Comparative transcriptional analysis between susceptible and resistant populations of Aedes (Stegomyia) aegypti (Linnaeus, 1762) after malathion exposure.

Aedes aegypti is an important vector of arboviruses, including dengue, chikungunya and Zika. The application of synthetic insecticides is a frequently used strategy to control this insect. Malathion is an organophosphate insecticide that was widely used in Brazil in the 1980s and 1990s to control the adult form of A. aegypti. In situations where resistance to currently used insecticides is detected, the use of malathion may be resumed as a control measure. Many studies have confirmed resistance to malathion, however, comparative studies of differential gene expression of the entire transcriptome of resistant and susceptible insects are scarce. Therefore, understanding the molecular basis of resistance to this insecticide in this species is extremely important. In this paper, we present the first transcriptomic description of susceptible and resistant strains of A. aegypti challenged with malathion. Guided transcriptome assembly resulted in 39,904 transcripts, where 2133 differentially expressed transcripts were detected, and three were validated by RT-qPCR. Enrichment analysis for these identified transcripts resulted in 13 significant pathways (padj < 0.05), 8 associated with down-regulated and 5 with up-regulated transcripts in treated resistant insects. It was possible to divide the transcripts according to the mechanism of action into three main groups: (i) genes involved in detoxification metabolic pathways; (ii) genes of proteins located in the membrane/extracellular region; and (iii) genes related to DNA integration/function. These results are important in advancing knowledge of genes related to resistance mechanisms in this insect, enabling the development of effective technologies and strategies for managing insecticide resistance.

Evaluation of Aedes aegypti control intervention with pyriproxyfen by lcWGS in Manacapuru, Amazonas, Brazil.

BACKGROUND: Ae. aegypti mosquitoes are considered a global threat to public health due to its ability to transmit arboviruses such as yellow fever, dengue, Zika and Chikungunya to humans. The lack of effective arboviral vaccines and etiological treatments make vector control strategies fundamental in interrupting the transmission cycle of these pathogens. This study evaluated Ae. aegypti mosquito populations pre- and post-intervention period with disseminating stations of the larvicide pyriproxyfen to understand its potential influence on the genetic structure and population diversity of these vectors. METHODOLOGY/PRINCIPAL FINDINGS: This study was conducted in Manacapuru city, Amazonas, Brazil, where 1,000 pyriproxyfen dissemination stations were deployed and monitored from FEB/2014 to FEB/2015 (pre-intervention) and AUG/2015 to JAN/2016 (post-intervention). Low-coverage whole genome sequencing of 36 individuals was performed, revealing significant stratification between pre- and post-intervention groups (pairwise FST estimate of 0.1126; p-value < 0.033). Tajima's D estimates were -3.25 and -3.07 (both p-value < 0.01) for pre- and post-intervention groups, respectively. Molecular diversity estimates (Theta(S) and Theta(Pi)) also showed divergences between pre- and post-intervention groups. PCA and K-means analysis showed clustering for SNP frequency matrix and SNP genotype matrix, respectively, being both mainly represented by the first principal component. PCA and K-means clustering also showed significant results that corroborate the impact of pyriproxyfen intervention on genetic structure populations of Ae. aegypti mosquitoes. CONCLUSIONS/SIGNIFICANCE: The results revealed a bottleneck effect and reduced mosquito populations during intervention, followed by reintroduction from adjacent and unaffected populations by this vector. We highlighted that low-coverage whole genome sequencing can contribute to genetic and structure population data, and also generate important information to aid in genomic and epidemiological surveillance.

Assessing pyrethroid resistance in Aedes aegypti from Cordoba Colombia: Implications of kdr mutations.

Resistance to insecticides is one of the great challenges that vector control programs must face. The constant use of pyrethroid-type insecticides worldwide has caused selection pressure in populations of the Aedes aegypti vector, which has promoted the emergence of resistant populations. The resistance mechanism to pyrethroid insecticides most studied to date is target-site mutations that desensitize the voltage-gated sodium channel (VGSC) of the insect to the action of pyrethroids. In the present study, susceptibility to the pyrethroid insecticides permethrin, lambda-cyhalothrin, and deltamethrin was evaluated in fourteen populations from the department of Córdoba, Colombia. The CDC bottle bioassay and WHO tube methods were used. Additionally, the frequencies of the F1534C, V1016I, and V410L mutations were determined, and the association of resistance with the tri-locus haplotypes was examined. The results varied between the two techniques used, with resistance to permethrin observed in thirteen of the fourteen populations, resistance to lambda-cyhalothrin in two populations, and susceptibility to deltamethrin in all the populations under study with the CDC method. In contrast, the WHO method showed resistance to the three insecticides evaluated in all populations. The frequencies of the mutated alleles ranged from 0.05-0.43 for 1016I, 0.94-1.0 for 1534C, and 0.01-0.59 for 410L. The triple homozygous mutant CIL haplotype was associated with resistance to all three pyrethroids evaluated with the WHO bioassay, while with the CDC bioassay, it was only associated with resistance to permethrin. This study highlights the importance of implementing systematic monitoring of kdr mutations, allowing resistance management strategies to be dynamically adjusted to achieve effective control of Aedes aegypti.

