QTL mapping of genome regions controlling temephos resistance in larvae of the mosquito aedes aegypti

Introduction: The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations. Methodology/Principal Findings: Quantitative Trait Loci (QTL) controlling temephos survival in Ae. aegypti larvae were mapped in a pair of F3 advanced intercross lines arising from temephos resistant parents from Solidaridad, México and temephos susceptible parents from Iquitos, Peru. Two sets of 200 F3 larvae were exposed to a discriminating dose of temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad6Iquitos (SLD6Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of México. Conclusions/Significance: Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome

Resistance to permethrin in Aedes aegypti (L.) in Northern México.

Diagnostic dose (DD) of permethrin was determined in Aedes aegypti (L.), from three states (Coahuila, Sonora, and Tamaulipas) in northern Mexico. After exposing 10 groups of 100 females to the DD obtained and producing 50% mortality, individuals were divided into two categories: survivors and dead. Mosquitoes in each of these categories were dissected to separate the head, thorax, and abdomen. Biochemical tests were done on the head and thorax to determine activity by resistance-related enzymes including: α and β-esterases, mixed-function oxidases (MFOs), glutathione-S-transferase (GST) and insensitive acetyl cholinesterase (iAChE). Results were compared with those for a susceptible strain of Ae. aegypti from New Orleans. A population from Coahuila showed iAChE as the only enzyme activity that surpassed the threshold established with the susceptible strain, however, this mechanism is not associated with resistance to pyrethroids, but rather with resistance to organophosphate and carbamate insecticides. For the populations from Tamaulipas, none of the mechanisms studied were important in conferring resistance to permethrin. MFOs were present at elevated levels of activity, appearing as the main detoxifying mechanism, in the population from Sonoita, Sonora state

Physical positions of markers and QTL affecting hours until death (H) and survival (D).

<p>Physical markers correspond to the system described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003177#pntd.0003177-Timoshevskiy2" target="_blank">[52]</a>.</p

Proportion surviving among larvae plotted against the three possible genotypes at each of the markers found to be significantly associated with the DOA phenotype (Table 2).

<p>Iq/Iq = both alleles inherited from the Iquitos parent, Iq/SLD = heterozygous for alleles inherited from both Iquitos and Solidaridad parents, SLD/SLD = both alleles inherited from the Solidaridad parent. Error bars are Bayes 95% highest density intervals (HDI), credible differences exist when the 95% HDI fail to overlap. For the SLD×Iq cross, A) shows the relationship among genotypes at four loci on chromosome 1 and proportion surviving, B) is the relationship among genotypes at four loci on chromosome 2 and proportion surviving, and C) indicates the relationship among genotypes at four loci on chromosome 3 and proportion surviving. For the Iq×SLD cross, D) shows the relationship among genotypes at one locus on chromosome 1, two loci on chromosome 2 and 3 loci on chromosome 3 and proportion surviving.</p

Analysis of Variance (ANOVA) of the hours until death among the three genotype classes.

<p>The means among the three classes are listed as are the probabilities estimated in the ANOVA. Probabilities from the ANOVA are listed in the sixth column. The last column indicates whether the allele inherited from the SLD F_S3 P₁ parent conferred resistance while the allele inherited from the Iq P₁ parent were susceptible.</p><p>Analysis of Variance (ANOVA) of the hours until death among the three genotype classes.</p

Hours until death among larvae plotted against the three possible genotypes at each of the markers found to be significantly associated with the HTD phenotype (Table 1).

<p>Iq/Iq = both alleles inherited from the Iquitos parent, Iq/SLD = heterozygous for alleles inherited from both Iquitos and Solidaridad parents, SLD/SLD = both alleles inherited from the Solidaridad parent. The second column corresponds to chromosomes 1, 2, and 3 in the Iq×SLD cross. Error bars are Bayes 95% highest density intervals (HDI), credible differences exist when the 95% HDI fail to overlap. For the SLD×Iq cross, A) shows the relationship among genotypes at six loci on chromosome 1 and HTD, B) is the relationship among genotypes at four loci on chromosome 2 and HTD, and C) indicates the relationship among genotypes at four loci on chromosome 3 and HTD. For the Iq×SLD cross, D) shows the relationship among genotypes at three loci on chromosome 1 and HTD, E) is the relationship among genotypes at four loci on chromosome 2 and HTD, and F) indicates the relationship among genotypes at five loci on chromosome 3 and HTD.</p

Fisher's Exact Test (FET) of proportions surviving past 16 hours among the three genotypes.

<p>The means in each of the three genotypes are listed. Probabilities from the Exact Test are listed in the sixth column. The last column indicates whether the allele was inherited from the SLD F_S3 P₁ parent conferred resistance while the allele inherited from the Iq P₁ parent was associated with susceptibility.</p><p>Fisher's Exact Test (FET) of proportions surviving past 16 hours among the three genotypes.</p

Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México

Background: Aedes aegypti, the ‘yellow fever mosquito’, is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5–10 mg of permethrin in bottle bioassays. Methods and Findings: A total of 81 field collections containing 3,951 Ae. aegypti were made throughout Me´xico from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay.Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990’s to 2006–2009 in several states including Nuevo Leo´n in the north, Veracruz on the central Atlantic coast, and Yucata´n, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003–2004 the overall frequency of Ile1,016 had increased ,100-fold to 2.7% (60.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (61.15% CI95; n = 473). This was followed in 2007–2009 by a sudden jump in Ile1,016 frequency to 33.2% (61.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007–2008 collections, which ranged from 45.7% (62.00% CI95) in the state of Veracruz to 51.2% (64.36% CI95) in the Yucata´n peninsula and 14.5% (62.23% CI95) in and around Tapachula in the state of Chiapas. Spatial heterogeneity was also evident at smaller geographic scales. For example within the city of Chetumal, Quintana Roo, Ile1,016 frequencies varied from 38.3%–88.3%. A linear regression analysis based on seven collections from 2007 revealed that the frequency of Ile1,016 homozygotes accurately predicted knockdown rate for mosquitoes exposed to permethrin in a bioassay (R2 = 0.98). Conclusions: We have recorded a dramatic increase in the frequency of the Ile1,016 mutation in the voltage-gated sodium channel gene of Ae. aegypti in Me´xico from 1996 to 2009. This may be related to heavy use of permethrin-based insecticides in mosquito control programs. Spatial heterogeneity in Ile1,016 frequencies in 2007 and 2008 collections may reflect differences in selection pressure or in the initial frequency of Ile1,016. The rapid recent increase in Ile1,016 is predicted by a simple model of positive directional selection on a recessive allele. Unfortunately this model also predicts rapid fixation of Ile1,016 unless there is negative fitness associated with Ile1,016 in the absence of permethrin. If so, then spatial refugia of susceptible Ae. aegypti or rotational schedules of different classes of adulticides could be established to slow or prevent fixation of Ile1,016