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Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

ABSTRACT: 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.

SUBMITTER: Chalegre KD

PROVIDER: S-EPMC3416613 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

Chalegre Karlos Diogo de Melo KD Romão Tatiany Patrícia TP Tavares Daniella Aliny DA Santos Eloína Mendonça EM Ferreira Lígia Maria LM Oliveira Cláudia Maria Fontes CM de-Melo-Neto Osvaldo Pompílio OP Silva-Filha Maria Helena Neves Lobo MH

Applied and environmental microbiology 20120706 17

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. ...[more]

PMID: 22773633

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Project description: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 cqm1 REC and cqm1 REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1 REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1 REC-2 , suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1 REC-2 and track its origin in field populations where cqm1 REC was previously identified.MethodsThe screening of the cqm1 REC-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 cqm1 REC . Full-length sequencing of the cqm1 REC-2 and selected cqm1 samples was performed to identify further polymorphisms between these alleles.ResultsThe cqm1 REC-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 cqm1 REC . The full-length sequences from these two cqm1 REC-2 copies were almost identical to the cqm1 REC-2 derived from the resistant colony but displayed more than 30 SNPs when compared with cqm1 and cqm1 REC . The cqm1 REC and cqm1 REC-2 resistant alleles were found to be associated with two distinct sets of wild-type cqm1 variants found in field populations.ConclusionsThe cqm1 REC-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 cqm1 REC-2 can be selected in field populations, although its low frequency and distribution in Recife suggest that cqm1 REC-2 presents a lower risk of selection compared to cqm1 REC .

Project description:BackgroundThe study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive exposure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratory-selected colonies resistant to this bacterium. One such colony, RIAB59, was selected after regular long-term exposure of larvae to the L. sphaericus IAB59 strain. This strain is characterized by its ability to produce the well known Binary (Bin) toxin, and the recently characterized Cry48Aa/Cry49Aa toxin, able to kill Bin-resistant larvae. Resistance to Bin is associated with the depletion of its receptor, Cqm1 α-glucosidase, from the larvae midgut. This study aimed to identify novel molecules and pathways associated with survival of the RIAB59 larvae and the resistance phenotype.MethodsA transcriptomic approach and bioinformatic tools were used to compare the profiles derived from the midguts of larvae resistant and susceptible to L. sphaericus IAB59.ResultsThe RNA-seq profiles identified 1355 differentially expressed genes (DEGs), with 673 down- and 682 upregulated transcripts. One of the most downregulated DEGs was cqm1, which validates the approach. Other strongly downregulated mRNAs encode the enzyme pantetheinase, apolipoprotein D, lipases, heat-shock proteins and a number of lesser known and hypothetical polypeptides. Among the upregulated DEGs, the top most encodes a peroxisomal enzyme involved in lipid metabolism, while others encode enzymes associated with juvenile hormone synthesis, ion channels, DNA binding proteins and defense polypeptides. Further analyses confirmed a strong downregulation of several enzymes involved in lipid catabolism while the assignment of DEGs into metabolic pathways highlighted the upregulation of those related to DNA synthesis and maintenance, confirmed by their clustering into related protein networks. Several other pathways were also identified with mixed profiles of down- and upregulated transcripts. Quantitative RT-PCR confirmed the changes in levels seen for selected mRNAs.ConclusionsOur transcriptome-wide dataset revealed that the RIAB59 colony, found to be substantially more resistant to Bin than to the Cry48Aa/Cry49Aa toxin, developed a differential expression profile as well as metabolic features co-selected during the long-term adaptation to IAB59 and that are most likely linked to Bin resistance.

Project description:BackgroundThe resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain.MethodsThe activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined.ResultsEnzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters.ConclusionsOur dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation.

Dataset Information

Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

Publications

Novel mutations associated with resistance to Bacillus sphaericus in a polymorphic region of the Culex quinquefasciatus cqm1 gene.

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