Optimizing black soldier fly larvae (BSFL) production requires a better understanding of how diet composition shapes growth, nutrient allocation, and metabolic efficiency. This study investigated whether respiratory measurements could serve as predictive, non-invasive tools for BSFL performance on diets differing in protein-to-carbohydrate (P:C) ratio. Larvae were reared for seven days on four experimental diets, with growth, survival, gas exchange (O2 consumption, CO2 production, and respiratory exchange ratio (RER)), and body composition (crude protein and lipid contents) measured across seven replicates per treatment. O2 consumption correlated strongly with final larval biomass, while RER was closely associated with protein and lipid deposition. Larvae on low-protein, high-carbohydrate diets accumulated proportionally more lipid, whereas protein-rich diets increased crude protein content but required higher energetic investment. This demonstrates that lipid-rich larvae grew more efficiently in energetic terms per unit accumulated biomass than protein-rich larvae. Across diets, gas exchange variables reliably reflected growth dynamics, metabolic activity, and nutrient assimilation. Integrating respirometry into rearing systems could enable real-time monitoring of insect biomass yield and nutrient composition.

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.

The kissing bug Rhodnius prolixus, a primary vector of Chagas disease, serves as a model for understanding insect physiology and vector-pathogen interactions. While the role of male reproductive factors in influencing female fertility is well-established in many insects, little is known about male fertility in R. prolixus. Our study examines the role of two apolipoproteins, RPRC010099 and RPRC015421, in male fertility and their potential function as carriers of eicosanoid hormones. Using RNA interference (RNAi), we silenced the genes for both apolipoproteins in male R. prolixus. The silencing of RPRC010099 led to a significant reduction of ∼60% in the number of eggs laid by mated females, while silencing RPRC015421 resulted in a ∼20% reduction. Further analysis, including molecular docking and enzyme-linked immunosorbent assays (ELISA), confirmed that these proteins are transferred from males to females during copulation and have a high affinity to eicosanoid signaling molecules, such as prostaglandin E2. Our findings demonstrate that RPRC010099 and RPRC015421 are important male fertility factors that promote female oviposition and probably act as carriers for eicosanoids. This work not only identifies a novel reproductive role for apolipoproteins but also contributes to a broader understanding of male-female interactions in insect reproduction.