Study On Imidacloprid Resistance In Aphis Gossypii Caused By NACHRs?1 Submit 81 Amino Acid Point Mutation

The cotton aphid, Aphis gossypii (Glover) (Homoptera:Aphididae), is one of the worldwide polyphagous pests that can damage many agricutural crops. It is also the most important cotton seedling pest in different cotton production regions in China. It is hard to control this pests due to the high reproductivity and short generation time. Imidacloprid, as the first commercial neonicotinoid pesticide, has been widely used to control some important sucking pests, including aphids. Due to the extensive and unscientific use of imidacloprid, imiddacloprid resistance has become more and more serious problems in the field.The imidacloprid resistance mechanisms have revealed both target-site mutations and enhanced metabolic detoxification by cytochrome P450 monooxygenases. The target mutation was an important mechanism of imidacloprid resistance, especially related to the high level of resistance. Previous studies have showed that the amino acid at 81 position in the loop D region of the nicotinic acetylcholine receptor (nAChRs) ?1subunit of insects (77 position in rat) is conserved residue, which can interacts directly with the nitro of imidicloprid. Comparison of the nucleotide sequence of nAChRs ?1subunit (Ag?1) genes from resistant and susceptible strain of A. gossypii, we have found that a single point mutation in the loop D region of the resistant strain, causing an arginine to threonine substitution (R81T). The nAChR ?1-subunit R81T mutation in the field strain of Myzus persicae also have been identified. However, the molecular mechanisms of imidacloprid resistance due to the mutation have not clear.In this paper, The quantitative PCR was used to study the different expression levelsof nAChRs subunit between resistant and susceptible strains of cotton aphid. Co-immunoprecipitation and western blot analysis were used to confirm the ? and ? subunit combinations in vivo of cotton aphid. The voltage-clamp technique was used to compare the difference of binding ability of acetylcholine and imidacloprid to recombinant hybrid nAChRs, containing point mutations and non-point mutations, expressed in Xenopus oocytes, The 3D model of nAChRs subunits (?3?1) of cotton were build by homology modeling. The difference of hydrogen number between wild-and mutant type nAChRs of cotton aphid was compared by molecular docking. Specific results are as follows:The quantitative PCR was used to study the expression level of different nAChRs subunits in resistant and susceptible strains. The results showed that the expression level of nAChRs subunits in resistant strains is lower than in susceptible strains, but the down regulationg levels isnot high. We think that, for resistant strains, the lower expression levels of nAChRs subunit genes may have contributed to reduce the amount of potential target after the excitatory synapses, indirectly led to cotton aphid resistance to imidacloprid. Polyclonal antisera specific for each submit of cotton aphid were raised against bacterially expressed fusion proteins containing the large cytoplasmic loop of Ag?1 (R348-G441), Ag?2 (P359--C445), Ag?3 (I350-S451), Ag?4-1 (I344-R480) and Ag?1 (K349-E441) The aphid head membrane proteins were extracted by detergent, the following co-immunoprecipitations were executed with each a subunit antibodies and Ag?1 antibody, and the immunoprecipitation product were analysised by immunoblot. The results showed that Ag?3 and Ag?1 could combined with each other. So we conduct that Ag?3 and Ag?1 can combine togetherto generate a functional nAChRs in vivo of cotton aphid.The full length gene of Aga3 (gene number:EF626657.1) and R?2T77(gene number:L31622) with the threonine at 81 position were cloned into pT7TS vector.Then the plasmid of R?2R77 was obtained by site-directed mutagenesis with the amino acidsmutated from threonine to arginine at 77 position. The each recombinant plasmid were linearizated using restriction endonuclease enzyme, and the cRNA were generated by vitro transcription. The cRNA of Ag?3 was mixtured formerly with the cRNA of R?2T77 or R?2R77 as 1:1 (m/m) ratio, then the mixture of Ag?3 R?2T77 (mutant type) and Ag?3 R?27 (wild-type) was injected into the Xenopus oocyte seprately. The dose response of two recombinant hybrid receptors to imidacloprid and acetylcholine were detected by voltage clamp respectively. The results showed that there were no significant differences of maximum curent (Imax) and EC50 values in response to acetylcholine in oocytes expresiing Ag?3 R?2T77 and Ag?3 R?2R77. The significant difference was observed in Imax and EC50 values in response to imidacloprid in oocytes expressing the two recombinant hybrid receptors (p< 0.01). The significant shifts in EC50 values to imidacloprid were observed in oocytes expresiing Aga3 R?2R77, when conpared with oocytes expresiing Aga3 R?2177.These results illustrated that the amino acid residues mutated from arginine to threonine at 81 position may lead to increased resistance to imidacloprid due to the decreased binding ability of nAChRs to imidacloprid in insects.The Aphis gossypii ?3?1 interface homology model was built by the Swiss-model web server by using nAChRs 2BG9 of Torpedo marmorata as a template based on sequence alignments.2BG9 chain D and chain C backbone atoms were used as a scaffold to assemble the individual chainsa3 and ?1 of Aphis gossypii, respectively. The model side chains and established interface homology models were optimized by using the Swiss PDB viewer and the Swiss-model web server, respectively. The ?3?1R81 and?3?1T81 was built independently. Then the molecular docking were performed to explore the interaction between imidacloprid and nAChR models by using the AutoDock 4.0 softwere. The results showed that both the direct interaction was observed between R81 or T81 and imidacloprid. Two putative hydrogen bonds were found between the nitro group of imidacloprid and the guanidine moiety on R81, but only one putative hydrogen bond was observed in the interaction between imidacloprid and T81. The distance of two hydrogen bonds (1.9 A and 2.1 A) in imidacloprid and R81 interaction was much shorter than that of one hydrogen bond (3.3 A) in imidacloprid and T81. These results indicated that the nAChR of cotton aphid with no amino acid mutation at 81 position (R 81) of ?1 subunit could bind closer and more tight with imidacloprid, when compared with the amino acid mutation at 81 position (T 81). These results might give evidence that the amino acid mutation at 81 position causing an arginine to threonine substitution (R81T) of nAChRs ?1 subunit could lead to increased resistance to imidacloprid in lead to increased resistance to imidacloprid in Aphis gossypii.