Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus
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. 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 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, ovary contraction resistance to 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 from cypermethrin-susceptible ticks. We also show that ovaries from populations that carry the kdr associated SNPs still contract at cypermethrin concentrations that completely block susceptible ovary contraction. The configuration of the experimental system excludes a xenobiotic detoxification mechanism. Conclusions: This is the first report that presents physiological evidence that the G184C, the C190A, and the T2134A mutations in the para-sodium channel have a phenotype in R. microplus. These results confirm that these SNPs confer cypermethrin resistance in this organism by avoiding presynaptic blockage that in turn cause 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 rapidly detect kdr-like cypermethrin resistant tick strains.