AKH Signaling System Is Involved In The Regulation Of The Susceptibility Of Bactrocera Dorsalis To Malathion

The oriental fruit fly,Bactrocera dorsalis,as a quarantine pest has become one of the most important pests because of it's high reproduction,better adaptability,strong flight ability and some other characteristics.In recent years,due to the unscientific and unreasonable use of the pesticides,B.dorsalis has developed serious insecticide resistance.As a kind of small molecule protein secreted by insect nervous system,neuropeptides has the characteristics of microscale and high efficiency.With the further research on neuropeptide signaling system,it has been reported that the neuropeptide signaling system is involved in the regulation of insecticide s susceptibility.Therefore,the development of novel synergist targetting neuropeptide signaling systems will become a new idea for reduction of pesticides in the future.In this study,qPCR was used to detect the expression patterns of 39 neuropeptide precursor genes under different stresses as well as in malathion resistant strain?MR?and malathion susceptible strain?MS?.We found that the expression levels of AKH and AKHR significantly increased in MR and by malathion induction.Subsequently,through RNA interference?RNAi?and bioassay,it has been proven that the AKH signaling system plays an important role regulating the susceptibility of malathion in B.dorsalis.Moreover,the enzymatic activity assay,qPCR and high performance liquid chromatogra?HPLC?were used to clarify the mechanism of AKH signaling system involved in regulating the malathion susceptibility of B.dorsalis.Our data will enrich the understanding of the function of the AKH signaling system.In addition,it can provide a reference for the study on the neurophysiological mechanisms of the insect susceptibility to pesticides.The main results of this study are as follows:1 The expression patterns of neuropeptides of B.dorsalis under different environment stressIn this study,qPCR was used to analyze the expression patterns of 39neuropeptide genes in B.dorsalis under different environment stress(40?for 1 h,0?for 1 h,desiccation for 12 h,starvation for 6 h and malathion(LC₅₀)treatment for24 h).The results showed that the expressions of most neuropeptides were significantly increased upon different stresses in B.dorsalis.Under heat treatment,the expressions of ETH and ILP2 were significantly increased.However,eight genes including ITP2,Myosuppressin,NPLP,Orcokinin B,PDF,RYamide,sNPF and Sulfakinin were significantly increased in low temperature treatment.Responding to starvation,the transcript level of GPA2 was significantly decreased,but the other 11genes including CCAP,CCHamide-2,ILP2,ITG-like peptide,ITP2,NPLP,Neuroparsin,Orcokinin B,PDF,sNPF and Sulfakinin were significantly increased.The expression levels of four genes including AST-C,CNMamide,ILP6 and ILP7significantly increased upon to desiccation.After malathion treatment,ten genes,including AKH?AST-A?CCHamide1?CCHamide2?ILP5?ILP6?Orcokinin B?PTTH?SIFamide and Tachykinin were significantly increased.2 The expression patterns of neuropeptides in malathion resistant and susceptible strains of B.dorsalisThe expression patterns of 39 neuropeptides in malathion resistant and susceptible strains of B.dorsalis were analyzed by using qPCR.The results showed that the expressions of AKH,AST-A,ETH,ILP2,Leucokinin,NPLP,Neuroparsin,Orcokinin B,PDF,RYamide,Sulfakinin,Trissin and sNPF were significantly increased in MR strain in comparison with MS.However,the expressions of AST-CC,CCAP,CNMamide,EH and SIFaimde were significantly decreased in MR strain.3 RNAi and phenotypic analysis of AKH and AKHRThe expressions of AKHR in MR and MS strain were silenced by RNAi,then followed by a malathion treatment with LC₅₀0 dose to evaluate the susceptibility of the flies.The results showed that after the injection of dsRNA,the expression level of AKHR decreased by 78.88%and 78.61%in MR and MS,respectively.After silencing AKHR,the results showed that the mortality rate was significantly increased by53.09%in MR,while the mortality rate in MS was significantly increased by 33.54%.4 The mechanism of AKH signaling system regulating the susceptibility of B.dorsalis to malathionBased on enzymatic activity assay,we tested the activity of detoxification enzyme genes after silencing AKHR.The result showed that the activities of detoxification enzymes were not significantly different after silencing AKHR comparing to control.As expected,HPLC results indicated that the metabolism ability of malathion in B.dorsalis was significantly reduced after interfering AKHR.In addition,the qPCR results showed the expression of GSTd10 and CarE6 that are associated with malathion metabolism in B.dorsalis were significantly declined.Based on our data,we speculated that AKH secreted from corpora cardiac?CC?,translocates nervous system/haemolymph to fat body,and then triggers the downstream signal transduction by activating on AKHR.Subsequently,the expressions of two enzymes?GSTd10 and CarE6?detoxify the malathion are positively regulated.Thus,the detoxification capacity of B.dorsalis to malathion will be enhanced.However,it is still not clear which downstream signaling pathway is triggered to regulate the expression of the two important detoxification enzyme genes?GSTd10 and CarE6?,and the molecular regulatory mechanism is worth further study.