| Sphingolipids are one kind of lipid groups with complex structures and diverse functions.Ceramides are the most important sphingolipid species.They are localized in the central position of sphingolipid metabolism and act not only as structural compounds of cellular membrane,but also as second messengers to initiate physiological changes during organism's development,growth,differentiation and apoptosis.The hydrolysis and synthesis of ceramides were regulated by the enzyme of ceramidase(CDase)and ceramide synthase(CerS),respectively.The profiles,content and metabolism of sphingolipids in Nilaparvata lugens(Stal)(the brown planthopper,BPH),the most important insect pests of rice in Asia,were investigated in this study.The biological functions of CDase and CerS in the sphingolipid metabolism were further explored.The main results were as follows:1.The sphingolipid profiles had been characterized in BPH,and the ceramide content in different developmental stages of BPH had been measured.We profiled 8 classes of sphingolipids in the BPH,including sphingosine(SPH),dihydrosphingosnie(dSPH),sphingosine-1-phosphate(S-1-P),dihydrosphingosine-1-phosphate(dS-1-P),ceramide(Cer),dihydroceramide(dCer),sphingomyline(SM)and ceramide phosphate(Cer-P).Two types of sphingo-backbones,C16 and C18,were identified and the later was the dominant specie in BPH.C18 sphingobase,together with fatty acid CoA whose carbon length ranged from 14 to 24,were ultilized by BPH to condense into different ceramide varieties.The content of ceramides was significant more abundant than the dihydroceramides,and C20:0-ceramide and C22:0-ceramide were found to be the dominant species.The content of ceramide in different developmental stages of BPH,including the eggs,the 1st to 5th instar nymphs,the female and male adults,were analyzed by HPLC-MS/MS.The results showed that ceramides content within per gram of fresh weight decreased with the development of BPH.2.The sphingolipid metabolic genes were identified and the gene expression was profiled in different developmental stages and different tissues of BPH.The results showed that the categories of sphingolipid metabolizing genes and sphingolipid pathways were relatively conserved,while the numbers of gene copies differed with other species.Two homologous sequences of serine palmitoyl transferase gene(SPT),one ceramidase gene(CDase),two isforms of ceramide synthase gene(CerS),seven phingomyeline synthases/ceramide cholinephosphotransferase genes(SMS),four sphingimyelinase genes(SMase)were isolated from BPH genome and their functions were predicted by phynogenetic analysis.The qPCR data showed that most of sphingolipid metabolizing genes were highly expressed in the BPH adults,especially in the female adults.Moreover,most genes showed higher transcript levels in the reproductive organs(ovary and spermary)than in other tissues.These findings suggested that sphingolipid metabolism was more active during the development of adults and may participated in the reproduction process.3.Accumulation of ceramides induced by knockdown of NICDase might arrest BPH oogenesis and inhibit BPH reproduction via initiating BPH ovariole apoptosis.The transcript of NICDase(CDase of BPH)was negatively correlated with ceramide content during different developmental stages of BPH by Pearson Correlation analysis.To investigate the function of ceramides during the BPH development,we knocked down NICDase through RNA interference(RNAi)and conducted the bioassay after RNAi.The results showed that NlCDase-silenced females had no change in the survival rate,while they showed reduction of oviposition by 35%on rice seedlings,decrease of hatching rate of eggs by 27%and poor viability of offsprings comparing with control individuals injected with dsRNA-GFP.Analyzed by HPLC-MS/MS,the content of ceramides,especially C20:0-ceramide and C22:0-ceramide,were significantly accumulated after the the blocking of NICDase.The females laid fewer eggs after feeding with exogenic C20:0-ceramide for 3 days,suggesting that excess ceramides may arrest ovarian development of BPH.The dissection of BPH ovaries from 1st to 5th day after dsRNA injection,showed that the development of BPH ovaries was suppressed with delayed maturity and fewer numbers of ovarioles since the 3rd day.Meanwhile,the expression of six vitellogenin-relevant genes was decreased after RNAi,especially for the gene vitellogenin receptor.Moreover,the suppression of superoxide dismutase(SOD)activity,elevation of Caspase3 activity and the apoptosis of BPH ovaries were provoked by silencing NICDase.In addition,the structural changes of mitochondrial and nuclear membrane in the BPH ovarian cells were demonstrated by electric microscopy.In conculsion,accumulation of ceramides induced by the ablation of NICDase may arrest the ovarian development of BPH and inhibit BPH reproduction through initiating the apoptosis of BPH ovarioles.4.Reduction of ceramides induced by the ablation of NlCerS severely inhibited BPH oogenesis.Ceramide synthase(CerS),a key enzyme in the sphingolipid de novo pathway,produced ceramides through the condensation reaction of sphingsines and fatty acid-CoA with different carbon length.Two homologous cDNA sequences of CerS,NICerS and NICerS-like,were identified from BPH genome.NICerS were highly expressed in the female adults and ovaries while NICerS-like was ubiquitously displayed in all stages and tissues of BPH.To investigate the functions of CerS,NICerS and NICerS-like genes were manipulated through RNA interference(RNAi).Comparing with control which injected with dsRNA-GFP,silencing of NICerS or NICerS-like genes neither exihibted significant difference of mortality in 5 days.NlCerS-silenced BPH females had shorter lifespan,lower body weight and abnormal ovaries,while NICerS-like-silenced BPH showed no difference in these phenotypes.Six vitellogenin-relevant genes showed significant transcriptal decrease after the ablation of NICerS while no expression difference after the suppression of NlCerS-like.The ovarian malformation was found in the NlCerS-silenced BPH instead of NlCerS-like-silenced BPH.The BPH females fed with fumonisin B1(FBI),an inhibitor of gene CerS,for 3 days had reduced oviposition.Above results suggested that NICerS gene was contributed to the development of BPH ovary.HPLC-MS/MS analysis showed that ceramides(product)decreased while sphingosines(substrate)increased after the block-down of NICerS.Meanwhile,the suppression of SOD activity,elevation of Caspase3 activity and the apoptosis of BPH ovaries were provoked by silencing NICerS.Moreover,electric microscopy analysis showed that the abnormality of ovariole structure,including damaged nucleus membrane and decreased mitochondrial crest,may explained its apoptosis reaction.In summary,the reduction of ceramides induced by the ablation of NICerS was severely inhibited rather than benefiting for BPH ovarian development.In conclusion,this study investigated the functions of ceramide and its metabolic enzymes.We focused on characterizing the functions of NICDase and NICerS in the BPH development.Silencing of NICDase inhibited the hydrolysis of ceramides,elevated ceramides level,induced apoptosis of BPH ovarian cells and arrested the oogenesis and reproductive process of BPH.While the silencing of NICerS ablated the synthesis of ceramide,increased the ceramides level,shortened BPH lifespan and triggered the apoptosis and malformation of BPH ovaries.These findings suggested that the homostasis of ceramides maintained by numerous sphingolipid enzymes was essencial for the normal developmental process and higher or lower ceramides both would cause cellular damage,thus controlled the biological process of the organisms. |
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