Organophosphorus Resistance Mechanisms Of Oriental Migratory Locust, Locusta Migratoria Manilensis (Meyen)

Locusta migratoria manilensis(Meyen) is one of the seriously endangering agricultural pests.Organophosphate(OP)insecticides have been used extensively to control the locust.In order to delaying organophosphorus resistance and carrying out effective management strategies of different populations of L.migratoria manilensis,the objectives of this study were to evaluate the resistance spectrum of the field locust populations using the bioassay to OPs and investigate major biochemical and molecular mechanisms of resistance to OPs by the modem molecular biology technology.Elucidation of the resistance spectrum and resistance mechanisms in L.migratoria manilensis will be significantly important in theory and practice.1.Resistance spectrum to selected OP insecticides and synergism of TPP, PBO and DEMBased on the scale,distribution,history of insecticides control and population characteristics of L.migratoria manilensis,the field populations of the locust were collected from Huanghua County of Hebei Province, Huangliu County of Hainan Province and Wudi County of Shandong Province.The sensitivity analysis or resistance spectrums to the representative OP insecticides were evaluated.The results showed that, compared with the susceptible strain(SS),the population of Wudi population (WD) was the most sensitive to malathion and Huanghua population(HH) showed 2.9-fold to phoxim,5.4-fold to chlorpyrifos of resistance levels. However,HH and Huangliu population(HL) developed 57.5-fold and 14.8-fold resistant to malathion.In conclusion,As a consequence of the various malathion selective pressures,there has been developed different resistance to malathion in different locusts populations and various resistant levels in the same locust population has been developed by the different OPs. Consequently,it is significantly necessary to evaluate the resistance spectrum and detect the effective resistance management strategy of the field locust populations.The malathion resistance of the HH was significantly diminished by TPP (synergism ratio:16.2) and DEM(3.3),but was unchanged by PBO(0.9).In contrast,none of these synergists significantly affected the toxicity of malathion in the SS.These results suggested that both ESTs and GSTs were involved in OP resistance in the HH,whereas P450s were unlikely to be involved in the resistance simply because PBO did not synergize the toxicity of malathion by the synergism of triphenyl phosphate(TPP),diethyl meleate (DEM) and piperonyl butoxide(PBO) on malathion in the HH and SS of the locust.2.Biochemical mechanisms of organophosphate resistance mediated by metabolic detoxification enzymes in L.migratoria manilensisThe metabolic detoxification enzymes(cytochrome P450 monooxygenases(P450s),general esterases(ESTs) and glutathione S-transferases(GSTs)) were biochemical characterized.The results showed that the mean of the ESTs activities in the HH was 2.1-3.2-fold significantly higher than that of the SS whenα-NA,α-NB andβ-NA were used as substrates,respectively.The GST activity was 1.2-2.0- fold significantly higher in the HH than in the LS when CDNB and DCNB were used as substrates,respectively.For P450 activity assay,there was no significant difference between the HH and SS of the locust.The non-denaturing PAGE analysis of ESTs in the HH displayed the similar bands but increased staining intensity compared with the LS.Therefore,ESTs and GSTs played major roles in conferring malathion resistance in the HH.Two organophosphorus inhibitors such as paraoxon and malaoxon were used to compare the sensitivity levels of carboxylesterases(CarEs) between the HL and WD.Significant differences were observed in pI₅₀ values between HL and WD for all the two OP inhibitors.Specifically,CarEs from the HL were 1.16- and 1.27- fold less sensitive to inhibition by paraoxon and malaoxon,respectively,than that from the WD based on the differences in the pI₅₀ values between the two populations.However,there were significant differences in CarEs specific activities between the WD and HL of the locust. CarEs specific activities in the WD were 1.75- and 1.50-fold higher than those in the HL whenα-NA andβ-NA were used as substrates,respectively. The increased CarEs specific activities in the WD appeared to