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Title: Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus

Author
item Temeyer, Kevin
item Pruett Jr, John
item Olafson, Pia

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2010
Publication Date: 4/16/2010
Citation: Temeyer, K.B., Pruett Jr, J.H., Olafson, P.U. 2010. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Veterinary Parasitology. 172:114-121.

Interpretive Summary: The Southern Cattle Tick is a vector for fatal diseases of cattle and was eradicated from the United States as a result of a massive program that took many decades. This tick remains endemic to Mexico, and reentry is prevented by constant vigilance in a program requiring acaricide treatment and inspection of all cattle entering at the U.S.-Mexico border. Increasing resistance of ticks to acaricides in Mexico and Latin America poses a threat to the Cattle Fever Tick Eradication Program (CFTEP) that has kept the tick from moving back into the United States. Successful continuation of acaricide tick control appears to require an understanding of the biochemical and genetic mechanisms used by the ticks to become resistant to acaricides, specifically, to the organophosphate (OP) acaricide that is the backbone of the current reentry barrier. Acetylcholinesterase (AChE) is a key enzyme in the nervous system, and may be inactivated by certain organophosphates (OPs) used as pesticides. This study demonstrated that the Southern Cattle Tick has at least three different AChEs that are present in the tick brain. We found that all three of the AChE genes contain a large number of mutations. Expression and biochemical characterization of cloned AChEs verified that at least two of the AChEs can become resistant to OP-inactivation as a result of mutations present in ticks. In addition, this study suggests that the genes encoding the AChEs may be present in multiple copies, possibly allowing the ticks additional options to evade OP-inactivation. This study is the first to conclusively demonstrate that the Southern Cattle Tick possesses multiple active AChE enzymes, all of which are expressed in the tick brain. The physiological functions of the various AChE enzymes remains to be elucidated, however, it is clear that the potential mechanisms of OP-resistance are far more complex than previously thought.

Technical Abstract: Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3,were previously identified as presumptively encoding acetylcholinesterases, but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as acetylcholinesterases (AChEs, E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl-ß-methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine concentrations as high as 40 mM, however, product inhibition was apparent at 10-100 µM choline in agreement with properties reported for the catalytic domain of Anopheles gambiae acetylcholinesterase-1. Substrate affinity and Vmax were highest for rBmAChE1, and one BmAChE1 construct (Tx11, an organophosphate-insensitive construct from strain Tuxpan), was insensitive to paraoxon and had a Vmax near that of rBmAChE2. To date, recombinant constructs of BmAChE1 and BmAChE3 with reduced sensitivity to organophosphate inhibition have been cloned from organophosphate-resistant strains. The presence of at least three genes expressing acetylcholinesterases strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character.