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The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ' Detox Chip' microarray during five generations of temephos selection. We selected for temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione- S-transferase ( eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of temephos selection. While expression of many carboxyl/cholinesterase esterase ( CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase ( CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione- S-transferase ( GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to temephos. We show that temephos-selected strains show no cross resistance to permethrin and that genes associated with temephos selection are largely independent of those selected with permethrin in a previous study. [ABSTRACT FROM AUTHOR]

Changes in gene expression before, during and after five generations of permethrin laboratory selection were monitored in six strains of Aedes aegypti: five F2- F3 collections from the Yucatán Peninsula of Mexico and one F2 from Iquitos, Peru. Three biological replicate lines were generated for each strain. The response to selection was measured as changes in the lethal and knockdown permethrin concentrations (LC50, KC50) and in the frequency of the Ile1,016 substitution in the voltage-gated sodium channel ( para) gene. Changes in expression of 290 metabolic detoxification genes were measured using the ' Aedes Detox' microarray. Selection simultaneously increased the LC50, KC50 and Ile1,016 frequency. There was an inverse relationship between Ile1,016 frequency and the numbers of differentially transcribed genes. The Iquitos strain lacked the Ile1,016 allele and 51 genes were differentially transcribed after selection as compared with 10-18 genes in the Mexican strains. Very few of the same genes were differentially transcribed among field strains but 10 cytochrome P450 genes were upregulated in more than one strain. Laboratory adaptation to permethrin in Ae. aegypti is genetically complex and largely conditioned by geographic origin and pre-existing target site insensitivity in the para gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance. Attempts to identify one or a few metabolic genes that are predictably associated with permethrin adaptation may be futile. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Tapachula, Mexico, a tropical city, is an endemic area for dengue, in addition to several outbreaks in the last decade with chikungunya and zika. As part of the migratory corridor from Central to North America and the risks of scattered infectious diseases that this implies, the identification and distribution of potential disease vectors in and around residential areas are essential in terms of entomological surveillance for the prevention of disease outbreaks. The identification of mosquito species of medical importance coexisting in houses and cemeteries in Tapachula and two semiurban sites in southern Chiapas was investigated. Adult mosquitoes were collected from May to December 2018, resting inside and outside houses and in the tombstones and fallen tree leaves in cemeteries. A total of 10,883 mosquitoes belonging to three vector species were collected across 20 sites; 6738 were from neighborhood houses, of which 55.4% were Culex quinquefasciatus , 41.6% Aedes aegypti , and 2.9% Ae. albopictus . Aedes aegypti was the most common mosquito resting inside houses (56.7%), while Ae. albopictus and Cx. quinquefasciatus were mostly found resting outside houses (75.7%). In the cemeteries, Cx. quinquefasciatus (60.8%) and Ae. albopictus (37.3%) were the most abundant, while Ae. aegypti (1.9%) was the least abundant. This is the first report to identify adults of three major disease vector species coexisting in the domestic environment of urban and semiurban sites and Ae. albopictus adult resting inside of urban houses in Mexico. It would be opportune to consider comprehensive strategies that can be applied in this region to control the three species at the same time and avoid outbreaks of the diseases they transmit.

Among many medically important pathogens, arboviruses like dengue, Zika and chikungunya cause severe health and economic burdens especially in developing countries. These viruses are primarily vectored by mosquitoes. Having surmounted geographical barriers and threat of control strategies, these vectors continue to conquer many areas of the globe exposing more than half of the world's population to these viruses. Unfortunately, no medical interventions have been capable so far to produce successful vaccines or antivirals against many of these viruses. Thus, vector control remains the fundamental strategy to prevent disease transmission. The long-established understanding regarding the replication of these viruses is that they reshape both human and mosquito host cellular membranes upon infection for their replicative benefit. This leads to or is a result of significant alterations in lipid metabolism. Metabolism involves complex chemical reactions in the body that are essential for general physiological functions and survival of an organism. Finely tuned metabolic homeostases are maintained in healthy organisms. However, a simple stimulus like a viral infection can alter this homeostatic landscape driving considerable phenotypic change. Better comprehension of these mechanisms can serve as innovative control strategies against these vectors and viruses. Here, we review the metabolic basis of fundamental mosquito biology and virus-vector interactions. The cited work provides compelling evidence that targeting metabolism can be a paradigm shift and provide potent tools for vector control as well as tools to answer many unresolved questions and gaps in the field of arbovirology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ratnayake, Chotiwan, Saavedra-Rodriguez and Perera.)

