Detection of an allele conferring resistance to Bacillus sphaericus binary toxin in Culex quinquefasciatus populations by molecular screening

Abstract

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 alpha-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 alpha-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.

FIG. 1.

Culex quinquefasciatus samples were collected in three areas of Pernambuco state, located in northeastern Brazil. Fazenda Nova is a nontreated rural district located 180 km from the RMA; both the nontreated area of Peixinhos and the Bacillus sphaericus-treated area of Água Fria are located in the RMA.

FIG. 2.

DNA fragments amplified from the Culex quinquefasciatus cqm1 and cqm1_REC alleles of susceptible and Bacillus sphaericus-resistant larvae, respectively. The diagnostic PCR produces distinct profiles for susceptible homozygote, resistant homozygote, and susceptible heterozygote larvae (lanes 1, 2, and 3, respectively). No fragments were amplified from the samples with Aedes aegypti DNA or without DNA (lanes 4 and 5, respectively). On the left, molecular size (M) markers are shown in base pairs.

FIG. 3.

Analysis of α-glucosidases from susceptible (S) and Bacillus sphaericus-resistant (R) Culex quinquefasciatus larvae. In-gel assays were performed using crude extracts from individual larvae. Samples were separated by 8% semidenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and submitted to enzymatic detection. Catalytic bands were visualized under a UV transilluminator, and the arrow indicates the 83-kDa band corresponding to the Cqm1 α-glucosidase.