Molecular diversity of genes related to biological rhythms (period and timeless) and insecticide resistance (Na V and ace-1) in Anopheles darlingi.

BACKGROUND: Malaria is a public health concern in the Amazonian Region, where Anopheles darlingi is the main vector of Plasmodium spp. Several studies hypothesised the existence of cryptic species in An. darlingi, considering variations in behaviour, morphological and genetic aspects. Determining their overall genetic background for vector competence, insecticide resistance, and other elements is essential to better guide strategies for malaria control. OBJECTIVES: This study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation in An. darlingi populations from Amazonian localities in Brazil and Pacific Colombian region. METHODS: We amplified, cloned and sequenced fragments of genes related to behaviour: timeless (tim) and period (per), and to insecticide resistance: voltage-gated sodium channel (Na V ) and acetylcholinesterase (ace-1) from 516 An. darlingi DNA samples from Manaus, Unini River, Jaú River and Porto Velho - Brazil, and Chocó - Colombia. We discriminated single nucleotide polymorphisms (SNPs), determined haplotypes and evaluate the phylogenetic relationship among the populations. FINDINGS: The genes per, tim and ace-1 were more polymorphic than Na V . The classical kdr and ace-1 R mutations were not observed. Phylogenetic analyses suggested a significant differentiation between An. darlingi populations from Brazil and Colombia, except for the Na V gene. There was a geographic differentiation within Brazilian populations considering per and ace-1. CONCLUSIONS: Our results add genetic data to the discussion about polymorphisms at population levels in An. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.

The redox-sensing gene Nrf2 affects intestinal homeostasis, insecticide resistance, and Zika virus susceptibility in the mosquito Aedes aegypti.

Production and degradation of reactive oxygen species (ROS) are extensively regulated to ensure proper cellular responses to various environmental stimuli and stresses. Moreover, physiologically generated ROS function as secondary messengers that can influence tissue homeostasis. The cap'n'collar transcription factor known as nuclear factor erythroid-derived factor 2 (Nrf2) coordinates an evolutionarily conserved transcriptional activation pathway that mediates antioxidant and detoxification responses in many animal species, including insects and mammals. Here, we show that Nrf2-mediated signaling affects embryo survival, midgut homeostasis, and redox biology in Aedes aegypti, a mosquito species vector of dengue, Zika, and other disease-causing viruses. We observed that AeNrf2 silencing increases ROS levels and stimulates intestinal stem cell proliferation. Because ROS production is a major aspect of innate immunity in mosquito gut, we found that a decrease in Nrf2 signaling results in reduced microbiota growth and Zika virus infection. Moreover, we provide evidence that AeNrf2 signaling also controls transcriptional adaptation of A. aegypti to insecticide challenge. Therefore, we conclude that Nrf2-mediated response regulates assorted gene clusters in A. aegypti that determine cellular and midgut redox balance, affecting overall xenobiotic resistance and vectorial adaptation of the mosquito.

Liaisons dangereuses: cross-border gene flow and dispersal of insecticide resistance-associated genes in the mosquito Aedes aegypti from Brazil and French Guiana.

BACKGROUND: In recent years, South America has suffered the burden of continuous high impact outbreaks of dengue, chikungunya and Zika. Aedes aegypti is the main mosquito vector of these arboviruses and its control is the only solution to reduce transmission. OBJECTIVES: In order to improve vector control it is essential to study mosquito population genetics in order to better estimate the population structures and the geneflow among them. METHODS: We have analysed microsatellites and knockdown resistance (kdr) mutations from a trans-border region in Amazonia between the state of Amapá (Brazil) and French Guiana (overseas territory of France), to provide further knowledge on these issues. These two countries have followed distinct vector control policies since last century. For population genetic analyses we evaluated variability in 13 well-established microsatellites loci in Ae. aegypti from French Guiana (Saint Georges and Cayenne) and Brazil (Oiapoque and Macapá). The occurrence and frequency of kdr mutations in these same populations were accessed by TaqMan genotype assays for the sites 1016 (Val/Ile) and 1534 (Phe/Cys). FINDINGS: We have detected high levels of gene flow between the closest cross-border samples of Saint-Georges and Oiapoque. These results suggest one common origin of re-colonisation for the populations of French Guiana and Oiapoque in Brazil, and a different source for Macapá, more similar to the other northern Brazilian populations. Genotyping of the kdr mutations revealed distinct patterns for Cayenne and Macapá associated with their different insecticide use history, and an admixture zone between these two patterns in Saint Georges and Oiapoque, in accordance with population genetic results. MAIN CONCLUSIONS: The present study highlights the need for regional-local vector surveillance and transnational collaboration between neighboring countries to assess the impact of implemented vector control strategies, promote timely actions and develop preparedness plans.