Impact of Long-Term Pyriproxyfen Exposure on the Genetic Structure and Diversity of Aedes aegypti and Aedes albopictus in Manaus, Amazonas, Brazil.

Aedes aegypti and Aedes albopictus are responsible for transmitting major human arboviruses such as Dengue, Zika, and Chikungunya, posing a global threat to public health. The lack of etiological treatments and efficient vaccines makes vector control strategies essential for reducing vector population density and interrupting the pathogen transmission cycle. This study evaluated the impact of long-term pyriproxyfen exposure on the genetic structure and diversity of Ae. aegypti and Ae. albopictus mosquito populations. The study was conducted in Manaus, Amazonas, Brazil, where pyriproxyfen dissemination stations have been monitored since 2014 up to the present day. Double digest restriction-site associated DNA sequencing was performed, revealing that despite significant local population reductions by dissemination stations with pyriproxyfen in various locations in Brazil, focal intervention has no significant impact on the population stratification of these vectors in urban scenarios. The genetic structuring level of Ae. aegypti suggests it is more stratified and directly affected by pyriproxyfen intervention, while for Ae. albopictus exhibits a more homogeneous and less structured population. The results suggest that although slight differences are observed among mosquito subpopulations, intervention focused on neighborhoods in a capital city is not efficient in terms of genetic structuring, indicating that larger-scale pyriproxyfen interventions should be considered for more effective urban mosquito control.

First detection of V410L kdr mutation in Aedes aegypti populations of Argentina supported by toxicological evidence.

BACKGROUND: Aedes aegypti (L.) is the main vector of dengue, yellow fever, Zika, and chikungunya viruses in many parts of the world, impacting millions of people worldwide each year. Insecticide-based interventions have been effective in controlling Aedes mosquito populations for several years, but in recent times, resistance to these compounds has developed, posing a global threat to the control of this mosquito. METHODS: Ovitraps were used to collect A. aegypti eggs in the cities of Tartagal and San Ramón de la Nueva Orán (Salta), Puerto Iguazú (Misiones), and Clorinda (Formosa). World Health Organization (WHO)-impregnated papers with the discriminating concentration (DC) of permethrin, 5X, 10X and pirimiphos methyl were used for the toxicological bioassays. We also genotyped each sample for the three kdr single nucleotide polymorphisms (SNP): V410L, V1016I, and F1534C in individual TaqMan quantitative PCR (qPCR) reactions. RESULTS: All investigated A. aegypti populations were highly resistant to permethrin, as the mortality percentage with the permethrin 10×DC remained below 98%. However, all populations were 100% susceptible to pirimiphos-methyl. Kdr genotyping demonstrated the presence of the V410L mutation for the first time in Argentina in all the populations studied. A prevalence of the triple mutant genotype (LL + II + CC) was observed in the northeastern cities of Clorinda (83.3%) and Puerto Iguazú (55.6%). CONCLUSIONS: This study demonstrates for the first time the presence and intensity of resistance to permethrin in different populations from Argentina, and correlates the observed phenotype with the presence of kdr mutations (genotype).

Molecular basis of RNA recombination in the 3'UTR of chikungunya virus genome.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Genomic Analysis of Aedes aegypti in the Northern Chihuahuan Desert of Texas and Mexico.

