High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Authors: KLAFKE, GUILHERME - Department Of Energy; Miller, Robert; Tidwell, Jason; Thomas, Donald; SANCHEZ, DANIELA - University Of Texas Rio Grande Valley; FERIA ARROYO, TERESA - University Of Texas Rio Grande Valley; Perez De Leon, Adalberto - Beto

Submitted to: International Journal for Parasitology: Drugs and Drug Resistance
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2019
Publication Date: 3/13/2019
Citation: Klafke, G.M., Miller, R., Tidwell, J.P., Thomas, D.B., Sanchez, D., Feria Arroyo, T.P., Perez De Leon, A.A. 2019. High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). International Journal for Parasitology: Drug and Drug Resistance. 9:100-111. https://doi.org/10.1016/j.ijpddr.2019.03.001.
DOI: https://doi.org/10.1016/j.ijpddr.2019.03.001

Interpretive Summary: The southern cattle fever tick (SCFT), scientifically known as Rhipicephalus microplus, is a common pest that parasitizes livestock in tropical and subtropical areas of our planet and is considered the most economically important tick species having a negative impact on beef and milk production systems. Keeping the SCFT eradicated from the United States depends on the quarantine of infested premises and the systematic treatment of cattle with acaricides, that is pesticides killing ticks, to suppress tick populations. Acaricide resistance has a genetic basis and its development among SCFT populations is a risk associated with the intense use of acaricides to treat infestations in livestock herds. Surveillance for acaricide resistance is critical to design strategies that mitigate risks for its development and spread. The development of a DNA-based test to identify mutations in the SCFT genome associated with resistance to pyrethroids, which is a class of pesticidal compounds commonly used for their acaricidal properties, is reported herein. SCFT that are resistant to pyrethroids can be identified rapidly with this molecular test. Knowledge of these mutations associated with resistance can inform decisions to avoid the inefficient use of pyrethroids, and can be applied to promote more sustainable approaches to manage SCFT infestations.

Technical Abstract: The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is the most economically important ecto- parasite of cattle worldwide. A limitation for sustainable control and eradication is the emergence of acaricide resistance among tick populations. Molecular diagnostic tools offer the opportunity to detect resistance rapidly, which can be complemented with confirmatory bioassays with larvae and adult ticks that are more resource and time consuming to generate. Synthetic pyrethroid resistance is one of the most prevalent and well-studied forms of resistance in arthropods, being linked with target site alterations in the sodium ion channel gene. Here, we report research on a novel molecular method to detect mutations in the para-sodium channel gene of R. microplus associated with acaricide resistance that is based on quantitative PCR high-resolution melt (HRM) analysis. Genomic DNA fragments of domains II and III of the para-sodium channel gene were amplified by real-time PCR in the presence of EVA®Green dye to test resistant and susceptible reference ticks from the U.S., Brazil, and Mexico. Larval packet tests with discriminating doses and a modified lethal time analysis were performed to confirm resistance to permethrin, cypermethrin, deltamethrin, and flumethrin in laboratory strains. Tick spe- cimens collected from cattle that were inspected at the United States Port-of-Entry at the Texas-Mexico border were also genotyped. Previously described mutations associated with pyrethroid resistance (T170C, C190A, G184C, and T2134A) were successfully detected by qPCR-HRM in different genotypes and confirmed by se- quencing. A novel non-synonymous SNP located at domain III (C2136A) and the G215T mutation in domain II, previously described only in Asian R. microplus and R. australis, were also detected with the HRM and confirmed by sequencing. This technique could be adapted for high-throughput screening, detection, and discovery of allele-specific mutations in cattle tick outbreak populations to inform eradication strategies in the USA. This knowledge could also be applied to integrated control programs in other parts of the world where R. microplus is endemic and where similar SNPs have been identified associated with pyrethroid resistance. This study high- lights the existence of several mutations in the para-sodium channel gene in different combinations in field populations of R. microplus from Mexico.