Functional Characteristics Of Fmo In Resistant Spodeptera Exigua And VGSC In Drosophila Melanogaster

The beet armyworm(Spodoptera exigua)is an important leaf-feeding pest on vegetables and other crops.Due to relied on chemical pesticides for prevention and control solely on a long-term,the pest has already generated high levels of resistance to traditional insecticides,including organophosphorus,pyrethroids and carbamate agents,and new insecticides such as chlorantraniliprole,indoxacarb,and metaflumizone.Metaflumizone is a novel semicarbazone sodium channel blocker,which is considered as a good substitute for traditional insecticides.The toxicity and mechanisms of the related resistance of Spodopetra to metaflumizone are still insufficient.This study was initiated from the investigation of the resistance mechanism of S.exigua to metaflumizone.We firstly found that Spodoptera exigua in Huizhou of Guangdong Province produced high levels of resistance to metaflumizone,flavin-dependent monooxygenase(FMO)was involved in the biochemical resistance of metaflumizone as a novel drug resistance mechanism.Through investigating the molecular characteristics,enzyme activity and mRNA expression levels in the susceptible/resistant population,in vitro expression and metabolic activity of the insecticide of Spodoptera exigua FMO,we confirmed S.exigua FMO was related with the biochemical resistance to metaflumizone.During the investigation of voltage-gated sodium channel(VGSC)and metaflumizone target resistance,due to the failure to obtain the full-length VGSC sequence of S.exigua,finally we selected VGSC of Drosophila melanogaster to perform the biological expression in vitro,then studied of the insecticide susceptibility of VGSC transcripts and the effects of gene mutations on VGSC gating characteristics and drug sensitivity systematically,it improved understanding of metaflumizone target protein and insecticide resistance.1.S.exigua generated metabolic resistance to metaflumizone.Using leaf dipping method,we determined that the population of S.exigua from Huizhou,Guangdong province generated 922-fold and 60-fold resistance,which were considered as extremely high level and high level of resistance,respectively.Synergist tests showed that the synergistic ratios of the esterase’s(EST’s)synergist DEF on the HZ11 and HZ12 populations were 5.7-and 3.4-fold,respectively;and the synergistic ratios of the FMO’s synergist methimazole on the HZ11 and HZ12 populations were 3.1-and 1.9-fold,respectively;however,the effects of PBO,a synergist of cytochrome P450,and DEM,a synergist of glutathione-S-transferase(GST),were not observed significantly.The detoxification enzyme activity assays showed that the activities of EST and FMO in HZ12 were significantly higher(2.8 and 1.4-fold)than those of Lab-sus,and further increased in the resistant population which was screened with metaflumizone(reaching the 3.4-and 1.5-fold to Lab-sus,).The activities of P450 and GST in the HZ 12 population were not observed to be significantly different from Lab-sus.Therefore,it is believed that the ESTs played a major role in the biochemical resistance of S.exigua to metaflumizone in Huizhou population.FMO also participates in it,P450 might play a weak role,while GST was not involved in the resistance.2.The molecular characteristics and expression patterns of SeFMOThe full-length sequences of three FMO genes(SeFMO)of S.exigua were obtained by PCR-RACE amplification.The full legnth of SeFMO1 was 1837 bp,encoding 454 amino acids;The full legnth of SeFMO2 was 2277 bp,encoding 453 amino acids;The full legnth of SeFMO3 was 1617 bp,encoding 431 Amino acids.Sequence analysis showed that SeFMO had basic domains of FMO,which were FAD-binding site,NADPH-binding site and FMO-identifing sequence.Phylogenetic analysis showed that the FMO1 and FMO2 of Lepidoptera species were evolved from the same ancestor,while FMO3 of Lepidoptera was clustered with the FMO genes of Orthoptera and Diptera.The real-time fluorescence quantitative PCR was used to detect the expression patterns of SeFMO.The results showed that SeFMO had the characteristics of tissue-specific expression and developmental-specific expression.it showed that higher levels of expression in midgut,fat bodies,malpighian tubules,and older larvae.FMO microsomal enzyme activity assay results correspond to mRNA expression levels and relatively high activity in midgut and fat bodies.Meanwhile,the activity of FMO increased followed by the increase of the larval instar,reaching the highest activity(4.814 nmol/min/mg protein)in 5th instar larvae.The fat body cell line of S.exigua were treated by 11 insecticides in vitro.After 24 h,a few insecticides were observed to slightly increase the expression of SeFMO,indicating that the expression of SeFMO could be induced by exogenous insecticides.Since detailed reports on the insect FMO activity were scarce,we had established the optimum conditions for the determination of the FMO enzyme activity of S.exigua:the optimum pH was 9.0,the optimum temperature was 35℃,the total protein concentration in the reaction system was 0.05-0.2 mg/mL,and optimal reaction time was within 20 minutes.The optimal temperature and the optimum pH for the reconstitution of the