| The beet armyworm,Spodoptera exigua(Hübner,is a world-wide important polyphagous pest.Chemical control is the main way against this pest,which leads to develop high resistance to many insecticides.For this reason,the traditional insecticides,such as organochlorines,organophosphorus,carbamate and pyrethroid,have already lost their effective control to S.exigua.Now the newer insecticides,such as emamectin benzoate,tebufenozide,indoxacarb,chlorfenapyr and spinosad et al,are intensively used in the field.So these newer chemical are also faced with the resistance problem.In this study the resistance status of nine insecticides was investigated in the field populations from different vegetable producing areas and a near-isogenic resistance strain(WH-EB)to emamectin benzoate was established.The cross-resistance,resistance inheritance mode and fitness cost of WH-EB were inspected.Based on the synergism and enzyme activity comparison,no difference was found between the resistant and susceptible strain(WH-S).The GluC1α and GABAal of S.exigua are proved to be the target sites of emamectin benzoate by RNAi,but there were no mutation and mRNA expression difference between WH-EB and WH-S.After screening the data of the transcriptome sequencing,it indicated that WH-EB was infected by Nosema bombycis.Further more NbABCGl of N.bombycis was proved to be associated with the resistance to emamectin benzoate.This is the first time to find the mutualism between S.exigua and N.bombycis for the resistance of emamectin benzoate.This result reveals a new approach for insect to develop resistance to insecticide.1.Monitoring of insecticide resistance in S.exigua field populationsThe current resistance status of nine insecticides was investigated in 18 field populations collected from seven provinces of China during 2009-2013.Compared with the susceptible strain WH-S,some field populations developed various levels of resistance to eight of the nine insecticides tested,emamectin benzoate(4-to 403-fold),indoxacarb(2-to 41-fold),spinosad(5-to 38-fold),chlorantraniliprole(2-to 44-fold),tebufenozide(2-to 87-fold),chlorfluazuron(3-to 31-fold),cypermethrin(79-to 1240-fold),and chlorpyrifos(8-to 3,080-fold),but no significant resistance was detected to chlorfenapyr(0.1-to 7-fold).This indicates that chlorfenapyr has no cross-resistance with these other currently used insecticides.Five consecutive years resistance screen at two places shows that resistance patterns are different between populations from Luhe(Jiangsu Province)and Fengxian(Shanghai),which are 300 km apart.Resistance levels to chlorpyrifos were much higher in populations from Luhe(877-to 3,080-fold)than Fengxian(8-to 110-fold).Fengxian populations developed moderate levels of resistance to tebufenozide(13-to 87-fold),but no resistance in Luhe populations(2-to 6-fold).It is suggested that local insecticide selection determined resistance patterns although S.exigua has long-distance migratory potential.Adaptive resistance management tactics(such as rotations)should be designed and implemented based on the resistance patterns of S.exigua for each geographic area.2.Cross-resistance and inheritance of resistance to emamectin benzoate in the beet armywormThrough repeated backcrossing to a susceptible strain(WH-S)and selection with emamectin benzoate,the trait conferring resistance to emamectin benzoate in a field-collected population of S.exigua(moderately resistant to emmamectin benzoate and strongly resistant to pyrethroids and indoxacarb)was introgressed into WH-S to generate a near-isogenic resistant strain(WH-EB).Comparing with WH-S,the WH-EB strain developed 1,110-fold resistance to emamectin benzoate and a high level of cross-resistance to abamectin(202-fold),with low levels of cross-resistance to cypermethrin(10-fold)and chlorfluazuron(7-fold),but no cross-resistance to representatives of another six different classes of insecticides(chlorantroniliprole,chlorfenapyr,indoxacarb,spinosad,tebufenozide and chlorpyrifos).Resistance to emamectin benzoate in WH-EB was autosomal,incompletely dominant,and polygenic.Limited cross-resistance in WH-EB indicates that