Weisellas Paramesenteroides Facilitates Drosophila Systemic Growth By Ecdysone And Insulin Signaling Pathways

Objective:This paper focuses on isolating and identifing lactic acid bacteria Weisellas paramesenteroides,and investigating its promoting effects on the development and growth of Drosophila melanogaster.This study also explores its mechanism of growth promoting effects.Methods:Bacterium was isolated from the gut of D.melanogaster with MRS selective medium and identified by Gram staining and BLAST analysis of 16S rRNA gene.Germ free,conventional reared and gnotobiotic flies were prepared.The developmeantal duration of D.melanogaster was quantified by counting the time to puparium formation and adult eclosion.The growth rate was calculated as the body surface area of larvae.The expression levels of genes involved in ecdysone(dib,E74B and PTTH)and insulin signaling(DILP2,DILP3 and InR)of D.melanogaster were assayed by quantitative real-time PCR in the time course.The glucose level in the hemolymph was measured by glucose oxidase assay.The number of mitotic cells in fly gut was checked by immunofluorescence staining.Results:1.W.paramesenteroides was isolated from fly gut.One strain of W.paramesenteroides was isolated and identified by BLAST analysis of 16S rRNA gene.This strain was a Gram-positive bacterium with rod-shaped using Gram staining.W.paramesenteroides metabolized common simple sugars:fructose,maltose,sucrose and glucose and the p H value of mudium declined at the end of fermentation.2.W.paramesenteroides efficiently colonized the gut of Drosophila.W.paramesenteroides was incubated to the GF flies,and the mount of per individual internal bacterial load in various growth stages of the gut of Drosophila was about 10~3-10~4.The mount of bacterial load of 1g corresponding culture medium of Drosophila was about 10~8.The mount of internal bacterial load of the progenies of Drosophila adults mono-associated with W.paramesenteroides was about 10~2-10~3.The mount of bacterial load of 1g corresponding culture medium of progenies was about 10~6.3.W.paramesenteroides promoted the growth of Drosophila by accelerating growth rate.On rich food(1%yeast),the time to CR puparium formation was 6.6 d,significantly differing from that of GF(9.3 d).The time to W.paramesenteroides-associated(WP)puparium formation was 6.3 d,which was comparable to GF ones.This growth-promoting effect was more dramatical in poor medium(0.5%yeast,0.25%yeast)than that in rich one.Consistently,W.paramesenteroides association resulted in earlier emergence of adults compared to GF siblings.No significant differences of growth rate were observed among GF,CR and WP larvae on rich diet(4%yeast).However,the growth rate of CR flies was 1.7-fold higher than that of GF ones in case of poor diet(0.5%yeast),the growth rate of WP fly was1.6-fold higher than that of GF larvae,but no significant differences of growth rate were observed among CR and WP larvae.4.W.paramesenteroides activated ecdysone and insulin signaling pathway.The peak expression of dib,E74B and PTTH in WP flies appeared earlier and steeper than that in GF flies.The peak expression in CR flies were similar to WP flies.We added mock or 20-hydroxyecdysone in GF flies food.The delay of GF pupae formation was efficiently rescued.The levels of DILP2 and DILP3 in WP individuals were higher than GF flies.The levels of InR and concentration of serum sugar in WP individuals were lower than GF flies.The results of CR flies were similar to WP flies.5.Gut microbiota enhanced the number of mitotic cells in gut.The number of mitotic cells in CR flies gut was more than in GF flies,but no significant differences were observed among WP and GF flies.Conclusion:W.paramesenteroides is the symbiotic bacterium of Drosophila,shortens the duration of the growth phase and accelerates the growth rate.W.paramesenteroides promotes proper moulting and metamorphosis by activating the ecdysone signaling pathway.W.paramesenteroides accelerates the growth rate and promotes the Drosophila development by activting the insulin signaling pathways.