FMO pure enzyme solution in vitro are 8.5-9.0 and 37℃,which have the same tendency as those of the microsomal enzyme solution.3.The relationship between SeFMO and metaflumizone resistanceWe have confirmed that FMO is related to resistance of metaflumizone through a series of studies.Firstly,in the metaflumizone screening strain(HZ12-meta),FMO enzyme activity was significantly higher than that of HZ 12 original population.The expression level of each SeFMO gene in HZ12-meta was significantly higher than that of the susceptible population(Lab-sus)in most age groups and tissues.Metaflumizone had a weak induction effect on the expressions of SeFMO,the induction effects for SeFMO1 and SeFMO2 were 1.6-and 1.5-fold,respectively.Therefore,we found that the improvement of FMO activity was related to the induction of metaflumizone.Secondly,we expressed SeFMOs in vitro using Bac-to-Bac baculovirus expression system.The enzymatic activities of rSeFMOs to 19 compounds were monitored by NADPH oxidation.The S-oxidation activity of rSeFMO protein is higher than that of N-oxidation activity,the activity of rSeFMOs to N-oxidation substrate dimethylamine was 1.99,2.07 and 4.22 nmol/min/mg protein respectively;The activity of rSeFMO to S-oxidation substrate methimazole were 5.83,4.14 and 4.98 nmol/min/mg protein.The S-oxidation activity of the rSeFMO enzyme was higher than that of the N-oxidation.The activity of rSeFMOs to metaflumizone(8.05、19.09and 12.19 nmol/min/mg protein)and to lambda-cyhalothrin(9.60,6.98 and 6.44 nmol/min/mg protein)were higher than model substrates.While the activities to other insecticides were relatively low,basically between 1-3 nmol/min/mg protein.The results of the rSeFMO activity assay showed that SeFMO was an important detoxification metabolic enzyme for insecticides,especially for metaflumizone.The results of the determination of recombinant FMO activity showed that SeFMO is metabolically active to insecticides,especially metaflumizone,and is an important detoxification metabolic enzyme.It is believed that the metabolic activity of FMO to metaflumizone is related to resistance of metaflumizone.4.The diversity of sensitivities in different voltage-gated sodium channel variants.We studied the function and characteristics of the voltage-gated sodium channel of D.melanogaster,systematically.Firstly,the bioassay showed that the sensitivities of W1118 D.melanogaster strain to six insecticides were:transfluthrin>dimefluthrin>metofluthrin>deltamethrin>permethrin>DDT,the LD50 were 0.059,0.094,1.37,2.62,9.37,and 56.99 ng,respectively.We expressed 29 sodium channel transcripts of D.melanogaster in Xenopus oocytes,recorded the relevant sodium currents by bipolar voltage clamp technique.We measured the sensitivities of the 29 sodium channel transcripts to transfluthrin and metofluthrin.The DmNav22 had the highest sensitivity to two agents,and the sodium channel modification percentages were 89.34±10.22%and 38.25±4.25%,respectively.The sensitivities of most sodium channel transcripts to transfluthrin are higher than that of metofluthrin,which corresponds to the bioassay results.There were a few differences in sensitivities between the different transcripts to same agent,and the percentage of relative susceptible transcripts was lower than that of the relative resistant transcripts.The analysis of the sensitivities of different transcripts of Drosophila sodium channels to insecticides complemented the understanding of the characteristics of VGSC which was identified as the target of metaflumizone.5.The I1545M and G1571R mutation mediated sodium channel resistance.Gene mutations in the insect voltage-gated sodium channels are important causes of insecticide resistance.D.melanogaster strain Ocd5 has produced an extremely high level of resistance to DDT(more than 1000-fold),and we detected two mutations I1545M and G1571R in VGSC,which are related with insecticide resistance.By using site-directed mutagenesis,we introduced the I1545M,G1571R,and I1545M/G1571R(double mutant)into the DmNav2-4 variant,and then expressed corresponding channels in Xenopus oocytes.The V1/2 and slope for the activation of DmNav2-4 were measured,which were-18.9±0.3 mV and 3.8±0.2 mV,respectively;the fast inactivation factor V1/2 and slope k for DmNav2-4 were-44.9±0.2 mV and 4.8±0.3 mV,respectively.The I1545M mutation affected the activation(V1/2=-7.8±0.2 mV)and fast inactivation(V1/2=-48.7±0.3 mV),while the G1571R mutation just affected the fast inactivation of sodium channels(V1/2=-49.3±0.3 mV).The double mutant channel also changed both activation(V1/2=-5.0±0.5 mV)and fast inactivation(V1/2=-53.7±0.2 mV).The I1545M and G1571R mutants attenuated the DDT inhibition of the sodium channel inactivation current and attenuated the continuous sodium currents.The double mutants further increased this effect.Similarly,after treatment with permethrin and deltamethrin,the I1545M and G1571R mutants reduced the modification of sodium channels at three concentrations,and the double mutants further enhanced the resistance to sodium channels.Therefore,we confirmed that I1545M and G1571R significantly reduced the susceptibility of VGSC in Drosophila melanogaster,which resulting in VGSC resistance to DDT and pyrethroid insecticides.