Background: Insecticide use continues as the main strategy to control Aedes aegypti, the vector of dengue, Zika, chikungunya, and yellow fever. In the city of Tapachula, Mexico, mosquito control programs switched from pyrethroids to organophosphates for outdoor spatial spraying in 2013. Additionally, the spraying scheme switched from total coverage to focused control, prioritizing areas with higher entomological-virological risk. Five years after this strategy had been implemented, we evaluated the status and variability of insecticide resistance among Ae. aegypti collected at 26 sites in Tapachula., Methodology/principal Findings: We determined the lethal concentrations at 50% of the tested populations (LC50) using a bottle bioassay, and then, we calculated the resistance ratio (RR) relative to the susceptible New Orleans strain. Permethrin and deltamethrin (pyrethroids), chlorpyrifos and malathion (organophosphates), and bendiocarb (carbamate) were tested. The frequencies of the substitutions V1016I and F1534C, which are in the voltage-gated sodium channel and confer knockdown-resistance (kdr) to pyrethroid insecticides, were calculated. Despite 5 years having passed since the removal of pyrethroids from the control programs, Ae. aegypti remained highly resistant to permethrin and deltamethrin (RR > 10-fold). In addition, following 5 years of chlorpyrifos use, mosquitoes at 15 of 26 sites showed moderate resistance to chlorpyrifos (5- to 10-fold), and the mosquitoes from one site were highly resistant. All sites had low resistance to malathion (< 5-fold). Resistance to bendiocarb was low at 19 sites, moderate at five, and high at two. Frequencies of the V1016I ranged from 0.16-0.71, while C1534 approached fixation at 23 sites (0.8-1). Resistance profiles and kdr allele frequencies varied across Tapachula. The variability was not associated with a spatial pattern at the scale of the sampling., Conclusion/significance: Mosquito populations respond to selection pressure at a focal scale in the field. Spatial variation across sites highlights the importance of testing multiple sites within geographical regions., Competing Interests: The authors have declared that no competing interests exist. Author Americo D. Rodriguez was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

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., Competing Interests: The authors have declared that no competing interests exist. The findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the National Institutes of Health. Author Americo Rodriguez was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

Aedes aegypti is a major vector of Zika, dengue, and other arboviruses. Permethrin adulticidal spraying, which targets the voltage-gated sodium channel (VGSC), is commonly done to reduce local mosquito populations and protect humans from exposure to arbovirus pathogens transmitted by this dangerous pest. Permethrin resistance, however, is a growing problem and understanding its underlying molecular basis may identify avenues to combat it. We identified a single G:C polymorphism in pre-miR-33 that was genetically associated with permethrin resistance; resulting isoforms had structural differences that may affect DICER-1/pre-miRNA processing rates. We then assessed the effects of overexpression of pre-miR-33 isoforms on permethrin toxicological phenotypes, VGSC transcript abundance and protein levels for two genetically related mosquito strains. One strain had its naturally high permethrin resistance levels maintained by periodic treatment, and the other was released from selection. VGSC protein levels were lower in the permethrin resistant strain than in the related permethrin-susceptible strain. Overexpression of the G-pre-miR-33 isoform reduced VGSC expression levels in both strains. To further elucidate changes in gene expression associated with permethrin resistance, exome-capture gDNA deep sequencing, genetic association mapping and subsequent gene set enrichment analysis revealed that transport genes, in particular, were selected in resistant versus susceptible mosquitoes. Collectively, these data indicate that miR-33 regulates VGSC expression as part of a nuanced system of neuronal regulation that contributes to a network of heritable features determining permethrin resistance.

The threat of mosquito-borne diseases continues to be a problem for public health in subtropical and tropical regions of the world; in response, there has been increased use of adulticidal insecticides, such as pyrethroids, in human habitation areas over the last thirty years. As a result, the prevalence of pyrethroid-resistant genetic markers in natural mosquito populations has increased at an alarming rate. This review details recent advances in the understanding of specific mechanisms associated with pyrethroid resistance, with emphasis on features of insecticide detoxification and the interdependence of multiple cellular pathways. Together, these advances add important context to the understanding of the processes that are selected in resistant mosquitoes. Specifically, before pyrethroids bind to their targets on motoneurons, they must first permeate the outer cuticle and diffuse to inner tissues. Resistant mosquitoes have evolved detoxification mechanisms that rely on cytochrome P450s (CYP), esterases, carboxyesterases, and other oxidation/reduction (redox) components to effectively detoxify pyrethroids to nontoxic breakdown products that are then excreted. Enhanced resistance mechanisms have evolved to include alteration of gene copy number, transcriptional and post-transcriptional regulation of gene expression, as well as changes to cellular signaling mechanisms. Here, we outline the variety of ways in which detoxification has been selected in various mosquito populations, as well as key gene categories involved. Pathways associated with potential new genes of interest are proposed. Consideration of multiple cellular pathways could provide opportunities for development of new insecticides.

