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.

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.

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.

Evaluation of Long-lasting insecticidal nets (LLINs) for malaria control in an endemic area in Brazil.

BACKGROUND: Most cases of malaria in Brazil are concentrated in the Amazon region. One of the vector control alternatives recommended by the WHO is the long-lasting insecticidal net (LLIN). This tool is used in the nine federal states of the Brazilian Legal Amazon, where LLINs are essential for reducing vector density and disease transmission as they prevent contact between the mosquito and the individual. The objective of this study was to evaluate the residuality and use of LLIN insecticides in different health regions in a city located in the Brazilian Amazon. METHODS: A total of 17,027 LLINs were installed in the third, fifth and ninth health regions of the municipality of Porto Velho, Rondonia State, Brazil. The LLINs were of two types: Olyset (permethrin), for around the bed, and Interceptor (alphacypermethrin), for around hammocks. The residuality of 172 LLINs was evaluated using cone bioassays to verify the mortality rate of the mosquito Nyssorhynchus darlingi, over a period of 2 years. Structured questionnaires on the acceptance and use of LLINs were distributed to the participating population (n = 391), covering a total sample of 1147 mosquito nets. The mortality rate was evaluated both in terms of days after LLIN installation and the type of insecticide used. Statistical analyses were based on analysis of variance (ANOVA) and Chi-square and were performed using the SPSS statistical program. RESULTS: For the Ny. darlingi mosquito, Interceptor-type LLINs showed residual efficacy, with mortality rates ≥ 80% during the 2-year study period, as determined by the WHO. In contrast, Olyset-type LLINs were associated with a reduction in mortality rates, with 76% and 45% mortality rates in the last two assessments, which occurred during the last 6 months of the study period. Based on the structured questionnaires, the acceptance rate, i.e. percentage of individuals accepting the permanence of the 1147 LLINs sampled, in the three health regions of Porto Velho was 93.8% (of 1076 LLINs). CONCLUSION: The alphacypermethrin-impregnated LLIN was more effective than the LLIN impregnated with permethrin. The results indicate that the correct use of mosquito nets-and consequently the protection of the population-needs to be supported by health promotion actions. These initiatives are considered to be essential for the success of this vector control strategy. New studies that consider the monitoring of the placement of mosquito nets are necessary to provide effective support in the correct use of this methodology.

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.

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.

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.

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.

High larvicidal efficacy of yeast-encapsulated orange oil against Aedes aegypti strains from Brazil.

BACKGROUND: Botanical substances such as essential oils (EOs) have demonstrated insecticidal properties and are a valid option for vector control. However, free EOs are unreliable as mosquito larvicides due their easy degradation by environmental exposure to ultraviolet light and higher temperatures. Here, we assessed the efficacy of a mosquito larvicide based on orange oil in a yeast-based delivery system against Aedes aegypti strains with different resistance status towards chemical neurotoxic insecticides. This larvicide preparation was physicochemically characterized in a previous report. METHODS: Larvae of four Ae. aegypti strains from different regions of Brazil and different resistance profiles for deltamethrin (pyrethroid) and temephos (organophosphate) were tested against yeast-encapsulated orange oil (YEOO) in laboratory conditions for measurement of LC50 and LC90 values. The same assays were performed with the Belo Horizonte strain under environmental conditions (natural light and temperature). The resistance profiles of these strains were compared to the Rockefeller reference strain in all conditions. RESULTS: YEOO was found to be a highly active larvicide (LC50 < 50 mg/L) against all Ae. aegypti strains tested in both laboratory conditions (LC50 = 8.1-24.7 mg/L) and environmental conditions with natural light and temperature fluctuation (LC50 = 20.0-49.9 mg/L). Moreover, all strains were considered susceptible (RR < 5) to YEOO, considering resistance ratios calculated based on the Rockefeller strain. The resistance ratios were only higher than 2.5 for LC90-95 of Belo Horizonte in the laboratory, probably due the higher heterogeneity associated with older egg papers (> 5 months). CONCLUSION: YEOO demonstrates high larvicidal activity against Ae. aegypti strains with resistant phenotypes for deltamethrin (PY) and temephos (OP). This larvicidal activity suggests the potential for the development of YEOO as an alternative intervention to synthetic insecticides in integrated vector management programs, for populations with resistance to commonly used insecticides.

