Beyond whole-organism assays: Compartmentalized multi-biomarkers in earthworm gut for cadmium and pyrene...

Soil pollution poses a growing threat to ecosystems worldwide, underscoring the critical need for bioindicators. Earthworms are recognized sentinels of soil contamination, yet current earthworm biomarker studies predominantly rely on whole-organism analyses, overlooking compartment-specific responses. This study addresses this gap by systematically investigating the heterogeneous distribution and pollutant-induced dynamics of digestive enzymes (e.g., cellulase, protease), antioxidant defenses (e.g., superoxide dismutase, catalase), and microbial functional genes (e.g., ureC, amoA) across gut tissue and gut content of Metaphire guillelmi exposed to cadmium (Cd: 0-20 mg kg^-1) and pyrene (Pye: 0-100 mg kg^-1). Results revealed compartment-specific biomarker responses: cellulase activity in gut content was 2.7-13.4 times higher than in gut tissue. Protease and alkaline phosphatase exhibited hormesis responses under Pye exposure, with protease peaking at 8.73 mg min^-1 g^-1 FW in gut content at 10 mg kg^-1 Pye. Conversely, cadmium exposure reduced protease activity by 26-60% in tissue and 18-30% in gut content. Antioxidant responses also diverged significantly. Gut tissue displayed fluctuating superoxide dismutase/catalase activities, while gut content demonstrated elevated lipid peroxidation under Cd exposure. Microbial functional gene analysis revealed that gut content was enriched in nitrogen-cycling genes (43-53% vs. 33.8-36.8% in bulk soil), with 21 genes (including high-abundance ureC) upregulated specifically under Pye exposure. These findings establish gut content as a critical ecotoxicological compartment and identify its enzymatic profiles, oxidative damage markers, and pollutant-responsive microbial genes as novel candidates for soil biomonitoring. This work advances the vermicompartment-specific biomarkers for early-warning detection in contaminated soils.