Heterologous Expression and Biochemical Characterisation of Fourteen Esterases from Helicoverpa armigera

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.This study was supported by an Australian Postgraduate Award and Top-up Scholarship from the Cotton Catchment Cummunities CRC to Claire Farnsworth and the China Scholarship Council to Yongqiang Li. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, \u3ci\u3eSchizaphis graminum\u3c/i\u3e (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

Changes in DNA methylation are associated with loss of insecticide resistance in the peach-potato aphid Myzus persicae (Sulz.)

AbstractInsecticide resistance in peach-potato aphids arises from the overproduction of one of two very closely related esterases (E4 or FE4) that detoxify insecticidal esters, and this is associated with amplification of E4-related DNA sequences. Some highly resistant aphid clones can spontaneously lose their elevated esterase and hence their resistant phenotype. We report here that such aphids also lose their elevated E4 mRNA whilst retaining their amplified genomic sequences. We have also shown that the amplified E4-related sequences are highly methylated at MspI sites in all resistant aphid clones examined, but not in those that have lost resistance