Studies On Chlorpyrifos And Deltamethrin Resistance In Small Brown Planthopper,Laodelphax Striatellus (Fall(?)n) And Possible Mechanisms

The small brown planthopper(SBPH),Laodelphax striatellus(Fallen),is an important pest in the family of Delphacidae of Hemiptera which is mainly distributed in the temperate and subtropical regions.It can attack a variety of cereal crops including rice,corn,wheat,oat and barley.The damage is inflicted not only by direct feeding but also by transmitting several plant viruses such as rice stripe virus and black-streaked dwarf virus.In the past,L.striatellus was a secondary pest which was generally not controlled.However,L.striatellus has frequently outbroken in Jiangsu and Zhejiang Provinces in recent more than ten years,and it has brought huge economic losses in the local agricultural production.SBPH rely mainly on treatment of chemical control.At present,SBPH has developed resistance to a variety of pesticide,and resistance lead to lower control effect of drug.This is one of the factors of serious harm of L.striatellu,so the resistance of SBPH need to be resolved.Chlorpyrifos is a organophosphate insecticide which act on acetylcholinesterase while deltamethrin is a pyrethroid insecticide which act on the voltage gate sodium channel.Both insecticides are currently main insecticides for controlling ice and wheat field L.striatellus population which have excellent control effect.In this paper,the selected chlorpyrifos and deltamethrin resistant strains and recovery susceptible strain and field strain were used as test materials.We systematically study resistance characteristics of chlorpyrifos and deltamethrin,including resistance selection,susceptibility restoration,resistance risk assessment,resistance stability,cross-resistance and analyze transcriptome of detoxification enzyme genes and determine related detoxification genes in different two kinds of drug.These findings provide a theoretical basis to clarify the evolution mechanism of resistance to insecticides and draw up effective control measures.1.The resistant and susceptible strain selection and resistance risk assessment in L.striatellusCompared to the indoor recovery susceptible strain,a 158.58-fold chlorpyrifos resistant strain(JH-chl)and a 1072.0-fold deltamethrin resistant strain(JH-del)were established after laboratory selection of 25 and 30 generations with chlorpyrifos and deltamethrin based on field-collected Laodelphax striatellus population from Jianhu county,Jiangsu province in this study.During the first fourteen consecutive generations of selections,chlorpyrifos resistance was developed slowly,but followed by a rapid increase from the fifteenth generation forward.In continual selection process with deltamethrin,the deltamethrin resistance of JH-del strain had been steadily increased,there is no float.In the meantime,the chlorpyrifos end deltamethrin susceptibility of the unselected population recovered steadily until the twentieth generation and then stabilized.The resistance realized heritability to chlorpyrifos and deltamethrin were 0.102 and 0.061 in JH-chl and JH-del strains,respectively.Dose of pesticide kill 70%-80%of the population,10-fold resistance increase to chlorpyrifos and deltamethrin in L.striatellus need 20-22 and 22-27 generations,respectively.The both resistance risks to chlorpyrifos and deltamethrin in L.striatellus were low,but the resistance development rate to chlorpyrifos was higher than deltamethrin.It was believed that the actual selection force of chlorpyrifos and deltamethrin to the L.striatellus were smaller.At present chlorpyrifos and deltamethrin to L.striatellus still have control effectiveness,we can take the rotation of pesticides to delay resistance and prevent the emergence of high chlorpyrifos and deltamethrin resistant strains in order to resistance management purposes.2.The cross-resistance of resistant strains and resistance stability in L.striatellusThe chlorpyrifos and deltamethrin resistant strains exhibited cross-resistance to other common insecticides in L.striatellus.The chlorpyrifos resistant strain showed significant cross-resistance to other other cholinesterase inhibitor insecticides(methomyl,carbosulfan,diazinon and acephate)and had some cross-resistance to pyrethroid insecticide such as deltamethrin.No cross-resistance to most different class insecticides such as imidacloprid,abamectin,emamectin benzoate,pymetrozine buprofezin and hexaflumuron