Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus
Authors: Cossío-Bayúgar Raquel , Miranda-Miranda Estefan , Ibañez Francisco Martínez , Padilla Verónica Narváez , Reynaud Enrique 
Published: June 2020

Abstract
Background: Acaricide resistance is a central problem for the control of the cattle tick Rhipicephalus microplus. Genetic changes that confer resistance get fixed in the population, however, the physiological effects and phenotypes of these mutations are not always well understood or characterized. Single Nucleotide Polymorphisms (SNPs) in the para-sodium channel gene that confer cypermethrin knockdown resistance (kdr) were identified in several arthropods, and homologous mutations have been reported in R. microplus populations. To our knowledge, R. microplus populations that have these homologous SNPs have been associated and correlated with pyrethroid resistance but there is no direct physiological evidence that their presence does confer kdr in this organism. Methods: Resistance profiles from resistant and susceptible strains used in this work were obtained using the larval packet discriminating dose assay. The relevant genomic regions of the para-sodium channel of these strains were amplified using standard PCR; SNPs were detected by sequencing the corresponding amplicons. Ovary response to cypermethrin exposure/treatment was evaluated using videometrical analysis.Results: In this work we present historical evidence that the pyrethroid resistance trait is stable in a resistant reference strain after many years without selection, thus suggesting that the primary resistance mechanism is caused by mutations fixed in the population. In our experimental system, the mechanism that allows the maintenance of the contraction of the ovary after treatment with pyrethroids, is likely to be mediated by a change in the structure of the presynaptic para-sodium channel. We found that the resistant strain has the G184C, the C190A and the T2134A mutations in the para-sodium channel gene. SNPs G184C and T2134A have been reported to be genetically linked in resistant populations and are always found together. These mutations were confirmed to be absent in the susceptible strain used as control. Finally, using videometric analysis, we demonstrate that cypermethrin blocks ovary contraction in cypermethrin-susceptible ticks. We also show that ovaries from populations that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block ovary contraction in the susceptible strain. The configuration of the experimental system excludes a xenobiotic detoxification mechanism. Conclusions: This is the first report that presents physiological evidence that the presence of the G184C, the C190A, and the T2134A mutations in the para-sodium channel correlates with the ability of maintaining muscle contractility in R. microplus when exposed to cypermethrin. These results confirm that these SNPs may confer cypermethrin resistance in this organism by avoiding presynaptic blockage that in turn causes flaccid muscle paralysis that is characteristic of this acaricide. This work also demonstrates that the videometric assay that we previously validated can be used to detect more rapidly than other assays that involve larval mortality kdr-like cypermethrin resistant tick strains, because adult preengorged females can be directly assayed after they are collected on the field without waiting until eggs are laid and the larvae eclose.