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

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

10.21203/rs.2.24147/v1 ◽

2020 ◽

Author(s):

Raquel Cossío-Bayúgar ◽

Estefan Miranda-Miranda ◽

Francisco Martínez Ibañez ◽

Verónica Narváez Padilla ◽

Enrique Reynaud

Keyword(s):

Sodium Channel ◽

Pyrethroid Resistance ◽

Experimental System ◽

Rhipicephalus Microplus ◽

Cattle Tick ◽

Knockdown Resistance ◽

Primary Resistance ◽

Channel Gene ◽

Sodium Channel Gene ◽

Physiological Evidence

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.

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

10.21203/rs.2.24147/v3 ◽

2020 ◽

Author(s):

Raquel Cossío-Bayúgar ◽

Estefan Miranda-Miranda ◽

Francisco Martínez Ibañez ◽

Verónica Narváez Padilla ◽

Enrique Reynaud

Keyword(s):

Sodium Channel ◽

Pyrethroid Resistance ◽

Experimental System ◽

Rhipicephalus Microplus ◽

Susceptible Strain ◽

Cattle Tick ◽

Knockdown Resistance ◽

Channel Gene ◽

Sodium Channel Gene ◽

Physiological Evidence

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.

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Characterization of the M918T sodium channel gene mutation associated with strong resistance to pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer)

Bulletin of Entomological Research ◽

10.1017/s0007485307005524 ◽

2007 ◽

Vol 98 (2) ◽

pp. 183-191 ◽

Cited By ~ 30

Author(s):

I. Eleftherianos ◽

S.P. Foster ◽

M.S. Williamson ◽

I. Denholm

Keyword(s):

Sodium Channel ◽

Myzus Persicae ◽

Pyrethroid Resistance ◽

Type I ◽

Pyrethroid Insecticides ◽

Potato Aphid ◽

Channel Gene ◽

Sodium Channel Gene ◽

Wide Range ◽

Peach Potato Aphid

AbstractRecent advances in the characterisation of insect sodium channel gene sequences have identified a small number of point mutations within the channel protein that are implicated in conferring target-site resistance to pyrethroid insecticides (so-called knockdown resistance or kdr). The L1014F (leucine-to-phenylalanine) mutation located in the centre of segment 6 of the domain II region (IIS6) of the sodium channel (the so-called kdr trait) has been detected in the peach-potato aphid, Myzus persicae (Sulzer), and is considered to be the primary cause of pyrethroid resistance in this species. Here we report on the characterisation of a second mutation, M918T (methione-to-threonine), within the nearby IIS4–S5 intracellular linker (the so-called super-kdr trait) in a field clone also possessing L1014F, with both mutations present in heterozygous form. The resistance phenotype of M. persicae clones possessing various combinations of L1014F and M918T to a wide range of pyrethroids (both Type I and II) was assessed in leaf-dip bioassays and to lambda-cyhalothrin applied at up to ten times the recommended field rate as foliar sprays to aphids feeding on whole plants. Bioassay results demonstrated that presence of both mutations was associated with extreme resistance to all the pyrethroids tested relative to aphids lacking the mutations. Furthermore, this resistance well exceeded that shown by aphids that were homozygous for L1014F but lacking M918T. However, pre-treatment with piperonyl butoxide in the leaf-dip bioassays failed to suppress pyrethroid resistance in aphids carrying one or both of the mutations. The relevance of these findings for monitoring and managing pyrethroid resistance in M. persicae populations in the field is discussed.

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