There are major public health concerns regarding the spread of mosquito-borne diseases such as dengue, Zika, and chikungunya, which are mainly controlled by using insecticides against the vectors, Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse). Pyrethroids are the primary class of insecticides used for vector control, due to their rapid knockdown effect and low toxicity to vertebrates. Unfortunately, continued use of pyrethroids has led to widespread insecticide resistance in Ae. aegypti; however, we lack information for Ae. albopictus-a sympatric species in Chiapas since 2002. In this study, we evaluated the permethrin resistance status of Ae. albopictus collected from Mexico and Texas. We also selected for permethrin resistance in the laboratory and investigated the potential mechanisms conferring resistance in this species. Knockdown resistance mutations, specifically F1534C, in the voltage-gated sodium channel gene, and increased activity of detoxifying enzymes were evaluated. Low levels of permethrin resistance (<2.4-fold) were observed in our field populations of Ae. albopictus and the F1534C mutation was not detected in any of the sites. Low levels of resistance were also observed in the artificially selected strain. There was significantly higher cytochrome P450 activity in our permethrin-selected and nonselected strains from Mexico compared to the control strain. Our results suggest the Ae. albopictus sampled from 2016 are mostly susceptible to pyrethroids. These results contrast with the high levels of permethrin resistance (>58-fold) found in Ae. aegypti from the same sites in Mexico. This research indicates the importance of continued monitoring of Ae. albopictus populations to prevent resistance from developing in the future., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The study of fitness costs of insecticide resistance mutations in Aedes aegypti has generally been focused on life history parameters such as fecundity, mortality, and energy reserves. In this study we sought to investigate whether trade-offs might also exist between insecticide resistance and other abiotic stress resistance parameters. We evaluated the effects of the selection for permethrin resistance specifically on larval salinity and thermal tolerance. A population of A. aegypti originally from Southern Mexico was split into two strains, one selected for permethrin resistance and the other not. Larvae were reared at different salinities, and the fourth instar larvae were subjected to acute thermal stress; then, survival to both stresses was compared between strains. Contrary to our predictions, we found that insecticide resistance correlated with significantly enhanced larval thermotolerance. We found no clear difference in salinity tolerance between strains. This result suggests that insecticide resistance does not necessarily carry trade-offs in all traits affecting fitness and that successful insecticide resistance management strategies must account for genetic associations between insecticide resistance and abiotic stress resistance, as well as traditional life history parameters.

Background: Drastic increases of dengue fever (DF) over the past few years have prompted studies on the development of resistance to insecticides in the mosquito vector, Aedes aegypti (Linnaeus). In Sri Lanka control of the vector population is essentially achieved using larvicides (temephos) and adulticides (principally pyrethroids). The present study investigates resistance to commonly used insecticides and underlying mechanisms of Ae. aegypti in selected sites in Sri Lanka., Methods: In this study, susceptibility to three commonly used adulticides (malathion, permethrin and deltamethrin) and the larvicide temephos were tested for Ae. aegypti sampled from five localities in Sri Lanka using WHO dose diagnostics tests. In addition, we performed dose-response tests for permethrin to determine lethal concentrations (LCs) with CDC bottle bioassays. An assessment of the activity of metabolic detoxifying enzymes (multifunction oxidases (MFOs), glutathione S-transferases (GSTs) and esterases) and determination of frequency of the kdr mutations (F1534C, V1016G and S989P) were also carried out to ascertain the associated resistance mechanisms. Kdr genotype frequencies were compared with samples collected from the same sites in 2015 to determine the change of allele frequencies over the years., Results: The present study revealed resistance in all Ae. aegypti populations studied, with low mortality percentages for both permethrin (10-89%) and deltamethrin (40-92%). Dose response tests revealed highest resistance ratios (RR) for permethrin and temephos from Colombo district whereas Puttalum district exhibited the lowest. High frequencies of the 1534C allele (0.052-0.802) were found in the study sites in 2017. Comparison with samples collected in 2015 revealed a substantial increase in this allele. The activity of MFOs and p-nitro phenyl-acetate esterase was significantly greater in most Sri Lankan populations in comparison to that of the New Orleans (NO) susceptible strain. In contrast, the activity of α-esterase and β-esterase was similar or lower than that in the NO strain., Conclusions: Aedes aegypti from Sri Lanka is resistant to pyrethroid insecticides showing rapid selection for kdr mutations and varying metabolic mechanisms. Continued monitoring of vector populations is crucial to mitigate the development of resistance to commonly used insecticides and in turn, controlling the vector population.