Background: Aedes aegypti, is the primary vector of dengue, Chikungunya, Zika, and yellow fever viruses. Both natural and human-impacted landscapes have selective pressures on Ae. aegypti, resulting in strong genomic structure even within close geographical distances. Materials and Methods: We assess the genetic structure of this medically important mosquito species at the northern leading edge of their distribution in Southwestern USA. Ae. aegypti were collected during 2017 in the urban communities of El Paso and Sparks, Texas (USA) and in the city of Ciudad Juárez, Mexico. Results: Thousands of nuclear loci were sequenced across 260 captured Ae. aegypti. First, we recovered the genetic structure of Ae. aegypti following geography, with all four major collection communities being genetically distinct. Importantly, we found population structure and genetic diversity that suggest rapid expansion through active-short distance dispersals, with Anapra being the likely source for the others. Next, tests of selection recovered eight functional genes across six outliers: calmodulin with olfactory receptor function; the protein superfamily C-type lectin with function in mosquito immune system and development; and TATA box binding protein with function in gene regulation. Conclusion: Despite these populations being documented in the early 2000s, we find that selective pressures on specific genes have already occurred and likely facilitate Ae. aegypti range expansion.

Genetic variability of Aedes aegypti (Diptera: Culicidae) in El Salvador and Honduras: presence of a widespread haplotype and implications for mosquito control.

BACKGROUND: This study examined population genetics of Aedes aegypti in El Salvador and Honduras, two adjacent countries in Central America. Aedes aegypti is associated with yellow fever, dengue, chikungunya, and Zika. Each year, thousands of cases of dengue are typically reported in El Salvador and Honduras. METHODS: In El Salvador, collections were obtained from five Departments. In Honduras, samples were obtained from six municipalities in four Departments. Mitochondrial DNA cytochrome oxidase I (COI) was sequenced, and consensus sequences were combined with available sequences from El Salvador to determine haplotype number, haplotype diversity, nucleotide diversity, and Tajima's D. A haplotype network was produced to examine the relationship between genotypes. RESULTS: In El Salvador, there were 17 haplotypes, while in Honduras there were 4 haplotypes. In both El Salvador and Honduras, Haplotype 1 is most abundant and widespread. In El Salvador, haplotype H2 was also widespread in 10 of 11 sampled municipalities, but it was not present in Honduras. The capital of El Salvador (San Salvador) and the eastern region of ES had the highest haplotype diversity of regions sampled. CONCLUSIONS: Haplotype 1 and H2 each belong to different phylogenetic lineages of Ae. aegypti. The most geographically widespread haplotype (H1) may have been present the longest and could be a remnant from previous eradication programs. These data may contribute to future control programs for Ae. aegypti in the two countries.

Aedes aegypti Knockdown Resistance Mutations and Dengue Virus Infection in Haiti.

Haiti is home to approximately 11 million people and has a high incidence of vector-borne disease, including more than 70,000 cases of dengue per year. Vector control is difficult in Haiti and adulticide spray of malathion is the main method of control employed during the outbreak of disease although pyrethroids are used in both bed net campaigns and in widely available aerosol cans for personal use. However, limited pathogen or insecticide resistance surveillance data are available for making operational decisions. In this study, we assessed Aedes aegypti from serial surveillance collections from 3 locations for the presence of dengue virus serotypes 1-3 (DENV1-3) by polymerase chain reaction and assessed, by melt curve analysis, samples from 10 locations in 2 departments for the presence of two mutations (V1016I and F1534C), that in combination, are linked to strong pyrethroid insecticide resistance. Only one of the 32 tested pools was positive for the presence of dengue virus. The two knockdown resistance (kdr) mutations were present in all locations. The 1016I mutation frequency varied from 0.29 to 0.91 and was in all sites lower than the 0.58-1.00 frequency of the 1534C mutation. We also observed that the genotype homozygous for both mutations (IICC), which has been linked to strong pyrethroid resistance, varied from 13 to 86% in each population. Notably, 3 locations - Ti Cousin and Christianville in Ouest department and Camp Coq in Nord department had more than 30% of the tested population without the presence of kdr mutations. These results indicate that the kdr markers of pyrethroid resistance are present in Haiti, at high frequency in several locations and, based on previous studies linking kdr genotypes and phenotypic resistance, that operational interventions with pyrethroids are not likely to be as effective as expected.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein.

BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

Western diet consumption by host vertebrate promotes altered gene expression on Aedes aegypti reducing its lifespan and increasing fertility following blood feeding.

