Multiple single nucleotide polymorphisms in the para-sodium channel gene lead to pyrethroid resistance in Rhipicephalus microplus ticks from North America

Rhipicephalus microplus is an invasive tick that vectors Babesia bovis and B. bigemina, the protozoans that cause cattle fever. Acaricide resistant R. microplus populations are a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically based on mutations in domains I, II, and III of voltage-gated sodium channels. We investigated permethrin resistance in ticks collected from Texas (n=1,457) and Mexico (n=31) by estimating resistance levels in tick collections using larval packet discriminating dose (DD) assays. We also genotyped field collected ticks at three single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that are associated with resistance. We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. Although we expected to observe a domain III SNP (T2134A) known to occur in North America, we only found it in three U.S. collections. A much more common SNP (19 of 21 U.S. field collections) was the C190A domain II mutation, which has never before been described in North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexican field collections that is associated with super-knockdown resistance in insects. We found a significant correlation (r=0.81) between the proportion of individuals from field collections that carried any two resistant SNPs and percent survivorship of their F1 larvae in DD assays. Our findings suggest that multiple mutations in the para-sodium channel gene can independently lead to pyrethroid resistance in R. microplus ticks.