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Abstract Background Knockdown resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of non-synonymous mutations in the voltage-gated sodium channel (vgsc) gene, and these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1016I and F1534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. Methods A multiplex PCR was developed to detect the V410L, V1016I and F1534C mutations in Ae. aegypti. The validation of the technique was carried out by DNA sequencing using field populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. Results The standardized protocol for multiplex end-point PCR was highly effective in detecting 15 genotypes considering the three mutations V410L, V1106I and F1534C, in 12 field populations of Ae. aegypti from Mexico. A complete concordance with AS-PCR and DNA sequencing was found for the simultaneous detection of the three kdr mutations. Conclusions Our diagnostic method is highly effective for the simultaneous detection of V410L, V1016I and F1534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

Abstract Background Insecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide’s target site or increased activity of detoxification enzymes. Methods We determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and β-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification. Results There was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5 vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3–3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5 vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed. Conclusions The high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes.

Background: Knockdown resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of non-synonymous mutations in the voltage-gated sodium channel (vgsc) gene, and these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1016I and F1534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. Methods: A multiplex PCR was developed to detect the V410L, V1016I and F1534C mutations in Ae. aegypti. The validation of the technique was carried out by DNA sequencing using field populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. Results: The standardized protocol for multiplex end-point PCR was highly effective in detecting 15 genotypes considering the three mutations V410L, V1106I and F1534C, in 12 field populations of Ae. aegypti from Mexico. A complete concordance with AS-PCR and DNA sequencing was found for the simultaneous detection of the three kdr mutations. Conclusions: Our diagnostic method is highly effective for the simultaneous detection of V410L, V1016I and F1534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti. [ABSTRACT FROM AUTHOR]

BACKGROUND Recent outbreaks of dengue and Zika have emphasized the importance to effectively control Aedes aegypti, which vectors the viruses causing these diseases. Pyrethroid insecticides are primarily used to control adult A. aegypti, especially during disease outbreaks. However, pyrethroid resistance in A. aegypti is an increasing problem. Mutations in the voltage‐sensitive sodium channel (Vssc) are a common mechanism of pyrethroid resistance. The F1534C mutation is common and distributed globally in A. aegypti populations, but previous studies disagree about the role of this mutation in conferring resistance to pyrethroid insecticides. RESULTS: We isolated a congenic strain (1534C:ROCK) which was closely related to a susceptible strain Rockefeller (ROCK), but was homozygous for the 1534C Vssc allele. We determined resistance levels against eight insecticides that target the VSSC: six pyrethroids, DDT and DCJW (the bioactivated metabolite of indoxacarb). The resistance levels ranged from 7‐ to 16‐fold, and resistance was inherited as an incompletely recessive trait. We also found a novel 367I+1520I+1534C allele, in addition to the 1534C and 1520I+1534C alleles, in mosquitoes from Thailand. The T1520I mutation did not increase pyrethroid resistance beyond what was conferred by the F1534C mutation alone. CONCLUSION: The F1534C Vssc mutation is common in A. aegypti populations and confers 7‐ to 16‐fold resistance to pyrethroids, DDT, and DCJW in Aedes aegypti. These resistance levels are considerably less than previously reported for the S989P+V1016G mutations. Our results provide useful information for resistance management, specifically the levels of resistance conferred by the most common Vssc mutation in A. aegypti. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Background: Insecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide's target site or increased activity of detoxification enzymes. Methods: We determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and β-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification. Results: There was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3–3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed. Conclusions: The high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes. [ABSTRACT FROM AUTHOR]

BACKGROUND: Knockdown resistance is conferred primarily by non‐synonymous mutations that reduce pyrethroids binding to voltage‐gated sodium channels. In 2014, kdr mutation V1016I in Aedes aegypti populations resistant to pyrethroids was reported for the first time in Colombiα, in 2016 another kdr mutation, F1534C, and in 2018 the mutation V419L. Nine populations of A. aegypti, previously characterized as being resistant to λ‐cyhalothrin, deltamethrin, cyfluthrin and permethrin, were used for this study. Genomic DNA was used to determine genotypes by allele‐specific PCR for mutations V1016I and F1534C, and to determine their association with pyrethroid resistance. RESULTS: All the populations analyzed showed both mutations, with allelic frequencies of 0.07–0.35 for I1016 and 0.47–0.88 for C1534. A percentage of co‐occurrence of mutant homozygotes I1016/C1534 of 5.3% was detected. A significant positive correlation was found between the frequency of the genotype I1016 and the resistance to permethrin, λ‐cyhalothrin and cyfluthrin, but not to deltamethrin; on the other hand, the correlation was not significant for the C1534 genotype and the four pyrethroids evaluated. No significant correlation was found between the frequencies of the mutations V1016I and F1534C. CONCLUSIONS: Both mutations V1016I and F1534C are present in A. aegypti populations of the Colombian Caribbean, and although the frequency of F1534C exceeds V1016I, the latter was correlated to resistance to pyrethroid insecticides. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the sample processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection. [Display omitted] • Development of rapid low-resource, kdr detection test. • Rapid sample processing of mosquitoes for kdr detection. • Test format using Recombinase Polymerase Amplification/Lateral flow dipsticks. • Achieved F1534C kdr detection in Aedes aegypti in 25 min. • Rapid F1534C test demonstrated 100% concordance with AS-qPCR melt curve analysis. [ABSTRACT FROM AUTHOR]