NFIL3-dependent regulation of NF-κB pathway mediates alginic oligosaccharide alleviation of nitrite immunotoxicity in Procambarus clarkii

Nitrite (NIT) is a prevalent environmental stressor in intensive aquaculture, known to induce significant oxidative damage and immunosuppression in aquatic species. Alginic oligosaccharide (AOS), a bioactive feed additive, has shown potential in mitigating such stresses, though its underlying mechanisms require further elucidation. This study was designed to investigate the protective effects of dietary AOS against NIT-induced gill damage and immunosuppression in the Procambarus clarkii and to explore the associated molecular pathways. Healthy crayfish were continuously exposed to 15 mg/L sodium nitrite (NIT) and fed diets supplemented with varying concentrations of AOS (0% AOS + NIT, 0.15%, 0.3%, and 0.45%) for 7 days. A multi-faceted analysis was conducted, incorporating histopathology, biochemical assays, transcriptome sequencing (RNA-seq), and gene silencing via RNA interference (RNAi). The results demonstrated that AOS supplementation significantly ameliorated NIT-induced histopathological alterations in gill tissue and effectively suppressed apoptosis. Biochemically, AOS administration enhanced the overall antioxidant capacity of gill tissue, as indicated by increased activities of enzymes such as catalase (CAT) and glutathione peroxidase (GSH-Px), alongside decreased levels of oxidative damage markers including hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA). Concurrently, AOS significantly boosted the activities of key immune-related enzymes, namely acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). Transcriptomic analysis revealed that AOS markedly regulated the expression of genes associated with the NF-κB signaling pathway. Furthermore, RNAi-mediated knockdown of the NFIL3 gene confirmed its pivotal role, as silencing NFIL3 attenuated the antioxidant and immunoenhancing effects conferred by AOS and compromised its protective efficacy on gill tissue integrity. In conclusion, this study demonstrates that dietary AOS supplementation enhances antioxidant defenses and immune function in P. clarkii , thereby alleviating NIT-induced gill injury and immunosuppression, primarily through the modulation of the NFIL3 / NF-κB signaling axis. These findings provide a solid theoretical foundation for the potential application of AOS as a functional feed additive in the sustainable aquaculture of P. clarkii.