Culex quinquefasciatus field populations subjected to treatment with Bacillus sphaericus did not display high resistance levels

Silva-Filha, Maria Helena Neves Lobo; Chalegre, Karlos Diogo de Melo; Anastacio, Daniela Bandeira; Oliveira, Cláudia Maria Fontes de; Silva, Sinara Batista da; Acioli, Ridelane Veiga; Hibi, Sumire; Oliveira, Débora Cardoso; Parodi, Eunice Santos Martini; Filho, Carlos Alberto Madeira Marques; Furtado, André Freire; Regis, Lêda

doi:10.1016/j.biocontrol.2007.10.002

Cited by 18 publications

(13 citation statements)

References 35 publications

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“…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.…”

Section: Discussioncontrasting

confidence: 65%

“…Samples from ROD and AZE, consisting of larva batches collected directly from breeding sites in these areas in 1999, were stored at Ϫ70°C. The treated area of Água Fria (AGU) has been exposed to B. sphaericus since 2003 (32), and egg rafts were collected in 2010 according to the methodology described above. Eggs were used to establish subcolonies maintained under laboratory conditions, and larvae from F1 or F2 progenies were stored at Ϫ70°C until use.…”

Section: Methodsmentioning

confidence: 99%

“…Utilization of B. sphaericus biolarvicides in RMA has been an important tool for controlling C. quinquefasciatus, which is the exclusive vector of the nematode Wuchereria bancrofti in Brazil (32). In view of the strategic role of this biolarvicide in RMA and the advantage provided by the knowledge available on the molecular basis of resistance, the goal of this work was to provide data on the frequency of the cqm1 REC allele through DNA screening, as well as to describe novel polymorphisms of the cqm1 gene which can disrupt the expression of the Cqm1 protein as a target site for Bin toxin.…”

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confidence: 99%

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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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Section: Discussioncontrasting

confidence: 65%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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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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“…The nontreated areas were Fazenda Nova, a rural district located 180 km from Recife, and Peixinhos, located in the RMA. The treated area of Á gua Fria is located in the RMA, 3 km away from Peixinhos, and has been subjected to the Filariasis Control Program, carried out by the municipality's Department of Health (32). The vector control subprogram includes the utilization, since 2003, of B. sphaericus-based larvicides in Culex breeding sites mapped in that area.…”

Section: Methodsmentioning

confidence: 99%

“…The Recife Metropolitan Area (RMA), located in the state of Pernambuco in northeastern Brazil, has been subjected to a vector control program to reduce C. quinquefasciatus density in districts where filariasis is endemic (32). 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.…”

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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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Susceptibility status of Culex quinquefasciatus (Diptera: Culicidae) populations to the chemical insecticide temephos in Pernambuco, Brazil

et al. 2013

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Culex quinquefasciatus field populations subjected to treatment with Bacillus sphaericus did not display high resistance levels

Cited by 18 publications

References 35 publications

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

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

Susceptibility status of Culex quinquefasciatus (Diptera: Culicidae) populations to the chemical insecticide temephos in Pernambuco, Brazil

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