title: A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its alpha-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 approximately 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 alpha-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 alpha-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.
authors.affiliation: Department of Entomology, Centro de Pesquisas Aggeu Magalhães/Fundação Oswaldo Cruz, Recife-PE, Brazil.
authors.forename: Tatiany Patrícia
authors.initials: TP
authors.lastname: Romão
authors.forename: Karlos Diogo
authors.initials: KD
authors.lastname: de Melo Chalegre
authors.forename: Shana
authors.initials: S
authors.lastname: Key
authors.forename: Constância Flávia Junqueira
authors.initials: CF
authors.lastname: Ayres
authors.forename: Cláudia Maria
authors.initials: CM
authors.lastname: Fontes de Oliveira
authors.forename: Osvaldo Pompílio
authors.initials: OP
authors.lastname: de-Melo-Neto
authors.forename: Maria Helena Neves Lobo
authors.initials: MH
authors.lastname: Silva-Filha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D001483:Base Sequence; D003465:Culex / Q000235:genetics / Q000502:physiology*; D041981:Gastrointestinal Tract / Q000033:anatomy & histology / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007305:Insecticide Resistance / Q000502:physiology*; D007306:Insecticides / Q000378:metabolism*; D007814:Larva / Q000033:anatomy & histology / Q000378:metabolism; D008969:Molecular Sequence Data; D009032:Mosquito Control; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D019476:Insect Proteins; D007306:Insecticides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C046134:binB protein, Bacillus sphaericus; D000520:alpha-Glucosidases