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Title: Sequence polymorphism in acetylcholinesterase transcripts and genotyping survey of BmAChE1 in laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus

item Temeyer, Kevin
item Olafson, Pia
item PRUETT, JOHN - Retired ARS Employee

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2012
Publication Date: 5/1/2012
Citation: Temeyer, K.B., Olafson, P.U., Pruett, J.H. 2012. Sequence polymorphism in acetylcholinesterase transcripts and genotyping survey of BmAChE1 in laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus. Journal of Medical Entomology. 49(3):555-562.

Interpretive Summary: Acetylcholinesterase (AChE) is a key enzyme in the central nervous system of animals and is the target for functional disruption by organophosphate nerve agents (humans), insecticides (insects), and acaricides (ticks). Many ticks and insects have developed resistance to organophosphate (OP) pesticides. This OP-resistance has frequently resulted from one or more changes (mutations) in the DNA sequence of the gene specifying production of AChE. Although mammals and some insects contain only a single gene for AChE, ticks have at least three different genes containing information on how and when to make AChE. This study reports that the tick genes that specify production of AChE contain a large number of sequence differences, some of which appear to be associated with OP-resistance. The study found that the ticks have multiple different copies for each of the AChE genes, which probably explains why previous searches to identify mutations in tick AChE were often unsuccessful. Identification of the specific sequence changes that appear to be associated with OP-resistance is important because it enables studies to test which mutations make AChE less sensitive to OP pesticides. The study enables development of DNA-based assays to test for the presence of specific mutations in tick populations. Together, these results are essential to enable development of rapid DNA-based tests to guide selection of successful tick control treatments.

Technical Abstract: BmAChE1, BmAChE2, and BmAChE3 cDNAs of Rhipicephalus (Boophilus) microplus were sequenced and found to exhibit significant polymorphism. A portion of the predicted amino acid substitutions in BmAChE1, BmAChE2 and BmAChE3 were found predominantly in organophosphate-resistant (OP-R) strains, but most did not correlate with resistant status. Multiple transcripts were observed from individual ticks, suggesting possible gene duplication or alternative splicing to produce more than two transcripts per individual. BmAChE1 transcript polymorphisms associating with OP-R status in laboratory strains were surveyed in laboratory and Mexican strains of R. microplus by sequencing BmAChE1 genomic DNA. Quantitative real-time PCR (qPCR) was used to determine copy numbers of BmAChE1 (8 copies/haploid genome), BmAChE2 (16 copies/haploid genome), and BmAChE3 (4 copies/haploid genome). Presence of at least three highly polymorphic amplified genes expressing AChE in tick synganglion suggested that ticks maintain a large and diverse assortment of AChE alleles available for rapid recombination and selection, which potentially reduces fitness costs associated with individual mutations. Elevated copy numbers for each of the BmAChEs may also explain previous failures to identify mutations resulting in insensitivity to OPs. It is clear that development of phenotypic resistance to OPs is highly complex and may be multigenic in character.