Characterisation of extracellular vesicles in insect cells and their role in interspecies communication

Extracellular vesicles (EVs) play a crucial role in intercellular, interspecies and interkingdom communication, facilitating the exchange of molecular information among diverse cells and organisms. Their ability to transport small RNAs enables them to modulate gene expression in recipient cells via the conserved regulation mechanism of RNA interference (RNAi). This property holds great promise for the development of sustainable, RNAi-based crop protection strategies. However, our knowledge of the molecular composition of insect-derived EVs remains limited. To address this, we isolated and characterized EVs from insect cell lines representing three orders: Coleoptera, Diptera and Lepidoptera. EVs were analysed by cryo-scanning electron microscopy (cryo-SEM), nanoparticle tracking analysis (NTA), and proteomics. EVs derived from different insect orders displayed comparable size distributions and morphological characteristics Proteomic analysis of Drosophila melanogaster EVs revealed a repertoire of EV-associated proteins, including orthologs of human EV markers, highlighting the evolutionary conservation of molecular components involved in EV-mediated processes across species. Conditioned medium transmission assays demonstrated that dsRNA-treated donor cells can transfer RNAi signals to naïve recipient cells. Fractionation experiments localized silencing activity specifically to EV-enriched (EV⁺) fractions, whereas EV-depleted (EV^-) fractions showed little to no silencing activity. EV-mediated RNAi transmission was observed within species, between related species within the same order, and, in one direction, across different insect orders. By integrating proteomic data of D. melanogaster with functional transmission assays in multiple insect cell lines, this study provides experimental evidence that insect EVs are conserved and capable of mediating RNAi signal transfer across taxonomic boundaries. These findings contribute to the understanding of systemic RNAi in insects and provide a foundation for exploring EV-based mechanism in RNAi-driven pest control strategies.