Roles Of P450s And Carboxylestrase In Permethrin Resistance In Culex Quinquefasciatu

The mosquito Culex quinquefasciatus is a primary vector of West Nile encephalitis,eastern equine encephalitis,Saint Louis encephalitis,and lymphatic filariasis pathogens.Pyrethroids such as permethrin is still currently the most widely used insecticide for the indoor control of mosquitoes worldwide and the only chemical recommended for the treatment of mosquito nets.Nevertheless,uncontrolled use of pyrethroids has led the pyrethroids resistance in mosquito vectors to become a global problem,resulting ineffective vector control activities.Cytochrome P450s and carboxylestrase have long been of particular interests because of their critical role in the detoxification and/or activation of xenobiotics such as drugs,pesticides,plant toxins,and are involved in the insecticide resistance.In our pevious study,we have found that multiple P450s and some carboxylestrase are overexpressed in permethrin resistant strains,however,their roles in metabolic resistance of permethrin have not been investigated yet.In this study,we investigated the functional roles of some P450 genes and carboxylestrase genes,which are potentially involved in the permethrin resistance in culex quinquefasciatus.The results are as belows:1.Permethrin induction of multiple P450 genes involved in the permethrin resistance in culex quinquefasciatusIn this study,we investigated the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes,Culex quinquefasciatus.The gene expression in 7 of the 19 P450 genes in the HAmCqG8 strain,increased more than 2-fold after exposure to permethrin at an LC50 concentration compared to their acetone treated counterpart.Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain were also induced more than doubled after exposure to an LC50 dose of permethrin.Three P450 genes,CYP9J34,CYP9M10 and CYP9J45,were induced by permethrin both in HAmCqG8 and MAmCqG6 strain.These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms,providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.2.Optimization of co-expression conditions when using baculovirus expression system to co-express CYP and CPRWe used baculovirus-insect expression system to co-express CYP9M10 with CPR,and we optimized the protein expression conditions based on the P450 content and catalytic activity measured by 7-ethoxycoumarin substrate(ECOD activity).The results showed that the combination of 0.1mM 5-ALA and 0.02mM Fe3+ or lug/mL hemin and 0.1mM 5-ALA allowed CYP9M10/CPR had higher ECOD activity than other conditions.The hightest ECOD activity was exhibited by co-expression of CYP9M10 with MOI of 0.5 or 0.2 and CPR with MOI of 0.05 or 0.02 in suspension culture(fixed ratio of 10).This expression profile also exhibit an expression mode for expression of other P450s.To our knowledge,it is the first time for co-expression Culex.Quinquefasciatus P450 protein and its co-factor CPR in baculovirus-insect expression system.3.In vitro expression of CYPs and permethrin metabolism,revealing the roles in prmethrin resistance in C.quinquefasciatusUsing baculovirus expression system,we totally co-expressed 8 CYP with cytochrome P450 reductase(CPR),respectively in insect Spodoptera frugiperda(SF9)cells,characterized enzymatic activity and metabolic ability of them to permethrin and 3-phenoxybenzoic alcohol(PBOH)and 3-phenoxybenzaldehyde(PBCHO),and conducted cytotoxicity assays.MTT assay results revealed that CYP9M10 and CYP6AA7 could play a role in detoxifying permethrin,PBOH and PBCHO in the insect cells.HPLC chromatographic analysis revealed that microsomes CYP9M10/CPR and CYP6AA7/CPR could metabolize permethrin(catalytic rate:0.49 pmol/mim/pmol P450 and 0.56 pmol/min/pmol P450,respectively)and PBOH was the metabolite detected in this study.PBOH and PBCHO can also be metabolized further by microsomes CYP9M10/CPR and CYP6AA7/CPR,which are the metabolites of permethrin demonstrated by some studies.PBCOOH was the ultimate metabolite both in the metabolism of PBOH and PBCOOH.Bioassay resuts of resistant and susceptive strains revealed that PBOH and PBCHO are more toxic to misquote larvaes than PBCOOH with the LC50 value ratio being 18 fold,and the resistant strain was more tolerant to PBOH and PBCHO than susceptive strain,which indicated that the metabolism of PBOH and PBCHO in mosquitoes by CYPs would also play great role in permethrin reisitance.The metabolisms of permethrin by other expressed P450 proteins also have been conducted.MTT results indicated that CYP9J34,CYP9J33,CYP9J40,CYP9J45,CYP6BZ2,CYP6P14 could play a role in detoxifying permethrin in the insect cells.HPLC chromatographic analysis revealed that all these expressed P450 isomers could metabolite permethrin,at the catalyric rate of 51.1,44.5,53.4,35.4,50.7,50.9 pmol/min/mg protein,respectively.Our results strongly demonstrated the role of these 8 P450s in metabolism of insecticide permethrin,the molecular basis of resistance development in mosquitoes.4.Functional study of three carboxylestrase in the permethrin resistance in Culex.quinquefasciatusWe used the real time PCR to examine the relative expression level of three carboxylestrase genes in resistant strain GAmCqG8 and suspentive strain S-Lab,and fond that these three genes are all up-regulated in HAmCqG8 strains compared to S-Lab strain;the expression level of them are all much higher in larvae than that in female ad male adult mosquitoes.We used baculovius expression system expressed these three carboxylestrase and the a-naphthyl acetate activity results showed that CPIJ018231 had excellent activity toward substrate a-naphthyl acetate,while CPIJ018232 and CPIJ018233 had just one tenth of the activity of CPIJ018231.Cytotoxicity of permethrin results revealed that CPIJ018232 play significant role in detoxification of permethrin in sf9 cells than CPIJ018231 and CPIJ018233.CPIJ018233 showed the lowest role in detoxification of permethrin in sf9 cells.HPLC chromatographic analysis results showed that CPIJ 018232 metabolited the permethrin at the highest rate with 8.0 pmol/min/mg,CPIJ018231 metabolited the permenthim at 4.5 pmol/min/mg,CPIJ018233 metabolited the permethrin at the lowest rate with 3.5 pmol/min/mg.PBOH is the metabolite detected in this study.The docking modes of naphthyl acetate and pemethrin were consistent with our metabolism results.In conclution,our results demonstrated that some upregulated P450s,like CYP9M10,CYP 6AA7,CYP9J34,CYP9J33,CYP9J40,CYP9J45,CYP6BZ2,CYP6P14,and three carboxylestrase,CPIJ018231,CPIJ018232and CPIJ018233,could metabolize permethrin,resulting in the permethrin resistance in culex mosquitoes.