Species-Specific molecular mechanisms of DEHP toxicity in earthworms revealed by integrated transcriptomics and metabolomics.

Di(2-ethylhexyl) phthalate (DEHP) is pervasive in agroecosystems; however, species-specific toxic mechanisms in soil detritivores remain unresolved. Here, we investigated cross-level mechanisms of DEHP toxicity in two earthworm's species, Eisenia foetida and Metaphire guillelmi, using an integrative framework combining histopathology, residue analysis, transcriptomics and metabolomics. DEHP induced significant tissue injury in both species, with more pronounced lesions observed in M. guillelmi. Although M. guillelmi exhibited slightly lower DEHP residues than E. foetida, it showed stronger molecular disturbances, suggesting higher sensitivity independent of body burden. These differences likely reflect distinct ecological strategies influencing bioavailability and physiological responses. Transcriptomic analysis identified 2673 differentially expressed genes in M. guillelmi compared with only 138 in E. foetida, demonstrating markedly divergent transcriptional plasticity between the two species. Metabolomic profiling similarly revealed broader metabolic disruption in M. guillelmi (138 differentially expressed metabolites, (DMEs)) compared with E. foetida (30 DEMs). Integrated pathway analysis indicated that DEHP perturbed arachidonic acid metabolism and KEGG-annotated oxytocin signaling in E. foetida, whereas lipid, amino-acid, and purine/pyrimidine metabolism were primarily affected in M. guillelmi. These alterations were consistent with oxidative stress, immune imbalance, and disrupted energy metabolism. Convergent evidence from omics and histological analyses supports species-specific mechanisms underlying tissue injury, with M. guillelmi being more susceptible at the molecular level. Overall, these findings provide mechanistic biomarkers and adverse outcome pathway-based evidence for effect-driven soil health assessment and species-sensitive ecological risk evaluation within a One Health context.