Functional Omics Of Embryonic Serosal Cuticle And Two Nymphal Cuticle Related Proteins In Nilaparvata Lugens

Insects are the largest animal group in the world.Compared with other higher animals,insects are relatively small in size with higher fertility,simpler embryonic development and shorter growth duration.Formation,movement and degration of important embryonic outer membranes,amnion,serosa and yolk sac,are involved in whole insect embryogenesis.The embryo outer membranes vary greatly among different insects.Study showed the serosa would appear after the germ band formation in nearly all insects.The serosal cuticle is secreted by serosa cells and is located at the inner side of eggshell,thereby the protection for embryo from eggshell is enhanced,especially the embryonic desiccation resistance(EDR)is enhanced.The embryonic cuticle forms during the late period of the embryonic development in most insects,but the number of the embryonic cuticle formation varies in different insects.The brown planthopper(BPH),Nilaparvata lugens(Hemiptera:Delphacidae),is one of the most destructive insect pests of rice crops.The major cause of its outbreaks is the strong reproductive capacity.Up to date,we have no idea about whether the serosal cuticle or embryonic cuticle existing in the BPHs,and we are unaware about how many cuticle proteins are related to the serosal cuticle.In this study,firstly,we observed the fonnation of the serosal cuticle and embryonic cuticle in BPH.Then with the mass spectrometry(ms)analysis,transcriptome and expression profile data analysis,real-time fluorescence quantitative RT-PCR(RT-qPCR),RNA interference(RNAi),Transmission electron microscope(TEM)and Western blotting technologies,the serosal cuticle related proteins were studied.In addition,2 other important cuticle proteins which not belonging to the CPR family were identified.Results as follows:1.Observation of the serosal cuticle and the embryonic cuticle.With TEM,we found the serosal cuticle was formed during the early stage of embryonic development period,and degradation of serosal cuticle and an embryonic cuticle were observed during the late stage of embryonic development period.The serosal cuticle and the embryonic cuticle both contain chitin which forms lamellar structure.2.Identification of Nilaparvata lugens serosal cuticle related proteins(Nlscrps)and function analysis.The 72h eggshells(E72)were collected and analysed by Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Then the E72 proteins were compared with the proteins of eggshells from newly laid eggs(E0)and eggshells after hatching(ES).47 proteins were found within the E72 and ES protein databases while not belonging to the EO database.And 25 credible proteins related to the serosal cuticle formation were screened.Based on the transcriptome analysis and Fragments per Kilobase Million(FPKM)databases,13 proteins encoding genes from the 25 credible proteins encoding genes were found to be expressed specifically or highly during the 0-72h of egg period,we speculated the 13 proteins are serosal cuticle related proteins.Among the 13 proteins,3 proteins were identified as cuticle proteins belonging to the CPR family(NlugCpr2 and NlugCpr3 vwere already studied in our laboratory before,NlugCpr97 was a new one).The 3 CPRs would be discussed in the next section.Other 10 proteins encoding genes were temporarily named as Nlscrpl-10,respectively,and were then studied with different technologies such as RT-qPCR,RNAi and TEM.The results showed that 3 genes(Nlscrp2,Nlscrp4,Nlscrp5)were essential for the formation of serosal cuticle.With RNAi treatment for 5 genes(Nlscrp2,Nlscrp4,Nlscrp5,Nlscrp7,Nlscrp10),respectively,hatchabilities were all significantly reduced.3.Functional analysis of the 6 cuticle proteins(CPRs)related to the serosal cuticle.Based on the transcriptome analysis and Fragments per Kilobase Million(FPKM)databases,6 CPRs were found expressing highly or specifically in the early stage during embryogenesis.Among the 6 CPRs,NlugCpr2,NlugCpr3 and NlugCpr97 were belonging to the 13 proteins described in the section above.Previous study in our lab revealed that the hatchabilities were reduced significantly with the RNAi of 4 CPRs(NlugCpr1,NlugCpr3,NlugCpr8,NlugCpr90),respectively,while no effect was observed with the RNAi targeting NlugCpr2.In this study,no effect on embryo development was observed after RNAi of NlugCpr97,while RNAi using a mixture of dsRNAs targeting the other 5 CPRs,the serosal cuticle showed unnormal,and hatchability was reduced even to only 5.16%,which was much lower than the hatchabilities(33.95-75.87%)with RNAi against any one CPR of them.We speculated the 5 CPRs paly a role for the serosal cuticle formation in whole or in part and a complementary effect may exist in these cuticle proteins.。4.The function of the chitin synthetase and dopa-decarboxylase.With RT-qPCR,we found three expression peaks of the chitin synthetase(CHS1)during the embryonic development,corresponding to the chitin deposition of the serosal cuticle,embryonic cuticle and the 1st instar cuticle,respectively.No serosal cuticle was formed after RNAi against CHS1 with the newly emerged females,indicating that CHS1 is essential for the serosal cuticle formation.Dopa decarboxylase(DDc)is a pyridoxal-5-phosphate-dependent enzyme,which converts L-dopa to dopamine.No serosal cuticle was formed after RNAi against the ddc in BPHs(Nlddc)with the newly emerged females,suggesting that Nlddc is also indispensable for the formation of serosal cuticle.5.Identification of a novel cuticle protein NlCP21.92.Using transcriptome analysis of tissues of the BPH,we identified a gene tentatively designated NlCP21.92 that was expressed at high levels in the integument.Spatiotemporal expression profiling with RT-qPCR and Western blotting verified its integument-specific expression and showed periodic expression during molting.The open reading frame(ORF)was GC-rich and encoded a hydrophobic polypeptide.The polypeptide contained AAPA/V repeat motifs and other sequence features similar to several reported cuticular proteins but lacked an R&R consensus and other chitin-binding domains.Double-stranded RNA-mediated RNAi of the NlCP21.92 resulted in shrivelled body or molting difficulty phenotypes,and TEM revealed the corresponding ultrastructural defects.Immunohistochemicals taining(IHC)demonstrated that the NlCP21.92 protein was primarily located in the procuticle.Our results suggested that NlCP21.92 is a novel ungrouped cuticular protein essential for normal endocuticle formation.6.Identification and functional analysis of the protein Nlegf-like.Using the mass spectrometry(ms)analysis of cuticle casts of the BPH and transcriptome analysis of BPH tissues,we identified a gigantic gene(50922 bp,16973 aa)tentatively called Nlegf-like.Multiple transcripts were found.Nlegf-like encodes an integral membrane protein of 16973 amino acid residues with 260 EGF-like repeats and 16 Ca2+-binding EGF repeats type(cbEGFs)in the extracellular portion.Nlegf-like was highly expressed in the integument and tended to peak at the middle stage or late stage of each nymph instar.Phylogenetic analysis showed this gene was conserved in many other insects.Different double-stranded RNA-mediated RNAi targeting eight different regions of the Nlegf-like gene resulted in abnormal cuticle formation or molting and lethal phenotypes.TEM revealed that the newly formed endocuticle was significantly thinner for RNAi-treated BPHs with phenotype of contracted abdomen,or the old cuticle could not be digested sufficiently for those with phenotype of slender body shape or died with molting difficulty when compared with the control group.We suggested that the Nlegf-like is crucial for metabolism of the cuticle in BPH molting.