MOLECULAR BIOLOGY OF BOOPHILUS MICROPLUS
Location: Tick and Biting Fly Research
Title: R86Q, a mutation in BmAChE3 yielding a Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase
Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 16, 2007
Publication Date: November 15, 2007
Citation: Temeyer, K.B., Pruett Jr, J.H., Olafson, P.U., Chen, A.C. 2007. R86Q, a mutation in BmAChE3 yielding a Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase. Journal of Medical Entomology. 44(6):1013-1018.
Interpretive Summary: The southern cattle tick is a vector for disease-causing agents of cattle and was eradicated from the United States, yet remains endemic to Mexico. Reintroduction of the tick to the United States is prevented by organophosphate (OP)-acaricide treatment of imported cattle and vigilant monitoring of a narrow quarantine zone along the U.S./Mexico border. Development of OP- resistance in the southern cattle tick threatens the efficacy of the import barrier, and poses a threat to the U.S. cattle industry. OP-acaricides kill ticks by inhibiting acetylcholinesterase, an important enzyme in nerve transmission. It has long been known that mutations in insect acetylcholinesterase constitute a major mechanism for development of organophosphate resistance; however, until now, efforts to demonstrate a similar mechanism in ticks have proven unsuccessful. A newly discovered mutation was shown to produce a mutant form of acetylcholinesterase that was resistant to OP-acaricide. Assays to diagnose presence of the resistance mutation were developed and demonstrated that OP-resistant tick strains carried the mutation at increased frequency compared to susceptible strains of ticks, suggesting that the mutation is significant to development of resistance to OP-acaricides. However, the presence of the studied mutation alone does not appear to be sufficient to produce resistance, possibly due to the presence of multiple acetylcholinesterases in the tick. Development of OP-acaricide resistance may require accumulation of mutations, possibly in more than one of the acetylcholinesterases.
Mutations were identified in the sequence encoding the acetylcholinesterase, BmAChE3, in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to orgaonphosphorus acaricide. The mutation which appeared most frequently in the organophosphorus-resistant San Román strain but not in susceptible strains resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in mature BmAChE3. Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in an ixodid acetylcholinesterase resulting in organophosphate insensitivity. Two independent assays were developed to diagnose frequency of the R86Q mutation in BmAChE3 genomic DNA from eight laboratory colonized strains. Individual genotypes assigned using an allele-specific PCR test demonstrated greater than 90% agreement with respect to genotypes generated using a restriction fragment length polymorphism test of the same individuals. Use of the R86Q diagnostic assays demonstrated a higher frequency of the R86Q mutation in organophosphate-resistant tick strains, than was found in organophosphate-susceptible strains. We conclude that the R86Q mutation may be important in the development of OP-resistance but that there may be additional mutations in BmAChE3, or mutations in additional acetylcholinesterase genes, that contribute to expression of the OP-resistant phenotype.