Cytochrome P450 Reductase Mediates The Susceptibility Of Asian Citrus Psyllid Diaphorina Citri To Imidacloprid And Thiamethoxam

The Asian citrus psyllid Diaphorina citri Kuwayama is a serious pest that can cause great harm to citrus industry.It feeds from the phloem and causes sooty blotch,and more severely,it also transmits the Huanglongbing(HLB).HLB is a devastating disease on citrus worldwide.HLB has caused tremendous losses in many countries,including America,Brazil and China.Because there is no effective cure to HLB,choosing bacteria-free seedlings,managing D.citri and removing diseased plants in time are main strategies.Reducing D.citri populations can effectively slow down the spread of HLB.Chemical control,biological control and physical control are used to manage this species.Chemical control is the most widely used method.However,with the long-term and large-scale use of insecticides,D.citri has developed severe resistance to many kinds of insecticides in different areas.The research about the susceptibility of D.citri to insecticides will aid in the control of this species.Esterases,glutathione S-transferases and cytochrome P450 monooxygenases(P450s)are involved in insecticide resistance,and Nicotinamide adenine dinucleotide phosphate(NADPH)-cytochrome P450 reductase(CPR)is the primary member of P450 s.CPR transfers electrons to P450 s in the process of insecticide detoxification.In this study,The CPR gene of D.citri(DcCPR)was cloned and sequenced.The expression levels of DcCPR,were determined by reverse-transcription quantitative polymerase chain reaction(RT-qPCR)analysis,in different body parts/tissues and stages.RNA interference(RNAi)and eukaryotic expression system were used to explore the relationship between DcCPR and the susceptibility to imidacloprid and thiamethoxam.These studies will provide a theoretical basis for insecticide resistance mechanism of D.citri.The main results are as follows:1 Cloning and bioinformatics analysis of DcCPR in D.citriUsing CPRs sequence of Nilaparvata lugens,Laodelphax striatellus and Bemisia tabaci to blast the sequence of DcCPR in D.citri.The DcCPR ORF contained 2058 bp and encoded 685 amino acid residues,with a predicted protein p I of 5.37 and Mw of77.5 k Da.The key conserved domains of CPR proteins were also found in DcCPR,including the FMN-,FAD-,and NADP-binding domains;the three residues(R462,Y464,and S465)in the FAD-binding domain;four catalytic residues(S465,C637,D682,and W684)constituted the conserved active site.Alignment of the amino acid sequence with those of other insects revealed that the DcCPR shared high identity with some species,including Sogatella furcifera(70.9%),L.striatellus(70.5%),N.lugens(68.8%)and B.tabaci(68.4%).The phylogenetic analysis showed that DcCPR was located in the Hemiptera branch.This suggested a close evolutionary relationship between DcCPR and the other CPRs in Hemiptera.2 The expression patterns analysis of DcCPR in D.citriThe expression patterns of DcCPR in developmental stages(first instar nymphs,fifth instar nymphs,newly emerged adults,15-day-old adults)and different body parts/tissues(head,wings,legs,integument,Malpighian tubules,midgut,and fat body)were investigated using RT-qPCR.Among the developmental stages,the expression of DcCPR was highest in first instar nymphs and lowest in 15-day-old adults.The midgut had the highest expression level among the body.3 The function research of DcCPR in D.citriThe double-stranded RNA(dsRNA)was fed to D.citri in an artificial diet.After feeding on ds CPR for 72 h,the DcCPR messenger RNA level in D.citri adults decreased by 68.4%;the specific activities of DcCPR protein significantly reduced by41.6%,the subsequent western-blot analysis also showed that DcCPR content significantly decreased 37%,consistent with the results of the specific activity test.Meanwhile,the specific activities of P450 were also significantly reduced by 44.7%.After DcCPR was silenced,the mortality was significantly increased 27.9% with imidacloprid(20.435 mg/L)and 37.9% with thiamethoxam(5.334 mg/L),respectively.In a eukaryotic expression assay,a Bac-to-Bac baculovirus expression system was used to produce DcCPR in Sf9 cells successfully,and the cells expressing DcCPR had higher viability when exposed to imidacloprid or thiamethoxam.In summary,these results indicate that DcCPR mediates the susceptibility of D.citri to imidacloprid and thiamethoxam.