composition and phylogenetic diversity. In a 21-day mesocosm experiment we assessed the responses of a simplified freshwater food web comprising of moss and seven species of benthic macroinvertebrate shredders and grazers (mayflies, stoneflies, caddisflies and amphipods) to four treatments in a full factorial design: control, increased water temperature, wastewater and a multiple stressor treatment combining increased temperature and wastewater. Physiological responses varied among taxa, with species-specific sensitivities observed in survival and lipid content. The lowest total lipid content was observed in caddisflies and a mayfly subjected to multiple stressor treatment. The effects of stressors were reflected in the altered metabolic pathways and lipid metabolism of the individual taxa, with differential treatment effects also observed between taxa. A notable decrease in phylogenetic diversity was observed across all experimental communities. Gammarus fossarum demonstrated a high susceptibility to environmental stressors at the genetic level. Hence, while commonly used indicators of ecosystem health (e.g. community composition) remained stable, molecular indicators (e.g. phylogenetic diversity, metabolome and lipidome) responded readily to experimental treatments. These findings underscore the vulnerability of macroinvertebrates to environmental stressors, even over relatively short exposure periods. They highlight the importance of molecular indicators in detecting immediate ecological impacts, offering valuable information for conservation strategies and understanding the ecological consequences in freshwater