Dual Oxidase Duox and Toll-like Receptor 3 TLR3 in the Toll Pathway Suppress Zoonotic Pathogens Through Regulating the Intestinal Bacterial Community Homeostasis in Hermetia Illucens L

Black soldier fly (BSF; Hermetia illucens L.) larvae can convert fresh pig manure into protein and fat-rich biomass, which can then be used as aquafeed for select species. Currently, BSF is the only approved insect for such purposes in Canada, USA, and the European Union. Pig manure could serve as a feed substrate for BSF; however, it is contaminated with zoonotic pathogens (e.g., Staphylococcus aureus and Salmonella spp.). Fortunately, BSF larvae inhibit many of these zoonotic pathogens; however, the mechanisms employed are unclear. We employed RNAi, qRT-PCR, and Illumina MiSeq 16S rDNA high-throughput sequencing to examine the interaction between two immune genes (Duox in Duox-reactive oxygen species [ROS] immune system and TLR3 in the Toll signaling pathway) and select pathogens common in pig manure to decipher the mechanisms resulting in pathogen suppression. Results indicate Bsf Duox-TLR3 RNAi increased bacterial load but decreased relative abundance of Providencia and Dysgonomonas, which are thought to be commensals in the BSF larval gut. Bsf Duox-TLR3 RNAi also inactivated the NF-κB signaling pathway, downregulated the expression of antimicrobial peptides, and diminished inhibitory effects on zoonotic pathogen. The resulting dysbiosis stimulated an immune response by activating BsfDuox and promoting ROS, which regulated the composition and structure of the gut bacterial community. Thus, BsfDuox and BsfTLR3 are important factors in regulating these key gut microbes, while inhibiting target zoonotic pathogens.