Mechanisms Of Chlorpyrifos Resistance In Apolygus Lucorum

Transgenic Bt cotton has shown great promise for control of the major pest, cotton bollworm, in cotton field since 1997. Mirids, however, have gradually developed to be the major pests and damaged many crops and vegetables because of the changes of farming system, cotton varieties and pest management. Chemical control is becoming the most important management measure for mirids. Chlorpyrifos, one of organophosphorate pesticides is applied regularly to control Apolygus lucorum, one of major species of mirids in cotton field. Excessive and unreasonable use of chemicals will inevitably result in resistance to the insecticides used against target insects.Few researches about resistance monitoring or resistance mechanisms were carried on for Chinese mirid populations. In the present study, resistance machanisms in A. lucorum to chlorpyrifos was investigated systematically. Relationship between metabolic enzymes and resistance of A. lucorum to chlorpyrifos was evaluated by comparing enzyme activities and synergistic effects. The relationship between target AChE and resistance to chlorpyrifos were determined by measuring kinetic parameters, comparing amino acids of the AChE gene and AChE mRNA expressing levels, and detecting gene mutation frequencies. It is the first report that A216S mutation in ace1 is assocated with chlorpyrifos resistance in A. lucorum. The results will provide valuable information for molecular detection of resistance to chlorpyrifos and designing adapted resistance management tactics for A. lucorum.1. Selection of the resistance strain of A. lucorum to chlorpyrifos (BZ-R)A resistant strain of Apolygus lucorum to chlorpyrifos (BZ-R) was obtained by 23 generations of selection on a weakly resistant field-caught strain (BZ), which was collected from Binzhou, Shandong province in 2009. The LC50 of chlorpyrifos to BZ-R was 75.35 mg·L-1. The resistance factor in BZ-R was 10-fold compared with BZ strain, and 59.8-fold when compared with a susceptible laboratory strain (SLF).2.Relationship between metabolic enzymes and chlorpyrifos resistance in A. lucorumThe activities of esterases (ESTs), glutathine S-transferases (GSTs), and mixed function oxidases (MFOs) were detected and compared among BZ-R, BZ, and SLF strains. The activities of ESTs, GSTs, and MFOs in BZ-R strain were 1.7,1.6, and 0.8-fold as that in SLF strain. The activity data, however, did not show significant difference between BZ-R and SLF strains. The resistance in BZ-R was not suppressed by the EST inhibitor S,S,S,-tributylphosphorotrithioate (DEF), the MFO inhibitor piperonyl butoxide (PBO), or by the GST depleter diethyl maleate (DEM). These results showed that metabolic enzymes were not involved in chlorpyrifos resistance in the BZ-R strain of A. lucorum.3.The relationship between target enzyme AChE and chlorpyrifos resistance in A. lucorumThe activity and kinetics of acetylcholinesterase (AChE) in three strains of A. lucorum were detected. The activity of acetylcholinesterase in BZ-R was lower than that in SLF. There were significant difference for the kinetics of acetylcholinesterase between BZ-R and SLF strains. The Km value in BZ-R was 3 times as in BZ, the Vmax value only 1/3 times as in SLF. These results showed the affinity and activity of acetylcholinesterase to substrate in BZ-R had decreased, suggesting the AChE gene (ace1) may have changed in structure.The ace1, one of acetylcholinesterase genes and related to OP resistance were cloned and sequenced. The ORF of ace1 contained 1791 bases and encoded 596 amino acid residues and shared 90% of similarity of ace1 in Cyrtorhinus lividipennis. The sequences of ace1 encoded all key AChE motifs required for catalytic function, such as the catalytic triad, nucleophilic elbow, oxyanion hole and chlorine binding site.The relative expression of acel in BZ-R, BZ, and SLF strains were analyzed using RT-qPCR. The relative expression of ace1 did not give significant difference among all three strains. A216S mutation resulted from the substitution of single nucleotide (G646T), however, was found in ace1 allele. This mutation located at oxyanion hole site and near to the Ser, one of three catalytic active sites. The frequency of A216S mutation of ace1 in SLF, BZ, and BZ-R strains were 0.21,0.34,1.0, respectively. These results showed the A216S mutation in acel resulted in the changes of AChE structure, which led to reducing affinity to chlorpyrifos in AChE and developing a high level resistance to chlorpyrifos in BZ-R strain.