Constância Flávia Junqueira Ayres

Insecticide resistance in Aedes aegypti populations from Ceará, Brazil

BackgroundOrganophosphates 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 Ceará 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 Ceará and use biochemical and molecular assays to characterise resistance mechanisms.ResultsResistance 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.ConclusionsOur results demonstrate that two A. aegypti populations from Ceará 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.

Resistance to the organophosphate temephos: mechanisms, evolution and reversion in an Aedes aegypti laboratory strain from Brazil.

Insecticide resistance is one of the main problems in vector control programs. Because insects have developed resistance to all classes of available chemical insecticides, a proper surveillance and management of resistance in areas where these compounds are being utilized is crucial for the success of control programs. Since the mechanisms and molecular bases of resistance are various, they must be characterized to allow efficient monitoring strategies. Here we report the establishment of an Aedes aegypti strain resistant to temephos, named RecR, selected under laboratory conditions. The parental A. aegypti population was obtained from eggs collected in an area where temephos had been used for 8 years, and presented a baseline resistance ratio (RR) of 7. After 17 generations under selective pressure, the RR has increased to 180. Biochemical assays indicate that metabolic mechanisms are involved on temephos resistance in the selected strain. These experiments showed that, compared to the susceptible colony Rockefeller, RecR present higher activity of glutathione S-transferases (GSTs), alpha- and beta-esterases, and, to a lesser degree, mixed function oxidases (MFO). At the 14th or 17th generations, there was no cross resistance of these insects to deltamethrin, cypermethrin and malathion, while a low resistance level (RR=3) was observed for pyriproxyfen, a juvenile hormone analogue. Experiments on resistance reversal, performed through three different field simulated schemes using the resistant strain, showed that temephos susceptibility can be recovered. The establishment of an A. aegypti colony resistant to temephos is extremely valuable for a deeper understanding of resistance mechanisms and thus for further improvements in control strategies against this vector. With the urgent need on improving methodologies to monitor resistance, molecular studies such as microarrays, and resistant colonies such as RecR will certainly hasten such studies.

Identification of Zika virus vectors and implications for control

Artigo liberado em acesso aberto como parte do acordo para tornar publico todos os dados produzidos sobre o virus zika - Compartilhamento de dados em emergencias de saude publica - http://www.wellcome.ac.uk/News/Media-office/Press-releases/2016/WTP060169.htm

A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its α‐glucosidase receptor in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the ≈ 60‐kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino‐acid sequence confirmed its identity as an α‐glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19‐nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full‐length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N‐terminal 45‐kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense‐mediated decay pathway of mRNA degradation. In‐gel assays confirmed that a single α‐glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N‐terminus of the receptor.

Genetic Differentiation of Aedes aegypti (Diptera: Culicidae), the Major Dengue Vector in Brazil

Abstract In 2000, Brazil reported 180,137 cases of dengue, ≈80% of the total in the Americas. However, little is known about gene flow among the vector populations in Brazil. Random amplified polymorphic DNA (RAPD) was used to study the genetic structure of Aedes aegypti in 15 populations from five states, with a range extending 2,800 km. An analysis of 47 polymorphic RAPD loci estimated gene flow at the macro- (different states) and micro- (different cities) geographical levels. Genetic polymorphism was high (HS = 0.274), and high levels of genetic differentiation existed both between different states (GST = 0.317) and between cities or neighborhoods in each state (GST = 0.085–0.265). These values are higher than those described for any other populations of A. aegypti.

The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for management

BackgroundAedes aegypti is the vector of dengue virus, and its control is essential to prevent disease transmission. Among the agents available to control this species, biolarvicides based on Bacillus thuringiensis serovar israelensis (Bti) are an effective alternative to replace the organophosphate temephos for controlling populations that display resistance to this insecticide. The major goal of this study was to determine the baseline susceptibility of Brazilian Ae. aegypti populations to Bti, taking into account their background in terms of larvicide exposure, status of temephos resistance and the level of activity of detoxifying enzymes involved in metabolic resistance to insecticides.MethodsPopulation samples were established under insectarium conditions. Larval susceptibility to temephos and Bti was evaluated through bioassays and lethal concentrations of these compounds were determined. Biochemical assays were performed to determine the specific activity of five detoxifying enzymes in these samples.ResultsFourteen populations were characterized and, except for one case, all displayed resistance to temephos. Most populations were classified as highly resistant. The populations also showed increased activity of one or more detoxifying enzymes (glutathione-S-transferases, esterases and mixed function oxidases), regardless of their temephos resistance status. All populations analyzed were susceptible to Bti, and the lethal concentrations were similar to those detected in two laboratory susceptible colonies. The response to Bti showed little variation. A maximum resistance ratio of 2.1 was observed in two untreated populations, while in two Bti-treated populations, the maximum resistance ratio was 1.9. No positive correlation was found between temephos resistance, increased activity of detoxifying enzymes, and susceptibility to Bti.ConclusionsData from this study show that all populations were susceptible to Bti, including twelve untreated and two treated populations that had been exposed to this agent for more than ten years. The temephos resistance and increased activity of detoxifying enzymes observed in thirteen populations was not correlated with changes in susceptibility to Bti. Our data show a lack of cross-resistance between these two compounds; thus, Bti can be used in an integrated control program to fight Ae. aegypti and counteract the temephos resistance that was found among all populations analyzed.

