MOLECULAR BIOLOGY OF BOOPHILUS MICROPLUS
Location: Tick and Biting Fly Research
Title: BACULOVIRUS EXPRESSION OF BMACHE3, A CDNA ENCODING AN ACETYLCHOLINESTERASE OF BOOPHILUS MICROPLUS (ACARI:IXODIDAE)
Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 15, 2006
Publication Date: June 30, 2006
Citation: Temeyer, K.B., Pruett Jr., J.H., Untalan, P.M., Chen, A.C. 2006. Baculovirus expression of BmAChE3, a cDNA encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae). Journal of Medical Entomology. 43(4):707-712.
Interpretive Summary: The Southern cattle tick, the vector of bovine babesiosis, was eradicated from the United States in 1943. However, because the tick is prevalent in Mexico, the potential for re-introduction into the U.S. exists. The Cattle Fever Tick Eradication Program, maintained by APHIS/VS, is the only barrier to tick reentry and success of that program is dependent upon the effectiveness of the organophosphate (OP) pesticide, coumaphos. Reports of tick resistance to coumaphos have driven the need for investigations into the mechanisms by which the tick becomes resistant. The target for coumaphos toxicity in the tick is an enzyme called acetylcholinesterase (AChE), which is involved in nerve transmission. Basically, resistance to OPs is believed to be the result of alterations in the chemical structure of AChE that allows the tick to survive OP exposure. There are three putative AChEs that have been reported in the literature. We copied the genetic information for one of the AChEs from the tick, introduced this information into insect cells in culture, and allowed the insect cells to produce functional tick AChE. This allows us ample AChE to study the possible mechanism(s) of OP-resistance in the tick. Information gained from these studies will allow us to design diagnostic tools to manage the development of OP-resistant ticks in the field.
The complete cDNA sequence encoding a Boophilus microplus acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors, eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable to that of adult, OP-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE