Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico

Table 1 Multiple single nucleotide polymorphisms (SNPs) identified in the exons encoding domains II and III of the para -sodium channel gene in Rhipicephalus microplus from Texas and Mexico

Assay	Locus (Accession #)	Bp	SNP	AA	Reference	Notes
Domain II	C190A [GenBank:KM073929]	190	C/A	Leucine/Isoleucine	Morgan et al. [25]	Three SNPs in priming site (184 bp, 189 bp, 190 bp) may cause this assay to fail
Domain II ( *super-kd* r)	T170C [GenBank:KM073928]	170	T/C	Methionine/Threonine	Williamson et al. [29], Current Study	One SNP in priming site (148 bp) may cause this assay to fail
Domain III	T2134A [GenBank:KM073935]	2134	T/A	Phenylalanine/Isoleucine	He et al. [27]	One SNP in priming site (2130 bp) may cause this assay to fail
na	C148T [GenBank:KM073932]	148	C/T	Leucine/Phenylalanine	Current Study	Present in Rm10, Rm12, Rm13, Rm40, Rm44, Rm47, Rm48, and Rm56 (all susceptible populations)
na	G184C [GenBank:KM073930]	184	G/C	Glycine/Arginine	Current Study	Present in Rm70, Rm71, Rm74, Rm75, Rm76, Rm77, San Felipe, and B&H Ranch collections (occurs only in individuals from resistant populations that also carry one or two copies of the domain III T2134A resistance SNP)
na	C189A [GenBank:KM073931]	189	C/A	Silent	Current Study	Present in Rm64, Rm65, Rm67, and San Felipe collections (occurs with resistant genotypes only)
na	C190G [GenBank:KM073933]	190	C/G	Leucine/Valine	Current Study	Present in Rm77 (present in collections that also contain C190A and T2134A, unsure of its role in resistance)
na	C2130T [GenBank:KM073934]	2130	C/T	Silent	Current Study	Present in Rm05, Rm13, Rm36, Rm38, Rm53, Rm56, Rm65, Rm66, Rm67, Rm68, Rm75, Rm76, and Rm77 (occurs in susceptible and resistant populations)

We designed Melt-MAMA qPCR assays [38] for three SNP positions. Two assays distinguish susceptible versus resistance SNPs previously described in the exons for domain II C190A and domain III T2134A [25],[27]. A third assay was designed for the putative super-kdr site (domain II T170C) observed in our study. Five other SNPs were identified from Sanger sequencing but their contribution to pyrethroid resistance in arthropods is unknown. The “Notes” column provides details on how these five SNPS may interfere with Melt-MAMA assays designed for the three resistance sites. In the case of ambiguous qPCR results, we validated all SNPs via Sanger sequencing. All SNP positions are based on annotations from the R. microplus para-sodium channel gene mRNA sequence (putative sodium channel accession# [GenBank:AF134216.2]).

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