Background: Resistance to pyrethroid insecticides in Aedes aegypti has become widespread after almost two decades of the frequent use of these pesticides to reduce arbovirus transmission. Despite this resistance, pyrethroids continue to be used because they are relatively inexpensive and have low human toxicity. Resistance management has been proposed as a way to retain the use of pyrethroids in natural populations. A key component of resistance management is the assumption that negative fitness is associated with resistance alleles such that resistance alleles will decline in frequency when the insecticides are removed. At least three studies in Ae. aegypti have demonstrated a decrease in pyrethroid resistance once the insecticide has been removed., Methods/principal Findings: The present study aims to evaluate variation in the loss of pyrethroid resistance among newly established laboratory populations of Ae. aegypti from Mexico. Eight field collections were maintained for up to eight generations, and we recorded changes in the frequencies of the mutations at the V1,016I locus and at the F1,534C locus in the voltage-gated sodium channel gene (VGSC). I1,016 and C1,534 confer resistance. We also examined resistance ratios (RR) with type 1 and 2 pyrethroids., Conclusions/significance: We demonstrate that, in general, the frequency of the Ae. aegypti pyrethroid-resistance alleles I1,016 and C1,534 decline when they are freed from pyrethroid pressure in the laboratory. However, the pattern of decline is strain dependent. In agreement with earlier studies, the RR was positively correlated with the frequencies of the resistance allele I1,016 and showed significant protection against permethrin, and deltamethrin, whereas F1,534C showed protection against permethrin but not against deltamethrin., Competing Interests: The authors have declared that no competing interests exist.

Association mapping of factors that condition pyrethroid resistance in Aedes aegypti has consistently identified genes in multiple functional groups. Toward better understanding of the mechanisms involved, we examined high throughput sequencing data (HTS) from two Aedes aegypti aegypti collections from Merida, Yucatan, Mexico treated with either permethrin or deltamethrin. Exome capture enrichment for coding regions and the AaegL5 annotation were used to identify genes statistically associated with resistance. The frequencies of single nucleotide polymorphisms (SNPs) were compared between resistant and susceptible mosquito pools using a contingency χ2 analysis. The -log10(χ2 p value) was calculated at each SNP site, with a weighted average determined from all sites in each gene. Genes with -log10(χ2 p value) ≥ 4.0 and present among all 3 treatment groups were subjected to gene set enrichment analysis (GSEA). We found that several functional groups were enriched compared to all coding genes. These categories were transport, signal transduction and metabolism, in order from highest to lowest statistical significance. Strikingly, 21 genes with demonstrated association to synaptic function were identified. In the high association group (n = 1,053 genes), several genes were identified that also genetically or physically interact with the voltage-gated sodium channel (VGSC). These genes were eg., CHARLATAN (CHL), a transcriptional regulator, several ankyrin-domain proteins, PUMILIO (PUM), a translational repressor, and NEDD4 (E3 ubiquitin-protein ligase). There were 13 genes that ranked among the top 10%: these included VGSC; CINGULIN, a predicted neuronal gap junction protein, and the aedine ortholog of NERVY (NVY), a transcriptional regulator. Silencing of CHL and NVY followed by standard permethrin bottle bioassays validated their association with permethrin resistance. Importantly, VGSC levels were also reduced about 50% in chl- or nvy-dsRNA treated mosquitoes. These results are consistent with the contribution of a variety of neuronal pathways to pyrethroid resistance in Ae. aegypti., Competing Interests: The authors have declared that no competing interests exist.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Background: Dengue is a serious arboviral disease in Sri Lanka with a large number of dengue fever (DF) cases every year. Control of the primary vector Aedes aegypti depends upon larval habitat source reduction and insecticide application. However, increases in the number of reported cases suggest the inefficiency of current control strategies and the possibility of resistance to currently used insecticides. Early detection of mutations in the voltage-gated sodium channel (vgsc) gene that confer knockdown resistance (kdr) to pyrethroid insecticides is important in resistance management in vector populations., Results: Resistance to pyrethroid insecticides was detected in the three populations studied. Polymerase chain reaction was used to detect the presence of two kdr mutations F1534C and V1016G. During this process a S989P mutation was also detected in pyrethroid-resistant Ae. aegypti populations. These mutations were found to be widespread and frequent in the collections studied., Conclusions: To our knowledge, this study reveals for the first time the presence of V1016G and S989P mutant alleles in the vgsc of Sri Lankan Ae. aegypti populations. The spread of the mutant alleles throughout the country poses a threat of increased resistance to pyrethroids. Long-term insecticide applications and indiscriminate use of pyrethroids has led to the evolution of resistance. More strategic and diverse strategies, including novel insecticides with new modes of action and community participation, should be engaged for Ae. aegypti control.

