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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY AND PHYSIOLOGY OF BITING FLIES AFFECTING LIVESTOCK

Location: Tick and Biting Fly Research

Title: Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): Baculovirus expression, biochemical properties and organophosphate insensitivity

Authors
item Temeyer, Kevin
item Brake, Danett
item Schlechte, Kristie

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 8, 2012
Publication Date: May 1, 2012
Citation: Temeyer, K.B., Brake, D.K., Schlechte, K.G. 2012. Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): Baculovirus expression, biochemical properties and organophosphate insensitivity. Journal of Medical Entomology. 49(3):589-594.

Interpretive Summary: Horn flies are a serious and economically significant pest of cattle. Efforts to control horn flies using chemical pesticides are increasingly difficult due to development of pesticide resistance in fly populations. Diazinon is a member of the organophosphate (OP) class of pesticides that disrupts the fly nervous system by targeting the enzyme acetylcholinesterase (AChE). Diazinon-resistant flies were previously shown to contain a mutation (G262A) in AChE predicted to alter the structure of the enzyme so that it would be less sensitive to OP insecticides. A new study used recombinant DNA technology to clone and express a recombinant form of the horn fly AChE with and without the G262A mutation to characterize biochemical properties of the horn fly AChE enzyme and the effects of the G262A mutation. Results of the study determined the biochemical properties of the horn fly AChE and confirmed that the G262A mutation significantly reduced sensitivity of the enzyme to OP insecticide. This study substantiates previous work and suggests that two additional AChE mutations may also contribute to insecticide resistance in horn flies. Elucidation of the mechanisms of fly resistance will allow development of rapid tests to identify mutations present in fly populations that will help guide selection of effective means of pest control.

Technical Abstract: This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (Km 31.3 X 10-6M) over butyrylthiocholine (Km 63.4 X 10-6M) and inhibition at high substrate concentration. Enzyme activity was inhibited by eserine (2.3 X 10-10M), BW284c51 (3.4 X 10-8M), malaoxon (3.6 X 10-8M), and paraoxon (1.8 X 10-7M), and was relatively insensitive to ethopromazine (1.1 X 10-6M) and iso-OMPA(4.1 X 10-4M). rHiAChE containing the G262A mutation exhibited decreased substrate affinity for both acetylthiocholine (Km 40.9 X 10-6M) and butyrylthiocholine (Km 96.3 X 10-6M), and exhibited 8-fold decreased sensitivity to paraoxon, and approximately (1.5-3)-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates. It is suggested that altered expression or maturation of HiAChE at the neural synapse may contribute to increased tolerance or resistance to OP.

Last Modified: 12/21/2014
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