Study On Monitoring And Mechanisms Of Imidacloprid Resistance In Soybean Aphid,Aphis Glycines (Matsumura)

Soybean aphid(Aphis glycines Matsumura) is one of the most important pests on soybean. Imidacloprid is the key insecticide which has been widely used for control of this pest since1990s. So study on imidacloprid resistance is very important for sustainable control of A. glycines. In this paper, the resistance survey, resistance selection, insect fitness analysis, cross-resistance and realized heritability were carried out to evaluate the risk for A. glycines to develop high resistance to imidacloprid. With selected susceptible strain and resistant strains, the biochemical and molecular mechanisms for the resistance were also analyzed. Some valuable breakthroughs have been achieved.1. Establishment of the relative susceptible baseline data to common insecticides and resistance monitoring in field populations of A. glycinesThe susceptibility of A. glycines to7common insecticides was determined with laboratory susceptible strain and the baseline data were obtained. Based on these data, five field populations (Jinan, Cangzhou, Chifeng, Changchun, Harbin) collected from main soybean producing provinces of China in2010and2011were monitored for resistance to imidacloprid, and the field population of Harbin collected in2010and2011were monitored for resistance to7common insecticides. The results revealed that the aphid of Changchun had developed low level of resistance to imidacloprid, and the other four areas had developed medium level of resistance to imidacloprid. Except imidacloprid, Lambda-cyhalothrin and Deltamethrin, A. glycines had no developed resistance to other insecticides.2. Imidacloprid resistance selected and resistance risk assessment in A. glycinesAn imidacloprid-resistant strain of A. glycines was developed by continuous selection for19generations with imidacloprid in laboratory. During the selection, imidacloprid resistance was found with a phased increase. From the1st to6th generation, the resistance developed very slowly, the rapid increase occurred from7th to16th generation, and leaving17th to19th as resistance developing plate. The estimate of realized heritability of19th generation showed that resistant ratio increased10-fold was only needed6-8generations when the continuous selection was used for A. glycines with imidacloprid. It was likely that the resistance level of A. glycines to imidacloprid would increase further if the selection had continued.3. The influence of imidacloprid resistance on the relative fitness of A. glycinesThe relative fitness of resistant strain (T9and T18) had been tested by constructing life tables and taking susceptible strain as control. The results showed that two resistant strains had obvious disadvantages in their development and reproduction. The relative fitness of resistant strain was determining by intrinsic rate of increase (rm) with susceptible strain as standard. The result showed that the relative fitness of resistant strain was decreased dramatically.4. Cross-resistance of the imidacloprid-resistant strain of A. glycinesThe cross-resistance of the imidacloprid-resistant strains (T14and T18) of A. glycines to6common insecticides was measured. The results showed that A. glycines with resistance to imidacloprid had no obvious cross-resistance to organophosphorus insecticides, and significant cross-resistance existed to the neonicotinoid insecticides and pyrethroid insecticides.5. Biochemical mechanisms for imidacloprid resistance in A. glycinesThe biochemical mechanisms of imidacloprid resistance in A. glycines were studied by synergism test and detoxification enzyme analysis. The results showed that triphenyl phosphate and piperonyl butoxide had significant synergism on imidacloprid in both resistant and susceputible strains, and the synergism ratio of triphenyl phosphate was much higher than the synergism ratio of piperonyl butoxide. Diethyl mateate did not show any synergism on imidacloprid in both strains. The results of detoxification enzyme analysis in two strains showed that the activity of carboxylesterase in resistant strain was much higher than the susceptible strain (2.1-fold), however the activity of glutathione S-transferase in resistant strain had no significantly enhanced. Thus, it was concluded that the enhancement of the activity of carboxylesterase should play an important role in the imidacloprid resistance of A. glycines.6. Cloning of nAChR genes from A. glycinesNicotinic acetylcholine receptors (nAChRs) are the target of imidacloprid which belong to neonicotinoid insecticides. With RT-PCR and RACE technique,6different subunits of nAChR had been successfully cloned from A. glycines, include5a subunits and1β subunit. They were named as Aglal, Agla2, Agla3, Agla4, Agla5and Aglβl, and their GenBank accession number were JQ690271, JF775487, JQ690273, JN681173, JQ690274and JN681174, respectively. The Aglal was1456bp in length, and the open reading frame encoded427amino acids; the Agla2was2463bp in length, and the open reading frame encoded592amino acids; the Agla3was2158bp in length, and the open reading frame encoded537amino acids; the Agla4was1792bp in length, and the open reading frame encoded532amino acids; the Agla5was1850bp in length, and the open reading frame encoded549amino acids; the Aglβl was2406bp in length, and the open reading frame encoded509amino acids. One alternative splice was found with the clone genes. Homology analysis demonstrated that these6subunits of nAChR had high amino acid sequence identity with other insect nAChR subunits previously reported. Polymorphism analysis demonstrated that there is no point mutation exited in resistant and susceptible strains. This work facilitated the further studies on natural subunit composition, function of nAChR and the molecular mechanisms of target resistance for neonicotinoid insecticides.7. Cloning and expression of carboxylesterase gene from A. glycinesThe carboxylesterase gene was cloned by using RT-PCR and RACE methods, and it was named as AgCarE. The AgCarE was1946bp in length, and the open reading frame encoded526amino acids, the GenBank accession number was JF970181. The AgCarE possesses a catalytic triad, consisting of a Ser, a His and a Glu residue (Ser186, Glu313and His434), which is characterized by carboxylic esters (EC:3.1.1.-). The AgCarE gene had been recombined into pET21b vector. Then the HIS fusion protein was expressed by the induction of IPTG. The result of SDS-PAGE and Western blot analysis showed that the AgCarE protein in A. glycines was expressed in E. coli BL21which induced by IPTG, and its MW was found to be about59kD, similar as the predicted result. The carboxylesterase activity of AgCarE was characterized with the a-NA as a substrate and its enzyme activity of AgCarE was analyzed.8. Relative expression of carboxylesterase mRNA in resistant and susceptible strains of A. glycinesThe relative expression of carboxylesterase mRNA in resistant and susceptible strains of A. glycines were estimated by Ct comprison method. It was found that the relative expression of carboxylesterase gene in resistant strain had much higher than the expression in the susceptible strain (2.25-fold). Combination of the previous results in biochemical mechanism analysis and enzyme activity analysis of AgCarE revealed that the over-expression of carboxylesterase might associate with the high resistance to imidacloprid in A. glycines.