Bacterial Extracellular Vesicles from Chromobacterium subtsugae and Bacillus thuringiensis as Cell-Free Bioinsecticidal Nanocarriers Against the Soybean Pest Euschistus heros

Bacterial extracellular vesicles (bEVs) are membrane-enclosed nanoparticles that transport bioactive cargo and mediate interactions between bacteria and their environment. Although bEVs are increasingly recognized as natural delivery systems, their potential application in plant pest biocontrol remains poorly explored. Here, we provide proof-of-concept evidence that isolated bEVs from two entomopathogenic bacteria, Chromobacterium subtsugae and Bacillus thuringiensis var. kurstaki, exert insecticidal activity against the soybean pest Euschistus heros. Isolated bEVs were characterized by tunable resistive pulse sensing, nano-flow cytometry, transmission electron microscopy, SDS-PAGE, MALDI-TOF mass spectrometry, and label-free quantitative proteomics. C. subtsugae bEVs displayed a proteome clearly remodeled relative to the soluble protein fraction, with enrichment of outer- membrane, secretion-associated, proteolytic, and membrane-active proteins. MALDI-TOF analysis detected a violacein-associated ion selectively in the C. subtsugae bEV fraction, supporting vesicular association of this hydrophobic bioactive metabolite. In survival assays, C. subtsugae bEVs strongly reduced E. heros nymph survival (HR = 4.0, p < 0.0001), whereas the corresponding soluble protein fraction was inactive (HR = 1.2, p = 0.50). In contrast, B. thuringiensis bEVs and soluble protein fractions produced similar moderate activity (both HR = 2.1), consistent with their largely overlapping proteomic profiles. Cry1Ab was detected mainly in the B. thuringiensis soluble fraction rather than selectively enriched in bEVs. Together, these findings support a multi-component cargo model in which C. subtsugae bEVs combine vesicle-associated violacein with enriched protein cargo, establishing bacterial EVs as promising natural nanocarriers for next-generation, cell-free bioinsecticides against Cry-resistant hemipteran pests such as E. heros.