Molecular Detection of Toxicodynamic and Metabolic Factors Associated with the Pyrethroid and Carbamate Resistance in Myzus persicae (Sulzer)

Abstract

The green peach aphid (Myzus persicae) is an economically serious pest of agricultural and horticultural crops all over the world and developed resistance almost all kinds of insecticides. In this study, target site mutations were investigated by sequencing the voltage-gated sodium channel (vgsc) and acetylcholinesterase (ace) genes in 7 and 10 local strains collected in 2010 and 2011, respectively. In addition, leaf dip bioassay was conducted to determine LC50 values of bifenthrin and methomyl. Based on the mutation survey, quantitative sequencing (QS) was employed for investigation of resistance allele frequencies in local strains. Mutation survey of the vgsc revealed that M918L and L1014F mutations were present in local strains. Especially, M918L mutation showed a higher correlation than L1014F between the resistance allele frequency and actual resistance level. Thus, QS using newly identified M918L mutation should facilitate the detection and monitoring of pyrethroid resistance levels in the field. As putative mutations for target site insensitivity to carbamates, frequencies of A301S and S431F mutations in ace were investigated by sequencing. However, only S431F mutation was revealed in local strains. QS was employed to detect S431F mutation frequency. Correlation analysis of mutation allele frequency versus actual resistance level revealed that S431F mutation itself does not appear to play a significant role. To identify whether higher expression of M. persicae carboxylesterase (CbE) E4 is due to gene duplication, gene copy number was determined by quantitative real-time PCR. To determine CbE E4 quantity, Western blotting in conjunction with activity staining were conducted. Correlation analysis was carried out to investigate the possible connection between CbE expression level and its gene copy number, activity and protein quantity. Qualitative changing from point mutation was considered and mutation survey was conducted by PCR. However, new mutation G134C was revealed in oxyanion hole. To predict the functions of this mutation, altered CbE E4 3D structure was predicted and correlation analysis was conducted using sequence chromatogram of G134C mutation. Finally, we expect that altered CbE E4 might play a significant role to hydrolyze and/or sequestrate the carbamate insecticide in M. persicae.