123 | Characterization of extracellular vesicles from the insect Hermetia illucens, a potential source of bioactive molecules

Sharon Catrin Raimondi¹, Kristian Riolo¹, Domenico Savastano², Sabrina Oliva¹, Jessica Maria Abbate¹, Antonio Leonardi¹, Alessia Irrera³, Annalisa Pinsino⁴, Alessia Giannetto¹ | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; ²Progetto Hermetia, Gioia Tauro, Reggio Calabria, Italy; ³CNR IMM sede di Messina, Messina, Italy; ⁴Institute of Translational Pharmacology IFT National Research Council CNR, Palermo, Italy.

Extracellular vesicles (EVs) are small membrane-enclosed vesicles known to contribute to intercellular communication in and among all domains of life. Increasing evidence has indicated their potential for diverse biotechnological applications. In insects, reports on EV studies are still in their infancy, as well as standardized procedures for the isolation of EVs from insect hemolymph. Hermetia illucens (Black Soldier Fly, BSF) is an insect species that has attracted increasing scientific interest due to its remarkable physiological adaptations and its ability to synthesize a wide range of bioactive compounds. During the larval stage, H. illucens thrives on decomposing organic substrates, an ecological niche that has driven the evolution of a highly efficient immune system capable of producing antimicrobial peptides, antioxidant molecules, and other biologically active compounds to cope with decomposing organic growth substrates. Based on these characteristics, we hypothesized that a part of this bioactive molecule repertoire produced during the larval stage may be conveyed through an intercellular communication system mediated by EVs released into the hemolymph. To date, the presence, morphology, and biochemical features of EVs in H. illucens remain entirely unexplored. This study reports the first isolation and preliminary characterization of EVs from the hemolymph of H. illucens larvae. EVs were isolated using differential ultracentrifugation and commercial EV isolation kits and subsequently characterized by multiple approaches, namely bicinchoninic acid assay (BCA), SDS-PAGE, immunoblotting, nanoparticle tracking analysis (NTA), and scanning electron microscopy (SEM). Preliminary results demonstrate that H. illucens releases EVs into the hemolymph. The EV populations displayed morphological and dimensional features consistent with canonical extracellular vesicles described in other invertebrates (EVs markers, size diameters), as demonstrated by SDS-PAGE, NTA, and SEM, supporting the existence of a previously unrecognized vesicle‑mediated communication system in this species. These findings lay the groundwork for future studies aimed at elucidating the molecular cargo of H. illucens EVs and assessing their potential biological activity on human cell lines. Overall, the identification of EVs in H. illucens advances the understanding of insect biology and also highlights their potential as a novel and sustainable source of bioactive molecules, thus opening new perspectives for promising biotechnological applications in the near future.
Studies were supported by the MUR-PNRR PRIN 2022 Project SURPRISE (GA P2022LASKT).