Identification and Characterization of Insect Peptides with Antibacterial Activity

Antimicrobial peptides (AMPs) constitute a chemically and structurally heterogeneous family of molecules produced by a wide range of living organisms, including plants, fish, amphibians, mammals, and insects. Their expression is particularly high in organisms that are frequently exposed to microbial invasions, where they play a key role in innate immune responses. Insects, in particular, represent one of the richest natural sources of AMPs. Over their long evolutionary history, they have developed a highly efficient immune system in which AMPs play a central role in defense against pathogens, allowing them to colonize a wide range of habitats. In recent years, interest in AMPs has significantly increased due to the rise in bacterial strains resistant to conventional antibiotics, positioning these peptides as potential therapeutic alternatives for infections caused by resistant pathogens. In this study, we investigated the antimicrobial activity of peptides extracted from the hemolymph of Hermetia illucens larvae (Diptera, Stratiomyidae), an insect known for its high expression of AMPs. Hemolymph samples were collected from larvae infected with Escherichia coli (Gram-negative) and Micrococcus flavus(Gram-positive), as well as from uninfected larvae, and subsequently treated by organic solvent precipitation. Antimicrobial activity was assessed through microbiological assays, including agar diffusion tests and microdilution assays, which the peptides demonstrated significant activity against pathogenic bacterial strains, including antibiotic-resistant strains. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for each experimental condition. Mass spectrometry analysis identified 33 antimicrobial peptides, 13 of which were differentially expressed in response to bacterial infection. The two selected peptides, MO_Ab1Lin and MO_Ab4, were chemically synthesized via solid-phase synthesis, which enabled their structural and functional characterization. The synthesized peptides demonstrated significant inhibitory potential in antimicrobial activity assays. Additionally, sequence analysis of the synthesized peptides showed a high degree of homology with peptides from the defensin family. In parallel, a cloning approach was developed to express two other peptides in a heterologous system (Pichia pastoris), with the goal of enhancing production and enabling a more detailed characterization of their biological activity. The results of this study highlight the potential of AMPs from H. illucens, both natural and synthetic, as promising candidates for the development of new antimicrobial therapies, particularly in the fight against antibiotic-resistant pathogens.