Biochemical and molecular mechanism of prothiofos-resistance in the diamondback moth, Plutella xylostella (L.)

Abstract

A prothiofos-resistant diamondback moth (DBM-R) strain has been established from the susceptible (DBM-S) strain over 100 generations under the selective pressure of the prothiofos. The resistance level of the DBM-R strain against prothiofos was 20- to 70-fold greater than that of the DBM-S strain in the leaf-dipping assay. To determine the resistance mechanism of the DBM-R strain, enzyme activities of acetylcholinesterase (AChE), general esterase and glutathione Stransferase (GST), usually responsible for insecticide resistance in arthropods, were examined. AChE activity in the DBM-R strain showed 1.2-fold higher than that of the DBM-S strain, whereas there was no significant difference in GST activity. The activity of general esterase in the midgut of DBM-R strain was 2 times higher than that of the DBM-S strain, but no different pattern of general esterase was detected in non-denaturing polyacrylamide gel electrophoresis. In contrast to low affinity of resistant AChE for the substrate, Vmax was 1.2-fold higher than that of susceptible AChE. In semiquantitative RT-PCR, the transcript level of resistant AChE gene was approximately 8% more than that of susceptible AChE gene. These suggest that increased amount of AChE is related to prothiofos-resistance in the DBM.R strain. Kinetic analysis of the resistant AChE with paraoxon (phenyl organothiophosphate) revealed 7-fold reduced ki, indicating AChE insensitivity to paraoxon, the same group with prothiofos, was increased. Furthermore, this result proposes that resistant AChE may result from structural alteration. In order to determine whether prothiofos- resistance of DBM is attributed to altered AChE (such as point mutation) or AChE gene amplification, AChE gene (cDNA and genomic DNA) from both strains was cloned and sequenced. Resistant and susceptible AChE gene showed polymorphism in nucleotide analysis, resulting in the substitution of amino acids in several positions.