The Analysis Of Bacterial Diversity In The Gut Of Hermetia Illucens Larvae And Characterization Of A Novel Bacterial Strain Of Flavobacteriaceae

Black soldier fly belongs to the suborder of Diptera,Stratiomyidae,Hermetia.It distributes widely in the world.BSF larvae are easy to raise and can degrade organic waste efficiently.Therefore,it has great potential in agriculture and environmental treatment and could bring high socioeconomic and ecological performance.At present,researches on BSF are mainly focused on the transformation of organic waste,while study on its intestinal microorganisms is scarce.In the view of the natural habitat environment of BSF,its gut microflora may play an important role in its growth and development.In order to understand the interactions between the gut microflora and the BSF and to find some functional strains from the gut of BSF,we analyzed the diversity of gut bacteria based on 16S rRNA gene clone library and the culturable aerobic and facultative anaerobic bacteria from the gut of BSF larvae.In addition,a new strain T-61T was indentified by polyphasic taxonomy approach.These works are important to further elucidate the interactions between gut bacteria and BSF larvae.The main results are as follows:1.Phylogenetic analysis of the diversity of intestinal bacteria in the gut of BSF larvaeIn order to understand the bacterial diversity,phylogenetic analysis based on the 16S rRNA genes of the intestinal bacteria was performed.The results showed that:the main intestinal bacteria of the BSF larvae were Proteobacteria,Actinobacteria,Bacteroidetes and Frimicutes.The number of bactreia in Proteobacteria is the most abundant with the propotion of 43%,followed by Actinobacteria with 26%,Bacteroidetes with 17%,Fruticutes with 12%.The propotion of the other unclassified bacteria is 3%.2.The diversity anlysis of culturable aerobic and facultative anaerobic bacteria in the gut of BSF larvaeThe whole gut of 3-4th instrar larvae of BSF was aseptically dissected and used for bacterial suspension.Bacteria were isolated and purified with 1/3 TSB medium from serial diluted bacterial suspensions.A total of 69 aerobic and facultative anaerbic bacteria were isolated and indentified with 16S rRNA genes.The 16S rRNA gene sequences of their representative strains were compared with known bacterial sequences in GenBank.Clustering anlaysis showed that,these 69 islolated strains were classified into 11ARDRA types.Phylogenetic analysis showed that they belong to the genus Morganella,Procidencia,Bacillus,Staphylococcus,Microbacterium,Proteus,,Exiquobacterium,Eenterococcus and Myroides.Among them,the number of bacteria in the phylum of Proteobacteria is the most abundant.3.Characterization of strain T-61T with polyphasic taxonomy approachThe morphology of the colonies of strain T-61T on TSB agar plates was characterized as yellow,round,slightly raised,with intact edges and a smooth surface,pigment production is weak positive,aerobic,non-spore-forming,non-motile rods.Gram staining was negative.The bacteria presented irregular short rod shape under the scanning electron microscope.It is approximately 1.0-1.25μm×0.4-0.5μm.Phylogenetic analysis of 16S rRNA gene sequences showed that strain T-61Twas closely related to Myroides guanonis with sequence similarity of 93.1%,and probably a new bacterium of the genus of family Flavobacteriaceae.The optimum growth temperature of strain T-61T was 35℃,the optimum growth pH was 8.0 and it was able to grow at salinities of 0-5%(m/v).Oxidase and catalase activity were negetive.No hydrolysis of starch,casein,Tween 80.Hydrogen sulfide was produced,V-P reaction was negative,indole was not produced,nitrate can not be reduced to nitrite.In addition,esterase lipase(C8),esterase,alkaline phosphatase,Leucine aramidase,valine aramidase,and some other hydrolytic enzymes are positive,which was somehow different from the two reference strains.The main respiratory quinone type is MK-7,which was different from other strains of the family of Flavobacteriaceae.The main polar lipid profile included PE(Phosphatidyl ethanolamine),three Amino lipids(AL1,AL2,AL3),three unknown lipid Lipids(L1,L2,L3)and APL(Amino phospholipids).The main fatty acid components were iso-C15:0,iso-C17:0 3-OH,Summed Feature9.The genomic G+C mol%content was 39.5 mol%,which was slightly higher than the other strains of the genus Myroides.Based on the above experimental results,it is concluded that strain T-61T represents a new genus of the family Flavobacteriaceae.Because strain T-61T was isloated from the gut of BSF,for which the name is Guizishania hermetiae gen.nov.sp.nov.T-61T is the type species of the genus Guizishania.4.Whole genome sequencing and analysis of strain T-61TThe genome of strain T-61T consisted a complete chromosome(3109169bp)with out plasmids.A total of 2976 protein-encoding genes predectied with a total length of 2764662 bp,accouting for 88.9%of the whole genome sequence.The genome contains 62 tRNA and 10 rRNA.A total of 50 tandem repeats were predicted.The whole genome was annotated with COG,GO and KEEG.Functions and drug resistance genes,virulence gene prediction,and bioinformatics analysis were performed on its whole genome data to explain the drug resistance,virulence and evolutionary relationships of the strain at the genome level.