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Applied and Environmental Microbiology, February 2009, p. 1044-1049, Vol. 75, No. 4
0099-2240/09/$08.00+0 doi:10.1128/AEM.02032-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening
Karlos Diogo de Melo Chalegre,1
Tatiany Patrícia Romão,1
Liliane Barbosa Amorim,1
Daniela Bandeira Anastacio,1
Rosineide Arruda de Barros,1
Cláudia Maria Fontes de Oliveira,1
Lêda Regis,1
Osvaldo Pompílio de-Melo-Neto,2 and
Maria Helena Neves Lobo Silva-Filha1*
Department of Entomology,1 Department of Microbiology, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Recife-PE 50670-420, Brazil2
Received 2 September 2008/ Accepted 15 December 2008
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 (cqm1REC) 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 cqm1REC 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 cqm1REC 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 cqm1REC 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.
* Corresponding author. Mailing address: Department of Entomology, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Av. Moraes Rego s/n, Cidade Universitária, Recife-PE 50670-420, Brazil. Phone: 55-81-21012553. Fax: 55-81-21012516. E-mail: mhneves{at}cpqam.fiocruz.br
Published ahead of print on 19 December 2008.
Applied and Environmental Microbiology, February 2009, p. 1044-1049, Vol. 75, No. 4
0099-2240/09/$08.00+0 doi:10.1128/AEM.02032-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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