emamectin benzoate can be rotated with other classes of insecticides to which it does not show cross-resistance in order to delay the evolution of resistance in S.exigua.The incompletely dominant nature of resistance in S.exigua may explain the rapid evolution of resistance to emamectin benzoate in the field,and careful deployment of this chemical within a resistance management program should be considered.3.The relative fitness in emamectin benzoate resistance strainFitness costs associated with abamectin benzoate resistance were examined in WH-S,WH-EB,CM,CM-EB strains.Comparison on growth characteristics,the larval period in WH-EB is 0.2 days less than WH-S,the larval period in CM-EB is 0.3 days less than CM,but the difference is not significant.The female pupa weight of WH-EB,CM and CM-EB strains are significantly higher than that of WH-S,there are no significant difference at the pupal period,the pupation rate,the male pupa weight between these strains.The survival rate of pupa in WH-EB is significantly lower than others.In terms of reproductive characteristics,the mean eggs laid per female is between 700-900 in WH-S,WH-EB and CM-EB strains,this in CM train is higher to 1000 eggs.The egg hatching rate of four strains is 50%to 60%,there was no significant difference.The net reproductive rate in WH-S,WH-EB,CM and CM-EB strains is 165.2,175.8,189.7 and 217.8,respectively.By the use of net reproductive of rate in WH-S or CM strains in artificial diet as control,the relative fitness of WH-EB and CM-EB is 1.06 and 1.15,no significant difference with control strains.4.Metabolism and target mechanisms of emamectin benzoate in WH-EBThe synergistic effects of PBO,DEM and DEF on emamectin benzoate were determined in the WH-EB and WH-S strain,and the results showed that the three inhibitors did not synergize emamectin benzoate against the two strains(SR<2-fold).In order to evaluate the role of metabolic detoxification mechanisms in WH-EB,the activities of monooxygenases,esterases and glutathione S-transferases were measured lin the three strains(WH-S,WH-EB and CM).Comparing with the WH-S strain,metabolic enzyme activities in WH-EB were 0.7～1.6-fold,there was no significant difference.Enzyme activity results are consistent with those of the synergism experiment,which indicates that metabolic detoxification is not involved in emamectin benzoate resistance in WH-EB.SeGluCla and SeGABAα1 were cloned by RT-PCR and RACE.After feeding dsRNA of the two genes,mRNA expression in the larva was dropped by 69%and 77%,and the lethality of emamectin benzoate was also decline.This means that both GluClα and GABAα1 subunits are the target sites of emamectin benzoate in beet armyworm.But no mutation and mRNA expression difference were found between WH-EB and WH-S.5.Nosema bombycis ABCG1 gene plays a role in emamectin benzoate resistance of beet armywormIt was found that the sequences of N.bombycis in WH-EB by comparing the transcriptome sequencing data of WH-S with WH-EB.The microsporidian spores could be found under microscope in the larva of WH-EB and it is identified that it is N.bombycis by SSU and IGS sequence identity.The moth of WH-EB was fed with honey water with fuumagillin in two generations,the number of spores in larva dropped by 60%and resistance to emamectin benzoate decreased.When the larvae were infected by N.bombycis,resistance to emamectin benzoate increased to 17-fold.All these results indicate that the infection of N.bombycis is an important reason of emamectin benzoate resistance in WH-EB strain.Verapamil,the ABC transporter inhibitor,has 6-fold synergism to emamectin benzoate in WH-EB.According to the transcriptome data,there are 8 ABC transporter members in Nosema bombycis and the NbABCGl has the highest expression.SeABCG1 and SeABCBl were cloned and no expression difference was found between WH-EB and WH-S.After feeding dsRNA of NbABCG1,mRNA expression of SeABCG1 in the larva was no change but mRNA expression of NbABCGl decreased and the sensibility to emamectin benzoate increased.Thus,the ABC transporter ABCG1 of N.bombycis plays a role in emamectin benzoate resistance of beet armyworm. |
