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 activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound ␣-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1 REC ) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1 REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1 REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through ␣-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1 REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.Bacillus sphaericus Neide is considered the most successful microbial larvicide to date for the control of mosquito species from the Culex pipiens (Diptera: Culicidae) complex (20). B. sphaericus biolarvicides commercially available are based on highly toxic strains characterized by their ability to express the binary (Bin) protoxin, a crystal protein produced in large amounts during sporulation (7). This heterodimer is formed by the BinA (42-kDa) and BinB (51-kDa) subunits that act in synergy to produce larvicidal activity upon Culex larvae (3, 23). The BinB subunit is responsible for the recognition and binding of the toxin to specific receptors on the midgut epithelium surface, while BinA is primarily responsible for the toxic effects, but first the crystal has to be ingested by the larvae and the protoxin must be processed into toxin by the midgut (7). The Bin toxin receptor in C. pipiens (Cpm1) and Culex quinquefasciatus (Cqm1) is a 60-kDa ␣-glucosidase attached to the epithelial cell membrane by a glycosylphosphatidylinositol anchor (9, 30, 31). The action of the Bin toxin on Culex larvae relies on its specific binding to those membrane-bound receptors (24). Disruption of the interaction between the toxin and the midgut is the major mechanism underlying resistance, and it has already been reported from different laboratory-or fieldselected colonies (25,26,27,3...

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