Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance, recovery, and death.

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site-the voltage-gated sodium channel (VGSC)-and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.

Evaluation of commercial aerosol insecticides for control of Aedes aegypti susceptible or resistant to pyrethroids.

OBJECTIVE: To evaluate indoor use of commercial aerosols for dengue vector mosquito control, and estimate the number of treatable houses per can. MATERIALS AND METHODS: Four aerosol products containing combinations of pyrethroids (two containing propoxur and one containing synergists too), were evaluated with mosquitoes in a room of a Tapachulastyle house. Eight cages containing 20 insecticide susceptible or resistant females were hung from tripods, another set was placed in sheltered areas of the room. From the entrance of the room, one of 4-9 concentrations was sprayed for each aerosol, leaving the mosquitoes for 30 min after sprayed. Mortality was recorded after 24 h and lethal concentrations were calculated. RESULTS: Aerosol A had the highest LC50, with 0.308 g for mosquitoes hanging from tripods and 0.453 g for sheltered mosquitoes; followed by aerosols C, D and B, with statistical differences between types of exposure. CONCLUSIONS: Aerosols B-D could spray 20-25 3-room houses (56 m3-room), killing all resistant mosquitoes. Aerosols may become a good tool for indoor mosquito control, if the optimal concentration and correct spray method are used.

[Insecticide resistance in Aedes aegypti and Ae. albopictus (Diptera: Culicidae) populations from Tapachula, Chiapas, Mexico]. / Resistencia a insecticidas en Aedes aegypti y Aedes albopictus (Diptera: Culicidae) de Tapachula, Chiapas, México.

OBJECTIVE: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México. MATERIALS AND METHODS: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively. RESULTS: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species. CONCLUSIONS: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur.

OBJETIVO: Determinar la resistencia a insecticidas en Ae. aegypti y Ae. albopictus de Tapachula, Chiapas, México. MATERIAL Y MÉTODOS: Se utilizaron ovitrampas para obtener huevos de mosquitos Aedes y se realizaron pruebas de susceptibilidad (CDC) y ensayos enzimáticos con la primera generación. RESULTADOS: Aedes aegypti mostró resistencia a deltametrina, permetrina, malatión, clorpirifos, temefos y a bendiocarb (CARB), mientras que Aedes albopictus a malatión y en menor grado a cloripirifos, temefos, permetrina y deltametrina. Ambas especies mostraron altos niveles de enzimas como citocomo P450 y glutatión S-tranferasa, mientras que los niveles de esterasas variaron por especie y sitio muestreado. Se detectó acetilcolinesterasa insensible a insecticidas en ambas especies. CONCLUSIONES: En un hábitat urbano de Tapachula, Chiapas, México donde se aplica control con insecticidas Ae. aegypti y Ae. albopictus sólo son susceptibles al propoxur.

[Indoor thermal fogging of the mixture of flupyradifurone and transflutrin on Aedes aegypti mosquitoes susceptible and resistant to pyrethroids in southern Mexico]. / Nebulización térmica intradomiciliar de la mezcla de flupyradifurona y transflutrina en mosquitos Aedes aegypti susceptibles y resistentes a piretroides en el Sur de México.

OBJECTIVE: To evaluate the efficacy of thermal fogging of a mixture of flupyrafirudone (26.3 g/L) and transfluthrin (52.5 g/L) against dengue, Zika y chikungunya Aedes mosquito vectors. MATERIALS AND METHODS: Groups of 15 caged Ae. aegypti (susceptible and pyrethroid resistant) mosquitoes were placed in living room, kitchen and bedroom inside houses, after which a dose of 2 and 4 mg/m3 of flupyradifurone and transfluthrine, respectively, was applied as thermal fog. After one hour of exposure mosquitoes were transferred to the laboratory and mortality was recorded after 24 h. RESULTS: The mixture killed 97 to 100% of mosquitoes from the strains and the efficacy was similar independently of their place within the premises. CONCLUSIONS: The mixture of flupyrafirudone and transfluthrin applied as thermal fog is a promising tool to control Aedes mosquito populations independently of the pyrethroid-insecticide resistance status.

OBJETIVO: Evaluar la efectividad de la mezcla de flupyradifurona 26.3 g/L y transflutrina 52.5 g/L aplicada como niebla térmica a mosquitos Aedes vectores de virus dengue, Zika y chikungunya. MATERIAL Y MÉTODOS: Se colocaron grupos de 15 mosquitos de Ae. aegypti (susceptibles y resistentes a piretroides) dentro de jaulas, en sala, recámara y cocina. Posteriormente, se aplicó la mezcla de flupyradifurona y transflutrina dentro de las viviendas a una dosis de 2 y 4 mg/m3, respectivamente. RESULTADOS: La mezcla de flupyradifurona y transflutrina causó mortalidades de 97 a 100% sobre las cepas de mosquitos Aedes y su efectividad fue la misma en los diferentes compartimentos de las viviendas. CONCLUSIONES: La mezcla de flupyradifurona y transflutrina, aplicada en niebla térmica, es una herramienta prometedora para el control de poblaciones de mosquitos Aedes independientemente de su estado de resistencia a insecticidas.

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.

Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus.

