Study On The Diversity Of Intestinal Bacteriae And Their Main Biological Functions In Lucilia Sericata

Lucilia sericata,belonging to Calliphorinae,Calliphoridae,Diptera,is a common medical vector insect worldwide.Generally,it can mechanically transmit various pathogenic microorganisms.The larvae can breed in rancid wounds and sores furuncle of the body,or grow in digestive tract,wound,five sense organs,and so on.This cause many infectious diseases,such as human digestive tract fly maggot diseas,nose fly myiasis,and fly maggot disease of livestock and poultry,which seriously threaten human health and livestock production.However,the larva has the necrophage,which also has great application value in the field of environmental purification and forensic entomology.Nowadays,there was no related research that whether the intestinal bacteria community influenced the distribution and omnivority of L.sericata.In this study,change law and the main biological function of intestinal bacteria of L.sericata at each stage was investigated through analyzing their intestinal bacteria community composition.The main results were as follows.1 Intestinal bacteria composition in various stages of L.sericataThe intestinal bacteria of L.sericata at stages of spawn,larvae,pupa and adult with no diet were determined by traditional pure culture method and high-throughput sequencing technology with metagenome.Total 37 species of bacteria were isolated after screening in intestinal tract.And 25 strains of intestinal bacteria at larvae stage,26 strains at pupa stage,5 strains at adult stage with no diet.However,none bacterium was separated at spawn stage.Staphylococcus sciuri,Staphylococcus gallinarum,Providencia rettgeri and Corynebacterium variabile were isolated during these three stages.Specially,some unique bacteria was screened at different stages,for example,Leucobacter holotrichiae,Enterococcus saccharolyticus,Enterococcus faecium,Lactococcus lactis,Raoultella ornithinolytica,Lampropedia puyangensis,Myroides injenensis,Bacillus megaterium,Sphingobacterium changzhouense were isolated at larvae stage.Brachybacterium paraconglomeratum,Corynebacterium glutamicum,Sphingobacterium spiritivorum,Brevundimonas diminuta,Pseudomonas putida,Providencia stuartii,Achromobacter insolitus,Serratia liquefaciens,Bacillus wiedmannii,Bacillus haynesii,Exiguobacterium aurantiacum,and Acinetobacter soli were specific strains at pupa stage.Staphylococcus lentus,Staphylococcus lentus was specific strains at adult stage.Metagenomic sequence showed that 468682 high quality sequences of intestinal bacteria of L.sericata were obtained.The length of these sequences was most 393 bp.And the predominant bacteria were Providencia,Proteusand Myroides.2 The digestive enzyme activity of intestinal bacteria of L.sericataThe activity of amylase,protease,cellulase and lipase of these 37 strains were determined through plate screening.And 32 strains accounting for 86.5% could secrete digestive enzymes.This indicated that intestinal bacteria of L.sericata had a higher ability to produce digestive enzymes.Among them,the bacteriae produced protease,lipase and amylase were 16,23 and 17 strains,respectively.Klebsiella oxytoca and Staphylococcus sciuri could produce these three enzymes.And there were 21 bacteria which could produce two enzymes.S.changzhouense,Alcaligenes faecalis,R.ornithinolytica,P.rettgeri,L.holotrichiae,E.faecium,C.glutamicum,P.stuartii,S.liquefaciens,Brevundimonas diminuta,and A.insolitus can secrete amylase and lipase.B.megaterium and B.wiedmannii can produce protease and amylase.E.saccharolyticus,L.puyangensis,Arthrobacter arilaitensis,M.Sorbitolivorans,S.gallinarum,Providencia vermicola,E.aurantiacum,and Corynebacterium variabile can produce lipase and amylase.3 Intestinal bacteria of L.sericata affect its spawning and larvae feedingThrough oviposition choice experiments,K.oxytoca,L.holotrichia,Bacillus licheniformis,Myroides odoratu,and Myroides odoratimimus had significant induction to spawning of L.sericata.L.holotrichiae and B.licheniformis is the endemic species at larvae and pupa stage,respectively.The other three bacteria were found both of larval stage and pupal stage.Besides,A.faecalis,R.ornithinolytica,Acinetobacter haemolyticus,and C.variabile had significant inhibition on oviposition.R.ornithinolytica and A.haemolyticus were specific strains of larvae stage.A.faecalis and C.variabile widely presented at larvae stage and pupa stage.Other strains had no distinct effect on spawning of L.sericata.Furthermore,9 intestinal bacteriae,A.faecalis,K.oxytoca,S.changzhouense,C.glutamicum,L.puyangensis,P.rettgeri,P.vermicola,P.stuartii,Kurthia gibsonii,had obvious induction to feeding of L.sericata larvae.And S.changzhouense had the strongest induction.On the contrary,E.saccharolyticus and B.paraconglomeratum were resistant to the feeding of the larvae.The other strains had no effect on the feeding of larvae.In all the bacteriaee,only K.vulcanca had obvious induction to the spawning,as well as larvae feeding.K.vulcanca exists as the larval stage and pupal stage of the mercerous green fly.4 The influence of intestinal bacteria on their gene expression profilesAfter intestinal bacteriae of L.sericata larvae were completely removed using diverse antibiotics,the RNA of larvae samples were extracted and purified.Then a ibrary was constructed.Based on Illumina Hi Seq Sequencing platform,transcriptome paired-end sequencing was conducted using the second Generation Sequencing technology(NGS).The obtained sequencing data and differential expressed genes were analyzed by bioinformatics.The results showed that 124365 Unigene was obtained after assembled.These genes mean length was about 697.34 bp and the length of N50 was 1158 bp.Compared with the samples without any disposal,there were 8711 differential expressed genes in the intestinal bacteriaee removed gram-negative bacteriaee.And the up-regulation and down-regulation genes were 6756 and 1955,respectively.There were16301 differential expressed genes in the intestinal bacteriaee removed gram-positive bacteriaee.And the up-regulation and down-regulation genes were 13535 and 2766,respectively.Similarly,there were 13598 differential expressed genes in the intestinal bacteriaee removed gram-negative bacteriaee.And the up-regulation and down-regulation genes were 10661 and 2937,respectively.Moreover,there were 12959 differential expressed genes in the intestinal bacteriaee removed gram-negative bacteriaee between the intestinal bacteriaee removed gram-negative bacteriaee and removed gram-positive bacteriaee.And the up-regulation and down-regulation genes were 4291 and 8668,respectively.There were 7702 differential expressed genes in the intestinal bacteriae removed gram-negative bacteriae between the intestinal bacteriae removed gram-negative bacteriae and removed all bacteriae.And the up-regulation and down-regulation genes were 3848 and 3854,respectively.There were 7485 differential expressed genes in the intestinal bacteriae removed gram-negative bacteriae between the intestinal bacteriae removed gram-negative bacteriae and removed all bacteriae.And the up-regulation and down-regulation genes were 1414 and 6071,respectively.