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 BmAChE proteins expressed in the baculovirus system demonstrated that each of the three R. (B.) microplus rBmAChEs have enzymatic properties consistent with designation as functional acetylcholinesterases. Complementary DNAs (cDNAs) for each of the three BmAChEs were cloned and sequenced from individual adult tick synganglia excised from OP-susceptible and OPresistant strains. The data revealed the presence of multiple transcript sequences within individual ticks 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 possible gene duplication or amplification 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 the presence or absence of sequence polymorphisms. Baculovirus expression of rBmAChE1 and rBmAChE3 proteins containing some of the predicted amino acid sequence polymorphisms resulted in production of OP-insensitive AChE, demonstrating that at least some OP-resistant individuals contain mutations that reduce OP-inhibition for at least two of the three known BmAChEs. RNA interference was utilized to silence in vivo expression of the BmAChE genes in adult ticks, resulting in tick mortality if all three BmAChEs were silenced simultaneously, strongly suggesting that the BmAChE proteins functionally complement one another in vivo. Together, the results presented provide strong evidence that OP-resistance in R. microplus is at least partially mediated by a combination of the expression of multiple genes encoding acetylcholinesterase, mutations in BmAChEs resulting in OP-insensitivity, gene duplication, and maintenance of allelic diversity, including both OP-sensitive and OPinsensitive alleles within individual ticks. The authors propose that these factors may mitigate fitness costs that might otherwise result from BmAChE mutations, and demonstrate the extreme complexity of OP-resistance in R. (B.) microplus. It is hoped that elucidation of the complex interactions among the multiple BmAChEs and their physiological roles may enable development of new opportunities for tick control.