Gene_locus Report for: haeir-ACHE

This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (Km 31.3 microM) over butyrylthiocholine (Km 63.4 microM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 x 10(-10) M), BW284c51 (3.4 x 10(-8) M), malaoxon (3.6 x 10(-9) M), and paraoxon (1.8 x 10(-7) M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 x 10(-6) M) and iso-OMPA (4.1 x 10(-4) M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (Km 40.9 microM) and butyrylthiocholine (Km 96.3 microM), and exhibited eight-fold decreased sensitivity to paraoxon, and approximately 1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.

Horn flies, Haematobia irritans, a major cattle pest in the USA, cause substantial economic losses and current control methods rely heavily on insecticides. Three horn fly populations were evaluated for insecticide susceptibility to permethrin, β-cyfluthrin, and diazinon. Susceptibility was variable by population, with the greatest resistance exhibited by a 66-fold resistance ratio (RR) to permethrin and >14-fold RR to diazinon. Mechanisms of resistance were determined using molecular techniques and enzymatic assays. The knockdown resistance (kdr) genotype (L150F) associated with pyrethroid resistance, and a G262A mutation in acetylcholinesterase, previously associated with organophosphate resistance, were found in all field populations evaluated. Insensitivity of diazoxon at the acetylcholinesterase (AChE) target site was significantly different in horn flies from one of the field sites. For metabolic detoxifying enzymes, cytochrome P450 nor general esterases showed a significant difference between field strains and a laboratory susceptible strain. Pyrethroid resistance was likely due to the presence of the L150F mutation in the population. In vitro studies targeting the AChE enzyme did not support the notion that the G262A mutation was the sole cause of resistance to organophosphates, and, therefore, the exact resistance mechanism to diazinon was not able to be confirmed.

This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (Km 31.3 microM) over butyrylthiocholine (Km 63.4 microM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 x 10(-10) M), BW284c51 (3.4 x 10(-8) M), malaoxon (3.6 x 10(-9) M), and paraoxon (1.8 x 10(-7) M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 x 10(-6) M) and iso-OMPA (4.1 x 10(-4) M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (Km 40.9 microM) and butyrylthiocholine (Km 96.3 microM), and exhibited eight-fold decreased sensitivity to paraoxon, and approximately 1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.