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

Stone, Nathan E.; Olafson, Pia U.; Davey, Ronald B.; Buckmeier, Greta; Bodine, Deanna; Sidak-Loftis, Lindsay C; Giles, J. R.; Duhaime, Roberta; Miller, Robert J.H.; Mosqueda, Juan; Scoles, Glen A.; Wagner, David M.; Busch, Joseph D.

doi:10.1186/preaccept-9495120713540941

Cited by 19 publications

(40 citation statements)

References 31 publications

(72 reference statements)

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“…Oligonucleotides for the para-sodium channel relevant domains were synthetized accordingly to the ones reported in (13). PCR was performed using hotstart procedure as follows: 5 minutes at 95°C before adding Taq DNApolymerase (Thermo Fisher Scienti c, Waltham, Massachusetts, United States) using the following ampli cation conditions, 30 seconds denaturation at 95°C, 30 seconds annealing at 59°C and 30 seconds polymerization at 72°C for 35 cycles and a nal extension cycle at 72°C for 7 minutes.…”

Section: Pcr Ampli Cation and Snps Determinationmentioning

confidence: 99%

“…Homologous mutations in R. microplus also correlate with pyrethoid resistance but in this organism, no measurable physiological effect that correlates with the presence of these mutations and resistance has been reported. The arthropod parasodium channel is structured into four homologous repeat domains (I-IV), each having six α-helical transmembrane segments (S1-S6) (11)(12)(13). The reported SNPs that cause amino acid substitutions and correlate with resistance are T170C, G184C, C190A, C190G in the II domain; there is also one characterized SNP (T2134A) that is in the III domain ( Table 1).…”

Section: Introductionmentioning

confidence: 99%

“…The reported SNPs that cause amino acid substitutions and correlate with resistance are T170C, G184C, C190A, C190G in the II domain; there is also one characterized SNP (T2134A) that is in the III domain ( Table 1). The G184C and the T2134A mutations occur only in resistant populations and are always genetically linked (13)(14)(15)(16)(17). In this study we analyzed an acaricide resistant reference tick strain that was originally isolated from heavily infested cattle with documented records of an intensive pryrethroid control background.…”

Section: Introductionmentioning

confidence: 99%

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Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

Cossío-Bayúgar

Miranda-Miranda

Ibañez³

et al. 2020

Preprint

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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.

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Section: Pcr Ampli Cation and Snps Determinationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

Cossío-Bayúgar

Miranda-Miranda

Ibañez³

et al. 2020

Preprint

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“…Since the second half of the 1990s acaricide resistance has been observed and since then it is very common against all commercially available products, resulting in serious economic losses [5,6]. Acaricide resistance is caused by several intrinsic and operational factors [7,8].…”

Section: Introductionmentioning

confidence: 99%

The current status of resistance to alpha-cypermethrin, ivermectin, and amitraz of the cattle tick (Rhipicephalus microplus) in Ecuador

et al. 2017

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Rhipicephalus microplus is widely distributed in tropical and subtropical areas of the world where livestock is a principal activity with great veterinary and economic importance. In Ecuador, this hematophagous ectoparasite has been observed between 0 and 2600 masl. One of the main tick control measures is the use of acaricides, which have been indiscriminately used worldwide and in Ecuador. In this country, no studies on acaricide resistance in Rhipicephalus microplus have been published. The current study aims to characterise the level of resistance of R. microplus against three main acaricides commonly used in Ecuador i.e. amitraz, alpha-cypermethrin and ivermectin to determine the level and pattern of dose-responses for R. microplus in 12 field populations (farms). The level of acaricide resistance was evaluated using three different bioassays: adult immersion test (AIT), larval package test (LPT) and larval immersion test (LIT), as recommended by the FAO. The predictive dose-responses were analysed by binomial logistics regression of the larval survival rate (resistance). In general, we found resistance of 67% for amitraz; 50% for alpha-cypermethrin and from 25 to 42% for ivermectin in the twelve field populations analysed. Resistance levels were studied in larval and adult bioassays, respectively, which were slightly modified for this study. For larval bioassays based on corrected mortality i.e. high (above 51%), medium (21–50%) and low (11–20%) resistance, percentages less than 10% were considered as susceptible. For the adult test, two resistance levels were used i.e. high (more than 76%) and medium (51 to 75%) resistance. Percentages lower than 50% were considered as susceptible. In this context, for larval bioassays, amitraz showed 21%, 38% and 8% for high, medium and low resistance, respectively. Alpha-cypermethrin presented 8%, 4 and 38% for high, medium and low resistance, respectively. Ivermectin presented 8%, 25% and 8% for high, medium and low resistance, respectively. For adult tests with amitraz 50% and 17% of the field populations showed average and high resistance, with evidences of average resistance to alpha-cypermethrin in 50% of the samples and average resistance against ivermectin in 25% of the farms. No statistical difference amongst the three bioassays was found and acaricide resistance was confirmed by logistic regression analysis; hence resistance (dose-responses) in each field populations differed, depending on the choice of the acaricide, frequent usage, frequency of treatment and farm management. The effective estimated dose needed to eliminate 99% of ticks is higher than dose recommended by the manufacturer. In conclusion, amitraz showed the highest resistance followed by ivermectin and alpha-cypermethrin and reveals differences on resistance in each individual field population. This information is important in order to establish the monitoring of resistance on each farm individually, contributing to the rational use of acaricides included in an integrated control program for R. m...

