Insecticide Resistance In Small Brown Planthopper, Laodelphax Striatellus (Fallen) And The Mechanisms

The small brown planthopper,Laodelphax striatellus(Fallen),is a worldwide important pest with an extensive host range especially infests important crops including rice,maize and wheat.It survives the winter with old nymphs in northern China,and breeds all the year in the southern.This pest formerly broke out only intermittently in China.Since the early 2000s,however,the small brown planthopper has reached large number every year,especially in Jiangsu Province,and large amount of insecticide have been used for combating its infestation on crops.As a result,many field populations of this pest have developed multiple resistances,which make more and more frequent control failures in field.In order to implementation of resistance management and efficient control, efforts have been made to declare its resistance and the mechanism,cross-resistance, realized heritability,and fitness costs in this work.Significant results have been obtained and summarized as follows:1 Insecticide resistance in field populations of L.striatellus Fallén(Homoptera: Delphacidae) the in China,and its possible mechanismsThe small brown planthoppers,the L.striatellus Fallén,were collected from 6 provinces in China and their resistance to common insecticides surveyed.The strains collected from Jiangsu province were found to be the most resistant,and showed high resistance to imidacloprid and moderate resistance to chlorpyrifos,acephate and deltamethrin(RF ranged 66-108,31-50,17-24 and 12-21,respectively).Another two strains collected from Fujian and Guangdong provinces also displayed moderate or low level resistance to imidacloprid,chlorpyrifos and acephate(RF ranged 27-29,10-12,and 9-13,respectively).However,the strains collected from Shandong and Yunnan provinces showed little resistance to any of the insecticides tested(RF＜6).The sensitivity of the strain collected from Hebei province(the most sensitive one) was similar to that of the susceptible strains previously reported.In all the strains tested,no obvious resistance to fipronil and carbosulfan was found,although the sensitivity of Jiangsu strains decreased by 5-8 fold.Biochemical analysis proved that the enhanced activity of cytochrome P450 monooxygenase(MFO) and esterase,as well as the AChE insensitivity,could all contribute to the multiple-resistance in this pest.2 Selection of resistance and susceptible strains of L.striatellus in laboratoryThe hoppers collected from Beijing and Jianhu county were subjected to sensitivity recovery by breeding without contacting any insecticides.After 12 generations,the susceptible strain(S) developed from Beijing population and its sensitivity to imidacloprid, fipronil,deltamethrin,chlorpyrifos,acephate and carbosulfan was little enhanced by 1.47, 2.23,1.39,1.56,2.49 and 2.23 folds,respectively.However,the resistant strain collected from Jianhu had a significant increase in its sensitivity to imidacloprid,fipronil, deltamethrin,chlorpyrifos,acephate and carbosulfan,with the resistance ratio(RR) decreasing from 108,6.90,21.0,34,24 and 6-fold to 10.4,1.54,2.34,2.07,4.16 and 1.04-fold,respectively.Except for the high resistance to imidacloprid,all medium and low level resistance was almost lost.On the other hand,selection with imidacloprid,fipronil, chlorpyrifos and carbosulfan for 12 generations in laboratory,the resistance strain of IMI-R, FIP-R,CHL-R and CAR-R were developed from Jianhu field strain(JH-F).The resistance of IMI-R,FIP-R,CHL-R and CAR-R strain to their own selection insecticide increased from 108,6.9,34 and 5.97-fold up to 281,33.1,283 and 52.2-fold,respectively.These results suggested that L.striatellus was easy to produce high level resistance to imidacloprid and chlorpyrifos as compared to carbosulfan and fipronil.Stopping use of insecticides could make sensitivity recover even to the sensitivity level,especially for those low or medium level resistances.3 Mechanisms for resistance of L.striatellus to chlorpyrifos and evaluation for the risk of cross-resistanceWhen CHL-R was selected from JH-F by chlorpyrifos treatment,its resistance to chlorpyrifos,acephate and carbosulfan increased by 5.35-,1.64- and 1.39-times, respectively.However,the resistance' ratio for fipronil,deltamethrin and imidacloprid decreased to only 0.708,0.679 and 0.304 times that of the original.These results suggest that cross-resistance might exist between chlorpyrifos and the AChE targeted insecticides, but not with fipronil,deltamethrin and imidaclorid.The synergism experiment revealed that PBO and TPP had significantly synergism to chlorpyrifos in both susceptible and resistant strains.The SR was 3.04-and 2.91-folds in S strain and 6.65-and 12.0-folds in CHL-R, respectively.These results were supported by the activities of detoxification enzymes which suggest involvement of both MFO and EST in the resistance mechanism.AChE from S-strain and CHL-R were partially characterized and examined for its possible roles in resistance to chlopyrifos.The kinetic parameters,K_m and V_m,for the enzymes from S-strain and CHL-R were found significantly different.The bimolecular rate constants(Ki) of chlorpyrifos oxon to AChE of CHL-R decreased by 2.26 folds and the concentration inhibiting 50%of AChE activity(1₅₀) increased 8.3-fold as compared with that of S-strain. This result suggested that insensitivity of AChE in the CHL-R should be a important factor conferring resistance to chlorpyrifos and other AChE targeting insecticides.4 Mechanisms for resistance of L.striatellus to imidacloprid and evaluation for the risk of cross-resistanceWhen IMI-R was developed from JH-F strain by imidacloprid treatment,no