Chronic carbonate alkalinity exposure impaired the mechanical properties of exoskeleton and the intestinal microbiota of juvenile Macrobrachium rosenbergii

Along with the global expansion of saline-alkali aquaculture, understanding the physiological adaptation of economically important species like Macrobrachium rosenbergii to alkaline stress is essential for sustainable aquaculture production. This study investigated the effects of chronic exposure to carbonate alkalinity (4 and 8 mmol/L) on the mechanical properties of the exoskeleton, chitin metabolism, and the intestinal microbiota of juvenile M. rosenbergii over four weeks. Nanoindentation results revealed that carbonate alkalinity significantly reduced the surface hardness and elastic modulus of both the carapace and abdominal segments, with the most severe mechanical weakening observed at 4 mmol/L. Scanning electron microscopy (SEM) revealed surface erosion and disruption of the organized exoskeletal structure, while energy-dispersive X-ray spectroscopy (EDS) showed a reduction in exoskeletal calcium abundance. Hemolymph calcium concentration also decreased following alkalinity exposure. These structural impairments were closely linked to a dose-dependent decrease in the FTIR-derived relative degree of chitin acetylation accompanied by altered expression of key chitin-metabolism genes, such as CHS2, Chit1, and Chit3. Furthermore, alkalinity stress compromised the internal intestinal physical barrier by causing thinning of the chitin-rich peritrophic membrane (PM). Although alpha diversity indices remained unchanged, the 8 mmol/L treatment showed the clearest adverse microbial signature, including enrichment of potentially opportunistic genera such as Vibrio and Pseudomonas . Redundancy analysis (RDA) further showed significant associations between intestinal Chit1 and Chit3 expression and the relative abundance of Bacteroidetes. Collectively, these results demonstrate that chronic carbonate alkalinity exposure alters exoskeletal mechanical properties, chitin metabolism, intestinal barrier integrity, and gut microbial composition in juvenile M. rosenbergii , with different biological endpoints exhibiting distinct stress-level response patterns.