BACKGROUND: The high prevalence of metabolic syndrome in low- and middle-income countries is linked to an increase in Western diet consumption, characterized by a high intake of processed foods, which impacts the levels of blood sugar and lipids, hormones, and cytokines. Hematophagous insect vectors, such as the yellow fever mosquito Aedes aegypti, rely on blood meals for reproduction and development and are therefore exposed to the components of blood plasma. However, the impact of the alteration of blood composition due to malnutrition and metabolic conditions on mosquito biology remains understudied. METHODS: In this study, we investigated the impact of whole-blood alterations resulting from a Western-type diet on the biology of Ae. aegypti. We kept C57Bl6/J mice on a high-fat, high-sucrose (HFHS) diet for 20 weeks and followed biological parameters, including plasma insulin and lipid levels, insulin tolerance, and weight gain, to validate the development of metabolic syndrome. We further allowed Ae. aegypti mosquitoes to feed on mice and tracked how altered host blood composition modulated parameters of vector capacity. RESULTS: Our findings identified that HFHS-fed mice resulted in reduced mosquito longevity and increased fecundity upon mosquito feeding, which correlated with alteration in the gene expression profile of nutrient sensing and physiological and metabolic markers as studied up to several days after blood ingestion. CONCLUSIONS: Our study provides new insights into the overall effect of alterations of blood components on mosquito biology and its implications for the transmission of infectious diseases in conditions where the frequency of Western diet-induced metabolic syndromes is becoming more frequent. These findings highlight the importance of addressing metabolic health to further understand the spread of mosquito-borne illnesses in endemic areas.

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.

Aedes albopictus infection by the native bacterium Elizabethkingia anophelis in Southern Mexico.

OBJECTIVE: To evaluate the presence of Elizabethkingia anophelis infection in Aedes albopictus wild populations of Southern Mexico. MATERIALS AND METHODS: Eight sites were selected to collect Aedes albopictus in the Soconusco region, Chiapas, females were analyzed to amplify the Gyrase B gene by PCR, the minimum infection rate of E. anopheliswas calculated and its species was determined by sequencing and phylogeny. RESULTS: The presence of E. anophelis was only observed in Huehuetán with a minimum infection rate of 37.8%. CONCLUSION: A local strain of E. anophelis was detected for the first time in Ae. albopictus from Chiapas and this bacterium could be considered a candidate for study as a probable control agent or as a vehicle for transgenesis.

Wolbachia dominance influences the Culex quinquefasciatus microbiota.

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

Genetic structure and kdr mutations in Aedes aegypti populations along a road crossing the Amazon Forest in Amapá State, Brazil.

Insecticide resistance in Aedes aegypti poses a significant threat to disease control. One form of resistance, caused by kdr mutations in the NaV gene, hinders vector control efforts in Brazil. Despite genetic differences typically accumulating among isolated populations, this mosquito can actively and passively disperse through human transportation. Our study investigated the genetic structure and spread of kdr mutations in Ae. aegypti populations across six localities in Amapá State, Brazil, within the Amazonian Forest. Using 12 microsatellite loci and qPCR methods, we assessed genetic structure and identified three common kdr mutations (V410L, V1016I, and F1534C). High prevalence of kdr alleles was observed in all localities, indicating widespread distribution in Amapá State. Microsatellite analysis revealed differentiation among mosquito populations, dividing them into two distinct clusters supported by Bayesian and DAPC analyses. Oiapoque, located along the northern border with French Guiana, exhibited the highest kdr frequencies and genetic differentiation compared to other localities. Our findings suggest genetic structure in Ae. aegypti populations in Amapá State, with some passive gene flow between clusters. The study underscores the need for continuous surveillance of Ae. aegypti populations to monitor the spread of insecticide resistance and inform effective vector control strategies.

Light and dark cycles modify the expression of clock genes in the ovaries of Aedes aegypti in a noncircadian manner.

Circadian oscillators (i.e., circadian clocks) are essential to producing the circadian rhythms observed in virtually all multicellular organisms. In arthropods, many rhythmic behaviors are generated by oscillations of the central pacemaker, specific groups of neurons of the protocerebrum in which the circadian oscillator molecular machinery is expressed and works; however, oscillators located in other tissues (i.e., peripheral clocks) could also contribute to certain rhythms, but are not well known in non-model organisms. Here, we investigated whether eight clock genes that likely constitute the Aedes aegypti clock are expressed in a circadian manner in the previtellogenic ovaries of this mosquito. Also, we asked if insemination by conspecific males would alter the expression profiles of these clock genes. We observed that the clock genes do not have a rhythmic expression profile in the ovaries of virgin (VF) or inseminated (IF) females, except for period, which showed a rhythmic expression profile in ovaries of IF kept in light and dark (LD) cycles, but not in constant darkness (DD). The mean expression of seven clock genes was affected by the insemination status (VF or IF) or the light condition (LD 12:12 or DD), among which five were affected solely by the light condition, one solely by the insemination status, and one by both factors. Our results suggest that a functional circadian clock is absent in the ovaries of A. aegypti. Still, their differential mean expression promoted by light conditions or insemination suggests roles other than circadian rhythms in this mosquito's ovaries.

