The carboxyl/cholinesterases of Helicoverpa armigera : a comparative genomic, proteomic and expression analysis of a diverse gene superfamily
Creators: name: Teese, Mark George
Publisher: name: The Australian National University
Year: 2010
Subjects: subject: SB945.H28 T44 2010
subject: Helicoverpa armigera Insecticide resistance
subject: Plant parasites Control
subject: Cholinesterase genes

description: Helicoverpa armigera is a major lepidopteran pest of crops in Southern Europe, Africa, Asia and Australasia. It can be controlled by several classes of conventional insecticides such as the organophosphates (OPs), carbamates and synthetic pyrethroids. There have been many reports, however, of field-derived or laboratory strains of H. armigera that survive otherwise toxic doses of these chemicals. In most cases, the mechanism for this resistance has not been elucidated to the molecular level. Most resistant strains show evidence of metabolic detoxification, where the insecticide is modified to a less toxic product through the action of detoxifying enzymes. The major gene families of detoxifying enzymes in insects are P450 monooxygenases, glutathione transferases and carboxyl/cholinesterases. Carboxyl/cholinesterases belong to the large superfamily of {u03B1}/{u03B2} hydrolase proteins. Their carboxylesterase activity is due to a two-step reaction that relies on a catalytic triad of a serine, histidine and an acid residue. OP and carbamate insecticides react with carboxyl/cholinesterases in a similar way to carboxylester substrates; however the second step of the reaction occurs very slowly, leaving the enzymes inhibited. OP resistance in insects has been shown to arise through mutations in the sequence encoding some carboxylesterases that increase the rate of this reaction. Alternatively, resistance can also arise through the over-expression of otherwise unaltered carboxyl/cholinesterases. In H. armigera and other heliothine pests, current evidence suggests that over-expression is the major mechanism by which carboxyl/cholinesterases contribute to resistance to conventional insecticides, rather than changes in catalytic properties of the enzymes caused by mutations within esterase coding regions. Studies have shown that insecticides can be synergised by esterase inhibitors and are metabolised in vivo in ways consistent with esterase hydrolysis. Also, resistant strains tend to show higher esterase activity than susceptible controls. Techniques to separate larval proteins have implicated several particular carboxylesterase isozymes in resistance in H. armigera, however as yet none of these proteins have been identified to their DNA. This study is the first comprehensive analysis of the carboxyl/cholinesterases from H. armigera larvae that examines both the DNA sequences and the carboxylesterases that they encode. Phylogenetic analysis of a data set of 39 carboxyl/cholinesterases from H. armigera revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori. B. mori orthologues were proposed for 31% of the sequences examined and there were several conserved clades of non-catalytic proteins. However low similarity was found between these lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. To identify .some of zones of esterase activity previously associated with resistance in H. armigera, I conducted a proteomic analysis of larval esterases that matched seven H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. Five of these sequences were matched to zones of activity on native PAGE previously associated with insecticide resistance. Ten carboxyl/cholinesterase sequences from H. armigera were then heterologously expressed in insect cells to obtain enough protein for further analysis. All showed evidence of catalytic actlvity. One carboxylesterase (016a) was attached to the plasma membrane by a glycosylphosphatidyl-inositol anchor. This finding may help to explain the proposed interaction between larval carboxylesterases and the Bacillus thuringiensis Cry1Ac toxin, which does not contain ester bonds but has a strong affinity for some GPI-anchored proteins. Preliminary kinetic analysis revealed that several of the expressed proteins had a k(cat) against OP substrates that exceeded the wildtype esterase E3 of Lucilia cuprina, however none equaled the turnover seen by the E3 mutants that convey resistance. These results reveal the baseline in H. armigera from which OP resistance can be evolved, either through the overexpression of the CCEs that already show significant OP turnover, or by mutations which increase the catalytic efficiency of these enzymes against OP insecticides.