Both Aedes aegytpi and Ae. albopictus are major vectors of 5 important arboviruses (namely chikungunya virus, dengue virus, Rift Valley fever virus, yellow fever virus, and Zika virus), making these mosquitoes an important factor in the worldwide burden of infectious disease. Vector control using insecticides coupled with larval source reduction is critical to control the transmission of these viruses to humans but is threatened by the emergence of insecticide resistance. Here, we review the available evidence for the geographical distribution of insecticide resistance in these 2 major vectors worldwide and map the data collated for the 4 main classes of neurotoxic insecticide (carbamates, organochlorines, organophosphates, and pyrethroids). Emerging resistance to all 4 of these insecticide classes has been detected in the Americas, Africa, and Asia. Target-site mutations and increased insecticide detoxification have both been linked to resistance in Ae. aegypti and Ae. albopictus but more work is required to further elucidate metabolic mechanisms and develop robust diagnostic assays. Geographical distributions are provided for the mechanisms that have been shown to be important to date. Estimating insecticide resistance in unsampled locations is hampered by a lack of standardisation in the diagnostic tools used and by a lack of data in a number of regions for both resistance phenotypes and genotypes. The need for increased sampling using standard methods is critical to tackle the issue of emerging insecticide resistance threatening human health. Specifically, diagnostic doses and well-characterised susceptible strains are needed for the full range of insecticides used to control Ae. aegypti and Ae. albopictus to standardise measurement of the resistant phenotype, and calibrated diagnostic assays are needed for the major mechanisms of resistance.

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

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

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The classification of esterases: an important gene family involved in insecticide resistance - A review

Organophosphates and pyrethroids are used widely in Brazil to control Aedes aegypti, the main vector of dengue viruses, under the auspices of the National Programme for Dengue Control. Resistance to these insecticides is widespread throughout Brazil. In Ceara the vector is present in 98% of districts and resistance to temephos has been reported previously. Here we measure resistance to temephos and the pyrethroid cypermethrin in three populations from Ceara and use biochemical and molecular assays to characterise resistance mechanisms. Resistance to temephos varied widely across the three studied populations, with resistance ratios (RR95) of 7.2, 30 and 192.7 in Juazeiro do Norte, Barbalha and Crato respectively. The high levels of resistance detected in Barbalha and Crato (RR95 ≥ 30) imply a reduction of temephos efficacy, and indeed in simulated field tests reduced effectiveness was observed for the Barbalha population. Two populations (Crato and Barbalha) were also resistant to cypermethrin, whilst Juazeiro do Norte showed only an altered susceptibility. The Ile1011Met kdr mutation was detected in all three populations and Val1016Ile in Crato and Juazeiro do Norte. 1011Met was significantly associated with resistance to cypermethrin in the Crato population. Biochemical tests showed that only the activity of esterases and GSTs, among the tested detoxification enzymes, was altered in these populations when compared with the Rockefeller strain. Our results demonstrate that two A. aegypti populations from Ceara are under strong selection pressure by temephos, compromising the field effectiveness of this organophosphate. Our results also provide evidence that the process of reducing resistance to this larvicide in the field is difficult and slow and may require more than seven years for reversal. In addition, we show resistance to cypermethrin in two of the three populations studied, and for the first time the presence of the allele 1016Ile in mosquito populations from northeastern Brazil. A significant association between 1011M et and resistance was observed in one of the populations. Target-site mechanisms seem not to be implicated in temephos resistance, reinforcing the idea that for the studied populations, detoxification enzymes most likely play a major role in the resistance to this insecticide.

/pdf/insecticide-resistance-in-aedes-aegypti-populations-from-3h3lde499k.pdf

Insecticide resistance in Aedes aegypti populations from Ceará, Brazil

Background: Most national dengue control programmes rely extensively on insecticides to control the mosquito vectors of this disease.
Objectives: The objective of this review is to describe current knowledge of the extent of insecticide resistance in dengue vectors and the potential impact of this resistance on control activities.
Methods: We searched Web of Science and PubMed for studies that included data on resistance to the four major classes of insecticides: organochlorines, carbamates, organophosphates and pyrethroids, in the dengue vectors Aedes aegypti and Aedes albopictus. Insecticide bioassy data were extracted from the published literature and the methods used to obtain, analyse and interpret this data were critically evaluated. Emphasis was placed on the two insecticide classes most widely used in dengue control, organophosphates and pyrethroids. The use of biochemical and molecular tools for resistance monitoring was also reviewed.
Results: 66 studies met our inclusion criteria and were uploaded on to a public databse (IRBase). There is a stong geographical bias in published studies with nearly half originating from three countries (Thailand, India and Brazil). Bioassay data demonstrates that resistance to the organophosphate temephos and to pyrethroids is widespread in Ae. aegypti and resistance has also been reported in Ae. albopictus. Assessing the impact of insecticide resistance on vector control is complicated by variations in the methodology used to measure and report resistance, and by the lack of studies into the epidemiological consequences of insecticicde resistance.
Conclusions: The lack of publicly accessible standardized data sets dcoumenting levels of insecticide resistance in many dengue endemic countries, and the absence of studies on the operational impact of resistance, preculdes a comprehensive analysis of the current global threat that insecticide resistance poses to dengue control. However, several countries with active resistance monitoring programmes have shown that insecticide resistance is reducing our ability to control dengue vectors. This situation is likely to worsen unless effective strategies are rapidly implemented to mitigate these effects.

