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Research Project: Detection of Ivermectin Target-site Resistance in Rhipicephalus Microplus

Location: Cattle Fever Tick Research

Project Number: 3094-32000-042-073-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Jul 15, 2021
End Date: Jun 14, 2023

1. To identify target-site insensitivity single nucleotide polymorphisms in the glutamate chloride channel gene of ivermectin-resistant strains of R. microplus. 2. To develop a real-time PCR high-resolution melting analysis (HRM) for the detection of mutation(s) in the glutamate chloride channel, the target-site of ivermectin. 3. To compare the expression of metabolic detoxification enzymes in R. microplus strains susceptible and resistant to ivermectin.

a. Source of ticks and resistance testing: Rhipicephalus microplus strains El Zamora and Yucatan, which are maintained at the USDA-ARS Cattle Fever Tick Research Laboratory, are known to exhibit cross-resistance to acaricides, including ivermectin. These strains will be subjected to the larval immersion test to determine the level of resistance to ivermectin (Klafke et al. 2006) and to facilitate phenotypic separation of ticks expressing this resistance (survivors of higher ivermectin concentrations are more likely to express the resistance alleles). b. Identification of target-site insensitivity single nucleotide polymorphisms (SNPs): RNA will be extracted from individual adult female ticks (susceptible and resistant) followed by cDNA synthesis using standard protocols. The segment containing the putative sequences of the GluCl-channel gene of R. microplus will be amplified using primers designed by Gassel et al. (2014) and the PCR products will then be subjected to Sanger sequencing for characterization of the GluCl-channel gene of R. microplus. The nucleic acid sequences from susceptible and resistant ticks will be translated and aligned to GluCl-channel protein sequences from Tetranicus urticae (Acari: Tetranychidae) to look for the single nucleotide polymorphisms associated with ivermectin resistance previously identified in this mite (Kwon et al. 2010). c. HRM assay: A HRM assay will be developed to genotype the different ivermectin resistance related polymorphisms in the GluCl-channel gene of R. microplus. The assay will use genomic DNA from individual adult females from susceptible and resistant strains and primers designed based on the partial sequence of the R. microplus glutamate-gated chloride channel gene. HRM results will be confirmed by Sanger sequencing. d. Detection of metabolic detoxification resistance: A RNA-seq comparative transcriptome approach will be used to compare the expression of metabolic detoxification enzymes (CytP450s, ABC transporters) in ivermectin-susceptible and –resistant strains of R. microplus.