The Study On Population Genetic Structure And Function Of Neuropeptide F And Receptors In Mythimna Separata

The oriental armyworm,Mythimna separata（Walker）,is one of the important agricultural pests in China.As its larval gluttony,seasonal and roundtrip migration,and strong fecundity has led to a large-scale reduction area of crop yieldsor even no harvest.Therefore,accurate prediction and effective prevention and control of M.separata outbreaks is a considerable ensure for crop production and food security.At present,the prevention and control of armyworms mainly dependson chemical agents,but it caused an increasingly serious matter of the resistance to insecticide and environmental problems.Therefore,there is urgently required for effective and green control strategies of M.separata.From the role of insect behavioral physiology,this study has created a new perspective in pest control on the feeding behavior,growth and development regulation mechanism.Neuropeptide as endogenous active substance involves in many physiological processes of insects by activating G protein-coupled receptor（GPCR）.Thus,Neuropeptide signaling system is considered as a potential target for pest management.Firstly,population structure of M.separata in China were studied based on microsatellite markers.The purpose of this study is to discover whether there is a genetic differentiation between populations in the eastern and western China and provide a basis for prediction.Secondly,based onRNA-seq,this study explored the sequences neuropeptide F,short neuropeptide F,and their receptor genes of M.separata that had been proved to be involved in insect feeding or reproductive behavior.RNA interference（RNAi）and RT-q PCR were used to explore the physiological functions of NPF and sNPF signaling systems in M.separata.The main results are as follows:1.Microsatellite-based analysis of genetic structure of M.separata in ChinaThe genetic structure of 20 natural populations from 9 provinces in China was examined based on microsatellite markers.The results indicated that 193 alleles were detected at 7polymorphic loci,and had high genetic diversity.Bayesian clustering analysis identified three groups whereas discriminant analysis of principal components determined ten clusters that were considered as two clear-cut clusters and one admixed group.However,little to moderate(0<F_ST<0.15)genetic differentiation was detected,and total genetic variation of the population mainly derived from individuals.There was no correlation（P=0.118）between genetic distance and geographical distance according to Mantel test.Gene flow（N_em>4）occurred frequently in most population pairs,and an asymmetrical migration rate was detected in several pairwise population comparisons.The bottleneck test manifested that few populations had experienced recent bottlenecks.Correspondingly,large-scale and long-distance migration of armyworms has led to low genetic differentiation and frequent gene exchange.This research not only confirms the previous findings on the wide range migration and dispersal at the molecular level,but also has great significance for studying genetic evolution and help to improve predictions of M.separata outbreaks in China.2.Identification and sequence analysis of neuropeptide F and its receptor genesBased onRNA-seq analysis,a total of 50 neuropeptide precursors were mined from M.separata,which contained the vast majority of known insect neuropeptides.Subsequently,neuropeptide NPF,NPY,and sNPF,and their receptor NPFR,sNPFR1,and sNPFR2 genes were cloned and acquired.Open reading frame of them were 246,282,549,1182,1401,and1305 bp,respectively.Multiple sequence alignment of amino acid indicated that mature neuropeptidesNPF,NPY,and sNPF had highly conserved motifs of RPRFamide,RGRYamide and RLRFamide,respectively.Three receptorsNPFR,sNPFR1,and sNPFR2had typical seven transmembrane domains and belonged to rhodopsin like GPCRs.Phylogenetic tree analysis illustrated that these neuropeptides and their receptors of M.separata and lepidoptera insects clustered into independent evolutionary branches,which were closely related.3.Spatiotemporal expression profile of neuropeptide F and its receptor genesThe analysis of gene temporal and spatial expression patterns highlighted that NPF,NPY,sNPF and receptorsNPFR,sNPFR1 and sNPFR2 genes were significantly and differentially expressed in different instar larvae,pupae and adults.Six genes mostly expressed in larval stage,followed by adult stage,and pupal stage were the lowest expression.It was found that the expression levels of NPF,NPY,and NPFR were the highest in midgut.In contrast,sNPF and sNPFR1 were highly expressed in the central nervous system（CNS）,followed by midgut.Six genes were expressed in various tissues of 1st day-old adults,and the differences were significant.Of