ORIGINAL ABSTRACT

Glyphosate is a widely used herbicide with increasing concern regarding its non-target impacts in coastal ecosystems and mariculture species. Here, we profiled acute physiological stress signatures of waterborne glyphosate exposure in the sea cucumber Apostichopus japonicus, integrating measured exposure concentrations, tissue residues, digestive and oxidative/innate immune biomarkers, and gut microbiota. After 24 h exposure, measured waterborne glyphosate confirmed the intended gradient (0.09 ± 0.02, 1.26 ± 0.09, and 4.49 ± 1.12 mg/L for low-, medium-, and high-dose treatments, respectively), and overt stress phenotypes with mortality occurred only at the high dose (36.67%), enabling separation of high-dose survivors (HS) and high-dose dead (HD) for downstream analyses. Tissue measurements showed low/background levels in controls, with compartment-specific distribution: the respiratory tree exhibited higher burdens at the medium dose, whereas coelomic fluid showed the highest burdens in HS at the 24 h endpoint. Functionally, most intestinal digestive enzymes were unchanged, but trypsin activity was consistently suppressed across exposed groups (p < 0.05). In coelomic fluid, oxidative stress responses were evident, with elevated MDA (L and M), reduced CAT (L, M, and HS), and reduced GSH-PX in HS (all p < 0.05), while SOD, GR, and lysozyme showed no significant changes. Gene sequencing of 16S rRNA (n = 3 per group) revealed significant shifts in community diversity/evenness (Shannon p = 0.0497; Simpson p = 0.0484) and beta diversity (PCo1 = 30.08%, PCo2 = 26.30%; PERMANOVA F = 1.816, p = 0.008), with LEfSe indicating discriminative taxa associated with exposure/outcomes. Collectively, these multi-level endpoints define an acute glyphosate stress signature in A. japonicus, linking internal dose distribution to oxidative disruption, impaired intestinal proteolysis, and microbiome restructuring.