Insecticide Metabolism And Expression Regulation Of The Cytochrome P450s In Locusta Migratoria

The migratory locust(Locust migratoria)is a paurometabolous insect and one of the most destructive agricultural pests in the world.Synthetic insecticides are often used to control L.migratoria in pest management programs.However,frequent applications of insecticides in a large scale have posed many environmental and ecological problems,including the development of insecticide resistance in the insect populations.One of the important mechanisms contributing to insect resistance is the enhancement of detoxification by detoxification enzymes.Cytochrome P450(P450)is one of the most important detoxification enzymes and plays an important role in the metabolism of endogenous and exogenous compounds,including chemical insecticides.With the completion of genome sequencing and the accumulation of transcriptome databases,more and more P450 genes were identified from L.migratoria.To date,at least 78 P450 genes have been identified and named in L.migratoria.With such a large number of P450 genes,elucidating their gene functions and understanding their regulatory mechanism have become an important focus of the current research.By using RNAi coupled with insecticides bioassay,our research team has found that some P450 genes are implicated in the detoxification of insecticides.However,in view of the complexity of the metabolic detoxification in insects and the shortcomings of RNAi,characterization of P450 enzymes at the biochemical level is crucial for better understanding the detoxification roles of P450 genes.On the other hand,the expression of P450 genes is regulated precisely and complexly to cope with the changing environment.Therefore,an in-depth understanding of their regulatory mechanisms is necessary for understanding the mechanisms leading to the induction of P450 genes by insecticides and the development of insecticide resistance in L.migratoria.1、Comparisons of microsomal cytochrome P450 content and enzymatic activity towards selected model substrates and insecticides in different tissues from the L.migratoriaMicrosomal P450 protein content and enzymatic activity in four major detoxification tissues(midgut,gastric caeca,Malpighian tubules and fat bodies)dissected from fifth-instar nymphs of L.migratoria were examined.The highest microsomal P450 protein content was found in the gastric caeca,followed by the midgut,Malpighian tubules and fat bodies.Microsomal P450 s showed the highest aromatic hydroxylation,O-dealkylation and O-dearylation activities towards six of the seven model substrates examined in the fat bodies.Although the gastric caeca showed the highest P450 protein content,the enzymatic activities towards six of the seven model substrates were the lowest in this tissue.Further,the midgut,gastric caeca and fat bodies showed significant metabolic activities towards two pyrethroid insecticides(deltamethrin and fluvalinate),but no significant activities towards the other four insecticides(malathion,chlorpyrifos,carbaryl and methoprene).It can be seen that the insect P450 enzyme activity is tissueand substrate-dependent.In addition,the research results also suggest that it is not informative to predict its enzyme activity based only on the total microsomal P450 protein content.2、Metabolism of recombinant Lm CYP6FD1 towards different model substrates and insecticidesLm CYP6FD1 and Lm CPR were heterologously co-expressed in Sf9 insect cells.The recombinant enzymes were assayed for metabolic activity towards six selected model substrates(Luciferin-H,Luciferin-Me,Luciferin-Be,Luciferin-PFBE,Luciferin-CEE and 7-ethoxycoumarin),and four selected insecticides(deltamethrin,chlorpyrifos,carbaryl and methoprene).Recombinant CYP6FD1 showed activity towards 7-ethoxycoumarin and Luciferin-Me,but no detectable activity towards the other Luciferin substrates.Furthermore,the enzyme efficiently oxidized deltamethrin to hydroxydeltamethrin through an aromatic hydroxylation in a time-dependent manner.These results provide direct evidence that CYP6FD1 is capable of metabolizing deltamethrin.3、Recombinant expression and functional analysis of Lm CYP6FE1Lm CYP6FE1 and Lm CPR were heterologously co-expressed in Sf9 insect cells.The recombinant enzymes were assayed for catalytic activity towards six selected model substrates(Luciferin-H,Luciferin-Me,Luciferin-Be,Luciferin-PFBE,Luciferin-CEE and 7-ethoxycoumarin),and five selected insecticides(deltamethrin,chlorpyrifos,carbaryl,methoprene and imidacloprid).The results showed that recombinant CYP6FE1 showed no detectable activity on these model substrates.However,the enzyme efficiently oxidized imidacloprid to hydroxyimidacloprid and contributed to the susceptibility of L.migratoria to imidacloprid.4、Characteristics and roles of cytochrome b5 in cytochrome P450-mediated oxidative reactions in L.migratoriaThe full-length c DNA sequence of Lm Cyt-b5 was sequenced after reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that Lm Cyt-b5 was closely related to the Cyt-b5 from Blattodea.Lm Cyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of Lm Cyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of Lm Cyt-b5 or silencing of both Lm Cyt-b5 and Lm CPR did not significantly change the total P450 activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of Lm CYP6FD1 with Lm CPR and Lm Cyt-b5 together in Sf9 cells significantly increased the catalytic activity of Lm CYP6FD1 toward 7-EC as compared with the coexpression of Lm CYP6FD1 with Lm CPR or Lm Cyt-b5 separately.These results suggest that Lm Cyt-b5 plays an important role in the catalytic reaction of Lm CYP6FD1 towards 7-EC.5、The transcription factor Cnc C regulates the expression of multiple genes involved in insecticide detoxification in L.migratoriaThe full-length c DNA sequence of Lm Cnc C was obtained by RT-PCR from L.migratoria.After suppression of Lm Cnc C expression,the susceptibility of L.migratoria to deltamethrin and imidacloprid increased.Comparative transcriptome sequencing was performed with L.migratoria injected with ds Cnc C and ds GFP.Nine genes either down-regulated or related to insecticide detoxification were revealed,which include five P450 genes(CYP6FD1,CYP6FD2,CYP6FE1,CYP6HL1 and CYP6MU1)and four UDP-glucuronosyltransferase(UGT)genes(UGT392A1,UGT392A2,UGT392B1 and UGT392C1).The role of these genes in detoxification of deltamethrin and imidacloprid was analyzed by RNAi followed by insecticides bioassay.The results showed that silencing of CYP6FD1 and UGT392C1 obviously enhanced the susceptibility of L.migratoria to deltamethrin,whereas silencing of CYP6FE1 and UGT392C1 increased the susceptibility of the nymph to imidacloprid.These results suggest that Lm Cnc C may contribute to the detoxification of deltamethrin and imidacloprid by regulating the expression of multiple detoxification genes.In summary,we characterized biochemical properties of microsomal P450 in the main detoxification organs from the migratory locust.Two CYP6 family P450 enzymes(CYP6FD1 and CYP6FE1)were heterologously expressed through Bac-to-Bac baculovirus expression system.We then systematically characterized the enzyme activity and their roles in insecticide metabolism.Finally,we examined the transcriptional response of Cnc C to the exposure of insecticides and its regulatory role for the selected detoxification genes.These results are expected to provide valuable information for further research to better understand the regulatory mechanisms of P450 genes,the physiological and biochemical characteristics of P450 enzymes,and the mechanism of insecticide detoxification in insects.