Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.