A Multidisciplinary Integrated Approach for the Identification and Characterization of the AMP Profile in Hermetia illucens Hemolymph

The growing threat of antimicrobial resistance necessitates alternative strategies to conventional antibiotics. Insects represent a promising source of antimicrobial peptides (AMPs) due to their potent innate immune responses. In this study, we investigated the hemolymph peptide extracts from Hermetia illucens larvae as a bioresource of infection-induced AMPs. Larvae were challenged with Gram-negative (Escherichia coli) and Gram-positive (Micrococcus flavus) bacteria, and hemolymph-derived peptides were extracted and fractionated by RP-HPLC. Peptide fractions were screened in vitro by agar diffusion assay against a panel of pathogenic strains. While extracts from uninfected larvae were essentially inactive, fractions 9–13 from infected larvae showed broad-spectrum antibacterial activity, including against multidrug-resistant clinical isolates such as carbapenem-resistant Klebsiella pneumoniae, suggesting an inducible immune response. Fractions were further analyzed by SDS-PAGE and LC-MS/MS to identify candidate AMPs, while shotgun proteomics on unfractionated extracts enabled quantitative comparison among conditions. Proteomics raw data are available via ProteomeXchange Consortium, with the dataset identifier PXD071144. In silico prediction using ProP 1.0, APD3, and CAMPR4 supported the antimicrobial potential of candidate peptides. Overall, our integrative approach demonstrates that bacterial infection induces a diverse and bioactive immunopeptidome in H. illucens, supporting its potential as a sustainable platform for the discovery of novel antimicrobial agents.