be associated with a 1.8-fold decreased susceptibility to malathion.The differences of CarEs activities may attribute to the locust differentiation between HL and WD due to the different locust breeding environment.It is not known whether or not the WD can be resistant to other OPs,the WD might be resistant to other OPs due to the increased Care activity.3.Bichemical mechanisms of organophosphate resistance mediated by target enzyme in L.migratoria manilensisThe biochemical characteristics of acetylcholinesterase(AChE,EC 1.1.1.7) were compared between the three populations and the susceptible strain of L. migratoria manilensis.AChE from the HH,HL and WD showed 4.04-,9.55-, and 1.99-fold significantly higher activity than that from the SS.Kinetic studies indicated that the K_m values of AChE from the three populations presented significantly difference compared with the SS.Inhibition kinetics revealed that AChE from the HH was 3.2-,2.2- and 1.1-fold less sensitive than that from the SS to malaoxon,chlorpyrfos oxon and phoxim respectively, AChE from the HL was 11.3- and 19.2-fold less sensitive to inhibition by malaoxon and chlopyrifos oxon,respectively,than that from the SS. Meanwhile,AChE from the WD was 4.8- and 2.4-fold less sensitive to inhibition by malaoxon and chlopyrifos oxon,respectively,in despite of 1.8-fold marginal resistance to malathion.Combined with the biochemical mechanisms of organophosphate resistance mediated by metabolic detoxification enzymes in L.migratoria manilensis,it was indicated that the malathion resistance in the HH was conferred by multiple mechanisms,including increased detoxification by ESTs and GSTs,and increased activity and reduced sensitivity of AChE to OP inhibition;the mild resistance to malathion in HL was associated with reduced sensitivity and increased catalytic activity of ACHE;the WD might be resistant to other OPs due to the decreased AChE sensitivity.Consequently, it was proposed that L.migratoria manilensis from different populations utilized different mechanisms,even including multiple mechanisms,to adapt to the OP insecticide selective pressure.4.Molecular mechanisms of organophosphate resistance in L.migratoria manilensisThree CarEs gene cDNA fragments of CarE1,CarE2 and CarE3 were cloned using the degenerate primers designed from conserved motifs in CarEs gene family and touchdown PCR program.According to the conservative regions(motifs) of CarEs and GSTs in insects,the database of expressing sequence tags(ESTs) in L.migratoria manilensis was searched, spliced,32 ESTs of CarEs and 12 ESTs of GSTs were obtained by bioinformatics methods.Total 34 CarEs gene fragments were produced by analyzing 32 ESTs of CarEs and 3 CarEs gene cDNA fragments cloned.The expression levels of these CarEs and GSTs gene were compared between the HH and SS using regular RT-PCR,then differential expression genes were further analyzed by real-time quantitative PCR.It was showed that CarE1, CarE4,CarE6,CarE12,CarE15,CarE19,CarE22,CarE23,CarE26,CarE27, CarE30 and CarE32 in the HH were expressed 3.29,1.13,8.78,3.16,3.17, 11.84,2.24,4.92,6.24,4.66,4.47 and 4.73- fold higher than that in the SS respectively;GST1,GST2,GST3 and GST12 in the HH were expressed 1.25, 1.33,2.47 and 1.61- fold higher than that in the SS respectively.These results indicated that higher expression level of CarEs and GSTs may contribute to the insecticides resistance of L.migratoria manilensis in HH.The gene cloning and Western blotting of acetylcholinesterase(ACHE) suggested that the AChE gene of insects from Orthoptera may be different from other insects.It will be necessary to be further confirmed. Elucidation of the resistance spectrum and resistance mechanisms in L. migratoria rnanilensis will help us to make knowledge-based selections of insecticides for locust control and resistance management in the field.In our study,we searched all of the CarEs and GSTs genes from the EST database of L.migratoria manilensis using the genome informatics(for example EST),screened out the CarEs and GSTs genes correlated with insecticide resistance and characterized these genes systematicly.Consequently,these findings were significant to help researchers detect and monitor insecticide resistance in L.migratoria manilensis,and to devise effective chemical control methods for this important insect pest in the field.