Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1 REC allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1 REC allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1 REC-D16 ) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1 REC . Yet a third resistant allele (cqm1 REC-D25 ), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening.T he utilization of biolarvicides based on Bacillus sphaericus requires monitoring strategies which can predict or prevent potential resistance selection among exposed mosquito populations. The binary (Bin) crystal toxin, which is the major active insecticidal factor found in commercial B. sphaericus strains, acts on mosquito larvae after ingestion, processing and binding to specific receptors located on the midgut epithelium (5, 24). Bin toxin displays high activity against larvae of the Culex pipiens complex and B. sphaericus has an excellent persistence under field conditions, which make this an effective biolarvicide for controlling these species in urban areas (17). However, the mode of action of Bin toxin relies entirely on its binding to a single class of midgut receptors which are glycosylphosphatidylinositol (GPI)-anchored ␣-glucosidases named Cpm1 and Cqm1 for Culex pipiens and Culex quinquefasciatus, respectively (7,29,30). Failure of toxins to bind to their midgut receptors has been described, in a wide range of target insects, as the primary resistance mechanism to insecticidal proteins from entomopathogenic bacteria (11,16,25). In the case of B. sphaericus, this is a critical aspect since resistance cases have also been reported after laboratory selection or field exposure (2,25,27,36,38,44).Investigation of the B. sphaericus resistance mechanisms has confirmed the essential role for the binding of Bin toxin to its receptors, since mutations within the cpm1/cqm1 genes, which are recessively inherited, are the major causes leading to the absence of ...