was observed.The deltamethrin resistant strain exhibited high levels of cross-resistance to same class insecticide such as fenvalerate and had low levels of cross-resistance to some cholinesterase inhibitor insecticides(chlorpyrifos,methomyl and carbosulfan).No cross-resistance to most different class insecticides such as diazinon,acephate,imidacloprid,abamectin,emamectin benzoate and buprofezin was found.The cross-resistance of the resistant strains in L.striatellus may involve in target insensitivity and metabolic detoxification enhancement.In addition,although high chlorpyrifos and deltamethrin resistant strains can not restore susceptibility,the resistance obviously decreased by stopping selection.It was believed that the resistance of chlorpyrifos and deltamethrin is not stable,and the insecticide resistance can be delayed by rotation of insecticides in agricultural production.3.The biochemical mechanism of chlorpyrifos resistance in L.striatellusThe synergistic effects of PBO,TPP and DEM on chlorpyrifos susceptibility were studied in vivo synergistic test in this chapter.No obvious synergistic effect was found in susceptible JHS strains.The PBO and TPP had slight synergistic effect in the field JHF strain.And the resistance of chlorpyrifos resistant strain was significantly suppressed by oxidase inhibitor PBO(2.81-fold)and esterase inhibitor TPP(3.47-fold),but no synergistic effect of DEM,a glutathione depletion agent,was found.Enzymatic activities were measured and compared among different strains.The activities of multifunction oxidase and esterase in JH-chl were 4.65-fold and 3.34-fold higher than that of JHS,respectively,while glutathione-S-transferase,alkaline and acid phosphatase activities had no significant change.No significant difference in activity of cytochrome P450,esterase,glutathione-S-transferase,alkaline and acid phosphatase was observed between JHF and JHS.The results of kinetics and inhibition test of acetylcholinesterase showed that the values of michaelis constant(Km)and maximum reaction rate(Vmax)increased 5.96-fold and 3.34-fold than that of JHS,and similar Km and Vmax values between JHF and JHS were observed.When the four insecticides(DDVP,profenofos,chlorpyrifos-oxon and acephate)were used as inhibitors of acetylcholinesterase,the values of median inhibitory concentration IC50 and inhibition constant Kj had no significant difference between between JHF and JHS,but the AChE of JH-chl was the least susceptible to the insecticide inhibitors indicated by higher IC50(3.27-8.27-fold)and lower Ki(0.17-0.35-fold).The above results suggested that the mechanism of chlorpyrifos resistance involves in multifunction oxidase and esterase activities increase,as well as reduced sensitivity of target acetylcholinesterase.4.The biochemical and target mechanism of deltamethrin resistance in L.striatellusThe synergistic effects of PBO,TPP and DEM on deltamethrin susceptibility were studied in vivo synergistic test in this chapter.PBO and TPP treatments reduced the RR of JH-del from 1072 to 441 and 576 with synergism ratio of 2.43 and 1.86,respectively,while DEM showed no apparent synergistic effect.The general EST activity toward a-NA was 15.23-,2.29-and 1.50-fold higher in JH-del(1072-fold),JH-del-G4(106-fold)and F-G1(63-fold)than that of JHS.P450 ECOD activity was significantly increased by 7.64-,2.84-and 2.19-fold in JH-del,JH-del-G4 and F-G1,respectively,when compared with that of JHS.GST activity towards CDNB and DCNB was similar among all strains.Between JH-del-G4 and JH-del(G30)the RR increased by～10 fold that accompanied by 6.7 and 2.7 fold increase in general EST and P450 activity,respectively.The increased EST and P450 activity in resistance strains also perfectly agreed with the synergism analysis.Target site mutation was checked between JH-del and JHS by amplifying and sequencing the L.striatellus para-homologous sodium channel gene.The low frequency of amino acid substitution(1/10)in resistant individuals suggested that it is unlikely to confer the high level resistance of JH-del.The expression of the voltage gate sodium channel gene was also studied between JH-del and JHS.The results showed that the expression exhibited no difference.Therefore,it was inferred that the voltage gate sodium channel was not associated with deltamethrin resistance,while P450 and esterase plays an important role in deltamethrin resistance.The molecular mechanism of detoxification enzymes need to be