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United States Department of Agriculture

Agricultural Research Service

Research Project: MINING THE GENOME OF RHIPICEPHALUS MICROPLUS TO DEVELOP NOVEL CONTROL TECHNOLOGY AND VACCINES

Location: Tick and Biting Fly Research

Title: Genetic factors potentially reducing fitness cost of organophosphate-insensitive acetylcholinesterase(s) in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Authors
item Temeyer, Kevin
item Tijerina, Mary
item Davey, Ronald
item Olafson, Pia

Submitted to: Acarology International Congress Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 11, 2011
Publication Date: December 7, 2011
Citation: Temeyer, K.B., Tijerina, M.A., Davey, R.B., Olafson, P.U. 2011. Genetic factors potentially reducing fitness cost of organophosphate-insensitive acetylcholinesterase(s) in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Acarology International Congress Proceedings. Zoosymposia 6:260–266.

Interpretive Summary: Tick resistance to organophosphate pesticides is believed to result in part, from mutations causing the enzyme, acetylcholinesterase, to become insensitive to the pesticide. As a result, the enzyme function to terminate nerve impulses by destruction of excess neurotransmitter is preserved; however, the mutation reducing sensitivity to pesticide may have detrimental effects on other functions of the enzyme, resulting in reduced fitness in the absence of the pesticide. Recent research has shown that these ticks contain three different acetylcholinesterase genes, and that multiple copies of each of the genes are present in organophosphate-resistant ticks. The different acetylcholinesterases can “fill-in” for one another as shown by selectively turning off, or “silencing” one or two of the genes without apparent effect on the ticks, however, if all three acetylcholinesterase genes are silenced at once, a high percentage of the ticks die, demonstrating the essential function of the acetylcholinesterases. The present research report proposes that ticks overcome the detrimental “fitness cost” associated with becoming resistant to organophosphate pesticide, by retaining copies of both the mutated and non-mutated forms of the acetylcholinesterase genes. According to the proposed model, the ticks preserve both the multiple functions of the non-mutated acetylcholinesterase, while at the same time, preventing nervous system collapse by producing organophosphate-insensitive (mutant) forms of the acetylcholinesterase enzyme.

Technical Abstract: Acaricidal activity of organophosphate (OP) and carbamate acaricides is believed to result from inhibition of acetylcholinesterase (AChE). Previous studies in Rhipicephalus (Boophilus) microplus demonstrated the presence of three presumptive AChE genes (BmAChEs). Biochemical characterization of recombinant BmAChEs expressed in the baculovirus system demonstrated that each of the three R. microplus rBmAChEs have enzymatic properties consistent with identification as functional acetylcholinesterases. Complementary DNAs (cDNAs) for each of the three BmAChEs were cloned and sequenced from individual adult tick synganglia excised from an OP-resistant strain. The data revealed the presence of multiple sequences within an individual tick for each of the BmAChEs, suggesting alternative mRNA splicing or expression of multiple alleles for each of the BmAChE genes. Quantitative real-time PCR provided evidence of elevated relative copy number for each of the BmAChE genes, and direct sequencing of genomic DNA provided evidence of structural BmAChE gene diversity with respect to presence or absence of introns, as well as presence or absence of sequence polymorphisms. Baculovirus expression of rBmAChE1 and rBmAChE3 constructs containing some of the observed sequence polymorphisms resulted in production of OP-insensitive AChE, demonstrating the presence of mutations resulting in reduced OP-inhibition for at least two of the three BmAChEs. RNA interference was utilized to silence expression of the BmAChE genes in adult ticks in vivo, resulting in tick mortality if all three BmAChEs were silenced simultaneously, but not if any two of the three were silenced, indicating that the BmAChEs functionally complement one another in vivo. It is proposed that deleterious effects of BmAChE mutations are mitigated by gene duplication and maintenance of allelic diversity, including both OP-sensitive and OP-insensitive alleles.

Last Modified: 7/30/2014
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