| The main concern of the present study was to assess the capability of some cytochrome P450 monooxygenase(P450s)for degrading insecticides in small brown planthopper,Laodelphax striatellus.L.striatellus is a key destructive rice pest in Southeast Asia.Since 1960s the incidence of this pest has become more serious and extensive studies have been carried out to develop control programs.As a vector pest of several plant viruses,it transmits important viral diseases in most of the rice growing areas of East Asia,causing a huge grain loss(up to 50%grain loss was reported due to infestation occurred by rice stripe virus disease)thereby threatening rice production and global food security.No doubt,a lot of money has been invested in preventing yield losses caused by L.striatellus.In China,L.striatellus has been considered as a serious pest from more than 40 years since it caused serious yield losses by transmitting virus diseases,such as rice stripe virus disease(RSV)and rice black-streaked dwarf virus disease(RBSDV),in the mid-1960s in the Yangtze Delta area of China.In recent years its field populations have developed variable resistance to different kinds of insecticides and now a day's resistance to insecticide has been a frequent incidence.Therefore,great effort has been devoted for several years to insecticide resistance research to combat this pest.Now it is well recognized that increased enzymatic detoxification is one of the major reason behind of this resistance and it is the most common and important mechanism for any insect pests to tolerate regularly applied insecticides.Indeed,P450s are the detoxification enzymes that play significant roles in the interactions of insects with insecticides and finally detoxify the insecticides.P450s are the ubiquitous hemoproteins in a living organism that use a variety of small and large molecules as substrates in enzymatic reactions.These P450s are an extremely important enzymatic system performed various physiological functions in insect body including the metabolism of insecticides and other endogenous compounds.In general,P450s are over-expressed in resistant insect strains and thought involved in insecticide detoxification that ensuing insecticide resistance.However,insecticide degradation activities of different P450s are not well studied.Insects as other organisms have multiple P450s so that it is difficult to analyze the functions of a single P450.Functional expression in vitro requires also fine techniques.Thus,no individual P450 was confirmed for its insecticide degradation activity until 1990s when P450 genes were expressed in vitro.Only a few of P450s have been identified for degrading any kind of insecticides.Thus,examples are not enough to study the substrate specificity of P450s.Based on the family characteristics of P450s and the resistance associating P450s reported previously,this study try to declare if the P450s(family 4)either resistance relevant or irrelevant,could degrade insecticides in L.striatellus.The P450 genes were selected and recombinantly expressed in Sf9 cells.Then,the expressed P450 proteins were checked by SDS-PAGE and CO-difference spectra analysis,enzyme activity were tested with traditional P450 activity probing substrates,and the insecticide degradation capability were confirmed by measuring substrate depletion and metabolite formation after incubation.The results make well understand of insecticide metabolism and resistance development,as it was found that besides the reported P450s from family 6 and 9,a member from family 4 can also degrade deltamethrin.Furthermore,it is not only resistance associating P450s,but also resistance irrelevant P450s could degrade insecticide.The work was summarized in detail as following.1.Deltamethrin is degraded by CYP439A1v3,,a cytochrome P450 over-expressed in resistant strain of Laodelphax striatellusTen P450s from other 5 insect species have been identified for their capability to degrade deltamethrin.In deltamethrin resistant L.striatellus strain,another four P450s like CYP353Dlv2,CYP6FU1,CYP6AY3v2,and CYP439Alv3 were found over-expressed and thought these are resistance associated.The first three have been checked for their capability in our laboratory and only CYP6FU1 was found to degrade deltamethrin.In this study,an investigation was conducted to confirm the capability of CYP439Alv3 to degrade deltamethrin.The CYP439Alv3 was first expressed in Sf9 cell line,and then,the activity of the expressed recombinant cytochrome P450 protein for degrading different common insecticides was tested by measuring substrate depletion and metabolite formation.As results,SDS-PAGE and CO-difference spectra analysis showed that a intact target cytochrome P450 protein was successfully expressed.Tests with probe substrates proved its enzyme activity,as p-nitroanisole,ethoxycoumarin and ethoxyresorufin were preferentially metabolized(specific activity 7.77 ± 1.21,1.32 ± 0.14 and 0.36 ± 0.36 mol min-1 mg protein-1,respectively)with only Luciferin-H EGE was not.Incubation with different