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.

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.

Effect of Larval Food Availability on Adult Aedes Aegypti (Diptera: Culicidae) Fitness and Susceptibility to Zika Infection.

Aedes (Stegomyia) aegypti (Linnaeus, 1762) is a mosquito species of significant medical importance. The use of this vector in research studies usually requires a large number of mosquitoes as well as rearing and maintenance in a laboratory-controlled environment. However, laboratory conditions may be different from field environments, presenting stressful challenges such as low food concentration, especially during larval stages, which may, in turn, impair vector biology. Therefore, we tested herein if larval food availability (0.004, 0.009, 0.020, and 0.070% diets) would affect overall adult insect fitness. We observed slower development in mosquitoes fed a 0.004% diet 15 d post-eclosion (DPE) and shorter mean time in mosquitoes fed a 0.020% diet (7 DPE). Larval diet and adult mosquito weight were positively correlated, and heavier females fed higher larval diets exhibited greater blood feeding capacity and oviposition. In addition, larval diet concentrations led to median adult lifespan variations (male/female in days-0.004%: 30 ± 1.41, 45 ± 1.3; 0.009%: 31.5 ± 1.33, 41 ± 1.43; 0.020%: 26 ± 1.18, 41 ± 1.45; 0.070%: 29 ± 1.07, 44 ± 1.34), reduced tolerance to deltamethrin (1 mg/m2) and changes in detoxification enzyme activities. Moreover, in the larval 0.070% diet, females presented higher Zika susceptibility (plaque-forming unit [PFU]: 1.218 × 106) compared with other diets (0.004%: 1.31 × 105; 0.009%: 2.0 × 105; 0.020%: 1.25 × 105 PFU). Altogether, our study demonstrates that larval diet restriction results not only in larval developmental arrest but also in adult fitness impairment, which must be considered in future assessments.

Assessing the efficacy of two new formulations of larvicide pyriproxyfen for the control of Aedes aegypti using dissemination stations in two sites of Rio de Janeiro city.

BACKGROUND: Aedes aegypti is the primary transmitter of several arbovirus with great impact in human health. Controlling vector mosquitoes is an essential and complex task. One promising control method is to use mosquitoes as a vehicle to disseminate tiny particles of juvenile-killing insecticides, such as pyriproxyfen (PPF), to breeding sites. OBJECTIVES: We aimed to investigate the capacity of Ae. aegypti to disseminate two new formulations of PPF in two sites of Rio de Janeiro city for assessment of the efficacy of these products. METHODS: Dissemination stations impregnated with powder and liquid new formulations of PPF were installed in two test sites. Ovitraps were used in the test sites and in a control site for monitoring the presence of Ae. aegypti throughout eggs collection. FINDINGS: Entomological indices indicated that the new formulations of PPF were efficient in reducing eggs abundance. Liquid formulation performed better than powder formulation. Ready-to-use formulations of PPF can be quickly applied in the field and can be replaced after a few months. MAIN CONCLUSIONS: New formulations of PPF associated with mosquito dissemination approach make a valuable vector control strategy, managing to cover places of difficult access for whatever reason. New formulations application requires less labour, being economically attractive.

Assessment of the susceptibility status of Aedes aegypti (Diptera: Culicidae) populations to pyriproxyfen and malathion in a nation-wide monitoring of insecticide resistance performed in Brazil from 2017 to 2018.