Evaluation of insecticide resistance in Aedes aegypti populations connected by roads and rivers: the case of Tocantins state in Brazil.

BACKGROUND: The longstanding application of insecticides for vector control without periodic monitoring of the population response to these chemicals can directly drive the selection of resistant populations of vector mosquitoes. Tocantins is the newest state of the Brazilian Federation. Despite a historically high number of dengue cases, studies and monitoring data concerning insecticide resistance in the state are lacking. OBJECTIVES: To verify the resistance status of Aedes aegypti from 10 populations distributed throughout the state connected by rivers and roads. METHODS: Between 50 and 150 ovitraps were installed in house gardens within each municipality. Collection points were established based on the importance of the towns and on geographic aspects. Dose response bioassays were performed in accordance with World Health Organization guidelines. Molecular assays were conducted to detect kdr mutations, which are related to pyrethroid resistance. FINDINGS: Of the 3,200 ovitrap paddles analysed, 25.8% contained eggs, with a total of 55,687 eggs collected. With the exception of Caseara, all evaluated populations were considered to be resistant to temephos. The data showed different levels of resistance to deltamethrin among the samples. Caseara and Guaraí showed the lowest RR95 values. On average, the NaVR1 kdr allele was most frequent (40.3%), followed by NaVS (38.1%), and NaVR2 (21.6%). Palmas, the capital of the state, had the highest frequency of kdr alleles (87.5%). MAIN CONCLUSIONS: With the exception of Palmas, the towns with the highest indexes (ovitrap positivity, number and density of eggs), as well with high levels of resistance and kdr alleles were located along the BR-153 road, indicating that the flow of people and cargo can contribute to the dispersion of the vector and potentially resistance. This study contributes substantially to knowledge regarding the insecticide resistance profile of Tocantins mosquito populations; the data generated via the study could facilitate the judicious use of insecticides by vector control programs.

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Impact of SumiLarv® 2MR on Aedes aegypti larvae: a multicenter study in Brazil.

BACKGROUND: Aedes aegypti is associated with dengue, Zika, and chikungunya transmission. These arboviruses are responsible for national outbreaks with severe public health implications. Vector control is one of the tools used to prevent mosquito proliferation, and SumiLarv® 2MR is an alternative commercial product based on pyriproxyfen for larval/pupal control. In this study, the residual effectiveness of SumiLarv® 2MR in different regions of Brazil was evaluated in simulated field conditions. METHODS: We conducted a multicenter study across four Brazilian states-Amapá, Pernambuco, Rio de Janeiro, and São Paulo-given the importance to the country's climatic variances in the north, northeast, and southeast regions and their influence on product efficiency. The populations of Ae. aegypti from each location were held in an insectary. Third-instar larvae (L3) were added every 2 weeks to water containers with SumiLarv® 2MR discs in 250-, 500- and 1000-l containers in Amapá and Rio de Janeiro, and 100-l containers in Pernambuco and São Paulo, using concentrations of 0.04, 0.08, and 0.16 mg/l. RESULTS: Adult emergence inhibition over 420 days was observed in all tests conducted at a concentration of 0.16 mg/l; inhibition for 308-420 days was observed for 0.08 mg/l, and 224-420 days for 0.04 mg/l. CONCLUSIONS: Sumilarv® 2MR residual activity demonstrated in this study suggests that this new pyriproxyfen formulation is a promising alternative for Aedes control, regardless of climatic variations and ideal concentration, since the SumiLarv® 2MR showed adult emergence inhibition of over 80% and residual activity greater than 6 months, a period longer than that recommended by the Ministry of Health of Brazil between product re-application in larval breeding sites.

