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 1011Met 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.

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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.

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.

BackgroundBacillus thuringiensis svar. israelensis (Bti) is an effective and safe biolarvicide to control Aedes aegypti. Its mode of action based on four protoxins disfavors resistance; however, control in endemic areas that display high mosquito infestation throughout the year requires continuous larvicide applications, which imposes a strong selection pressure. Therefore, this study aimed to investigate the effects of an intensive Bti exposure on an Ae. aegypti strain (RecBti), regarding its susceptibility to Bti and two of its protoxins tested individually, to other control agents temephos and diflubenzuron, and its profile of detoxifying enzymes.MethodsThe RecBti strain was established using a large egg sample (10,000) from Recife city (Brazil) and more than 290,000 larvae were subjected to Bti throughout 30 generations. Larvae susceptibility to larvicides and the activity of detoxifying enzymes were determined by bioassays and catalytic assays, respectively. The Rockefeller strain was the reference used for these evaluations.ResultsBti exposure yielded an average of 74% mortality at each generation. Larvae assessed in seven time points throughout the 30 generations were susceptible to Bti crystal (resistance ratio RR ≤ 2.8) and to its individual toxins Cry11Aa and Cry4Ba (RR ≤ 4.1). Early signs of altered susceptibility to Cry11Aa were detected in the last evaluations, suggesting that this toxin was a marker of the selection pressure imposed. RecBti larvae were also susceptible (RR ≤ 1.6) to the other control agents, temephos and diflubenzuron. The activity of the detoxifying enzymes α- and β-esterases, glutathione-S-transferases and mixed-function oxidases was classified as unaltered in larvae from two generations (F19 and F25), except for a β-esterases increase in F25.ConclusionsProlonged exposure of Ae. aegypti larvae to Bti did not evolve into resistance to the crystal, and no cross-resistance with temephos and diflubenzuron were recorded, which supports their sustainable use with Bti for integrated control practices. The unaltered activity of most detoxifying enzymes suggests that they might not play a major role in the metabolism of Bti toxins, therefore resistance by this mechanism is unlikely to occur. This study also highlights the need to establish suitable criteria to classify the status of larval susceptibility/resistance.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3246-1) contains supplementary material, which is available to authorized users.