BACKGROUND: Chemical mosquito control using malathion has been applied in Brazil since 1985. To obtain chemical control effectiveness, vector susceptibility insecticide monitoring is required. This study aimed to describe bioassay standardizations and determine the susceptibility profile of Ae. aegypti populations to malathion and pyriproxyfen, used on a national scale in Brazil between 2017 and 2018, and discuss the observed impacts in arbovirus control. METHODS: The diagnostic-doses (DD) of pyriproxyfen and malathion were determined as the double of adult emergence inhibition (EI) and lethal doses for 99% of the Rockefeller reference strain, respectively. To monitor natural populations, sampling was performed in 132 Brazilian cities, using egg traps. Colonies were raised in the laboratory for one or two generations (F1 or F2) and submitted to susceptibility tests, where larvae were exposed to the pyriproxyfen DD (0.03 µg/l) and adults, to the malathion DD determined in the present study (20 µg), in addition to the one established by the World Health Organization (WHO) DD (50 µg) in a bottle assay. Dose-response (DR) bioassays with pyriproxyfen were performed on populations that did not achieve 98% EI in the DD assays. RESULTS: Susceptibility alterations to pyriproxyfen were recorded in six (4.5%) Ae. aegypti populations from the states of Bahia and Ceará, with Resistance Ratios (RR95) ranging from 1.51 to 3.58. Concerning malathion, 73 (55.3%) populations distributed throughout the country were resistant when exposed to the local DD 20 µg/bottle. On the other hand, no population was resistant, and only 10 (7.6%) populations in eight states were considered as exhibiting decreased susceptibility (mortality ratios between 90 and 98%) when exposed to the WHO DD (50 µg/bottle). CONCLUSIONS: The feasibility of conducting an insecticide resistance monitoring action on a nation-wide scale was confirmed herein, employing standardized and strongly coordinated sampling methods and laboratory bioassays. Brazilian Ae. aegypti populations exhibiting decreased susceptibility to pyriproxyfen were identified. The local DD for malathion was more sensitive than the WHO DD for early decreased susceptibility detection.

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

Insecticide resistance is currently a threat to the control of Aedes agypti, the main vector of arboviruses in urban centers. Mutations in the voltage gated sodium channel (NaV), known as kdr (knockdown resistance), constitute an important selection mechanism for resistance against pyrethroids. In the present study, we investigated the kdr distribution for the Val1016Ile and Phe1534Cys alterations in Ae. aegypti from 123 Brazilian municipalities, based on SNP genotyping assays in over 5,500 mosquitoes. The alleles NaVS (1016Val+ + 1534Phe+), NaVR1 (1016Val+ + 1534Cyskdr) and NaVR2 (1016Ilekdr + 1534Cyskdr) were consistently observed, whereas kdr alleles have rapidly spread and increased in frequency. NaVS was the less frequent allele, mostly found in Northeastern populations. The highest allelic frequencies were observed for NaVR1, especially in the North, which was fixed in one Amazonian population. The double kdr NaVR2 was more prevalent in the Central-west and South-eastern populations. We introduce the 'kdr index', which revealed significant spatial patterns highlighting two to three distinct Brazilian regions. The 410L kdr mutation was additionally evaluated in 25 localities, evidencing that it generally occurs in the NaVR2 allele. This nationwide screening of a genetic mechanism for insecticide resistance is an important indication on how pyrethroid resistance in Ae. aegypti is evolving in Brazil.

Insecticide resistance, fitness and susceptibility to Zika infection of an interbred Aedes aegypti population from Rio de Janeiro, Brazil.

BACKGROUND: Aedes aegypti is a vector of high relevance, since it transmits several arboviruses, including dengue, chikungunya and Zika. Studies on vector biology are usually conducted with laboratory strains presenting a divergent genetic composition from field populations. This may impair vector control policies that were based on laboratory observations employing only long maintained laboratory strains. In the present study we characterized a laboratory strain interbreed with Ae. aegypti collected from five different localities in Rio de Janeiro (Aedes Rio), for insecticide resistance (IR), IR mechanisms, fitness and Zika virus infection. METHODS: We compared the recently established Aedes Rio with the laboratory reference strain Rockefeller. Insecticide resistance (deltamethrin, malathion and temephos), activity of metabolic resistance enzymes and kdr mutation frequency were determined. Some life table parameters (longevity, blood-feeding, number and egg viability) and Zika virus susceptibility was also determined. RESULTS: Aedes Rio showed resistance to deltamethrin (resistance ratio, RR50 = 32.6) and temephos (RR50 = 7.0) and elevated activity of glutathione S-transferase (GST) and esterases (α-EST and pNPA-EST), but not acetylcholinesterase (AChE). In total, 92.1% of males genotyped for kdr presented a "resistant" genotype. Weekly blood-fed females from both strains, presented reduced mortality compared to sucrose-fed mosquitoes; however, Aedes Rio blood-fed females did not live as long (mean lifespan: Rockefeller = 70 ± 3.07; Aedes Rio = 53.5 ± 2.16 days). There were no differences between strains in relation to blood-feeding and number of eggs, but Aedes Rio eggs presented reduced viability (mean hatch: Rockefeller = 77.79 ± 1.4%; Aedes Rio = 58.57 ± 1.77%). Zika virus infection (plaque-forming unit, PFU) was similar in both strains (mean PFU ± SE: Aedes Rio: 4.53 × 104 ± 1.14 × 104 PFU; Rockefeller: 2.02 × 104 ± 0.71 × 104 PFU). CONCLUSION: Selected conditions in the field, such as IR mechanisms, may result in pleiotropic effects that interfere in general physiology of the insect. Therefore, it is important to well characterize field populations to be tested in parallel with laboratory reference strains. This practice would improve the significance of laboratory tests for vector control methods.

