| Some symbionts can regulate insect reproduction by mediating cytoplasmic incompatibility,feminization,parthenogenesis,and male killing.In addition,the other symbionts can synthesize essential nutrients for the host and regulate insect reproduction.However,the underlying molecular mechanisms are largely unknown.Previous studies have shown that the symbionts Hamiltonella in the bacteriocytes of Bemisia tabaci MEAM1 induces the female biased offspring.However,the physiological and molecular mechanisms by which the Hamiltonella regulates the sex ratio of whitefly offspring are still unclear.Whiteflies are haplodiploid insects;fertilized eggs produce female adults and unfertilized eggs develop into male adults.The symbionts Hamiltonella carries all most of genes for the synthesis of various B vitamins.Nutritional metabolites(such as B9,folate)can regulate proteins post translational modifications,such as histone methylation and acetylation.Metabolites produced by some pathogens or gut microbiota can mediate proteins post translational modifications of host proteins.Meanwhile,post translational modifications of proteins can affect animal reproduction.Therefore,this study investigated whether Hamiltonella regulates post translational modification of whitefly proteins by synthesizing B vitamins,affecting fertilization of whitefly and thus regulating host sex ratio of offspring.The main findings are as follows:1.Effect of Hamiltonella elimination on symbiont titer,symbiont protein expression and gut microbiota in the whiteflyThe Hamiltonella was specifically eliminated by treating B.tabaci adults with an antibiotic cocktail.The Hamiltonella titer was reduced significantlly in Hamiltonella-cured(–HBt)whiteflies compared to Hamiltonella-infected(+HBt)whiteflies,while the titers of Portiera and Rickettsia remained similar.Proteome of symbionts in +HBt and –HBt adult whiteflies was compared by UPLC-MS/MS and the results indicates that Hamiltonella elimination by antibiotic treatment has negligible effect on protein expression of other two symbionts Rickettsia and Portiera in whiteflies.At the same time,the gut microbiota of +HBt and –HBt adult whiteflies was analyzed by 16 S r DNA amplicon sequencing.Gut bacterial communities in both +HBt and –HBt adult whiteflies were dominated by the phyla Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria.Additionally,both α diversity and β diversity of gut microbiota didn’t differ between +HBt and –HBt adult whiteflies.This data suggests that Hamiltonella elimination by antibiotic treatment has no effect on the composition and diversity of gut microbiota in the whitefly.2.Folate synthesized by the cooperation between Hamiltonella and the whitefly determines the sex ratio of the whiteflyThe percentage of female offspring of Hamiltonella-cured whiteflies is significantly lower compared to Hamiltonella-infected whiteflies.Supplementing B vitamins to Hamiltonella-cured whiteflies in artificial cocktail can restore the percentage of female offspring of Hamiltonella-cured whiteflies to the level of those Hamiltonella-infected whiteflies.Hamiltonella has almost complete folate synthesis pathway,but lacks alkaline phosphatase pho AB gene.The gene is encoded in the host B.tabaci.UPLC-MS/MS analyses showed that THF and SAM had decreased levels in –HBt compared to +HBt whiteflies.UPLC analyses showed that Met have elevated levels in –HBt compared to +HBt whiteflies.The percentage of female offspring was lower in –HBt compared to +HBt whiteflies that fed on a 30% sucrose diet.Supplementation of the 30% sucrose diet with folate increased the percentage of female offspring produced by –HBt whiteflies to the level observed in +HBt whiteflies.In addition,to determine the role of folate in whitefly sex ratio,pho AB,the key gene involved in folate synthesis,was silenced in adult whiteflies by microinjection with dspho AB.UPLC-MS/MS analyses showed that the levels of THF and SAM were decreased in dspho AB-injected adult females.UPLC analyses showed that Met had elevated levels in dspho AB-injected adult females compared to ds GFP-injected adult females.The percentage of female offspring was lower in dspho AB-injected compared to ds GFP-injected whiteflies that fed on a 30% sucrose diet.Supplementation of the artificial diet with folate restored the percentage of female offspring produced by dspho AB-injected whiteflies to the level observed in ds GFP-injected whiteflies.These data suggest that the folate synthesized by the cooperation between Hamiltonella and the whitefly pho AB affects the sex ratio of B.tabaci offspring.3.Hamiltonella deficiency affects the