title: The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina.
abstract: Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.
authors.affiliation: CESAR -- Centre for Environmental Stress and Adaptation Research, Genetics Department, University of Melbourne, Parkville 3052, Australia. z.chen@genetics.unimelb.edu.au
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Newcomb
authors.forename: E
authors.initials: E
authors.lastname: Forbes
authors.forename: J
authors.initials: J
authors.lastname: McKenzie
authors.forename: P
authors.initials: P
authors.lastname: Batterham
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D003001:Cloning, Molecular; D004175:Diptera / Q000201:enzymology* / Q000235:genetics; D017344:Genes, Insect; D007305:Insecticide Resistance / Q000235:genetics; D007306:Insecticides; D008969:Molecular Sequence Data; D009946:Organothiophosphorus Compounds; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep
chemical_list: D007306:Insecticides; D009946:Organothiophosphorus Compounds; D000110:Acetylcholinesterase