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“…El uso continuo de acaricidas organofosforados, formamidinas y piretroides ha seleccionado individuos resistentes a ellos, siendo la resistencia a piretroides sintéticos una de las más difundidas en las áreas donde R. microplus está presente (He, Chen, Davey, Ivie, & George, 1999;Miller, Davey, & George, 1999;Villarino, Wagner, & George, 2002;Rosario-Cruz et al, 2005;Jonsson, Cutulle, Corley, & Sedon, 2010;Andreotti et al, 2011;Domingues et al, 2012;Lovis et al, 2012). El blanco de los piretroides sintéticos (PS) son los canales del sodio dependientes del voltaje al impedir que estos se cierren y permitir la entrada continua de iones sodio, manteniendo la transmisión del impulso nervioso y generando un efecto de derribo por parálisis muscular conocido como "knock down" (Soderlund & Knipple, 2003;Hemingway, Hawkes, McCarroll & Ranson, 2004;Rinkevich, Du & Dong, 2013;Stone et al, 2014). El canal del sodio está formado por pequeñas proteínas integrales de membrana cuya estructura primaria la componen cuatro dominios (I-IV) con seis segmentos transmembranales cada uno (S1-S6), todos ellos necesarios para iniciar y propagar el potencial de acción a través de las células nerviosas (Hemingway et al, 2004;Brackenbury & Isom, 2011;Dong et al, 2014;Klafke et al, 2017).…”

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New polymorphism in the sodium channel gene of Rhipicephalus microplus tick (Ixodida: Ixodidae) resistant to pyrethroids

Rivera

Cespedes

Montes

2019

RBT

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Rhipicephalus microplus, garrapata común de los bovinos, transmite patógenos que afectan la producción ganadera en áreas tropicales. Para su control se han utilizado compuestos piretroides cuyo uso inapropiado ha generado resistencia en esta especie de garrapata. El estudio de los mecanismos de resistencia hacia piretroides en R. microplus, ha demostrado la presencia de mutaciones en genes relacionados con su acción, ya sea de enzimas que inactivan el compuesto o de los canales del sodio, sitio blanco del acaricida. En Colombia, la primera fase del presente proyecto encontró una mutación en una esterasa hidrolizante en poblaciones de campo de R. microplus resistentes a piretroides. Como se ha demostrado que la resistencia a químicos en esta garrapata es multigénica, en su segunda fase el proyecto tuvo como objetivo principal detectar un polimorfismo en la posición 2134 del gen del canal del sodio. Esta mutación sustituye una timina por una adenina, T2134A, y ha sido relacionada previamente en otros países con resistencia a piretroides en los individuos portadores. Para ello se colectaron 182 garrapatas R. microplus en dos fincas con historial de resistencia a este tipo de acaricidas y se sometieron a una prueba de inmersión de hembras en una solución de cipermetrina preparada a la dosis recomendada por uso en campo, obteniendo seis individuos susceptibles, 92 resistentes y 84 medianamente resistentes. Se extrajo el ADN de todos los especímenes mediante el método fenol–cloroformo y se proceso por PCR convencional empleando cebadores alelo específicos para amplificar un segmento del dominio IIIS6 del gen del canal del sodio. Los amplicones resultantes se detectaron por electroforesis en geles de agarosa al 2% teñidos con GelRed® y se visualizaron con luz ultravioleta. De igual manera se efectuó el secuenciamiento de 20 muestras obtenidas en los tres fenotipos susceptible, medianamente resistente y resistente y las secuencias resultantes fueron alineadas mediante el programa Mega 7 y comparadas con secuencias almacenadas en el GenBank®. Los análisis mostraron que la población de R. microplus estudiada no presentaba la mutación esperada sino una mutación diferente en el sitio 2134. En el ADN de garrapatas resistentes y medianamente resistentes se observó la sustitución de una timina por una citosina, T2134C, que a la traducción genera el cambio de una fenilalanina por una leucina, a diferencia de la mutación previamente identificada, que genera el cambio de una fenilalanina por una isoleucina. Esto demuestra la presencia de un polimorfismo que no ha sido reportado en otros estudios con esta especie de garrapata y que generaría un nuevo marcador molecular de resistencia a piretroides para poblaciones de este acaro. Palabras clave: garrapatas; resistencia a químicos; pesticidas; mutaciones; reacción en cadena de la polimerasa.

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

Cited by 19 publications

References 31 publications

Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

Physiological evidence that three known mutations in the para-sodium channel gene confer cypermethrin knockdown resistance in Rhipicephalus microplus

The current status of resistance to alpha-cypermethrin, ivermectin, and amitraz of the cattle tick (Rhipicephalus microplus) in Ecuador

New polymorphism in the sodium channel gene of Rhipicephalus microplus tick (Ixodida: Ixodidae) resistant to pyrethroids

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