Identification And Analysis Of Neuropeptide In Atrijuglans Hetaohei Yang (Lepidoptera: Oecophoridae)

Atrijuglans hetaohei Yang(Lepidoptera: Gelechioidea),is one of the major pests seriously damaging the walnut fruits.Previous studies about A.hetaohei focus on the life history,field damage,prevention and control techniques and morphological description of antennae and other organs.In order to understand the types and distribution of neuropeptide genes in A.hetaohei,RT-qPCR was conducted to select the most suitable reference genes and their numbers in different developmental stages and sexes,as well as in different tissues of larva.At the same time,32 neuropeptide genes and 40 neuropeptide receptor genes were identified,and used to measure the expression of neuropeptide genes and receptors between sexes and across different tissues by RT-qPCR.The main findings are as follows:1 Identification of reliable reference genes for RT-qPCR studies of A.hetaoheiEight candidate reference genes(GAPDH,?-Tubulin,TBP,Histone,UBC,AK,28 S and ELF)from the results of transcriptome data were used to examine these genes' expression stabilities across developmental stages and between sexes by softwares GeNorm,NormFinder,BestKeeper and RefFiner.Between sexes and across developmental stages,the stabilities of these reference genes are GAPDH > 28 s > histone > TBP >ELF > UBC9 >AK > ?-Tubulin,and GAPDH and 28 S can be used to adjust the data;across tissues,the stabilities of candidate genes are 28 S > histone > TBP > GAPDH > AK > ELF > UBC9 > ?-Tubulin,and the combination of 28 S,histone and TBP is better during the process of data correction.The results are of great significance for the analysis of target gene expression in A.hetaohei.2 Identification of the neuropeptides and neuropeptide receptors of A.hetaoheiThe neuropeptide and neuropeptide receptor genes were from the head transcriptome data of adult A.hetaohei,and then blasted with blastp in NCBI(https://blast.ncbi.nlm.nih.gov).A total of 32 neuropeptides were found,containing 12 full-ORF genes(AKH1,AT,DH31,DH45,FMRF,ITG,OKA,PBAN,Pro,SIF,sNPF and TK),10 genes(AKH2,AstB,CAPA,CCH2,PTTH,Bur,CRF,ETH,LK and NPFb)having 5'untranslated region,and CCH1,MS,NP and NPLP1 having 3'untranslated region.40 neuropeptide receptors were also found,including 9 full-ORF genes(A1,A4,A10,A12,A13,A15,A20,A23 and A26),NPY and SPR having 5'untranslated region.15 genes(AstR,A7,A8,A11,A17,A27,A29,A30,A32,DHR,MCHR,INR and Thyrotropin-releasing hormone Receptor)having 3'untranslated region.3 Expression of Neuropeptide and receptors in different tissues of adult A.hetaoheiThe expression of 22 neuropeptides and 22 receptor genes were quantitatively analyzed by RT-qPCR in different sex and tissues.AKH1,AstB,CCH1,CCH2,CCAP,ITG,ETH,MS,OKA and so on showed a significantly higher expression in head than that in thorax and abdomen in females;NP,FMRF and PTTH had the highest expression in male abdomen.The expressions of Bur,CAPA,DH31,NPLP1,PBAN and SIF are higher in female adults than that in male adults,Bur is higher in abdomen of female adults than that in male adult,NPLP1,PBAN and SIF are higher in female head than that in male.In terms of the expression of the neuropeptide receptors: 9 genes(A6-A,A8,A9,A11,A12 and so on)showed a higher expression in head of male than that of female;A1,A4,A10,and A13 had the highest expression in abdomen and in male adults;A20 showed higher expression in head and abdomen,A24 and DHR showed the highest expression in female head and thorax,respectively.In this study,we identified and analyzed the neuropeptides and receptor genes in the head of the moth were also identified and analyzed,the expression of which in different tissues of male and female adults were measured.The above results lay a foundation for further studies on the development and physiological signal regulation of A.hetaohei.