Insecticide resistance in dengue vectors

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

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Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost

In Brazil, Aedes aegypti resistance to temephos, used since 1967, was detected in several municipalities in 2000. Organophosphates were substituted by pyrethroids against adults and, in some localities, by Bti against larvae. However, high temephos resistance ratios were still detected between 2001 and 2004. Field-simulated assays confirmed a low temephos residual effect. Acethylcholinesterase and Mixed Function Oxidase profiles were not altered. In contrast, higher Esterase activity, studied with three substrates, was found in all examined populations collected in 2001. From 2001 to 2004, a slight reduction in α-Esterase (EST) and β-EST activity together with a gradual increase of p-nitrophenyl acetate (PNPA)-EST was noted. Gluthathione-S-transferase alteration was encountered only in the northeast region in 2001, spreading the entire country thereafter. In general, except for α-EST and β-EST, only one enzyme class was altered in each mosquito specimen. Data are discussed in the context of historic application of insecticides in Brazil.

Insecticide Resistance Mechanisms of Brazilian Aedes aegypti Populations from 2001 to 2004

Insecticides are still largely applied in public health to control disease vectors. In Brazil, organophosphates (OP) and pyrethroids (PY) are used against Aedes aegypti for years. Since 2009 Insect Growth Regulators (IGR) are also employed in the control of larvae. We quantified resistance to temephos (OP), deltamethrin (PY), and diflubenzuron (IGR) of A. aegypti samples from 12 municipalities distributed throughout the country, collected between 2010 and 2012. High levels of resistance to neurotoxic insecticides were detected in almost all populations: RR95 to temephos varied between 4.0 and 27.1; the lowest RR95 to deltamethrin was 13.1, and values higher than 70.0 were found. In contrast, all samples were susceptible to diflubenzuron (RR95 < 2.3). Biochemical tests performed with larvae and adults discarded the participation of acetylcholinesterase, the OP target, and confirmed involvement of the detoxifying enzymes esterases, mixed function oxidases, and glutathione-S-transferases. The results obtained were discussed taking into account the public chemical control component and the increase in the domestic use of insecticides during dengue epidemic seasons in the evaluated municipalities.

/pdf/resistance-status-to-the-insecticides-temephos-deltamethrin-1irc4z3xra.pdf

Resistance Status to the Insecticides Temephos, Deltamethrin, and Diflubenzuron in Brazilian Aedes aegypti Populations

BACKGROUND Aedes aegypti populations in Brazil have been subjected to insecticide selection pressures with variable levels and sources since 1967. Therefore, the Brazilian Ministry of Health (MoH) coordinated the activities of an Ae. aegypti insecticide resistance monitoring network (MoReNAa) from 1999 to 2012. OBJECTIVES The objective of this study was to consolidate all information available from between 1985 and 2017 regarding the resistance status and mechanisms of Brazilian Ae. aegypti populations against the main insecticide compounds used at the national level, including the larvicide temephos (an organophosphate) and the adulticide deltamethrin (a pyrethroid). METHODS Data were gathered from two sources: a bibliographic review of studies published from 1985 to 2017, and unpublished data produced by our team within the MoReNAa between 1998 and 2012. A total of 146 municipalities were included, many of which were evaluated several times, totalling 457 evaluations for temephos and 274 for deltamethrin. Insecticide resistance data from the five Brazilian regions were examined separately using annual records of both the MoH supply of insecticides to each state and the dengue incidence in each evaluated municipality. FINDINGS Ae. aegypti resistance to temephos and deltamethrin, the main larvicide and adulticide, respectively, employed against mosquitoes in Brazil for a long time, was found to be widespread in the country, although with some regional variations. Comparisons between metabolic and target-site resistance mechanisms showed that one or another of these was the main component of pesticide resistance in each studied population. MAIN CONCLUSIONS (i) A robust dataset on the assessments of the insecticide resistance of Brazilian Ae. aegypti populations performed since 1985 was made available through our study. (ii) Our findings call into question the efficacy of chemical control as the sole methodology of vector control. (iii) It is necessary to ensure that sustainable insecticide resistance monitoring is maintained as a key component of integrated vector management. (iv) Consideration of additional parameters, beyond the supply of insecticides distributed by the MoH or the diverse local dynamics of dengue incidence, is necessary to find consistent correlations with heterogeneous vector resistance profiles.

