Glyphosate (Gly), the world's most widely used herbicide in agriculture, can poison the red swamp crayfish, Procambarus clarkii, via spray drift and surface runoff into surface waters. However, there is a paucity of research on the mechanisms that affect crayfish tolerance to Gly at typical environmental concentrations. To address this research gap, we investigated the effects of Gly stress (0, 6, 12, 24, and 72 h) at different concentrations (0, 1.20, 3.60, 7.20, and 10.80 mg·L-1) on antioxidant enzyme activity in crayfish hepatopancreas. Furthermore, we analyzed the species' tolerance mechanism to Gly exposure at a typical environmental concentration (3.60 mg·L-1) based on integrative transcriptomics, metabolomics, and proteomics. The Gly concentration and exposure time affected the crayfish's antioxidant system, and interacted with each other (P < 0.01). Gly concentrations higher than 7.20 mg·L-1 and exposure times longer than 48 h caused oxidative stress. When the Gly concentrations were lower than 3.60 mg·L-1, crayfish tolerated Gly exposure within 72 h by self-regulating superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA). A multi-omics analysis revealed that crayfish upregulated the expression of amino acid metabolites (such as glutamate, proline, and lysine) and amino acid transformation-related genes (such as GlnA and P5CS) to tolerate Gly stress by enhancing the antioxidant capacity, ammonia‑nitrogen regulation, and energy supply of the organism. Metallothionein and polyadenylate-binding proteins, which are potential markers of Gly exposure, crucially influenced crayfish tolerance to Gly by synthesizing metalloenzymes and scavenging reactive oxygen species. This study revealed the Gly tolerance mechanism in crayfish and can provide a theoretical reference for commercial eco-farming in rice-crayfish integrated aquaculture systems.