Evaluation of resistance to pyrethroid and organophosphate adulticides and kdr genotyping in Aedes aegypti populations from Roraima, the northernmost Brazilian State.

BACKGROUND: Roraima, the northernmost State in Brazil, borders Venezuela and Guyana. Although mostly covered by the tropical forests, the urban centers of this state are highly infested with Ae. aegypti and are endemic for dengue, Zika and chikungunya. We accessed the insecticide resistance status of Ae. aegypti populations from the capital Boa Vista, two cities on international borders (Pacaraima and Bonfim) and Rorainópolis bordering Amazonas State, in order to evaluate the chemical control efficacy in these localities. METHODS: Tests with World Health Organization (WHO)-like tubes impregnated with the pyrethroid deltamethrin (0.05% and 0.12%) and the organophosphate malathion (0.7%) were conducted with Ae. aegypti from Boa Vista, Pacaraima, Bonfim and Rorainópolis, collected in 2016 and 2018. Genotyping of kdr mutations, related to resistance to pyrethroids, was performed for the SNP variations at sites 1016 and 1534 of the voltage gated sodium channel gene (NaV) with a TaqMan qPCR approach. RESULTS: Aedes albopictus was absent in our collections, and therefore only Ae. aegypti was tested. All Ae. aegypti populations were susceptible to 0.7% malathion in 2016; however, mortality dropped to under 90% in Bonfim and Pacaraima populations in 2018. All populations were resistant to 0.05% deltamethrin in both years. The time that 50% of females suffered knockdown (KdT50) under exposure to 0.05% deltamethrin was 3.3-5.9-fold longer in mosquitoes from the natural populations compared to the susceptible Rockefeller strain. Only the Pacaraima population (2018) remained resistant to 0.12% deltamethrin. Kdr genotyping revealed the absence of the wild-type NaVS haplotype (1016Val + 1534Phe) in the populations from Roraima, indicating that all tested insects had a genetic background for pyrethroid resistance. The double kdr NaVR2 haplotype (1016Ile + 15434Cys) was present in higher frequencies in all populations except for Rorainópolis, where this haplotype seems to have arrived recently. CONCLUSIONS: These results are important for the knowledge about insecticide resistance status of Ae. aegypti populations from Roraima and will help improve vector control strategies that may be applied to diverse localities under similar geographical and urban conditions.

Action of Metarhizium brunneum (Hypocreales: Clavicipitaceae) Against Organophosphate- and Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) and the Synergistic Effects of Phenylthiourea.

