The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the ≈ 60‐kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino‐acid sequence confirmed its identity as an α‐glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19‐nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full‐length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N‐terminal 45‐kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense‐mediated decay pathway of mRNA degradation. In‐gel assays confirmed that a single α‐glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N‐terminus of the receptor.

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...

The Cqm1 a-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 a-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1 REC , was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1 REC-2 , that was co-selected with cqm1 REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1 REC was found to predominate early on but later was replaced by cqm1 REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1 REC and cqm1 REC-2 . In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1 REC showed a higher frequency than cqm1 REC-2 . The replacement of cqm1 REC by cqm1 REC-2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.

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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 ...

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BackgroundThe Cqm1 α-glucosidase of Culex quinquefasciatus larvae acts as the midgut receptor for the binary toxin of the biolarvicide Lysinibacillus sphaericus. Mutations within the cqm1 gene can code for aberrant polypeptides that can no longer be properly expressed or bind to the toxin, leading to insect resistance. The cqm1REC and cqm1REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1REC-2, suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1REC-2 and track its origin in field populations where cqm1REC was previously identified.MethodsThe screening of the cqm1REC-2 allele was based on more than 2000 C. quinquefasciatus larvae from five localities in the city of Recife, Brazil and used a multiplex PCR assay that is also able to identify cqm1REC. Full-length sequencing of the cqm1REC-2 and selected cqm1 samples was performed to identify further polymorphisms between these alleles.ResultsThe cqm1REC-2 allele was found in field samples, specifically in two heterozygous individuals from a single locality with an overall frequency and distribution much lower than that observed for cqm1REC. The full-length sequences from these two cqm1REC-2 copies were almost identical to the cqm1REC-2 derived from the resistant colony but displayed more than 30 SNPs when compared with cqm1 and cqm1REC. The cqm1REC and cqm1REC-2 resistant alleles were found to be associated with two distinct sets of wild-type cqm1 variants found in field populations.ConclusionsThe cqm1REC-2 allele occurs in populations in Recife and was probably already present in the samples used to establish the laboratory resistant colony. The data generated indicates that cqm1REC-2 can be selected in field populations, although its low frequency and distribution in Recife suggest that cqm1REC-2 presents a lower risk of selection compared to cqm1REC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1347-2) contains supplementary material, which is available to authorized users.

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