Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico
Nathan E. StonePia U. OlafsonRonald B. DaveyGreta BuckmeierDeanna BodineLindsay C. Sidak‐LoftisJ GilesRoberta DuhaimeRobert J. MillerJuan MosquedaGlen A. ScolesDavid M. WagnerJoseph D. Busch
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Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.Keywords:
Knockdown resistance
Rhipicephalus microplus
SNP
Rhipicephalus
Parasitology
Background Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. Methods and Findings Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500–1000 m. Conclusions The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to cause serious problems in the control of dengue fever in Vietnam.
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Rhipicephalus (Boophilus) microplus is an economically significant pest of livestock in India. Allele-specific polymerase chain reaction (PCR) was employed to detect Phe→Ile substitution in the sodium channel gene in different populations of Rhipicephalus microplus collected from foothills (Kashipur, Pantnagar, Nagla and Dehradun) and high-altitude areas (Pithoragarh, Almora and New Tehri) of Uttarakhand state in northern India. The allele-specific PCR assay revealed 85% larvae to be homozygous susceptible, 3% to be homozygous resistant and 12% to be heterozygous. Homozygous resistant genotypes were recorded in two tick populations (Kashipur, 13% and Pantnagar, 4%); heterozygous genotypes in four tick populations (Kashipur, 45%; Pantnagar, 17%; Almora, 14%; and New Tehri, 3%) and 100% homozygous susceptible genotypic frequencies in three tick populations (Nagla, Dehradun and Pithoragarh). The highest rate of mutant allele (R) occurred in tick populations of Kashipur (35%) (p < 0.05), followed by Pantnagar (13%), Almora (7%) and New Tehri (1%). The results of the study suggested that frequent monitoring of the level of pyrethroid resistance was required to formulate any control strategy against ticks and hence extend the life of the existing acaricide(s).
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Pyrethroid insecticides stabilize the open state of insect sodium channels. Previous mutational, electrophysiological, and computational analyses led to the development of homology models predicting two pyrethroid receptor sites, PyR1 and PyR2. Many of the naturally occurring sodium channel mutations, which confer knockdown resistance (kdr) to pyrethroids, are located within or close to these receptor sites, indicating that these mutations impair pyrethroid binding. However, the mechanism of the state-dependent action of pyrethroids and the mechanisms by which kdr mutations beyond the receptor sites confer resistance remain unclear. Recent advances in protein structure prediction using the AlphaFold2 (AF2) neural network allowed us to generate a new model of the mosquito sodium channel AaNav1-1, with the activated voltage-sensing domains (VSMs) and the presumably inactivated pore domain (PM). We further employed Monte Carlo energy minimizations to open PM and deactivate VSM-I and VSM-II to generate additional models. The docking of a Type II pyrethroid deltamethrin in the models predicted its interactions with many known pyrethroid-sensing residues in the PyR1 and PyR2 sites and revealed ligand-channel interactions that stabilized the open PM and activated VSMs. Our study confirms the predicted two pyrethroid receptor sites, explains the state-dependent action of pyrethroids, and proposes the mechanisms of the allosteric effects of various kdr mutations on pyrethroid action. The AF2-based models may assist in the structure-based design of new insecticides.
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Abstract Target-site mutations and changes in insect metabolism or behavior are common mechanisms in insecticide-resistant insects. The co-occurrence of such mechanisms in a pest strain is a prominent threat to their management, particularly when alternative compounds are scarce. Pyrethroid resistance among stored grain weevils (i.e., Sitophilus spp.) is an example of a long-standing concern, for which reports of resistance generally focus on a single mechanism in a single species. Here, we investigated pyrethroid resistance in maize and rice weevils (i.e., Sitophilus zeamais and S . oryzae ), exploring potential knockdown resistance ( kdr ) mutations in their sodium channels (primary site for pyrethroid actions) and potential changes in their detoxification and walking processes. Resistance in pyrethroid-resistant rice weevils was associated with the combination of a kdr mutation (L1014F) and increases in walking and detoxification activities, while another kdr mutation (T929I) combined with increases in walking activity were the primary pyrethroid resistance mechanisms in maize weevils. Our results suggest that the selection of pyrethroid-resistant individuals in these weevil species may result from multiple and differential mechanisms because the L1014F mutation was only detected in Latin American rice weevils (e.g., Brazil, Argentina and Uruguay), not in Australian and Turkish rice weevils or Brazilian maize weevils.
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Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr), in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel.
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The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2-15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82-100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected.
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Background Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. Methodology/Principal findings V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. Conclusions/Significance Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.
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Abstract Knockdown resistance to DDT and the pyrethrins was first described in 1951 in the housefly ( Musca domestica L.). This trait, which confers reduced neuronal sensitivity to these insecticides, was subsequently shown to confer cross‐resistance to all synthetic pyrethroid insecticides that have been examined to date. As a consequence, the worldwide commercial development of pyrethroids as a major insecticide class over the past three decades has required constant awareness that pyrethroid overuse has the potential to reselect this powerful resistance mechanism in populations that previously were resistant to DDT. Demonstration of tight genetic linkage between knockdown resistance and the housefly gene encoding voltage‐sensitive sodium channels spurred efforts to identify gene mutations associated with knockdown resistance and understand how these mutations confer a reduction in the sensitivity of the pyrethroid target site. This paper summarizes progress in understanding pyrethroid resistance at the molecular level, with particular emphasis on studies in the housefly. Copyright © 2008 Society of Chemical Industry
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Rhipicephalus microplus is a hard tick that has a major impact on cattle health in tropical and subtropical regions because it feeds on cattle and is implicated in the transmission of pathogens that cause diseases such as bovine anaplasmosis and babesiosis. Presently, acaricides are used to control tick infestation but this is becoming increasingly less effective due to the emergence of tick strains that are resistant to one or more classes of acaricides. Anti-tick vaccines are a promising alternative to control tick infestation in cattle. The life-cycle and host preference of R. microplus, however, makes vaccine research in cattle costly and would therefore greatly benefit from an in vitro screening system. To this aim, a stacked 24-well in vitro feeding system was designed in which the blood meal was administered in a chamber on top of the compartment containing the ticks, exploiting their anti-gravitational tendency. Both compartments were separated by a special feeding membrane, which was made by applying a silicone mixture to a gold beater's skin (baudruche membrane) with a paint roller to create a slightly uneven surface of 17–40 μm variable thickness. To further stimulate feeding, the membrane was treated with bovine hair extract and the unit was placed at 37 °C with 90% RH and 5% CO2. Using this set-up with Rhipicephalus australis (formerly Rhipicephalus microplus), a larval engorgement rate of up to 71% could be achieved. The larvae could successfully feed on blood, but also on serum. The latter allows easy screening of the effect of sera that are raised against tick proteins on feeding. As an example, serum from cattle that were vaccinated with the Bm86 midgut protein of R. microplus significantly reduced larval engorgement rates by 42%. The in vitro feeding system's high throughput design and its ability to measure statistically significant anti-tick effects in sera from immunized cattle enables screening of multiple vaccine candidates in a cost-effective manner.
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