A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its α‐glucosidase receptor in Culex quinquefasciatus

Abstract

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.

Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening

“…The ␣-glucosidase assay was performed essentially as described previously (30). Individual larvae were homogenized in phosphate-buffered saline buffer and solubilized in sodium dodecyl sulfate-polyacrylamide sample buffer, lacking 2-mercaptoethanol, followed by electrophoresis on 8% acrylamide gels.…”

“…Among other control measures, B. sphaericus-based larvicides have been used, and one of the major concerns regarding their use as a microbial larvicide is monitoring the susceptibility of populations under selection. The cqm1 REC allele was identified in a C. quinquefasciatus colony derived from the RMA (30), and it is believed that this mutation may be present and subjected to B. sphaericus selection in field populations. Here we report the development of a diagnostic PCR approach based on the detection of cqm1 REC and its use to investigate the frequency of resistance in nontreated and treated C. quinquefasciatus populations.…”

“…The cpm1 GEO allele, detected in a C. pipiens laboratory-selected colony (GEO) from California, contains a nonsense mutation leading to the synthesis of a truncated protein lacking the glycosylphosphatidylinositol anchor and is therefore not able to localize to the apical membrane of the midgut epithelium (11,38). A 19-nucleotide deletion in the gene cqm1, here named cqm1 REC , was associated with the resistance of a C. quinquefasciatus laboratory colony (CqRL1/ 2362) originating from the city of Recife (Brazil), and this mutation prevents the expression of Cqm1 on the midgut brush border membranes (28,29,30). Two alleles were found in the C. pipiens BP population from France (8).…”

“…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).…”

Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae

“…After processing, Bin toxin recognizes and binds to specific receptors in the midgut of Bin-toxin-susceptible species through its subunit BinB (51 kDa), while the component BinA (42 kDa) confers toxicity and is likely to form pores in the cell membrane (7,25). The membrane-bound receptors of Bin toxin on the midgut of Culex quinquefasciatus larvae, Cqm1, were characterized as 60-kDa ␣-glucosidases (24). The mode of action of Cry48Aa/ Cry49Aa is still unknown, but a remarkable feature of this new two-component toxin is the capacity to overcome C. quinquefasciatus resistance to the Bin toxin (13,19,21).…”