Genetic diversity in Brazilian populations of Aedes albopictus

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations.

Spatial and temporal country-wide survey of temephos resistance in Brazilian populations of Aedes aegypti.

The organophosphate temephos has been the main insecticide used against larvae of the dengue and yellow fever mosquito (Aedes aegypti) in Brazil since the mid-1980s. Reports of resistance date back to 1995; however, no systematic reports of widespread temephos resistance have occurred to date. As resistance investigation is paramount for strategic decision-making by health officials, our objective here was to investigate the spatial and temporal spread of temephos resistance in Ae. aegypti in Brazil for the last 12 years using discriminating temephos concentrations and the bioassay protocols of the World Health Organization. The mortality results obtained were subjected to spatial analysis for distance interpolation using semi-variance models to generate maps that depict the spread of temephos resistance in Brazil since 1999. The problem has been expanding. Since 2002-2003, approximately half the country has exhibited mosquito populations resistant to temephos. The frequency of temephos resistance and, likely, control failures, which start when the insecticide mortality level drops below 80%, has increased even further since 2004. Few parts of Brazil are able to achieve the target 80% efficacy threshold by 2010/2011, resulting in a significant risk of control failure by temephos in most of the country. The widespread resistance to temephos in Brazilian Ae. aegypti populations greatly compromise effective mosquito control efforts using this insecticide and indicates the urgent need to identify alternative insecticides aided by the preventive elimination of potential mosquito breeding sites.

Climate Change Influences on the Global Potential Distribution of the Mosquito Culex quinquefasciatus, Vector of West Nile Virus and Lymphatic Filariasis

Rapid emergence of most vector-borne diseases (VBDs) may be associated with range expansion of vector populations. Culex quinquefasciatus Say 1823 is a potential vector of West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. We estimated the potential distribution of Cx. quinquefasciatus under both current and future climate conditions. The present potential distribution of Cx. quinquefasciatus showed high suitability across low-latitude parts of the world, reflecting the current distribution of the species. Suitable conditions were identified also in narrow zones of North Africa and Western Europe. Model transfers to future conditions showed a potential distribution similar to that under present-day conditions, although with higher suitability in southern Australia. Highest stability with changing climate was between 30°S and 30°N. The areas present high agreement among diverse climate models as regards distributional potential in the future, but differed in anticipating potential for distribution in North and Central Africa, southern Asia, central USA, and southeastern Europe. Highest disparity in model predictions across representative concentration pathways (RCPs) was in Saudi Arabia and Europe. The model predictions allow anticipation of changing distributional potential of the species in coming decades.

Fitness cost in field and laboratory Aedes aegypti populations associated with resistance to the insecticide temephos.

BackgroundThe continued use of chemical insecticides in the context of the National Program of Dengue Control in Brazil has generated a high selective pressure on the natural populations of Aedes aegypti, leading to their resistance to these compounds in the field. Fitness costs have been described as adaptive consequences of resistance. This study evaluated the biological and reproductive performance of A. aegypti strains and a field population resistant to temephos, the main larvicide used for controlling mosquitoes.MethodsComparative tests were performed with a resistant field population from the municipality of Arcoverde, Pernambuco State, Brazil, with a high rate of temephos resistance (RR = 226.6) and three isogenetic laboratory strains from the same origin (Araripina municipality, Pernambuco): RecR (RR = 283.6); RecRNEx (RR = 250.5), a strain under a process of resistance reversion; and RecRev (RR = 2.32), a reversed susceptible strain used as an experimental control.ResultsOur study revealed that the absence of selective pressure imposed by exposure to temephos, for five consecutive generations, led to a discrete reduction of the resistance ratio and the response of the detoxifying enzymes. Most of the 19 biological parameters were impaired in the resistant strains and field population. The analysis of the fertility life table confirmed the presence of reproductive disadvantages for the resistant individuals. Similarly, the longevity, body size, and total energetic resources were also lower for the resistant females, except for the last two parameters in the field females (Arcoverde). In contrast, the sex ratio and embryonic viability suffered no interference in all strains or population evaluated, regardless of their status of resistance to temephos.ConclusionsThe reproductive potential and survival of the resistant individuals were compromised. The parameters most affected were the larval development time, fecundity, net reproduction rate, and the generational doubling time. These fitness costs in the natural population and laboratory strains investigated are likely associated with maintaining the metabolic mechanism of resistance to temephos. Our results show that despite these costs, the highly temephos resistant populations can compensate for these losses and successfully overcome the control actions that are based on the use of chemical insecticides.