which,the NPF,NPY and NPFR expressed the highest levels in alimentary canal of M.separata adults.The sNPF had high abundance expression in head,while sNPFR1 and sNPFR2 were the highest expression levels in alimentary canal or fat body of adults.In addition,these genes had also a small amounts expression in testis and ovary.Further observation of the effects of mating and oviposition on gene expression found that,the expression levels of NPF,sNPF,sNPFR1,and sNPFR2 increased,while the levels of NPY and NPFR decreased in mated male.After laying eggs of female,the expression levels of NPFR and sNPFR1 enhanced significantly.It was assumed that these genes were also participated in the reproductive regulation of M.separata.4.Expression response of neuropeptide F and its receptor genes under stressDifferent stress treatments could significantly influence the expression levels of NPF,NPY,sNPF,and their receptor NPFR,sNPFR1 and sNPFR2 genes.Under starvation,the expression levels of NPF,NPY and NPFR in larvae and adults were up-regulated,while the levels of sNPF,sNPFR1 and sNPFR2 were down-regulated and six genes returned to the original level with refeeding,which indicating that the expression levels of six genes were closely associated to the feeding of M.separata.After treated with high and low temperatures,except for the down-regulation of sNPF expression,others elucidated an up-regulation tendency.Six genes of M.separata larvae were basically up-regulated by exposure to the different sublethal doses of chlorpyrifos,lambda-cyhalothrin and Bacillus thuringiensis.After the 3rd instar larvae were parasitized by Microplitis mediator,the transcription peaks of six genes appeared.These results provide supportive evidence that six genes play a vital role in the stress response of M.separata.5.Exploring the function of neuropeptide F and its receptor genes based onRNAiBy injected with dsRNA,the appropriate interference doses of sNPF and sNPFR1 were3μg for the 3rd instar larvae,while sNPFR2 was 5μg,with the silencing efficiency was30%～50%.For the 4th instar larvae,dose with 5μg dsNPY and 4μg dsNPFR were suitable forRNA interference（RNAi）,with the silencing efficiency was 20%～50%.After silencing of five genes,the relative consumption rate and growth rate of larvae decreased,which were presented in reduced food consumption,lower weight,smaller size and delayed development.The energy substance glycogen and total lipid decreased,and the content of trehalose increased.RNAi of newly emerged adults found that different doses of dsNPF and dsNPFR had a certain gene silencing efficiency,but were not stable.A dose of 4μg dsRNA had obvious silencing effect on NPY,sNPF and sNPFR1 genes,which led to 30%～60%down-regulation of gene expression.The interference efficiency of 6μg dssNPFR2 reached 40%in adults of M.separata.Gene silencing of sNPF and sNPFR2 caused decreased significantly of the number of eggs laid per female,while up-regulated expression of NPY,and sNPFR1had little impact on female egg-laying amounts of female.After silencing these four genes,there were no significant influence on the longevity of both female and male.It was further found that gene silencing of sNPFR2 resulted in significantly down-regulated expression levels of reproduction related genes JHAMT,FAMe T,Vg,and Vg R.In conclusion,NPF and sNPF signaling pathways regulate larval feeding,growth and development,and are related to energy metabolism.Furthermore,sNPF and its receptor sNPFR2 genes play an important function in female reproductive process of M.separata.6.Molecular simulation of the interaction between neuropeptide F and its receptorThe three-dimensional structure models of the receptor protein NPFR or sNPFRs and its polypeptide ligand NPF or sNPF were constructed by AlphaFold.On this basis,the interaction mode between them was visualized through molecular docking.It was found that three hydrogen bondsformed between Glu24-Tyr171,Tyr44-Tyr244 and Arg51-Ser300residues of NPF and its receptor NPFR,Arg39-Leu262,Pro52-Tyr303,and His53-Ser299residues between sNPF and ots receptor sNPFR1,and Pro52-Tyr339 between sNPF and its receptor sNPFR2 mighe be the main binding sites.It provides a reference for the design of agonists or antagonists targeting M.separata NPF and sNPF signaling system.In summary,this study makes it clear that there is no genetic differentiation among populations of M.separata in the eastern and western China,which complements the lack of research on population genetic structure and provides a basis for its scientific prediction.ByRNAi,it is determined that NPF,sNPF,and their receptor genes were closely correlated with the feeding and reproduction of armyworms.This aims to provide basis for creating new pest control agents with NPF and sNPF signal transduction system as control targets.