| The gut of insects is inhabited by diverse microorganisms,which affect the physiological functions in hosts such as development,energy metabolism,immune regulation,digestion and absorption of nutrients,as well as the behaviors such as movement,reproduction and aggression.The feeding behavior is critical for nutrient acquisition and energy supply for the body,and therefore for maintaining individual survival and population reproduction in insects.So far,gut microbes have been found to affect the feeding behavior of insects,such as foraging and feeding preferences.However,the underline molecular mechanism of gut microbes in regulating the host feeding behavior remains unclear.Insect’s neuropeptides are polypeptide endogenous active substances secreted by the Insect’s nervous system.They function as neurohormones,neurotransmitters,neuromodulators and other forms in the body to comprehensively regulate the various physiological and behavioral processes of insects.Previous studies have shown that a variety of neuropeptides played an important role in the feeding behavior of insects.Therefore,based on microbiology,aseptic biology and RNAi techniques,the effects of gut microbes on the food intake,neuropeptide genes and related metabolites were determined in Bactrocera dorsalis,one of the critical fruit and vegetable pests,to investigate whether gut microbes can regulate the food intake of B.dorsalis through neuropeptides and clarify the underling molecular dmechanism.The main results are as follows:1.Gut symbiont affects the food intake of B.dorsalisAfter eliminating gut microbiome by feeding antibiotics,the results showed that the total abundance of intestinal bacteria was significant decreased by 97.35%,and the food intake of B.dorsalis was significant decreased by 36.06%.Then we fed and replenished six gut commensal bacteria after antibiotic treatment,including Klebsiella michiganensis,Enterobacter hormaechei,Enterobacter cloacae,Enterobacter asburiae,Citrobacter freundii and Citrobacter koseri.Compared with the B.dorsalis after eliminating gut microbiome,the food intake of B.dorsalis after K.michiganensis supplementation was significantly increased by 24.01%,while the food intake of other strains of B.dorsalis was not significantly restored.These results indicating that the lack of gut microbiome significantly affect the food intake of B.dorsalis,and K.michiganensis is the key symbiotic bacteria that affecting the food intake of B.dorsalis.To explore how the symbiotic bacteria affect the food intake of B.dorsalis.We further fed the flies without gut bacteria with Heat-killed K.michiganensis,live K.michiganensis and fermentation supernatant of K.michiganensis,and studied the effects of these treatments on the food intake of B.dorsalis.The results showed that the food intake was significantly increased by 44.00%and restored to the same level as control group when compared with the flies without gut bacteria.Heat-killed K.michiganensis and fermentation supernatant of K.michiganensis had no significant effect on host food intake,indicating that only live K.michiganensis bacteria affect food intake of B.dorsalis.2.Gut symbiont affects the genes’ expression of neuropeptides and the synthesis of octopamineThe transcriptome data of adults treated with antibiotics and normally reared in the early stage of the laboratory were analyzed,and many differential neuropeptide genes were screened out,and further verified by real-time quantitative PCR.The results showed that compared with the normal group,after eliminating gut microbiome with antibitotic treatment,CCHamide-2(CCH-2)gene expression was significantly down-regulated by 53.02%,CCHamide-2 receptor(CCH-2R)gene was significantly down-regulated by 52.77%,Allatostatin-A(Ast-A)gene was significantly up-regulated by 30.85%,but Ast-A receptor gene did not change,Octopamine Receptor(OAR)gene expression was significantly increased by 55.10%,wheras there was no significant change of gene expression of Ast-A receptor,Tyrosine decarboxylase(Tdc)and tyramine betahydroxylase(Tβh)for octopamine synthesis.Since the studies on octopamine were mostly carried out at the substance level,we further detected the changes of octopamine signaling pathway related metabolitesin in the hemolymph.The results showed that the concentration of octopamine was significantly decreased by 28.33%,the concentration of tyramine,the precursor of octopamine synthesis,was significantly decreased by 45.07%,the concentration of tyramine β-hydroxylase,a key enzyme for octopamine synthesis,was significantly decreased by 16.33%in hemolymph after eliminating gut microbiome.These results indicating that eliminating gut microbiome significantly affected the gene expression of CCH-2,CCH2R,Ast-A and OAR,and the concentration of octopamine signaling pathway related substances.To further study the effect of intestinal symbiotic bacteria on the expression of neuropeptide genes,we replenished the above six symbiotic bacteria after