Insights into tolerance mechanisms of earthworms (Eisenia fetida) in copper-contaminated soils by integrating multi-omics analyses

Earthworms can resist high levels of soil copper (Cu) contamination and play an essential role in absorbing them effectively. However, the molecular mechanisms underlying Cu tolerance in earthworms are poorly understood. To address this research gap, we studied alterations of Eisenia fetida in antioxidant enzymes, gut microbiota, metabolites, and genes under varying levels of Cu exposure soils (0, 67.58, 168.96, 337.92 mg/kg). Our results revealed a reduction in antioxidant enzyme activities across all treatment groups, indicating an adaptive response to alleviate Cu -induced oxidative stress. Analysis of gut microbiota revealed a significant increase in the abundance of bacteria associated with nutrient uptake and Cu 2 + excretion under Cu stress. Furthermore, metabolomic analysis discovered an increase in certain metabolites associated with energy metabolism, such as pyruvic acid, L-malic acid, and fumaric acid, as Cu concentration escalated. These results suggested that enhanced energy supply contributes to the elevated tolerance of E. fetida towards Cu. Additionally, transcriptome analysis not only identified crucial detoxification genes ( Hsp70 , CTSL , GST , CHAC , and GCLC ), but also confirmed the critical role of glutathione metabolism as a key pathway in E. fetida Cu detoxification processes. These findings provide a new perspective on the molecular mechanisms of Cu tolerance in earthworms.