Esterases and helicoverpa armigera

Abstract

Pesticide use has been carefully regulated by the Australian cotton industry in an attempt to control resistance in the cotton bollworm (Helicoverpa armigera) but the species has still shown a propensity to develop resistance. This thesis examines the contribution of metabolic enzymes to resistance, with a focus on the carboxylcholinesterases (CCEs, or esterases). CCEs have been implicated in metabolic resistance to several insecticide classes (including SPs and OPs) in a range of species. The large number of recently radiated esterases found in those species within available sequenced genomes suggests the potential for them to play some role in xenobiotic detoxification. Of particular interest is the recently radiated 'Clade 1' (as proposed by Teese et al, 2010). Cytochrome P450s (P450s) are also crucial players in the metabolic detoxification story. P450s have been proposed to be involved in resistance through active site mutations, upregulation and/or duplication and amplification. In an attempt to characterise the esterases of H. amigera that might be involved in resistance I first used a baculovirus system to express 14 full length midgut esterases sequences. Thirteen of the enzymes yielded sufficient quantity to conduct OP and SP activity assays. All showed tight binding to OPs and low but measurable OP hydrolase activity. There were large differences between the isozymes in their activities against the nine SP isomers tested. The enzymes with the highest activity against the most insecticidal isomers were from regions of the native gel profile previously associated with resistance. I then looked at the relationship between resistance and metabolic detoxification in an Australia H. armigera strain selected for fenvalerate resistance over 24 generations. Bioassays with P450 and esterase inhibitors (PBO and DEF respectively) suggest a large effect of P450s and a smaller effect of esterases. An increased intensity of staining in specific zones was observed on native polyacrylamide gels stained for esterase activity. The increases in these zones were then associated with Clade 1 esterases by a native Western with Clade 1 specific antibodies. Clade 1 esteraes have previously been proposed to be important contributors to resistance. This thesis also examines the suggested contribution of esterases to resistance to the proteinaceous Bt toxins in Helicoverpa species. I compared 11 strains of H. armigera (nine variously resistant to the Cry1Ac, Cry2Ab and VIP3A toxins and two susceptible to all three) and two strains of H. punctigera (one resistant and one susceptible to Cry1Ac) in a series of bioassays and biochemical activity assays. In contrast to previously published findings, this study did not see increased esterase activity or isozyme intensities in the Cry1Ac resistant line tested. Nevertheless my study did agree with the earlier studies of esterases in relation to Cry1Ac resistance in that I found a strong synergistic effect of DEF on resistance to Cry1Ac resistance in vitro. While my data thus do not support all the previously presented lines of evidence for the involvement of esterase in Bt toxin resistance, it does support some of the evidence. Show more