Study On The Diversity Of Intestinal Bacteriae And Their Main Biological Functions In Blattella Germanica

ObjectiveThe objective is to study the diversity of the intestinal bacteria composition of Blattella germanica at different developmental stages,and to clarify the role of B.germanica intestinal bacteriae in assisting the digestion of food and resisting pathogenic fungi,as well as the influence on the host's individual development,population reprod-uction and functional gene expression.The intestinal bacteria of B.germanica will be developed the ecological control technology as the target and some the promising intestinal bacteriae will be researched and developed to functional microbial agents.MethodsThe diversity of intestinal bacteriae of B.germanica was analyzed by combining traditional isolation culture and high-throughput sequencing technology.The activity of digestive enzymes produced by the intestinal bacteriae of B.germanica was analyzed by the method of flat transparent circle.B.germanica intestinal bacteriae antagonistic to Bea-uveria bassiana were screened by plate confrontation method.The effect of intestinal bac-teriae on the individual development and population reproduction of B.germanica was stu-died by using two-sex life table.Transcriptome sequencing was used to study the effect of intestinal bacteriae of B.germanica on gene expression profile.Results(1)12 genera and 20 species of bacteria had been isolated by traditional culture method in different development stages in the B.germanica intestinal,16S rDNA high-throughput sequencing analysis showed that Blattabacterium was the dominant intestinal flora of B.germanica.Pseudomonas,Serratia,Bacillus and Enterococcus were detected in Blattella germanica at all developmental stages.(2)Among the intestinal bacteriae of B.germanica,7 species of bacteriae can produce digestive enzymes,accounting for 35%of the total bacteriae.They were Pseudomonas aeruginosa,Serratia marcescens,B.amyloliquefacien,Microbacterium oxidants,Chry-seobacterium massiliae,B.paramycoides and B.tequilensis.Among them,these 7 kinds of bacteriae had protease activity.Serratia marcescens,B.amyloliquefaciens and C.massiliae showed amylase activity.B.tequilensis,B.amyloliquefaciens and C.massiliae showed cellulase activity.Lipase producing bacteria were not screened.In the isolated bacteria,B.amyloliquefaciens and C.massiliae produced amylase,cellulase and protease simultan-eously.Serratia marcescens can produce both protease and amylase.B.tequilensis pro-duced both protease and cellulase.There were 7 kinds of bacteriae with digestive enzy-me activity in the intestinal bacteriae of B.germanica,accounting for 35%of the total bact-erial strains,Pseudomonas aeruginosa,Serratia marcescens,B.amyloliquefaciens,Micro-bacterium oxidants,C.massiliae,B.paramycoides and B.tequilensis.They all had protease producing.There were 3 kinds of amylase-producing bacteriae,accounting for 15%of the total bacterial species.There were 3 kinds of cellulose-producing bacteriae,accounting for15%of the total bacterial species.Lipase-producing bacteriae were not screened.Both B.amyloliquefaciens and C.massiliae were active in amylase,cellulase and protease prod-uction.S.marcescens had the activity of both protease and amylase.B.tequilensis has pro-tease and cellulase activity simultaneously.(3)P.aeruginosa,B.amyloliquefaciens and B.tequilensis isolated from the intestinal bacteriae of B.germanica could inhibit the spore germination and mycelial growth of B.bassiana.The germination rates of B.bassiana spores were 5.33±1.53%,58.00±3.00%and59.33±3.06%,respectively,after treatment with the three bacteriae,compared with97.67±0.58%in the control group.Among them,P.aeruginosa had the strongest inhibitory effect.(4)The average developmental duration of the nymphs of B.germanicus was pro-longed from 63.92±8.00 d to 122.07±13.70 d,the life span of female was shortened from57.43±21.42 d to 30.00±13.90 d,and that of male was shortened from 46.93±10.12 d to23.07±11.44 d.The intrinsic rate(r)decreased from 0.01549 to 0.0014,finite rate(?)decr-eased from 1.0156 to 1.0014,and net reproductive rate(R₀)decreased from 5.14 to 1.31,indicating that B.germanica's individual development and population reproduction were significantly inhibited after intestinal bacteria removal.(5)After removing intestinal bacteriae,there were a total of 4256 differentially expressed genes in the normal group and the drug feeding group's females.There were a total of 3332 genes in the normal group and the drug feeding group's males.Among these genes,there were 726 genes in both male and female.GO enrichment analysis showed that it had affected biological process,cellular component and molecular function of B.germ-anica after removing intestinal bacteria,KEGG pathway enrichment analysis showed the removal of intestinal bacteria mainly affected the metabolism of carbohydrates,amino acids and lipids and related growth and development pathways of B.germanica.Conclusion:The structure and composition of the intestinal bacteriae of B.germanica were chang-ed with the development stage.The intestinal bacteriae of B.germanica can assist B.germanica in the digestion of protein,amylase and cellulose,and also antagonize the infection of B.bassiana.B.germanica intestinal bacteriae may also affect their individual development and population reproduction by regulating the metabolism genes of carbo-hydrates,amino acids and lipids and the expression of genes related to growth and development.