Identification Of Neuropeptides And Their G Protein-Coupled Receptors From The Peanut Aphid Aphis Craccivora And Regulation Of Feeding Behavior By Neuropeptide F In Aphids

Neuropeptides are the most diverse signal molecules in insects,acting as neurohormones and neuromodulators to regulate insect physiology and behaviors.Most neuropeptides exert their functions through binding to G protein-coupled receptors(GPCRs)to initiate downstream signaling pathways.As promising targets for alternative insecticides,neuropeptide GPCRs provide potential pathways for pest control.To achieve this goal,we must thoroughly study and understand the insect neuropeptide system.Feeding behavior is of important significance for insect survival of both individual and population.The neuropeptide F(NPF)signaling system has been shown to regulate feeding in Drosophila melanogaster.However,little is known with regard to the functional roles of NPF signaling in aphids.In order to enhance our understanding of the neuropeptide system in aphids,we chose the peanut aphid Aphis craccivora,an important agricultural pest,as the research object.Based on RNA-seq,the tBlastn program was used to search for genes encoding the putative neuropeptide precursors and neuropeptide GPCRs.The structures of the putative neuropeptide precursors were analyzed and their posttranslational modifications were predicted.The phylogenetic analysis of neuropeptide GPCRs was also carried out and their ligands were predicted.The expression profiles for A.craccivora life stages of these genes were validated by quantitative real-time PCR(qRT-PCR).The biological function of the NPF signaling in aphids was further analyzed and in view of the fact that NPF signaling is highly conserved in aphids,we selected Acyrthosiphon pisum(parthenogenetic wingless aphids),which is larger in body size and more convenient for operation,as the study object.The temporal and spatial expressions of NPF and NPFR genes as well as their responses to nutritional status were detected by qRT-PCR.The relation between the NPF signaling and the feeding behavior of aphids was explored using RNA interference with injected dsRNA and electrical penetration graphs(EPGs).Finally,the binding sites of A.pisum NPF with its receptor NPFR were predicted by homology modelling and molecular docking analysis.The main results in this thesis are as follows:A total of 40 genes encoding neuropeptide precursors belonging to 32 neuropeptide families were identified from A.craccivora transcriptome,including allatostatin(Ast),myosuppressin(MS),insulin-like peptide(ILP),leucokinin(LK),tachykinin(TK)and short neuropeptide F(sNPF).These include a vast majority of insect neuropeptides that have been reported.Through software analysis and meanwhile according to known homologous neuropeptides,the signal peptides and the amidation,pyrophosphorylation and other modification sites were predicted from the candidate neuropeptide precursors.Finally,a total of 164 mature peptides and more than 60 bioactive mature neuropeptides were predicted.The expression patterns of neuropeptide genes in different ages and different wing types of A.craccivora were confirmed by expression profiling.Among the neuropeptide genes involved in feeding regulation,LK,NPF and sNPF were differentially expressed in the adults and the nymphs.AstA,AstB,AstCC,LK,MS,NPF and sNPF were differentially expressed in wingless and winged aphids.We identified a total of 46 genes encoding neuropeptide GPCRs.Among them,40 genes encoded family A GPCRs,including receptors for 21 families of neuropeptides and 7 orphan receptors;6 genes encoded family B GPCRs,including receptors for diuretic hormone 31(DH31),DH44 and pigment-dispersing factor(PDF).Compared with holometabolous insects such as D.melanogaster,the genes encoding sulfakinin(SK),corazonin(Crz),arginine-vasopressin-like peptide(AVLP),trissin and their corresponding receptors were not found in A.craccivora,indicating these four neuropeptide signaling systems were lost.This result agrees with that observed in A.pisum.In addition,the expression profiles of neuropeptide GPCR genes at different developmental stages were determined.The receptor genes involved in feeding regulation were significantly differentially expressed in different ages of the wingless or winged aphids,as well as in different wing types at the same age.The NPF transcript was mainly detected in the head but not in the gut of the wingless adults,whereas the NPFR transcript was mainly detected in both the gut and head and the level in the former was approximately twice of that in the latter.The expression levels of these two genes varied across developmental stages,which imply that this signaling might be related to the development of aphids.The feeding and nutritional status has a significant effect on NPF expression,but not on its receptor gene;the NPF transcript level was markedly up-regulated in starved aphids compared with satiated aphids,and recovered after re-feeding.Injection of dsRNA effectively silenced the NPF gene(interference efficiency reached 50%)in wingless adult aphids,but had little effect on the NPFR gene.After NPF was silenced,the amount of honeydew secreted by aphids was significantly reduced,indicating a decrease in food intake.Further analysis of the modification of aphid probing behavior on broad bean seedlings using EPGs revealed the apparent reduction of total duration in phloem ingestion in the RNAi group,which could directly explain the reduction in food intake.The reduction in phloem ingestion and the postponement of the first phloem activity indicated the decreased preference of aphids for phloem sap.The postponement of the initiation of penetration and the shortened total duration of probing also indicated a reduced appetite in the RNAi group.NPF silencing was also shown to reduce reproduction but not survival rate in adult aphids.In addition,the three-dimensional structure models of A.pisum NPFR and its endogenous ligand NPF were constructed respectively,using homology modeling.Based on these sturctures,their interaction was visualized using molecular docking analysis.The results show that the major binding sites of NPF-NPFR are Phe45-Leu5,Arg44-Asp6,Ser41-Phe194 and His1-Thr295.This result provides the basic information for designing agonists or antagonists targeting NPF signaling system of aphids.In general,RNA-seq is a powerful tool for discovering the genes encoding neuropeptides and their GPCRs in insects.The identification of neuropeptides and their receptors from A.craccivora provides a solid foundation for further functional analysis.It is also of great significance in developing potential targets of alternative control agent of aphids.Meanwhile,the NPF signaling system was shown to play a positive role in the regulation of feeding behavior in aphids.This study revealed the relation between the NPF signaling and the feeding behavior in aphids for the first time.