Validation of a Point Mutation of Acetylcholinesterase in Colorado Potato Beetle by Polymerase Chain Reaction Coupled to Enzyme Inhibition Assay

Abstract

A serine to glycine point mutation of the acetylcholinesterase (AChE, EC 3.1.1.7) gene was identified previously in an azinphos-methyl-resistant (AZ-R) strain of the Colorado potato beetle,Leptinotarsa decemlineata(Say). To validate this mutation, the polymerase chain reaction (PCR) amplification of specific alleles (PASA) was coupled to a functional assay for AChE in individual insects. The remaining activities of AChE after inhibition by 50 μMazinphos-methyl-oxon was 22.3% ± 6.5 and 48.0% ± 3.9 (mean ± SD,n= 13) for the beetles from the azinphos-methyl-susceptible (SS) and AZ-R strains, respectively. This pattern of AChE inhibition established that AChE from the AZ-R beetles were significantly less sensitive to azinphos-methyl-oxon inhibition than that from the SS beetles. Using a competitive PASA technique to diagnose the azinphos-methyl-resistance allele of the AChE gene in these insects, the resistance allele was found in all AZ-R beetles but was never found in the SS beetles. These results agree well with the level of susceptibility of the AChE in individual insects and support the contension that the serine to glycine mutation is responsible for the reduced sensitivity of the AChE to inhibition by azinphos-methyl-oxon in the AZ-R strain of beetles. The utilization of the PASA technique for resistance monitoring in Colorado potato beetle is also discussed.