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.

Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers -for which vaccines and effective 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 space-sprays 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 identified in Brazil and its effect on vgsc was confirmed by electrophysiology. Herein, 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 alongside V1,016I and F1,534C. Knowledge of the specific vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management.

The emergence of novel diseases spread by the Aedes aegypti mosquito in Jamaica and the Caribbean, has prompted studies on insecticide resistance towards effective management of the vector. Though Jamaica has been using the organophosphate insecticide malathion in its vector control program for more than 30 years, resistance to the pesticide has not been tested in over a decade. We analyzed resistance to malathion and the pyrethroid insecticide, permethrin on mosquitoes collected across St. Andrew, Jamaica, and analyzed the molecular basis of resistance. The Center for Disease Control (CDC) bioassay revealed that Ae. aegypti mosquitoes from St. Andrew, Jamaica were resistant to permethrin (15 μg/bottle) with mortalities at 0-8% at 30 minute exposure time, while contact with malathion (50 μg/bottle) revealed ≤ 50% mortality at 15 minutes, which increased to 100% at 45 minutes. The standard susceptible New Orleans (NO) strain exhibited 100% mortality within15 minutes. The activities of multifunction oxidases and p-nitro phenyl-acetate esterases were significantly greater in most Jamaican populations in comparison to the NO strain, while activities of glutathione-S-transferase, acetylcholinesterase, α-esterase and ß-esterase activity were relatively equal, or lower than that of the control strain. The frequency of knockdown resistance mutations in the voltage dependent sodium channel gene were measured. All collections were fixed for Cys1,534 while 56% of mosquitoes were Ile1,016/Val1,016 heterozygotes, and 33% were Ile1,016 homozygotes. Aedes aegypti from St. Andrew Jamaica are resistant to permethrin with variations in the mode of mechanism, and possibly developing resistance to malathion. Continued monitoring of resistance is critically important to manage the spread of the vector in the country.

Permethrin resistance is widespread in Aedes aegypti (L.), the main dengue, zika, and chikungunya virus vector in Latin America and the Caribbean. A common mechanism of resistance to pyrethroids-knockdown resistance (kdr)-is conferred through mutations in the insect's voltage-dependent sodium channel. In this mosquito, around 10 replacement substitutions in the voltage-gated sodium channel gene (vgsc) have been reported in pyrethroid-resistant strains. Two of these mutations, named Ile1,016 and Cys1,534, are widespread in mosquito populations from Latin America and the Caribbean. This study assessed the levels of permethrin resistance and the frequency of two kdr mutations in eight Ae. aegypti populations collected in Puerto Rico in 2013. Permethrin resistance factors ranged from 33-214-fold relative to the New Orleans reference strain. The frequency of kdr mutation Ile1,016 ranged from 0.65 to fixation (1.0), and for Cys1,534 frequencies varied from 0.8 to fixation. Alarmingly, two populations-Carolina and Caguas-reached fixation at both loci. Our results suggest that permethrin effectiveness for Ae. aegypti control is compromised in these collections from Puerto Rico., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Background: The V1016I and F1534C mutations in the voltage-gated sodium channel gene have been associated with resistance to pyrethroids and DDT in Aedes aegypti mosquitoes. A study was carried out to determine the frequency of I1016 and C1534 by real-time PCR in five natural populations of Ae. aegypti in Venezuela during 2008, 2010 and 2012, as well as in a strain selected with 0.14 µg of deltamethrin for 15 generations., Results: In natural populations, frequencies of I1016 varied between 0.01 and 0.37, and frequencies of C1534 between 0.35 and 1.0. In the Pampanito strain, the frequency of I1016 increased from 0.02 in F1 up to 0.5 in F15 and from 0.35 up to fixation for C1534 after selection with deltamethrin., Conclusion: The results showed that C1534 frequencies are higher than I1016 frequencies in natural populations of Ae. aegypti in Venezuela, and that deltamethrin selected the C1534 more rapidly than I1016., (© 2014 Society of Chemical Industry.)

