Physiological and Proteomic Insights into Melatonin-Mediated Regulation of Copper Toxicity in the Crayfish Procambarus clarkii

Copper (Cu) contamination in aquatic environments induces oxidative stress and structural damage to crustaceans. This study investigated the protective effects and associated mechanisms of exogenous melatonin (MT) against Cu-induced toxicity in Procambarus clarkii using integrated physiological, histopathological, proteomic, and molecular analyses. MT supplementation enhanced antioxidant defense by elevating SOD, CAT, and T-AOC activities, while reducing MDA accumulation, with peak effects observed at 24 h. MT also restored endogenous melatonin levels and regulated phosphatase activity, thereby maintaining immune and metabolic homeostasis. Histopathology showed reduced hepatopancreatic damage, characterized by reduced epithelial vacuolization and preserved basement membrane integrity. Proteomics suggested that MT modulates a multilayered network associated with detoxification, redox balance, and cellular homeostasis. Pathway enrichment showed that Cu exposure dysregulated proteins involved in mitochondrial biogenesis, ABC transporters, membrane trafficking, and apoptosis. MT administration partially counteracted these alterations and was associated with the regulation of glutathione metabolism, as well as reduced enrichment of lysosome- and apoptosis-related pathways. Quantitative RT-PCR results were consistent with the proteomic data. Overall, MT partially alleviated Cu-induced toxicity and was associated with enhanced antioxidant defense, improved cellular homeostasis, and metabolic regulation. Our study provides new molecular insights and suggests its potential application for mitigating metal toxicity in aquaculture.