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Title: Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins

Author
item Temeyer, Kevin
item Olafson, Pia
item BRAKE, DANETT - Former ARS Employee
item Tuckow, Alexander
item Li, Andrew
item Perez De Leon, Adalberto - Beto

Submitted to: Pesticide Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2013
Publication Date: 7/1/2013
Citation: Temeyer, K.B., Olafson, P.U., Brake, D.K., Tuckow, A.P., Li, A.Y., Perez De Leon, A.A. 2013. Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins. Pesticide Biochemistry and Physiology. 106:118-123.

Interpretive Summary: The southern cattle tick and the sand fly each feed on blood obtained from host animals. Both the tick and sand fly may acquire or transmit disease causing microorganisms during the process of obtaining blood from their host animals and are serious threats to animal and human health. One class of chemical acaricides and insecticides used to kill ticks, sand flies, and other pests works by inactivating an enzyme that is essential for function of the central nervous system of these pests. The enzyme targeted by this class of pesticides is called acetylcholinesterase. Pests may become resistant to chemical pesticides as a result of mutations that alter their acetylcholinesterase enzyme so that it becomes insensitive to the pesticide. Research reported here utilized gene cloning to produce the tick and sand fly acetylcholinesterase enzymes in cell cultures so that mutations in the enzymes that cause pesticide resistance could be identified. This knowledge will help scientists to develop rapid molecular tests to determine if the pests can be killed by specific types of pesticides to guide selection of effective control options.

Technical Abstract: Rhipicephalus (Boophilus) microplus (Bm) ticks are vectors of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. Sequencing and in vitro expression of Bm genes encoding AChE allowed biochemical characterization of three BmAChEs expressed in tick synganglion. BmAChE1, BmAChE2, and BmAChE3 exhibited substrate preference for acetylthiocholine, high substrate inhibition, and sensitivity to AChE-specific inhibitors. OP-insensitivity mutations were demonstrated in BmAChE1 and BmAChE3. Gene silencing suggested functional complementation of BmAChEs in vivo. BmAChE genes were amplified in copy number and multiple transcript polymorphisms were expressed in individual tick synganglia for each of the BmAChEs, suggesting allelic diversity within individuals. Studies also identified a gene encoding AChE of the sand fly, Phlebotomus papatasi, a vector of leishmaniasis in humans and animals. Expression of recombinant P. papatasi AChE (PpAChE) enabled biochemical characterization and identification of effective inhibitors that selectively target PpAChE,