resistance was found to increase obviously except for imidacloprid.The resistance to imidaclorid increased by 1.78 folds,and to carbosulfan did not change obviously(1.06-times). However,the resistance of IMI-R to deltamethrin,chlorpyrifos,acephate,and fipronil decreased from the original 29.6,52.9,59.6-and 15.4-fold to 24.3,41.6,28.1 and 4.80-fold, respectively.These results seems suggesting that no cross-resistance risk exist between imidaclorid and the insecticides tested.Analysis of detoxification enzymes found that MFO and EST in IMI-R were 3.05-and 7.81-times higher than that in JH-F strain,but GST activity is similar.This result suggested the enhancement of MFO and EST activity might contribute the resistance to imidacloprid in this pest.5 Mechanisms for resistance of L.striatellus to carbosulfan and evaluation for the risk of cross-resistanceCompared with JH-F strain,the resistance of CAR-R to carbosulfan has significantly increased(4.49 folds).However,the resistance to deltamethrin,chlorpyrifos,acephate, imidacloprid and fipronil decreased from 29.6,52.9,59.6,158-and 15.4-fold to 12.6,16.7,14.8,24.2-and 1.16-fold,respectively.These results suggest that no cross-resistance risk exist between carbosulfan and the tested insecticides.Analysis of the activities of detoxification enzymes suggested involvement of both MFO and EST in the resistance mechanism,which activities increased by 2.21-and 1.92-times.AChE from S-strain and IMI-R were partially characterized and examined for its possible roles in resistance with 3 inhibitors,acephate,carbosulfan.The bimolecular rate constants(Ki) in the CAR-R was significantly decreased by 4.98,4.39-and 3.11-fold when compared with S-strain.This result suggested that insensitivity of AChE in the CAR-R was probably a important factor conferring cross-resistance to other AChE targeting insecticides.6 Susceptibility recovering and cross-action among different insecticides in L. striatellusResistance dynamics were surveyed for the resistance selection strain and the susceptibility recovery strain in this study.It was found that when JH-F strain bred continuously without contacting any insecticides,its resistance to all insecticides tested decreased dramatically.When JH-F strain was selected with different insecticides,its resistance increased to all the selection insecticides and some non-selection insecticides which were traditionally thought cross-resistant insecticides.But to most non-selection insecticides its resistance decreased,which sometime was claimed as negative cross-resistance.However,the resistance of the selection strain to any insecticides tested did decrease so much as that of the recovery strain in the same period.This implys that a given insecticide dose not select cross-resistance to all insecticides,but it dose prevent the treated population from recovering its sensitivity to different insecticides.That is why insects collected from high resistant area always have high resistance or tolerance to different insecticides.Biochemical mechanism research revealed that all the resistant strain selected with any insecticides had higher activity of the detoxification metabolism enzyme. Thus,it is deduced that the enhancement of detoxification enzymes may be the mechanism for insecticide cross-action.7 Realized heredity and fitness cost of the resistance in L.striatellusRealized heredity and fitness cost were studied with 3 resistant strains of L.striatellus, IMI-R,CHL-R and CAR-R.The results showed that the realized heredity(h²) of the resistance to chlorpyrifos,carbosulfan and imidacloprid was 0.173,0.127 and 0.108, respectively.Supporting insecticide kills 70%～80%of the population,increase of 10-fold resistance will take 20～25 generations for chlorpyrifos,28～34 generations for carbosulfans and 33～40 generations for imidacloprid,respectively.There obviously existed some resistance risk for the 3 insecticides.Of which,the resistance risk of chlorpyrifos is significant stronger than that of imidacloprid and carbosulfan.The fitness tests suggested that Rec-F strain and BJ-S strain doesn't differ,but the 3 resistant strains showed significantly lower fitness.As compared with the BJ-S strain,the fitness index of CHL-R, IMI-R and CAR-R strain is only 0.372,0.325 and 0.434.Based on the low fitness of resistant strains and low realized heredity of the resistance,it was thought that this pest could not develop resistance fast in field,and implementation of a efficient resistance management should delay the resistance development.8 Clonning and sequence analysis of the L.striatellus insecticide target geneWith degenerate primers and half nest PCR technique,the cDNA fragments of 4αsubunits of the nicotinic acetylcholine receptor(nAChR) were cloned from L.striatellus (named Lsα1,Lsα2,Lsα3,Lsα4,respectively).Sequence analysis proved that all the cloned gene fragements have the typical structure ofαsubunit:2 adjacent cysteine,the loop consisting of 15 amino acids and formed by the disulfide bond and the amino acids interacting with the nicotine and theα-bungarotoxin.The most similar genes were those reported insect nAChRαsubunit genes,and the similarity was as high as 65%～99%.Adopting the same method,the two different cDNA fragments of acetylcholinesterase were cloned from L.striatellus(named as Ls.ace1 and Ls.ace2).Sequence homology analysis showed that Ls.acel was over 90%similarity with other insect typeâ… AChE genes (ace 1),and Ls.ace2 was above 80%with other insect typeâ…¡AChE genes(ace 2).However, the similarity between Ls.ace1 and Ls.ace2 was only 55%.This implied that these two fragements were cloned from the AChE genes of L.striatellus and this pest had at least two different AChE genes in vivo.