/pdf/resistance-to-temephos-and-deltamethrin-in-aedes-aegypti-2y8nf6tlo9.pdf

Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017.

In Brazil, insecticide resistance in Stegomyia aegypti (= Aedes aegypti) (Diptera: Culicidae) populations to pyrethroids and to the organophosphate (OP) temephos is disseminated. Currently, insect growth regulators (IGRs) and the OP malathion are employed against larvae and adults, respectively. Bioassays with mosquitoes from two northeast municipalities, Crato and Aracaju, revealed, in both populations, susceptibility to IGRs and malathion (RR95  ≤ 2.0), confirming the effectiveness of these compounds. By contrast, temephos and deltamethrin (pyrethroid) resistance levels were high (RR95  > 10), which is consistent with the use of intense chemical control. In Crato, RR95 values were > 50 for both compounds. Knock-down-resistant (kdr) mutants in the voltage-gated sodium channel, the pyrethroid target site, were found in 43 and 32%, respectively, of Aracaju and Crato mosquitoes. Biochemical assays revealed higher metabolic resistance activity (esterases, mixed function oxidases and glutathione-S-transferases) at Aracaju. With respect to fitness aspects, mating effectiveness was equivalently impaired in both populations, but Aracaju mosquitoes showed more damaging effects in terms of longer larval development, decreased bloodmeal acceptance, reduced engorgement and lower numbers of eggs laid per female. Compared with mosquitoes in Crato, Aracaju mosquitoes exhibited lower OP and pyrethroid RR95 , increased activity of detoxifying enzymes and greater effect on fitness. The potential relationship between insecticide resistance mechanisms and mosquito viability is discussed.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mve.12241

Insecticide resistance, associated mechanisms and fitness aspects in two Brazilian Stegomyia aegypti (= Aedes aegypti) populations

Background 
Resistance to pyrethroids and to the organophosphate temephos is widespread in Brazilian populations of the dengue vector, Aedes aegypti. Thereof, since 2009 Insect Growth Regulators are employed as larvicides, and malathion is used against adults.


Methodology/Principal findings 
We performed laboratory selection with malathion of two A. aegypti field populations initially susceptible to this organophosphate but resistant to temephos and deltamethrin. A fixed malathion dose inducing at least 80% mortality in the first generation, was used throughout the selection process, interrupted after five generations, when the threshold of 20% mortality was reached. For each population, three experimental and two control groups, not exposed to insecticides, were kept independently. For both populations, quantitative bioassays revealed, in the selected groups, acquisition of resistance to malathion and negative impact of malathion selection on deltamethrin and temephos resistance levels. In the control groups resistance to all evaluated insecticides decreased except, unexpectedly, to deltamethrin. Analysis of the main resistance mechanisms employed routine methodologies: biochemical and molecular assays for, respectively, metabolic resistance and quantification of the NaV pyrethroid target main kdr mutations at positions 1016 and 1534. No diagnostic alteration could be specifically correlated with malathion selection, neither with the unusual deltamethrin increase in resistance levels observed in the control groups.


Conclusions/Significance 
Our results confirm the multifactorial character of insecticide resistance and point to the need of high throughput methodologies and to the study of additional field vector populations in order to unravel resistance mechanisms.

/pdf/laboratory-selection-of-aedes-aegypti-field-populations-with-2w9x15i558.pdf

Laboratory selection of Aedes aegypti field populations with the organophosphate malathion: Negative impacts on resistance to deltamethrin and to the organophosphate temephos

Priscila Fernandes Viana-Medeiros

Papers

Insecticide Resistance Mechanisms of Brazilian Aedes aegypti Populations from 2001 to 2004

Resistance Status to the Insecticides Temephos, Deltamethrin, and Diflubenzuron in Brazilian Aedes aegypti Populations

Resistance to temephos and deltamethrin in Aedes aegypti from Brazil between 1985 and 2017.

Insecticide resistance, associated mechanisms and fitness aspects in two Brazilian Stegomyia aegypti (= Aedes aegypti) populations

Laboratory selection of Aedes aegypti field populations with the organophosphate malathion: Negative impacts on resistance to deltamethrin and to the organophosphate temephos