Study On Genes Of Cuticular Lipid Biochemical Pathway In The Brown Planthopper,Nilaparvata Lugens

Cuticular lipids are important cuticle components of various insects.As the outmost protective barrier,the surface lipid layer plays crucial roles in maintaining normal life activities.A common insect cuticular lipid biochemical pathway involves an enzymatic cascade including acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS).fatty acid desaturase(FAD),fatty acid elongase(ELO),Fatty acyl-CoA reductase(FAR)and P450 oxidative decarbonylase(CYP4G).However,the researches are limited in individual genes of only one or several gene families.The comprehensive and systematic research and understanding of insect cuticular lipid biosynthesis are still acking.The brown planthopper.Nilaparvata lugens(Stal)(Hemiptera:Delphacidae).an insect of incomplete metamorphosis,is the number one rice pest in Asia rice region.N.lugens lives in paddy fields,they should not only be able to adapt to the varying humidity,but also be able to walk and jump on water surface.Therefore,studies on the cuticular lipid biosynthesis of N.lugens will not only promote the research of cuticular lipids,but also contribute to developing new strategies for pest control.Moreover,the synthesis of cuticular lipid has rarely been studied in N.lugens.In this study,we first analyzed the main components of N.lugens cuticular lipids,then identified the genes of cuticular lipid biochemical pathway based on bioinformatics analyses and provided a comprehensive analysis of the gene functions base on RNA interference(RNAi),scanning electron microscope(SEM).GC/MS,3D volume reconstruction.etc.The main results are as follows:1.Analysis of the main components of N.lugens cuticular lipids.Data from GC/MS showed that N.lugens had a mixture of sixteen compounds of saturated unbranched n-alkanes(C14,C16-C20 and C22-C31).sixteen branched hydrocarbons(HCs)and six fatty acids(C14:0.C16:1,C16:0,C18:2,C18:1 and Cl 8:0).The results revealed that the main components determined the cuticle hydrophobicity were HCs.The present study provided a further understanding of the structure of insect cuticle and formed the basis for the research of cuticular lipid biosynthesis.2.Identification of ACC,FAS,FAD,ELO,FAR,WS and CYP4G gene families in N.lugens.Based on bioinformatics analyses of N.lugens genomic and transcriptomic databases,we identified 58 genes of seven gene families(only 1 in ACC gene family,3 in FAS,10 in FAD,20 in ELO,17 in FAR,5 in WS and 2 in CYP4G).We used expression profiles to find that the above genes are higly tissue-specific,and the developmental expression patterns showed high diversity.Forty-two of these genes were successfully confirmed by RT-PCR and sequencing.RNAi against nymphs showed that seventeen genes(NIACC,NlFAS1,NlELO2,3,7,8,9,10,12,16,NIFAR5,6,7,9,11,15 and CYP4G115)are essential for N.lugens and knowckdown any of them caused lethal phenotypes.The upstream genes of cuticular lipid biochemical pathway e.g.NIACC perturbed lipid metabolism,and seven key genes coding for ELO.FAR and CYP4G enzymes acting downstream were essential for cuticle waterproofing.Our present study provides an initial comprehensive analysis of the genes in cuticular lipid biochemical pathway in an insect,and may enrich the research achievements in the field of insect cuticular lipids.3.Four ELO and two FAR genes are essential for cuticle waterproofing.Systematic RNAi experiments demonstrated that NlIELO2、3、8、16 and NIFAR7、9 were required for the production of cuticular hydrocarbons(CHCs).Surface analysis by SEM and GC/MS quantification indicated that knockdown of NlIELO2,3,8,16 and NlFAR7,9 resulted in a smooth body surface and a decrease in CHC amounts.CHC deficiency,in turn,resulted in increased adhesion of water droplets and secreted honeydew to the animal surface and the inability of N.lugens to survive in paddy fields with varying humidity.Our results revealed that there existed an ultimate relationship among these six genes,CHC synthesis and cuticle surface waterproofing.These findings may serve as a basis for further biochemical studies on the functions of insect cuticle and the underlying molecular regulation mechanisms of cuticular lipid biosynthesis.It provides not only a new perspective for pest control by reducing the hydrophobicity of insect cuticle surface,but also important basis for the research and development of new insecticides targeting cuticle permeability barrier.4.Identification of genes in lipid biochemical pathway essential for survival.Knockdown of NIELO7.9.10 or 12 caused lethal phenotypes with a thin and wizened body and reduced lipids in the fat body.Knockdown of NIFAR5,6.11 or 15 was lethal and caused a slender body shape,while the old cuticles of the respective animals remained attached to the abdomen or failed to split open from the nota.Knockdown of NIELO1,3,4,7,8,9,10,11,12,18 and NIFAR1,4,5,6,8,9,11,13 additionally resulted in decrease of adult fertility.Moreover,RNAi against nymphs for NIACC,NIFAS and CYP4G115 caused high mortality.The present study illustrates the functional differences of the genes in lipid biochemical pathway and provides new potential targets for RNAi-based rice planthopper management.