Veterinary Parasitology 172 (2010) 114–121 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus Kevin B. Temeyer ∗ , John H. Pruett, Pia U. Olafson Knipling-Bushland U.S. Livestock Insects Research Laboratory, USDA-ARS, 2700 Fredericksburg Road, Kerrville, TX 78028, USA article info Article history: Received 5 March 2010 Received in revised form 8 April 2010 Accepted 9 April 2010 Keywords: Acetylcholinesterase AChE Cattle fever tick Acaricide resistance Acari Ixodidae abstract Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3, were previ- ously identified as presumptively encoding acetylcholinesterases (AChEs), but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recom- binant BmAChE1 constructs and single recombinant constructs of BmAChE2 and BmAChE3 were expressed in baculovirus. Biochemical characterization of the recombinant proteins supports classification of rBmAChE1, rBmAChE2, and rBmAChE3 as AChEs (E.C.3.1.1.7), as evidenced by (i) substrate preference for acetylthiocholine, (ii) inhibition by eserine, BW284c51, and the organophosphates (OPs) malaoxon and paraoxon, (iii) insensitivity to iso-OMPA, and (iv) rapid hydrolysis of acetyl--methyl-thiocholine. Unlike reports for insect AChEs, we did not observe substrate inhibition of activity at acetylthiocholine con- centrations as high as 40 mM, however, product inhibition was apparent at 10–100 M choline in agreement with properties reported for the catalytic domain of Anopheles gam- biae acetylcholinesterase-1. Substrate affinity and V max values were highest for rBmAChE1 proteins, and one rBmAChE1 enzyme (Tx11, derived from the OP-resistant strain Tuxpan), was insensitive to paraoxon and exhibited a greatly reduced V max near that of rBmAChE2. To date, recombinant BmAChE1 and BmAChE3 enzymes with reduced sensitivity to OP- inhibition have been cloned and expressed from OP-resistant strains. The presence of at least three genes expressing AChEs in R. (B.) microplus, at least two of which contain muta- tions expressed as OP-insensitive enzymes, strongly suggests that phenotypic resistance to OPs may be complex and multigenic in character. Published by Elsevier B.V. 1. Introduction The Southern cattle tick, Rhipicephalus (Boophilus) microplus, a vector of bovine babesiosis and anaplasmo- sis, was eradicated from the United States (Graham and Hourrigan, 1977), but remains endemic to Mexico. Re- establishment of R. microplus within the United States is prevented by the Cattle Fever Tick Eradication Program (CFTEP) including a permanent quarantine zone along the ∗ Corresponding author. Tel.: +1 830 792 0330; fax: +1 830 792 0314. E-mail address: Kevin.Temeyer@ars.usda.gov (K.B. Temeyer). southern border of Texas maintained by the Veterinary Services branch of the Animal and Plant Health Inspec- tion Service (APHIS) of the U.S. Department of Agriculture. All cattle imported from Mexico are required to be dipped in vats containing the organophosphate (OP), coumaphos (George, 1996). The CFTEP is considered to be in a near crisis condition with concerns of increasing OP resistance in Mexico (Santamaría and Fragoso, 1994; Fragoso et al., 1995), increasing numbers of tick outbreaks in the counties adjacent to the border with Mexico prompting tempo- rary expansion of the quarantine area by nearly a million acres in July of 2008, the recent finding of an OP-resistant outbreak strain within the U.S. quarantine zone (Miller 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2010.04.016