Parallel evolution of vgsc mutations at domains IS6, IIS6 and IIIS6 in pyrethroid resistant Aedes aegypti from Mexico

Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and efective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control of Ae. aegypti is challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid spacesprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements in vgsc have been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years across Ae. aegypti populations. Recently, a novel replacement V410L was identifed in Brazil and its efect on vgsc was confrmed by electrophysiology. Herein, we screened V410L in 25 Ae. aegypti historical collections from Mexico, the frst heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specifc vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management

Evolution of mutations associated with pyrethroid resistance and the reversal of resistance in Aedes aegypti

2019 Spring.Includes bibliographical references.Worldwide vector control has been relying heavily on pyrethroid insecticides to reduce Aedes aegypti Linnaeus populations. Pyrethroids are relatively inexpensive, have low vertebrate toxicity, and have been efficient in reducing mosquito populations. Constant use of pyrethroid insecticides, however, has driven mosquito populations to develop resistance over time. In this dissertation, we have tracked the evolution of three mutations in the voltage gated sodium channel (vgsc) that are associated with pyrethroid resistance Aedes aegypti populations in Mexican. These are 410, 1,016 and 1,534, corresponding to the position of amino acid substitutions in the vgsc. A valine at locus 410 (V410) confers susceptibility, while leucine (L410) confers resistance. A valine at locus 1,016 (V1,016) confers susceptibility, while isoleucine (I1,016) confers resistance. A phenylalanine at locus 1,534 (F1,534) confers susceptibility, while cysteine (C1,534) confers resistance. We performed a linkage disequilibrium analysis of the three mutations in Mexican collections from 2000–2016. In the first study, a linkage disequilibrium analysis was performed on I1,016 and C1,534 in Ae. aegypti collected in Mexico from 2000–2012, to test, in natural populations, for statistical associations between segment six (S6) in domains II and III of the vgsc. We estimated the frequency of the four di-locus haplotypes in 1,016 and 1,534: V1,016/F1,534 (susceptible), V1,016/C1,534, I1,016/F1,534, and I1,016/C1,534 (resistant). The susceptible V1,016/F1,534 di-locus haplotype went from near fixation to extinction, and the resistant I1,016/C1,534 di-locus haplotype increased in all collections from a frequency near zero, to frequencies ranging from 0.5–0.9. The V1,016/C1,534 di-locus haplotype frequency increased in all collections until 2008. After this year, the frequencies in two collections began to decrease, likely due to the fact that the I1,016/C1,534 di-locus haplotype frequency increased in all collections. However, the I1,016/F1,534 di-locus haplotype was rarely detected; for instance, it reached a frequency of only 0.09 in one collection and subsequently declined. Pyrethroid resistance in the vgsc gene appears to require the sequential evolution of two mutations. The I1,016/F1,534 di-locus haplotype appears to have low fitness, suggesting that I1,016 was unlikely to have evolved independently. Instead the C1,534 mutation evolved first but conferred only a low level of resistance. I1,016 in S6 of domain II then arose from the V1,016/C1,534 haplotype and was rapidly selected because double mutations confer higher pyrethroid resistance. This pattern suggests that knowledge of the frequencies of mutations in both S6 in domains II and III are important to predict the potential of a population to evolve kdr. Susceptible populations with high V1,016/C1,534 frequencies are at high risk for kdr evolution, whereas susceptible populations without either mutation are less likely to evolve high levels of kdr, at least over a 10 year period. In the second chapter we describe a novel replacement V410L that was initially detected in a pyrethroid resistant insectary strain from Brazilian Ae. aegypti populations. We screened V410L in 25 Ae. aegypti historical collections from Mexico. The first heterozygote appeared in 2002, and frequencies have increased in the last 16 years, along with I1,016 and C1,534. L410 showed a strong association between 1,534 and 1,016 mutations. Individuals with the triple homozygote resistant genotype had higher survival after pyrethroid exposure, 96% of the alive individuals had the triple homozygote resistant genotype after permethrin and 76% after deltamethrin treatment. The purpose of insecticide resistance management strategies is to minimize the selection for resistance to any one type of insecticide, or to help regain susceptibility in insect populations in which resistance has already arisen. A key component of resistance management assumes that there will be a negative fitness associated with resistance alleles, so that when insecticides are removed, resistance alleles will decline in frequency. In the third chapter we tested for the loss of pyrethroid resistance from eight field populations of Ae. aegypti, (six field collections from or near the city of Merida, and two collections from Tapachula and Acapulco in southern Mexico) to assess variation in the rate of loss of pyrethroid resistance. Collections were maintained for up to eight generations after pyrethroids were discontinued. We recorded changes in the frequencies of two kdr mutations, I1,016 and C1,534, and the analysis of resistance ratios (RR) with permethrin (pyrethroid type 1) and deltamethrin (pyrethroid type 2). In generations F3, F6, and F8, we also evaluated fecundity to test for parallel changes in a fitness trait during the eight generations. This was analyzed because a negative association between resistance and fecundity had previously been described in two studies [1, 2]. We demonstrate that the frequency of the Ae. aegypti pyrethroid resistance alleles I1,016 and C1,534 decline when pyrethroid pressure is removed in the laboratory; however, the pattern of decline is strain dependent. In agreement with earlier studies, fecundity was negatively associated with the frequency of resistance alleles

Coevolution of the Ile1,016 and Cys1,534 Mutations in the Voltage Gated Sodium Channel Gene of Aedes aegypti in Mexico.

