DEVELOPING AND USING MOLECULAR AND BIOCHEMICAL METHODS FOR THE DIAGNOSIS OF ACARICIDE RESISTANCE IN BOOPHILUS MICROPLUS
Location: Tick and Biting Fly Research
Title: ACETYLCHOLINESTERASE MUTATION IN DIAZINON-RESISTANT HAEMATOBIA IRRITANS (L.) (DIPTERA: MUSCIDAE)
Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 20, 2008
Publication Date: July 4, 2008
Citation: Temeyer, K.B., Li, A.Y., Lohmeyer, K.H., Chen, A.C., Olafson, P.U., Sanson, D.W., Foil, L.D. 2008. Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae). Veterinary Parasitology. 154(3-4):300-310.
Interpretive Summary: Flies are serious pests of humans and animals, and successful fly control has become more difficult to achieve due to increasing development of pesticide resistance among these pests. Adult horn flies are biting flies that feed on the blood of cattle. A convenient method for horn fly control on cattle has utilized ear tags impregnated with pesticide for slow release, but in recent years, this method has increasingly failed to provide long-term control of horn fly populations. Diazinon is an organophosphate pesticide, used in some ear tags, that targets the nervous system of insects. A new study of horn flies resistant to diazinon, found that they have a mutation in a gene that specifies production of a key enzyme of the insect nervous system that appears to be the target for diazinon toxicity. The same mutation was previously found in house flies resistant to diazinon, and the mutant enzyme, acetylcholinesterase (AChE) was shown to be insensitive to the toxic effects of diazinon. Knowledge of the mutation in the horn fly gene specifying AChE production allowed development of two new and rapid tests for the presence of the mutation in horn flies. These new tests allow rapid testing of horn fly DNA to determine if the horn flies harbor this diazinon-resistant form of the AChE gene. Knowledge of the pesticide-resistance status of horn flies enables cattle producers to make more informed choices among available pesticides to avoid the labor and expense of purchasing and applying a pesticide to which the horn flies are resistant, resulting in lower production costs, more effective fly control, and reduced release of toxic pesticides into the environment.
Acetylcholinesterase(AChE) cDNA from individual field collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that a point mutation previously shown to be associated with resistance to organophosphates in house flies and Drosophila was present in most of the diazinon-resistant horn flies, strongly suggesting that this cDNA encodes the acetylcholinesterase that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the encoded amino acid sequence at position 262. An allele-specific DNA-based PCR assay and an allele-specific PCR-RLFP assay were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays demonstrated the presence of the G262A mutation in 10 of 12 field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products and providing stronger support that this AChE is the target site for OP pesticide. The allele-specific assays will provide a useful tool for quantitative assay of the resistance allele in horn fly populations.