High Frequencies of kdr Mutation and Chikungunya Infection in Aedes aegypti Population from Minas Gerais, Brazil.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

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.

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.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos.

In Brazil, decades of dengue vector control using organophosphates and pyrethroids have led to dissemination of resistance. Although these insecticides have been employed for decades against Aedes aegypti in the country, knowledge of the impact of temephos resistance on vector viability is limited. We evaluated several fitness parameters in two Brazilian Ae. aegypti populations, both classified as deltamethrin resistant but with distinct resistant ratios (RR) for temephos. The insecticide-susceptible Rockefeller strain was used as an experimental control. The population presenting the higher temephos resistance level, Aparecida de Goiânia, state of Goiás (RR(95) of 19.2), exhibited deficiency in the following four parameters: blood meal acceptance, amount of ingested blood, number of eggs and frequency of inseminated females. Mosquitoes from Boa Vista, state of Roraima, the population with lower temephos resistance level (RR(95) of 7.4), presented impairment in only two parameters, blood meal acceptance and frequency of inseminated females. These results indicate that the overall fitness handicap was proportional to temephos resistance levels. However, it is unlikely that these disabilities can be attributed solely to temephos resistance, since both populations are also resistant to deltamethrin and harbour the kdr allele, which indicates resistance to pyrethroids. The effects of reduced fitness in resistant populations are discussed.

Enhanced procedures for mosquito identification by MALDI-TOF MS.

BACKGROUND: In the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling. METHODS: Three homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes. RESULTS: Relevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database. CONCLUSION: The results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.

Genome-wide Association Study Reveals New Loci Associated With Pyrethroid Resistance in Aedes aegypti.

Genome-wide association studies (GWAS) use genetic polymorphism across the genomes of individuals with distinct characteristics to identify genotype-phenotype associations. In mosquitoes, complex traits such as vector competence and insecticide resistance could benefit from GWAS. We used the Aedes aegypti 50k SNP chip to genotype populations with different levels of pyrethroid resistance from Northern Brazil. Pyrethroids are widely used worldwide to control mosquitoes and agricultural pests, and their intensive use led to the selection of resistance phenotypes in many insects including mosquitoes. For Ae. aegypti, resistance phenotypes are mainly associated with several mutations in the voltage-gated sodium channel, known as knockdown resistance (kdr). We phenotyped those populations with the WHO insecticide bioassay using deltamethrin impregnated papers, genotyped the kdr alleles using qPCR, and determined allele frequencies across the genome using the SNP chip. We identified single-nucleotide polymorphisms (SNPs) directly associated with resistance and one epistatic SNP pair. We also observed that the novel SNPs correlated with the known kdr genotypes, although on different chromosomes or not in close physical proximity to the voltage gated sodium channel gene. In addition, pairwise comparison of resistance and susceptible mosquitoes from each population revealed differentiated genomic regions not associated with pyrethroid resistance. These new bi-allelic markers can be used to genotype other populations along with kdr alleles to understand their worldwide distribution. The functional roles of the genes near the newly discovered SNPs require new studies to determine if they act synergistically with kdr alleles or reduce the fitness cost of maintaining resistant alleles.

On the use of inhibitors of 4-hydroxyphenylpyruvate dioxygenase as a vector-selective insecticide in the control of mosquitoes.

BACKGROUND: Blood-sucking insects incorporate many times their body weight of blood in a single meal. Because proteins are the major component of vertebrate blood, its digestion in the gut generates extremely high concentrations of free amino acids. Previous reports showed that the tyrosine degradation pathway plays an essential role in adapting these animals to blood feeding. Inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD), the rate-limiting step of tyrosine degradation, results in the death of insects after a blood meal. Therefore, it has been suggested that compounds that block the catabolism of tyrosine could act selectively on blood-feeding insects. Here, we evaluated the toxicity against mosquitoes of three HPPD inhibitors currently used as herbicides and in human health. RESULTS: Of the compounds tested, nitisinone (NTBC) proved to be more potent than mesotrione (MES) and isoxaflutole (IFT) in Aedes aegypti. NTBC was lethal to Ae. aegypti in artificial feeding assays [median lethal dose (LD50 ): 4.53 µm] and in topical application (LD50 : 0.012 nmol/mosquito). NTBC was also lethal to Ae. aegypti populations that were resistant to neurotoxic insecticides, and to other mosquito species (Anopheles and Culex). CONCLUSION: HPPD inhibitors, particularly NTBC, represent promising new drugs for mosquito control. Because they affect only blood-feeding organisms, they represent a safer and more environmentally friendly alternative to conventional neurotoxic insecticides. © 2021 Society of Chemical Industry.

