Study On The Effects Of Sublethal Concentration Of Insecticides On Juvenile Hormone Metabolic Enzymes And Insulin-like Genes Of Nilaparvata Lugens Stal(Hemiptera:Delphacidae)

The brown planthopper,Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)is an important pest on rice.Indoxacarb and chlorantraniliprole are two new insecticides which widely used in the production.Previous studies have shown that sublethal concentration of indoxacarb and chlorantraniliprole can inhibit the BPH reproduction.It has been widely recognized that juvenile hormone plays a critical role in ovarian development and JH is regulated by its metabolic enzymes.Insulin is an important kind of endocrine hormones in insects which regulates the physiology,biochemistry and reproduction of insects,and it is also closely related to JH.In this paper,we cloned the gene of juvenile hormone diol kinase(Jhdk).Difference of the gene expression in different stages of brachypterous BPH strains was analysis,and the relationship with juvenile hormone esterase and juvenile hormone epoxide hydrolase was studied using RNAi method.We also integrated the analysis of changes in NlIlp1-4 and 3 JH metabolic enzyme genes mRNA relative expression levels,further revealed the mechanism of the two insecticides inhibiting BPH reproduction.RT-PCR was used to amplify the full length Nljhdk cDNA from BPH.Compilation of the cDNA clones resulted in a 684 bp contiguous sequence containing a 558 bp ORF.The encoded 185 amino acid residues predict a protein with a calculated molecular mass of 21.57 kDa and a 4.54 isoelectric point.An amino acid sequence alignment shows that Nljhdk shares high amino acid identity with other JHDK homologues including Bdjhdk in Bactrocera dorsalis(JN25 6054,55.68%),Ldjhdk in Leptinotarsa decemlineata(KP295467,61.08%),and Msjhdk in Manduca sexta(AJ430670,51.35%).To gain the expression of Nljhdk in BPH,the mRNA levels of this gene was analyzed using qPCR in different periods of BPH.The expression pattern revealed that Nljhdk was expressed in all development periods tested with the lowest level in 4 instar and the highest in 5 instar.And Nljhdk relative expression level in male adults is higher than in female adults.Through the RNAi technology,we found that silent Nljhdk had no significant effect on Nljhe,while Nljheh expression level was up regulated.The sublethal effects of indoxacarb on BPH was studied,and the result showed that sublethal dose of indoxacarb could inhibit the expression of NlIlp2-4 in BPH ovaries,however,the expression level of Nlllp1 was stimulated with 20mg/L indoxacarb treatment.The expression level of Nljhe in ovaries was increased with the increase of indoxacarb concentration,which were increased by 52.92%,38.82%,105.5%,79.44%respectively,after the 3rd nymph exposed to 3,5,10,20 mg/L indoxacarb.With 1,3,5,10,20 mg/L indoxacarb treatment,Nljheh expression levels were reduced 34.09%,30.49%,48.21%,45.21%and 42.49%,respectively,compared with untreated insects.The expression levels of Nljhdk were up regulated only with 1,3mg/L indoxacarb treatment,no significant difference in other concentration treatments.After treated by sublethal dose of chlorantraniliprole,NlIlp1 mRNA relative expression level in ovaries increased by 26.88%with 15 mg/L concentration.The treatment with 3.75 mg/L chlorantraniliprole decreased NlIlp1 mRNA expression level by 7.9%,while 7.5,30,60 mg/L increased by 20.13%,28.12%and 47.77%.NlIlp3 mRNA relative expression level with 60 mg/L chlorantraniliprole treatment increased by 153.27%compared with control and there is no significant difference in other concentrations.The relative expression level of NlIlp4 decreased by 9.62%in 3.75 mg/L chlorantraniliprole concentration compared with control,while with 7.5,15,30,60 mg/L treatment,the levels increased by 51.24%,69.77%,52.63%and 95.17%,respectively.After treated by 15,30,60 mg/L chlorantraniliprole,the Nljhe mRNA relative expression levels in ovaries increased by 25.77%.24.12%and 72.45%compared with control,respectively.Nljheh mRNA relative expression levels in ovaries in 3.75,15,60 mg/L chlorantraniliprole concentration increased by 16.18%,23.09%,65%compared with untreated ones.With 15,60 mg/L chlorantraniliprole treatment,Nljhdk mRNA expression levels increased by 48.8%and 8.51%,which decreased in other concentration treatments.In this paper,we explored the mechanism of indoxacarb and chlorantraniliprole inhibiting the reproduction of brown planthopper,which provided theoretical basis for rational use of these two insecticides in paddy fields.