Dengue, yellow fever, Zika, and chikungunya arboviruses are endemic in tropical countries and are transmitted by Aedes aegypti. Resistant populations of this mosquito against chemical insecticides are spreading worldwide. This study aimed to evaluate the biological effects of exposure of pesticide-sensitive Ae. aegypti larvae (Rockefeller) to conidia of the entomopathogen, Metarhizium brunneum, laboratory strains ARSEF 4556 and V275, and any synergistic activity of phenylthiourea (PTU). In addition, to investigate the nature of any cross-resistance mechanisms, these M. brunneum strains were tested against the Rockefeller larvae and two temephos- and deltamethrin-resistant wild mosquito populations from Rio de Janeiro. Treatment of Rockefeller larvae with 106 conidia/ml of ARSEF 4556 and V275 fungal strains resulted in significant decreased survival rates to 40 and 53.33%, respectively (P < 0.0001), compared with untreated controls. In contrast, exposure to 104 or 105 conidia/ml showed no such significant survival differences. However, the addition of PTU to the conidia in the bioassays significantly increased mortalities in all groups and induced a molt block. Experiments also showed no differences in Ae. aegypti mortalities between the fungal treated, wild pesticide-resistant populations and the Rockefeller sensitive strain. The results show the efficacy of M. brunneum in controlling Ae. aegypti larvae and the synergistic role of PTU in this process. Importantly, there was no indication of any cross-resistance mechanisms between Ae. aegypti sensitive or resistant to pesticides following treatment with the fungi. These results further support using M. brunneum as an alternative biological control agent against mosquito populations resistant to chemical insecticides.

Insecticide resistance in Culex quinquefasciatus Say, 1823 in Brazil: a review.

Culex quinquefasciatus is a successful invasive species broadly distributed in subtropical regions, including Brazil. It is an extremely annoying mosquito due to its nocturnal biting behavior, in high-density populations and it is a potential bridge between sylvatic arbovirus from birds to man in urban territories. Herein, we present a review concerning the methods of chemical control employed against Cx. quinquefasciatus in Brazil since the 1950's and insecticide resistance data registered in the literature. As there is no specific national programme for Cx. quinquefasciatus control in Brazil, the selection of insecticide resistance is likely due in part to the well-designed chemical campaigns against Aedes aegypti and the elevated employment of insecticides by households and private companies. There are very few publications about insecticide resistance in Cx. quinquefasciatus from Brazil when compared to Ae. aegypti. Nevertheless, resistance to organophosphates, carbamate, DDT, pyrethroids and biolarvicides has been registered in Cx. quinquefasciatus populations from distinct localities of the country. Concerning physiological mechanisms selected for resistance, distinct patterns of esterases, as well as mutations in the acetylcholinesterase (ace-1) and voltage-gated sodium channel (NaV) genes, have been identified in natural populations. Given environmental changes and socioeconomical issues in the cities, in recent years we have been experiencing an increase in the number of disease cases caused by arboviruses, which may involve Cx. quinquefasciatus participation as a key vector. It is urgent to better understand the efficiency and susceptibility status to insecticides, as well as the genetic background of known resistant mechanisms already present in Cx. quinquefasciatus populations for an effective and rapid chemical control when eventually required.

Evaluation of the Persistence of Three Larvicides Used To Control Aedes aegypti In Arapiraca, Northeastern Brazil.

The chemical control of the mosquito Aedes aegypti is a great challenge worldwide, since several populations of this species are already resistant to traditional insecticides, such as temephos. In Brazil, alternative larvicides, such as Bacillus thuringiensis israelensis (Bti) and pyriproxyfen, have been used more recently. In this study we evaluated the persistence of pyriproxyfen (Sumilarv 0.5%G), 2 commercial formulations of Bti (Vectobac WDG and Vectobac G), and temephos (Fersol 1G) under field and simulated field conditions with treatments exposed to sun and shadow. In the field tests, the 2 formulations of Bti presented less persistence in the 8th wk of evaluation (46% and 37% positivity) compared with temephos (3.6% and 6.8% positivity) and Sumilarv (6.6% and 3.8% positivity) in containers exposed to the sun and shadow, respectively. In the simulated field trial, temephos and the 2 formulations of Bti presented high persistence (100% mortality at 8th wk) when applied in the water box and in deposits placed in the shade. In containers exposed to the sun, the persistence of these products was lower (>80% mortality by temephos after 4 wk, Bti formulations for 3 wk, and Sumilarv for 6 wk). Based on these data, however, Sumilarv presented better performance in the containers exposed to the sun than the 2 formulations of Bti (Vectobac G and Vectobac WDG), which were affected by the sun.