First national-scale evaluation of temephos resistance in Aedes aegypti in Peru.

BACKGROUND: The development of resistance against insecticides in Aedes aegypti can lead to operational failures in control programs. Knowledge of the spatial and temporal trends of this resistance is needed to drive effective monitoring campaigns, which in turn provide data on which vector control decision-making should be based. METHODS: Third-stage larvae (L3) from the F1 and F2 generations of 39 Peruvian field populations of Ae. aegypti mosquitoes from established laboratory colonies were evaluated for resistance against the organophosphate insecticide temephos. The 39 populations were originally established from eggs collected in the field with ovitraps in eight departments of Peru during 2018 and 2019. Dose-response bioassays, at 11 concentrations of the insecticide, were performed following WHO recommendations. RESULTS: Of the 39 field populations of Ae. aegypti tested for resistance to temephos , 11 showed high levels of resistance (resistance ratio [RR] > 10), 16 showed moderate levels of resistance (defined as RR values between 5 and 10) and only 12 were susceptible (RR < 5). The results segregated the study populations into two geographic groups. Most of the populations in the first geographic group, the coastal region, were resistant to temephos, with three populations (AG, CR and LO) showing RR values > 20 (AG 21.5, CR 23.1, LO 39.4). The populations in the second geographic group, the Amazon jungle and the high jungle, showed moderate levels of resistance, with values ranging between 5.1 (JN) and 7.1 (PU). The exception in this geographic group was the population from PM, which showed a RR value of 28.8 to this insecticide. CONCLUSIONS: The results of this study demonstrate that Ae. aegypti populations in Peru present different resistance intensities to temephos, 3 years after temephos use was discontinued. Resistance to this larvicide should continue to be monitored because it is possible that resistance to temephos could decrease in the absence of routine selection pressures.

Population genetics of an invasive mosquito vector, Aedes albopictus in the Northeastern USA.

The Asian tiger mosquito (Aedes albopictus) arrived in the USA in the 1980's and rapidly spread throughout eastern USA within a decade. The predicted northern edge of its overwintering distribution on the East Coast of the USA roughly falls across New York, Connecticut, and Massachusetts, where the species has been recorded as early as 2000. It is unclear whether Ae. albopictus populations have become established and survive the cold winters in these areas or are recolonized every year. We genotyped and analyzed populations of Ae. albopictus from the northeast USA using 15 microsatellite markers and compared them with other populations across the country and to representatives of the major global genetic clades to investigate their connectivity and stability. Founder effects or bottlenecks were rare at the northern range of the Ae. albopictus distribution in the northeastern USA, with populations displaying high levels of genetic diversity and connectivity along the East Coast. There is no evidence of population turnover in Connecticut during the course of three consecutive years, with consistent genetic structure throughout this period. Overall, these results support the presence of established populations of Ae. albopictus in New York, Connecticut, and Massachusetts, successfully overwintering and migrating in large numbers. Given the stability and interconnectedness of these populations, Ae. albopictus has the potential to continue to proliferate and expand its range northward under mean warming conditions of climate change. Efforts to control Ae. albopictus in these areas should thus focus on vector suppression rather than eradication strategies, as local populations have become firmly established and are expected to reemerge every summer.

Insecticide resistance and genetic structure of Aedes aegypti populations from Rio de Janeiro State, Brazil.

Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Aedes aegypti populations' susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations. All RJ Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.

Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan.

Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha-cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression.

Correction: Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans.

[This corrects the article DOI: 10.1371/journal.pntd.0005625.].