insecticides and subsequent HPLC,LC-MS and MS/MS analysis demonstrated that the expressed recombinant CYP439Alv3 could degrade deltamethrin to its less toxic metabolites i.e.4' hydroxy-deltamethrin by hydroxylation,but not for imidacloprid,buprofezin,chlorpyrifos,and fipronil.This study did not only confirm the function of the overexpressed CYP439Alv3 in deltamethrin resistance in L.striatellus,but also revealed the first insect cytochrome P450 monooxygenase of family 4 degrading deltamethrin,which should be helpful in both P450 specificity study and better insecticide resistance management.Otherwise,this study also lead to the conclusion that p-nitroanisole is a better probe substrate for mornitoring CYP439Alv3 activity and related deltamethrin resistance,but not for Luciferin-H EGE.2.Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellusP450s usually over-expressed in resistant strain were found involved in oxidative detoxification of insecticides.In this study,an investigation was conducted to confirm if resistance irrelevant P450s which were not over-expressed in the L.striatellus strains resistant to imidacloprid,deltamethrin and chlorpyrifos before,were capable of degrading insecticides.Three resistance irrelevant P450s viz.CYP417A2v2,CYP425A1v2,and CYP4DJ1 from CYP4 family of L.striatellus were randomly selected and expressed recombinant in Sf9 cells.When checked,CYP417A2v2 and CYP425Alv2 were found expressed successfully,while no target protein was found for CYP4DJ1.Furthermore,CO-difference spectra analysis and enzyme activity tests demonstrated that only CYP417A2v2 showed its intatct absorption spectrum and efficient catalytic activity out of the two expressed P450s.For catalytic activity tests,three traditional model probe substrates(p-nitroanisole,ethoxycoumarin,and ethoxyresorufin)and five insecticides(imidacloprid,deltamethrin,buprofezin,chlorpyrifos and fipronil)were assayed.Among the probe substrates screened,p-nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2(specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min-1 mg protein-1,respectively)and they may be potential diagnostic probes for this enzyme.For insecticides,only imidacloprid was efficiently degraded by CYP417A2v2.Incubation of imidacloprid with CYP417A2v2 of L.striatellus and subsequent HPLC,LC-MS and MS/MS analysis was done for imidacloprid depletion,metabolite formation and metabolite molecule.Thes results revealed that CYP417A2v2 could catalyte the formation of imidacloprid metabolites i.e.less toxic 4'-hydroxy imidacloprid by hydroxylation,and imidacloprid depletion paralleled with metabolite formation is,as expected,incubation time dependent.This result implies the exemption of P450s character that it is not always,all the P450s degrading insecticides being selected and over-expressed in resistant strains and the degrading P450s without mutations to up-regulate could be candidates during insecticide resistance evolution.These characterizations of individual insect P450s in insecticide degradation can provide insight for better understand of insecticide resistance development.3.Fipronil was found to be degraded by a cytochrome P450 monooxygenase CYP426A1 in Laodelphax striatellusSome other resistance irrelevant P450s from family 4 were further studied and CYP426A1 was found to degrade fipronil,which was also not reported before over-expressed in the strains resistant to imidacloprid,deltamethrin and chlorpyrifos.The same experiment procedure was employed and the CYP426A1 was first combinant expressed in Sf9 cells.Then,the expressed target protein was checked by SDS-PAGE and CO-difference spectra analysis.Insecticides incubation and subsequent HPLC analysis were used to confirm the enzyme degrading activity on different insecticides by checking the depletion of the parent fipronil and the formation of its metabolites.The results showed that the intact target P450 protein was successfully expressed in Sf9 cells.Enzyme activity tests with traditional model probe substrates found that CYP426A1 could preferentially metabolize p-nitroanisole(specific activity 5.21 ± 0.71 nmol min-1 mg protein-1),ethoxycoumarin(specific activity 1.01 ± 0.12 nmol min-1 mg protein-1)and ethoxyresorufin(0.73 ± 0.26 nmol min-1 mg protein-1),that may be potential diagnostic probes for this enzyme.Insecticide degrading experiments demonstrated that only fipronil was degraded by CYP426A1 and no enzyme catalyzing degradation was found on imidecloprid,deltamethrin,buprofizen and chlorpyrifos.The molecule of fipronil metabolite is still being analyzing.Though CYP426A1 was not found over-expressed in resistant strains before,the resistant strains of L.striatellus reported for P450 over-expression previously were all selected with insecticides other than fipronil.Thus,it is still not clear if CYP426A1 could be selected by fipronil and contributes to its resistance development.Further studies is still needed even its degradation capability has been confirmed in this study. |
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