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 ␣-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...

Novel Mutations Associated with Resistance to Bacillus sphaericus in a Polymorphic Region of the Culex quinquefasciatus cqm1 Gene

“…Susceptibility to B. sphaericus in two nontreated populations of IPO and JAB showed that RR values at the LC 90 were similar to that of the reference colony, and the slight variations in RRs found at the LC 50 are comparable to previous RRs reported for other nontreated mosquito populations, which have demonstrated the existence of natural variations in their B. sphaericus susceptibilities (4,20,34,36,37). The status of two other nontreated populations of ROD and AZE could not be analyzed; nevertheless, they were expected to be susceptible since B. sphaericus had not been used in these areas and they were also geographically isolated from the only two exposed areas in RMA at the time the samples were collected (28,31).…”

“…The treated population of AGU evaluated here showed a higher cqm1 REC frequency than those from the nontreated populations, which is consistent with B. sphaericus exposure in that area. However, this frequency has not increased compared to previous screenings performed in AGU (4,32), suggesting that the selection pressure might be low. This could be related with the introduction of Bacillus thuringiensis serovar israelensis (B. thurin- The premature translation stop codon originating from the deletions described here is boxed in bold.…”

“…The status of two other nontreated populations of ROD and AZE could not be analyzed; nevertheless, they were expected to be susceptible since B. sphaericus had not been used in these areas and they were also geographically isolated from the only two exposed areas in RMA at the time the samples were collected (28,31). The cqm1 REC allele was found in all nontreated populations analyzed, despite being originally identified in a laboratory-selected colony (4,29), highlighting the strategic importance of monitoring for this allele in RMA. This result contrasts with those observed for laboratoryselected cadherin alleles associated with Cry1Ac resistance and whose screening in field populations has not led to a positive detection (13,33).…”

“…For DNA isolation, individual fourth-instar larva samples were homogenized in DNAzol (Invitrogen), as recommended by the manufacturer, followed by precipitation with ethanol and DNA recovering in Tris-EDTA buffer. Allele-specific PCR (AS-PCR) was performed using specific primers, described in Chalegre et al (4), and reactions were carried out for 35 cycles with an annealing temperature of 60°C using a Biometra thermocycler. Amplification products were separated by electrophoresis on 2.5% agarose gels, and each assay included no-DNA samples and A. aegypti DNA samples as negative controls.…”

Insecticides for Vector-Borne Diseases: Current Use, Benefits, Hazard and Resistance

“…Cases of resistance to these alternative insecticides are still limited (Rivero et al, 2010) and the underlying mechanisms are only beginning to be identified (Chalegre et al, 2009;Darboux et al, 2007 Adapted from Rivero et al (2010). Metabolic resistance: EST, enhanced esterase activity; GST, enhanced glutatione-S-transferase activity; MOX, enhanced p450 monoxygenase activity.…”