Inheritance and Mechanism of Resistance to<i>Bacillus sphaericus</i>in<i>Culex quinquefasciatus</i>(Diptera: Culicidae) from China and Brazil

Oliveira, Cláudia Maria Fontes de; Silva-Filha, Maria Helena Neves Lobo; Nielsen-LeRoux, Christina; Pei, Guofeng; Yuan, Zhiming; Regis, Lêda

doi:10.1603/0022-2585-41.1.58

Cited by 37 publications

(34 citation statements)

References 30 publications

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“…Diagnostic bioassays were performed to overcome this constraint, and, conversely, it not only discriminated susceptible and resistant larvae but also demonstrated that frequency of resistant individuals has not decreased along the generations analyzed, in despite of the introduction of susceptible individuals at 1:9 or 1:1 ratios in Rev2362 and RevIAB colonies, respectively. This bioassay was suitable for screening resistant individuals in samples, whose presence could not be directly detected by multiple concentrations assays and it is an important tool for monitoring B. sphaericus resistance since alleles already studied in this model showed to be recessively inherited Oliveira et al, 2004;Amorim et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Availability of Cqm1 receptors on the midgut is crucial for larvicidal action of Bin toxin and its alteration, or absence, can lead to high levels of B. sphaericus resistance (Nielsen-Leroux et al, 1995;Darboux et al, 2002;Oliveira et al, 2004;Romão et al, 2006). Four alleles of the receptor gene were found to display mutations which prevent the production of functional membranebound proteins and, therefore, binding of Bin toxin to midgut epithelium (Darboux et al, 2002(Darboux et al, , 2007Romão et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools

Amorim

Barros

Chalegre

et al. 2010

Insect Biochemistry and Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools

Amorim

Barros

Chalegre

et al. 2010

Insect Biochemistry and Molecular Biology

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“…Each allele displays distinct resistance-associated mutations which result in potential transcripts for truncated soluble proteins lacking the GPI anchor (cpm1 GEO , cqm1 REC , cpm1 BP ) or for truncated GPI-anchored proteins which are still unable to bind Bin toxin due to the loss of 66 amino acids (cpm1 BP -del). The CqRL1/2362 colony, derived from eggs collected in the Recife Metropolitan Area (RMA; Brazil) and laboratory selected, displays high levels of resistance (resistance ratio [RR], Ͼ100,000) due to the failure of Bin toxin binding to microvillus receptors, and larvae from this colony were found to be homozygous for the cqm1 REC allele (26,27). Characterization of this allele showed a 19-nucleotide (nt) deletion which changes the frame of the protein coding sequence and originates a premature stop codon.…”

mentioning

confidence: 99%

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

Chalegre

Romão

Tavares

et al. 2012

Appl Environ Microbiol

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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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“…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).…”

mentioning

confidence: 99%

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

Chalegre¹,

Romão²,

Amorim³

et al. 2009

Appl Environ Microbiol

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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...

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Inheritance and Mechanism of Resistance toBacillus sphaericusinCulex quinquefasciatus(Diptera: Culicidae) from China and Brazil

Cited by 37 publications

References 30 publications

Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools

Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools

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

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

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