Proteomic analysis of hemolymph reveals immune response changes in silkworm pupae following treatment with Bacillus subtilis

The silkworm ( Bombyx mori ), a well-established model organism in the order Lepidoptera, is widely used to investigate insect immunity. Currently, our understanding of the silkworm’s innate immunity is primarily based on genomic and transcriptomic studies. To explore potential antimicrobial proteins/peptides (AMPs), this study infected silkworm pupae with the probiotic Bacillus subtilis and collected hemolymph for proteomic analysis. A total of 669 proteins were identified, of which 214 were significantly changed post-infection. GO and KEGG analyses revealed several immune- and metabolism-related processes and pathways, while protein interaction analysis identified clusters associated with immune-related functions as well as clusters associated with protein synthesis. Finally, we mapped the immune pathways of silkworm pupae in response to bacterial infection and found that immune recognition proteins were primarily upregulated in the early stage of infection, whereas immune effector proteins were upregulated in the late stages. This study provides a comprehensive proteomic profile of immune-related proteins in pupal hemolymph and valuable insights for the development and application of AMPs in the silkworm. • 214 hemolymph proteins were significantly changed in B. subtilis -infected silkworm pupae. • Stage-specific upregulation of immune proteins was detected after B. subtilis infection. • Immune and metabolic pathways were coordinately reprogrammed in response to bacteria. • Typical and potential immune-related proteins were screened from silkworm pupae. • Crosstalk among Toll, Imd and JNK pathways was indicated in antibacterial immunity.