Gut microbiota induces host midgut immune response and contributes to defending against Bt Cry1Ac protoxin in Ostrinia furnacalis

Insects’ gut microbiota plays essential roles in the development, digestion, immunity, and pathogen resistance of the host. Bacillus thuringiensis (Bt) is recognized for its ability to generate insecticidal protoxins to kill insect pests. However, systematic studies on the interplay between Bt toxins, host insect gut microbiota, and gut immunity are still lacking for Ostrinia furnacalis. Our findings demonstrate that the Bt Cry1Ac protoxin significantly induces the midgut immune responses, causes a dynamic shift in bacterial communities, and increases the total bacterial load within the midgut and hemocoel of O. furnacalis. In addition, the absence of the gut microbiota significantly increased the susceptibility of O. furnacalis larvae to Cry1Ac protoxin. The reintroduction of five intestinal commensal bacterial strains, OfG3 (Enterobacter cancerogenus), OfG4 (Kluyvera sichuanensis), OfG5 (Klebsiella variicola), OfG6 (Klebsiella grimontii), and OfG7 (Citrobacter freundii), markedly reduced the susceptibility of both axenic and normal O. furnacalis larvae to Bt Cry1Ac protoxin. Our study further demonstrated that the loss of the gut microbiota resulted in a significant down-regulated expression of most immune-related genes, and the introduction of five gut commensal bacteria strains (OfG3-OfG7) significantly up-regulated the expression levels of antimicrobial peptide (AMP) genes in the midgut of normal O. furnacalis larvae. These results suggest a critical role for the gut microbiota in modulating host midgut immunity and mitigating the toxicity of Bt Cry1Ac protoxin in O. furnacalis, offering valuable insights for improving Bt resistance management and developing novel biological control strategies for lepidopteran pests.