We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow., (© The American Society of Tropical Medicine and Hygiene.)

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 Solidaridad×Iquitos (SLD×Iq) 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.

The home, or domicile, is the principal environment for transmission of dengue virus (DENV) between humans and mosquito vectors. Community-wide distribution of insecticide-treated curtains (ITCs), mimicking vector control program-driven interventions, has shown promise to reduce DENV infections. We conducted a Casa Segura consumer product intervention study in Mérida, Mexico to determine the potential to reduce intradomicillary DENV transmission through ITC use in individual homes. Dengue virus infections in mosquitoes and in humans were reduced in homes with ITCs in one of two study subareas. Overall, ITCs reduced intradomicillary DENV transmission; ITC homes were significantly less likely to experience multiple DENV infections in humans than NTC homes. Dengue virus-infected Aedes aegypti females were reduced within the ITC homes where curtain use was highest. Some homes yielded up to nine infected Ae. aegypti females. This study provides insights regarding best practices for Casa Segura interventions to protect homes from intradomicillary DENV transmission.

We collected mosquito immatures from artificial containers during 2010-2011 from 26 communities, ranging in size from small rural communities to large urban centers, located in different parts of Yucatán State in southeastern México. The arbovirus vector Aedes (Stegomyia) aegypti was collected from all 26 examined communities, and nine of the communities also yielded another container-inhabiting Aedes mosquito: Aedes (Howardina) cozumelensis. The communities from which Ae. cozumelensis were collected were all small rural communities (<6,000 inhabitants) in the north-central part of Yucatán State. These new collection records for Ae. cozumelensis demonstrate that this mosquito has a far broader geographic range in the Yucatán Peninsula than previously known. Ae. cozumelensis immatures were collected from both residential premises and cemeteries, with specimens recovered from rock holes as well as various artificial containers including metal cans, flower vases, buckets, tires, and a water storage tank. The co-occurrence with Ae. aegypti in small rural communities poses intriguing questions regarding linkages between these mosquitoes, including the potential for direct competition for larval development sites. Additional studies are needed to determine how commonly Ae. cozumelensis feeds on human blood and whether it is naturally infected with arboviruses or other pathogens of medical or veterinary importance. We also summarize the published records for Ae. cozumelensis, which are restricted to collections from México's Yucatán Peninsula and Belize, and uniformly represent geographic locations where Ae. aegypti can be expected to occur., (© 2012 The Society for Vector Ecology.)

We describe a novel software application (QCal) that was developed for calculation of dose-response curves in insecticide resistance bioassays. QCal uses a logistic regression model to generate values for lethal dose/knockdown dose based on data from a bioassay entered into the application user interface. The application can be freely distributed to interested parties.

Background: Aedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock., Results: Herein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways., Conclusion: These results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects' neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Permethrin is an insecticide used to suppress Ae. aegypti adult populations but metabolic and target site resistance to pyrethroids has evolved in many locations worldwide. Quantitative trait loci (QTL) controlling permethrin survival in Ae. aegypti were mapped in an F(3) advanced intercross line. Parents came from a collection of mosquitoes from Isla Mujeres, México, that had been selected for permethrin resistance for two generations and a reference permethrin-susceptible strain originally from New Orleans. Following a 1-hr permethrin exposure, 439 F(3) adult mosquitoes were phenotyped as knockdown resistant, knocked down/recovered, or dead. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 22 loci with potential antixenobiotic activity including genes encoding cytochrome P450s (CYP), esterases (EST), or glutathione transferases (GST) and at 12 previously mapped loci. Seven antixenobiotic genes mapped to chromosome I, six to chromosome II, and nine to chromosome III. Two QTL of major effect were detected on chromosome III. One corresponds with a SNP previously associated with permethrin resistance in the para sodium channel gene and the second with the CCEunk7o esterase marker. Additional QTL but of relatively minor effect were also found. These included two sex-linked QTL on chromosome I affecting knockdown and recovery and a QTL affecting survival and recovery. On chromosome II, one QTL affecting survival and a second affecting recovery were detected. The patterns confirm that mutations in the para gene cause target-site insensitivity and are the major source of permethrin resistance but that other genes dispersed throughout the genome contribute to recovery and survival of mosquitoes following permethrin exposure.