Mechanism Of Protein Degradation By Black Soldier Fly Larva In Synergism With Gut Microorganisms

Hermetia illucens L.,commonly known as black soldier fly larvae（BSFL）.BSFL can convert various types of organic waste,mainly including livestock manure,food waste,etc.High quality insect protein can be harvested after the conversion of organic waste by BSFL,which is important for alleviating the crisis of animal feed protein shortage.Gut microbes play an important role in assisting BSFL to degrade proteins from organic waste,but the synergistic mechanism is still unclear.Due to the complex composition of organic waste,the correlation between BSFL and gut microbes in nutrient metabolism could not be further studied.Therefore,in this study,we constructed the germ-free BSFL（GF）model,gnotobiotic BSFL（GB）model,monomicrobial BSFL（IS）model,and a high protein artificial diet model with welldefined nutritional composition.We sequenced and analyzed the gut microbiome of GB,the gut metabolome,gut transcriptome of GB and GF,and the gut Dual RNA-seq of IS during the conversion artificial diet.We isolated a fungus Issatchenkia orientalis and named it IS-001,which is closely related to protein degradation metabolism,from the gut of the larvae that are converted high-protein artificial diet by the GB.Combined with the already published black soldier fly genomes,the comprehensive analysis of multi omics data is used to identify the key genes involved in the synergistic degradation of protein by BSFLand gut microbes,to deeply analyze the molecular mechanisms by which gut microbes promote the efficient protein degradation of BSFL,and to reveal the key metabolic pathways by which gut microbes regulate the protein degradation of BSFL.The main findings are as follows:1.Gut Amplicon research on gut microorganisms enhancing BSFL’s protein degradationThe protein degradation rate and major gut proteolytic enzymes activities of GBBSFL and GF-BSFL showed that GB exhibited 73.44%higher feed protein degradation rate than GF,and the GB gut activities of trypsin,peptidase,and pepsin were significantly higher than GF.On this basis,the analysis of GB-BSFL gut amplicon results showed that the gut microbes Pseudomonas spp.,Orbus spp.,and Campylobacter spp.were strongly correlated with amino acid metabolism.Dysgonomonas spp.had a strong correlation with protein digestion and absorption.Campylobacter spp.,Pediococcus spp.,and Lactobacillus spp.were strongly correlated with trypsin activity.Lactobacillus spp.and Bacillus spp.were strongly correlated with peptidase activity.Issatchenkia spp.was strongly correlated with pepsin activity.The above results indicated that some key gut microbes played an important role in protein degradation by BSFL.2.Combined transcriptome and metabolome to profile the synergetic degradation of protein machinery by BSFL and gut microbesBased on the analysis of GB-BSFL gut amplicon,the gut transcriptome and gut metabolome of GB-BSFL and GF-BSFL were analyzed.The results showed that,the differentially expressed genes and differential metabolites in the gut of GB-BSFL versus GF-BSFL were significantly enriched in the branched chain amino acid degradation metabolic pathway,tryptophan metabolic pathway.WGCNA analysis found that,δ-Trypsin is the major proteolytic enzyme of BSFL;One pepsin encoding gene,LOC119660302,was found in the hub genes of the WGCNA module,which showed a significant positive correlation with the BSF split protein M1 enhancer encoding gene LOC1 19650725 and the BSF uncharacterized protein encoding gene LOC119650726.The results of the correlation analysis between the microbiome and transcriptome of GB-BSFL gut showed that there are co-metabolic pathways between BSFL and its gut microbes on amino acid metabolism.Gut microbes Issatchenkia spp.,Orbus spp.,Lactobacillus spp.of BSFL exhibited significant positive correlations with branched chain amino acid degradation metabolism and tryptophan metabolism.3.Study on the synergetic degradation of protein between the gut fungus Isatchenkia orientalis IS-001 and host BSFLBSFL gut amplicon,transcriptome,and metabolome analysis results showed that the abundance of fungi Issatchenkia spp.increased significantly during the BSFL converted feed process.The gut fungus plays an important role in synergizing with the host in protein metabolism processes.On this basis,a strain of Issatchenkia orientalis is isolated from the gut of the GB-BSFL and named it IS-001.Through the analysis of the differentially expressed genes in the Dual RNA-seq of GF-BSFL with IS-001,the results showed that pepsin activity of BSFL gut was mainly from the provocation of I.orientalis IS-001;In co-occurrence network analysis,it was found that the BSF pepsin encoding gene LOC119660302 only exhibited a positive correlation with the IS-001 tRNA splicing endonuclease encoding gene C5L36₀E04100 at the 12th day of conversion artificial feed.The expression of the pepsin encoding gene LOC1 19660302 may be regulated by the IS-001 gene C5L36₀E04100.BSFL aldehyde dehydrogenase encoding gene LOC119659862,spermidine synthase encoding gene LOC119660560 and I.orientalis IS-001（S）-3-amino-2methylpropionic transaminase encoding gene C5L36₀C06340 are key genes for the synergetic metabolism of branched-chain amino acids between larvae and gut microbiota.BSFL trypsin encoding genes LOC1 19654741,LOC1 19654746,LOC1 19654747 and I.orientalis IS-001 acetyl CoA acyltransferase encoding gene C5L36₀A08380 are the key genes for the synergetic degradation of protein by larvae and gut microbiota.The gut microbe I.orientalis IS-001 promotes BSFL to obtain more amino acids from the diet and convert them into BSFL protein through the synergistic effect with the host in the branched chain amino acid degradative metabolism.And the expression of the above genes was verified by qPCR.In summary,through multi omics studies combined with GF-BSFL technology,this study found a synergistic correlation between gut microbiota and BSFL in protein degradation as well as amino acid metabolism;Promotes the degradation of proteins by BSFL key gut fungus I.orientalis IS-001 and key synergistic genes.The synergetic mechanism was revealed,which laid a theoretical foundation for improving the efficiency of protein degradation and promoting the resource utilization of livestock and poultry manure in the process of converting livestock and poultry manure and other organic solid wastes.