We tested a nootkatone product for insecticide activity against the most prominent vectors of Zika virus (ZIKV), Aedes aegypti, and Aedes albopictus. We tested the permethrin-resistant (PERM-R) Vergel strain of A. aegypti and the permethrin-susceptible (PERM-S) New Orleans strain of A. aegypti to determine if insecticide resistance affected their susceptibility to nootkatone. Bottle bioassays showed that the PERM-S strain (New Orleans) was more susceptible to nootkatone than the confirmed A. aegypti permethrin-resistant (PERM-R) strain, Vergel. The A. albopictus strain ATM-NJ95 was a known PERM-S strain and Coatzacoalcos permethrin susceptibility was unknown but proved to be similar to the ATM-NJ95 PERM-S phenotype. The A. albopictus strains (ATM-NJ95 and Coatzacoalcos) were as susceptible to nootkatone as the New Orleans strain. Bottle bioassays conducted with ZIKV-infected mosquitoes showed that the New Orleans (PERM-S) strain was as susceptible to nootkatone as the mock-infected controls, but the PERM-R strain was less susceptible to nootkatone than the mock-infected controls. Repellency/irritancy and biting inhibition bioassays (RIBB) of A. aegypti determined whether the nootkatone-treated arms of three human subjects prevented uninfected A. aegypti mosquitoes from being attracted to the test subjects and blood-feeding on them. The RIBB analyses data calculated the spatial activity index (SAI) and biting inhibition factor (BI) of A. aegypti at different nootkatone concentrations and then compared the SAI and BI of existing repellency products. We concluded that nootkatone repelled mosquitoes at a rate comparable to 7% DEET or 5% picaridin and has the potential to be an efficacious repellent against adult A. aegypti mosquitoes.

Mitochondrial metabolic genes provide phylogeographic relationships of global collections of Aedes aegypti (Diptera: Culicidae).

Phylogeographic relationships among global collections of the mosquito Aedes aegypti were evaluated using the mitochondrial Cytochrome C Oxidase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4) genes including new sequences from Sri Lanka. Phylogeographic analysis estimated that Ae. aegypti arose as a species ~614 thousand years ago (kya) in the late Pleistocene. At 545 kya an "early" East African clade arose that continued to differentiate in East Africa, and eventually gave rise to three lineages one of which is distributed throughout all tropical and subtropical regions, a second that contains Southeast Asian/Sri Lankan mosquitoes and a third that contains mostly New World mosquitoes. West African collections were not represented in this early clade. The late clade continued to differentiate throughout Africa and gave rise to a lineage that spread globally. The most recent branches of the late clade are represented by South-East Asia and India/Pakistan collections. Analysis of migration rates suggests abundant gene flow between India/Pakistan and the rest of the world with the exception of Africa.

Estimation of DENV-2 Transmission as a Function of Site-Specific Entomological Parameters from Three Cities in Colombia.

BACKGROUND: Measuring dengue virus transmission in endemic areas is a difficult task as many variables drive transmission, and often are not independent of one another. OBJECTIVES: We aimed to determine the utility of vectorial capacity to explain the observed dengue infection rates in three hyperendemic cities in Colombia, and tested hypotheses related to three variables: mosquito density, effective vector competence, and biting rate. METHODS: We estimated two of the most influential entomological variables related to cumulative vectorial capacity, which is a modification of the traditional vectorial capacity equation, of three Colombian mosquito populations. Laboratory studies were undertaken to measure vector competence and man biting rate of local mosquito populations. In addition, the assessment of cumulative vectorial capacity also incorporated site-specific estimations of mosquito density and the probability of daily survival from previous studies conducted in those cities. FINDINGS: We found that the biting rates and mosquito infection rates differed among populations of mosquitoes from these three cities, resulting in differences in the site-specific measures of transmission potential. Specifically, we found that using site-specific entomological measures to populate the cumulative vectorial capacity equation was best at recapitulating observed mosquito infection rates when mosquito density was discounted compared to when we incorporated site-specific density measures. CONCLUSIONS: Specific mosquito-biting rate is likely sufficient to explain transmission differences in these three cities, confirming that this parameter is a critical parameter when predicting and assessing dengue transmission in three Colombian cities with different field observed transmission patterns.

The Use of Insecticide-Treated Curtains for Control of Aedes aegypti and Dengue Virus Transmission in "Fraccionamiento" Style Houses in México.

Dengue, chikungunya, yellow fever, and Zika viruses transmitted by Aedes aegypti mosquitoes are major public health threats in the tropical and subtropical world. In México, construction of large tracts of "fraccionamientos" high density housing to accommodate population growth and urbanization has provided fertile ground for Ae. aegypti-transmitted viruses. We investigated the utility of pyrethroid-treated window curtains to reduce both the abundance of Ae. aegypti and to prevent dengue virus (DENV) transmission in fraccionamiento housing. Windows and doors of fraccionamiento homes in urban/suburban areas, where Ae. aegypti pyrethroid resistance associated with the Ile1016 knock down resistance (kdr) mutation in the voltage gated sodium channel gene was high, and in rural areas, where kdr resistance was low, were fitted with either insecticide-treated curtains (ITCs) or non-treated curtains (NTCs). The homes were monitored for mosquito abundance and DENV infection. ITCs reduced the indoor abundance of Ae. aegypti and the number of DENV-infected mosquitoes in homes in rural but not in urban/suburban study sites. The presence of non-treated screens also was associated with reduced numbers of mosquitoes in homes. "Super-infested" homes, yielding more than 50 mosquitoes, including DENV-infected mosquitoes, provide a significant public health risk to occupants, visitors, and people in neighboring homes.

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.

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 microg of permethrin in bottle bioassays. METHODS AND FINDINGS: A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México 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 León in the north, Veracruz on the central Atlantic coast, and Yucatán, 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 approximately 100-fold to 2.7% (+ or - 0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (+ or - 1.15% CI95; n = 473). This was followed in 2007-2009 by a sudden jump in Ile1,016 frequency to 33.2% (+ or - 1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007-2008 collections, which ranged from 45.7% (+ or - 2.00% CI95) in the state of Veracruz to 51.2% (+ or - 4.36% CI95) in the Yucatán peninsula and 14.5% (+ or - 2.23% 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 (R(2) = 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 México 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.