Comparative Transcriptomic Analysis of Insecticide-Resistant Aedes aegypti from Puerto Rico Reveals Insecticide-Specific Patterns of Gene Expression.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

First report on knockdown resistance mutations in wild populations of Aedes aegypti from Argentina determined by a novel multiplex high-resolution melting polymerase chain reaction method.

BACKGROUND: The mosquito Aedes aegypti is an urban vector of dengue and other arboviruses. During epidemics of these viruses, pyrethroid insecticides are used for the control of adult mosquitoes. The worldwide resistance of Ae. aegypti to these insecticides is a cause of failure of vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel. Point mutations in the gene coding for this channel, called knockdown resistance (kdr) mutations, are associated with pyrethroid resistance. Two kdr mutations, V1016I and F1534C, have increased in frequency in natural populations of Ae. aegypti in the Americas during the last decade. Their association with pyrethroid resistance has been largely demonstrated in field populations throughout the Americas, and in in vitro assays. Diagnostics for kdr polymorphism allow early detection of the spread of insecticide resistance, which is critical for timely decisions on vector management. Given the importance of resistance management, high-throughput methods for kdr genotyping are valuable tools as they can be used for resistance monitoring programs. These methods should be cost-effective, to allow regional-scale surveys. Despite the extensive presence of Ae. aegypti and incidence of dengue in Argentina, the presence, abundance, and distribution of kdr mutations in populations of this mosquito have yet to be reported for the country. METHODS: Aedes aegypti samples were collected as immature stages or adults from Buenos Aires Metropolitan Area and northern localities of Tartagal (Salta Province) and Calilegua (Jujuy Province). Immature stages were maintained in the laboratory until they developed into adults. A high-resolution melting assay, based on an analysis of melting temperatures, was developed for the simultaneous genotyping of V1016I and F1534C kdr mutations. We used this method to infer the presence and frequencies of kdr alleles in 11 wild populations from Argentina. RESULTS: We demonstrated the presence of kdr mutations in Ae. aegypti in Argentina in regions where this species is under different selection pressures due to the use of pyrethroids. The populations under analysis are located in geographically distant regions of the species' distribution in Argentina: the northern provinces of Salta and Jujuy and the Buenos Aires Metropolitan Area. Higher frequencies of resistant-associated alleles were detected in the northern region. We report a multiplex high-throughput assay based on a high-resolution melting polymerase chain reaction method for the simultaneous genotyping of V1016I and F1534C kdr mutations. This assay was shown to be cost-effective, and thus provides an interesting molecular tool for kdr genotyping in A. aegypti control campaigns. CONCLUSIONS: We report, to the best of our knowledge for the first time, the presence of kdr mutations in populations of Ae. aegypti from geographically distant locations of Argentina that differ with respect to their epidemiological situation and history of mosquito control. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the Americas. Given its affordability and short running time, this method can be used in control campaigns to monitor the presence and spread of kdr alleles. The information provided here is relevant for the rational design of control strategies in the context of integrated vector management.

Insecticide resistance levels and associated mechanisms in three Aedes aegypti populations from Venezuela.

BACKGROUND: The massive use of insecticides in public health has exerted selective pressure resulting in the development of resistance in Aedes aegypti to different insecticides in Venezuela. Between 2010 and 2020, the only insecticides available for vector control were the organophosphates (Ops) fenitrothion and temephos which were focally applied. OBJECTIVES: To determine the state of insecticide resistance and to identify the possible biochemical and molecular mechanisms involved in three populations of Ae. aegypti from Venezuela. METHODS: CDC bottle bioassays were conducted on Ae. aegypti collected between October 2019 and February 2020 in two hyperendemic localities for dengue in Aragua State and in a malaria endemic area in Bolívar State. Insecticide resistance mechanisms were studied using biochemical assays and polymerase chain reaction (PCR) to detect kdr mutations. FINDINGS: Bioassays showed contrasting results among populations; Las Brisas was resistant to malathion, permethrin and deltamethrin, Urbanización 19 de Abril was resistant to permethrin and Nacupay to malathion. All populations showed significantly higher activity of mixed function oxidases and glutathione-S-transferases (GSTs) in comparison with the susceptible strain. The kdr mutations V410L, F1534C, and V1016I were detected in all populations, with F1534C at higher frequencies. MAIN CONCLUSION: Insecticide resistance persists in three Ae. aegypti populations from Venezuela even in the relative absence of insecticide application.