BACKGROUND:Worldwide the mosquito Aedes aegypti (L.) is the principal urban vector of dengue viruses. Currently 2.5 billion people are at risk for infection and reduction of Ae. aegypti populations is the most effective means to reduce the risk of transmission. Pyrethroids are used extensively for adult mosquito control, especially during dengue outbreaks. Pyrethroids promote activation and prolong the activation of the voltage gated sodium channel protein (VGSC) by interacting with two distinct pyrethroid receptor sites [1], formed by the interfaces of the transmembrane helix subunit 6 (S6) of domains II and III. Mutations of S6 in domains II and III synergize so that double mutants have higher pyrethroid resistance than mutants in either domain alone. Computer models predict an allosteric interaction between mutations in the two domains. In Ae. aegypti, a Ile1,016 mutation in the S6 of domain II was discovered in 2006 and found to be associated with pyrethroid resistance in field populations in Mexico. In 2010 a second mutation, Cys1,534 in the S6 of domain III was discovered and also found to be associated with pyrethroid resistance and correlated with the frequency of Ile1,016. METHODOLOGY/PRINCIPAL FINDINGS:A linkage disequilibrium analysis was performed on Ile1,016 and Cys1,534 in Ae. aegypti collected in Mexico from 2000-2012 to test for statistical associations between S6 in domains II and III in natural populations. We estimated the frequency of the four dilocus haplotypes in 1,016 and 1,534: Val1,016/Phe1,534 (susceptible), Val1,016/Cys1,534, Ile1,016/Phe1,534, and Ile1,016/Cys1,534 (resistant). The susceptible Val1,016/Phe1,534 haplotype went from near fixation to extinction and the resistant Ile1,016/Cys1,534 haplotype increased in all collections from a frequency close to zero to frequencies ranging from 0.5-0.9. The Val1,016/Cys1,534 haplotype increased in all collections until 2008 after which it began to decline as Ile1,016/Cys1,534 increased. However, the Ile1,016/Phe1,534 haplotype was rarely detected; it reached a frequency of only 0.09 in one collection and subsequently declined. CONCLUSION/SIGNIFICANCE:Pyrethroid resistance in the vgsc gene requires the sequential evolution of two mutations. The Ile1,016/Phe1,534 haplotype appears to have low fitness suggesting that Ile1,016 was unlikely to have evolved independently. Instead the Cys1,534 mutation evolved first but conferred only a low level of resistance. Ile1,016 in S6 of domain II then arose from the Val1,016/Cys1,534 haplotype and was rapidly selected because double mutants confer higher pyrethroid resistance. This pattern suggests that knowledge of the frequencies of mutations in both S6 in domains II and III are important to predict the potential of a population to evolve kdr. Susceptible populations with high Val1,016/Cys1,534 frequencies are at high risk for kdr evolution, whereas susceptible populations without either mutation are less likely to evolve high levels of kdr, at least over a 10 year period

Frequencies of Ile1,016 and Cys1,534 alleles from 1999 to 2012 and their Bayesian 95% HDI.

<p>Frequencies of Ile1,016 and Cys1,534 alleles from 1999 to 2012 and their Bayesian 95% HDI.</p

Two models for the evolution of mutations in subunit 6 of domains II and III.

<p>Model 1 proposes that the 1,532 and 1,016 mutations occurred independently and became <i>cis</i> through crossing over. Model 2 instead proposes that 1,532 mutations occur first because 1,016 mutations confer low fitness. Ile1,016 mutations then arise on a Val1,016/Cys1,534 background.</p

Frequencies of (A) Ile1,016 and (B) Cys1,534 alleles from 2000 to 2012 in cities in Veracruz and Chiapas and their maximum and minimum frequencies among collections in each year.

<p>Frequencies of (A) Ile1,016 and (B) Cys1,534 alleles from 2000 to 2012 in cities in Veracruz and Chiapas and their maximum and minimum frequencies among collections in each year.</p

Locations of <i>Aedes aegypti</i> collections used in the present study.

<p>Locations of <i>Aedes aegypti</i> collections used in the present study.</p

Frequencies of the four potential dilocus haplotypes plotted by year.

<p>A) Frequency of the susceptible Val1,016/ Phe1,534 (VF) haplotype, B) Frequency of the Val1,016/Cys1,534 (VC) haplotype, C) Frequency of the Ile1,016/Phe1,534 haplotype and D) Frequency of the resistant Ile1,016/ Cys1,534 (IC) haplotype.</p

Pyrethroid susceptibility reversal in Aedes aegypti: A longitudinal study in Tapachula, Mexico.

Pyrethroid resistance in Aedes aegypti has become widespread after almost two decades of frequent applications to reduce the transmission of mosquito-borne diseases. Because few insecticide classes are available for public health use, insecticide resistance management (IRM) is proposed as a strategy to retain their use. A key hypothesis of IRM assumes that negative fitness is associated with resistance, and when insecticides are removed from use, susceptibility is restored. In Tapachula, Mexico, pyrethroids (PYRs) were used exclusively by dengue control programs for 15 years, thereby contributing to selection for high PYR resistance in mosquitoes and failure in dengue control. In 2013, PYRs were replaced by organophosphates-insecticides from a class with a different mode of action. To test the hypothesis that PYR resistance is reversed in the absence of PYRs, we monitored Ae. aegypti's PYR resistance from 2016 to 2021 in Tapachula. We observed significant declining rates in the lethal concentration 50 (LC50), for permethrin and deltamethrin. For each month following the discontinuation of PYR use by vector control programs, we observed increases in the odds of mosquitoes dying by 1.5% and 8.4% for permethrin and deltamethrin, respectively. Also, knockdown-resistance mutations (kdr) in the voltage-gated sodium channel explained the variation in the permethrin LC50s, whereas variation in the deltamethrin LC50s was only explained by time. This trend was rapidly offset by application of a mixture of neonicotinoid and PYRs by vector control programs. Our results suggest that IRM strategies can be used to reverse PYR resistance in Ae. aegypti; however, long-term commitment by operational and community programs will be required for success