histone H3 methylation profile in whiteflies and folate is responsible for the H3K9me3 modificationThe abundance of methylated histone H3 lysine sites,including H3K4me1-3,H3K9me1-3,H3K27me1-3,H3K36me1-3 and H3K79me1-3 was compared between Hamiltonella-infected and Hamiltonella-deficient whiteflies using western blot.Among the histone 3 lysine methylation markers,H3K9me3,H3K36me3 and H3K79me2 had drastically induced levels in –HBt whiteflies compared to +HBt whiteflies.To verify the role of folate in the changed level of H3K9me3 in whiteflies,+HBt and –HBt adult whiteflies as well as ds GFP-injected and dspho AB-injected whiteflies were fed on artificial diet with folate.Supplementation with folate restored the level of H3K9me3 in –HBt whiteflies to that observed in +HBt whiteflies.Likewise,supplementation with folate restored the level of H3K9me3 in dspho AB-injected whiteflies to that observed in ds GFP-injected whiteflies.To identify the genes associated with H3K9me3,Ch IP-seq was performed using the antibody for H3K9me3 followed by DNA sequencing.After peak calling,a total of 9722 peaks were significantly up-regulated,and 10881 peaks were significantly down-regulated in –HBt whiteflies compared to +HBt whiteflies.We further found that 3,145 and 3,399 genes were associated with significantly up-regulated and down-regulated H3K9me3 peaks,respectively,in –HBt whiteflies compared to +HBt whiteflies.The KEGG pathway enrichment analysis showed that five pathways were enriched for the genes associated with differential H3K9me3 peaks in –HBt whiteflies compared to +HBt whiteflies.Among the five enriched KEGG pathways,the c AMP signaling pathway and the AMPK signaling pathway regulate various aspects of mitochondrial function.These data indicate that Hamiltonella may influence the mitochondrion function of whiteflies.4.Hamiltonella deficiency leads to mitochondrial disfunction in whitefly ovariesThe level of ATP was significantly reduced in the ovaries of –HBt whiteflies compared to+HBt whiteflies.The abundance of mt DNA was also significantly reduced in the ovaries of –HBt whiteflies compared to +HBt whiteflies.The mitochondrial gene mitofusins including Mfn2 are key regulators of mitochondrial fusion and fission.Ch IP-seq revealed that Mfn2 is one of the mitochondrion function genes associated with the induced level of H3K9me3 in –HBt whiteflies compared to +HBt whiteflies.Thus,H3K9me3 modification could impact the expression of Mfn2 in whiteflies.A q RT-PCR verified that Mfn2 expression was significantly reduced in the ovaries of –HBt whiteflies compared to the +HBt whiteflies.Further,transmission electron microscope analysis showed that normal mitochondria with the intact membrane and well-defined cristae were observed in the ovarioles of +HBt whiteflies.In contrast,abnormal mitochondria with swollen morphology,disruptive membrane and broken cristae were present in the ovarioles of –HBt whiteflies.Supplementation of the 30% sucrose diet with folate increased the level of ATP and mt DNA in ovaries of –HBt whiteflies to the level observed in +HBt whiteflies.Additionally,transmission electron microscope analysis showed that folate supplementation reduced the number of abnormal mitochondria with broken cristae,swollen morphology,and disruptive membrane in the ovarioles of –HBt whiteflies.5.Mfn2 silencing represses mitochondrial function of whitefly ovaries,inhibits fertilization and reduces the percentage of female offspringMitofusin plays an important role in animal reproduction.Repressing mitofusin impairs germline development,oogenesis,egg development and female fertility in Drosophila.Mfn2 expression was significantly reduced in the ovaries of whiteflies after Hamiltonella curing.The level of ATP and mt DNA was significantly reduced in the ovarioles of ds Mfn2-injected adult females compared to the control whiteflies that were injected with ds GFP.The expression of the apoptosis marker protein caspase 3 was induced in the ovarioles of ds Mfn2-injected adult females compared to the control whiteflies that were injected with ds GFP.The fertilization rate was lower in ds Mfn2-injected adult females compared to ds GFP-injected adult females.Additionally,the percentage of female offspring was significantly reduced in ds Mfn2-injected adult females compared to ds GFP-injected adult females.This study finds that bacteriocyte symbionts drived folate regulates the ovary mitochondrial function by histone methylation modification,leading to a female-biased sex ratio offspring of the whitefly,which benefits symbiont vertical transmission. |
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