Generation of Full-Length cDNAs for Eight Putative GPCnR from the Cattle Tick, R. microplus Using a Targeted Degenerate PCR and Sequencing Strategy

Abstract

We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.

Mutation in the RmβAOR gene is associated with amitraz resistance in the cattle tick Rhipicephalus microplus

“…These findings have been reported elsewhere (14). Subsequently, as we have developed molecular tools for analyzing the R. microplus populations (9,(15)(16)(17), we examined population dynamics on a molecular genetic basis. The primary objectives of this work were to: (i) determine whether intensive selection with amitraz resulted in an increased frequency of the I61F SNP in the β-adrenergic octopamine receptor (RmβAOR); (ii) undertake a detailed analysis of the R. microplus population dynamics in the field in the first generations after admixture of two distinct strains of cattle tick (susceptible and multiple resistant); and (iii) characterize the relationship between resistance to amitraz and resistance to SPs derived from a single multiresistant strain of ticks.…”

“…This similarity is reflected by their pharmacology in that they are likely to mediate their effects via elevations in intracellular calcium concentrations (18). A putative αAOR (Rmα2AOR) has been isolated in the cattle tick (9). The second group includes the octopamine/tyramine receptors, which are preferentially activated by tyramine over octopamine in most cases (19,20).…”

“…The second group includes the octopamine/tyramine receptors, which are preferentially activated by tyramine over octopamine in most cases (19,20). The first octopamine-like receptor isolated in the cattle tick (7) was recently shown to group with the octopamine/ tyramine receptors on phylogenetic analysis of insect and arachnid octopamine receptors (9). The third group includes β-adrenergiclike octopamine receptors (βAOR), which show the greatest structural similarity to vertebrate β-adrenergic receptors.…”

“…Identification of amitrazresistance mutation. The βAOR was sequenced using gDNA prepared from a pool of 30 larvae for each of the NRFS and the Ultimo strains using the method described by Corley et al (9). PCR primers (F1 5′-AGTGCCCTC-TAGGCGATACA-3′ and R1 5′-TGTTGCTACGGTTGGCTATG-3′), designed using Primer3 software (24), were used to amplify a 1,644-bp fragment containing the entire coding sequence, which is a single exon.…”

“…Previous investigations of the role of the octopamine receptor in amitraz resistance in R. microplus (7,8) were not fruitful, but it seems likely that the studies investigated a tyramine receptor, rather than a true octopamine receptor (9). We have recently identified several G protein-coupled receptors in R. microplus (9) and identified polymorphisms in resistant populations in the β-adrenergic octopamine receptor (RmβAOR, SNP designated I61F), which we propose might be associated with resistance to amitraz in R. microplus.…”