antibiotic treatment,and detected the change of gene expression of CCH-2,CCH2R,Ast-A and OAR by qPCR.The results showed that compared with the group of eliminating gut microbiome,the expression levels of CCH-2 and CCH2R genes did not change significantly after the supplementation of six intestinal symbiotic bacteria;the expression level of Ast-A gene was significantly down-regulated by 43.07%and restored to normal leve after replenishing K.michiganensis,but there was no significant effect on the expression level after replenishing other symbiotic bacteria;the expression level of OAR gene was significantly down-regulated by 30.07%-50.62%and recovered to the normal level after replenishing K.michiganensis,E.cloacae,E.hormaechei and C.freundii respectively,indicating that the expression of OAR gene was affected by a variety of symbiotic bacteria.Since K.michiganensis can significantly affect the expression of AstA and OAR,we selected K.michiganensis for the next research.The results showed that the concentration of octopamine,tyramine and tyramine β-hydroxylase increased significantly by 30.24%,34.39%,and 34.66%,respectively when compared with those of the control group after K.michiganensis replenishment.These findings suggest that K.michiganensis affects the gene expression of Ast-A and octopamine receptor OAR,and the synthesis of octopamine signaling pathway related metabolites.3.Gut symbiont regulates the food intake of B.dorsalis through neuropeptide Ast-A geneSince gut microbes can affect food intake and the expression of neuropeptide Ast-A gene of B.dorsalis,in order to further explore the relationship between gut microbes,AstA gene and food intake,we carried out experiment as follows:In normal populations of B.dorsalis,functional studies of Ast-A gene in regulating the food intake were performed by RNAi technology.The results showed that at 2 days post Ast-A RNAi.,the expression of Ast-A gene was significantly decreased by 27.52%and the food intake was significantly increased by 21.30%when compared with the control group,these results indicating that Ast-A gene could inhibit food intake of B.dorsalis.The changes of lipid and carbohydrate after Ast-A RNAi were further detected.The results showed that the contents of triglyceride,glucose and trehalose were significantly increased by 144.47%,54.32%and 63.35%respectively at 2 days Ast-A RNAi.On the other hand,compared with normal population,Ast-A gene expression was significantly upregulated by 15.90%,and food intake was significantly decreased by 31.13%in flies without gut bacteria.To further investigate whether gut microbe regulates host food intake by affecting Ast-A gene expression,Ast-A RNAi was performed in flies of eliminating gut microbiome.The results showed after RNAi of Ast-A gene in flies of eliminating gut microbiome,compared with the ds-Egfp group,the gene expression level of Ast-A in the ds-Ast-A group was significantly down-regulated by 25.27%and restored to the normal flies level,the food intake was significantly increased by 39.16%and also restored to the normal flies level.The results showed that after the up-regulated expression of Ast-A gene caused by the elimination of intestinal microorganisms was knocked down by RNAi and returned to the expression level of normal flies,the food intake of B.dorsalis would not be reduced due to the elimination of gut microbiome,and there was no significant difference with the normal flies,suggesting that the lack of intestinal microorganisms would cause the up-regulated expression of Ast-A gene and then reduce the food intake of B.dorsalis.Since feeding status is very important to reproduction,we further studied the effects of Ast-A on the reproduction of B.dorsalis.The result showed that knockdown of Ast-A gene had no significant effect on ovarian development and fertility,survival rate and starvation resistance under normal total nutrition feeding.However,under the condition of half yeast feeding,due to nutrient deficiency,compared with the normal feeding group,the ovary development of B.dorsalis was delayed,the amount of total eggs was significant decreased by 64.43%,and the amount of daily eggs was significant decreased at 16 d,18d and 20 d;However,after knockdown of Ast-A gene in Half Yeast+Ast-A group,ovarian development was improved,the amount of total eggs was significantly increased by 1.13 folds,and the amount of daily eggs was significantly improved at 16 d and 18 d,and returned to the same level as full nutrition feeding group.These results suggest that knockdown of Ast-A gene may maintain the normal ovarian development and fecundity of B.dorsalis via promoting its food intake under nutritional deficiency condition.In summary,we concluded that the gut symbiont regulates the food intake of the host by affecting expression of the neuropeptide Ast-A gene in B.dorsalis,which is considered as the "gut symbiont-neuropeptide-food intake" regulatory pathway. |
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