Mitigating LPS-induced stress in Chinese mitten crab (Eriocheir sinensis) with P4' peptide-bearing Bacillus subtilis

The Chinese mitten crab (Eriocheir sinensis) is an important component in Chinese aquaculture. Due to its lacking adaptive immune system as a crustacean, it exhibits poor tolerance to environmental stresses, particularly the deleterious impact of lipopolysaccharide (LPS) from pathogenic bacteria during E. sinensis culture. In a previous study, we isolated LGSPDVIVIR (cmP4) peptide from cottonseed meal hydrolysate, having excellent antioxidant and immune-enhancing properties in vitro. Expressing this peptide abundantly as a tandem (a tandem of five cmP4 peptides, cmP4') using the Bacillus subtilis expression system, we aimed to investigate the effects of incorporating recombinant B. subtilis into diets on growth performance, acute oxidative stress, and hepatopancreatic injury induced by LPS injection in E. sinensis. Crabs were cultured for a period of 12 weeks on three diets: basal diet, basal diet supplemented with 10⁹ CFU/kg of unmodified B. subtilis, and recombinant B. subtilis, respectively. Results indicated that both B. subtilis species improved the growth performance of E. sinensis. Subsequent challenge with LPS at 400 μg/kg body weight for 6 h revealed that both B. subtilis groups exhibited improved antioxidant capacity, decreased oxidative stress indexes in hemolymph, enhanced mitochondrial membrane potential, and reduced hepatopancreatic damage compared to the single LPS-treated group. Notably, the recombinant B. subtilis had better performance, demonstrating superior effects. Specifically, compared with the single LPS-treated group, the oxidative stress indexes, mitochondrial membrane potential, and apoptosis-related gene expression in both B. subtilis groups followed a similar trend. However, the recombinant B. subtilis group displayed greater absolute changes in these indexes, a finding further supported by histopathological observations of the hepatopancreas. In conclusion, this study provides useful information for promoting the application of plant protein by-products in aquafeeds, promoting antimicrobial-free aquaculture practices for E. sinensis.