further studied.5.The identification of detoxification enzyme genes and screen of related resistance genes in L.striatellusIn this study,we obtained 80 P450,56 carboxylesterase,63 phosphoesterase and 13 glutathione-S-transferase gene transcripts by searching annotation detoxification genes from the L.striatellus transcriptome data.The cDNA fragments of 71 P450,39 carboxylesterase(CE),54 phosphoesterase(PE)and 12 glutathione-S-transferase(GST)were successfully cloned using RT-PCR technique from the field-collected L.striatellus strain.NCBI BLASTX analysis showed that the these like sequences have high levels of similarity to that of other insect species,which belonged to different detoxification enzyme gene families.The 71 P450s were systematically denominated by International P450 Commission,CE,PE and GST were tentatively named as LSCE1-LSCE56,LSPE1-LSPE63 and LSGST1-LSGST13.The transcriptional level of detoxification genes were compared among chlorpyrifos and deltamethrin resistant strains and susceptible strains using semi-quantitative RT-PCR technique.The results showed that the expression levels of CYP6AY3v2,CYP306A2v2,CYP353D1v2 and LSCE36 were up-regulated in the chlorpyrifos resistant strain and the expression levels of CYP439A1v3,CYP6AY3v2,CYP314A1v2,CYP6FU1,CYP353D1v2 and LSCE12 significantly up-regulated in deltamethrin resistant strain,the rest P450 family,phosphoesterase and glutathione-S-transferase genes were expressed similarly between resistant and susceptible strain.qRT-PCR was utilized for further validating findings.The expression ratios were 7.07-,6.87-,12.14-and 10.20-fold between JH-chl and JHS for CYP6AY3v2,CYP306A2v2,CYP353D1v2 and LSCE36.The four overexpressed genes showed no significant difference between low level chlorpyrifos resistant F-G1 strain and susceptible strain.The expression ratios of CYP439A1v3,CYP6AY3v2,CYP314Alv2,CYP6FU1,CYP353D1v2 and LSCE12 were 4.68-,24-,2.36-,16-,5.33-,and 11.10-fold higher in JH-del than that of JHS.The six overexpressed genes showed no significant difference between low level chlorpyrifos resistant F-G1 strain and susceptible strain.The overexpressed full-length genes were cloned using RACE technique and the full-length sequences were analyzed by sequence analysis software.The expression data of detoxification genes were accordant to the enzyme activity and synergist test.The gene copy number of these overexpressed detoxification genes revealed no change in chlorpyrifos and deltamethrin resistant strains.It was believed that these overexpressed detoxification genes were caused by gene transcription level enhancement rather than copy amplification in genome level.Therefore,these overexpressed detoxification genes play a key role in chlorpyrifos and deltamethrin resistance.6.The RNAi analysis of related resistant detoxification genes in L.striatellusIn order to evaluate roles of overexpressed detoxification genes in the resistance,the functional analysis via RNAi of related resistant detoxification genes was performed by using dsRNA feeding method.Toxicity determination was performed by using three concentrations of 100ng/pest,50ng/pest and 26 ng/pest in chlorpyrifos resistant strains after interference.Compared to the mortality of L.striatellus of feeding dsGFP and non-dsRNA nutrient solution,the chlorpyrifos resistance could be significantly reduced by feeding dsRNA of related chlorpyrifos resistant genes.Similarly,Toxicity determination was performed by using three concentrations of 48ng/pest,24ng/pest and 12ng/pest in deltamethrin resistant strains after interference.The deltamethrin susceptibility was increased significantly after feeding dsRNA of related deltamethrin resistant genes.The expression change of genes was checked by Quantitative PCR after feeding dsRNA of related chlorpyrifos and deltamethrin resistant genes.The expression level of chlorpyrifos resistance-associated genes wasn’t affected after feeding dsGFP.After dsRNA feeding,the expression level of CYP6AY3v2,CYP306A2v2,CYP353D1v2 and LSCE36 were greatly decreased by 56.8%,35.6%,49.77%and 48.02%while the expression level of CYP439A1v3,CYP6AY3v2,CYP314Alv2,CYP6FU1,CYP353D1v2 and LSCE12 were greatly decreased by 58.8%,67.35%,63.26%,47.39%,59.79%and 60.59%.Feeding dsRNA method can effectively silence target genes.These data suggested that overexpressed detoxification genes in chlorpyrifos and deltamethrin resistant strains specifically involved in the insecticide resistance.