A new allele conferring resistance to Lysinibacillus sphaericus is detected in low frequency in Culex quinquefasciatus field populations.

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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 .

Co-selection and replacement of resistance alleles to Lysinibacillus sphaericus in a Culex quinquefasciatus colony.

The Cqm1 α-glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α-glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory-resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC-2, that was co-selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut-bound Cqm1 proteins. Using a PCR-based assay to monitor the frequency of each allele during long-term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC-2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high-resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC-2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC-2. The replacement of cqm1REC by cqm1REC -2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.

Caracterização e detecção molecular de alelos de resistência ao biolarvicida Bacillus sphaericus em culex quinquefasciatus / Characterization and molecular detection of resistance alleles to the Bacillus sphaericus biolarvicide in Culex quinquefasciatus

A resistência de Culex quinquefasciatus à toxina inseticida (Bin) de Bacillus sphaericus (Bsp) pode estar associada a uma falha da ligação da toxina com os receptores Cqm1, localizados no microvilli intestinal das larvas através de uma âncora GPI. Mutações no gene cqm1 podem impedir a expressão de proteínas Cqm1 funcionais e gerar um alto nível de resistência. O objetivo deste trabalho foi caracterizar e avaliar a frequência de alelos de resistência (r) em populações e colônias de C. quinquefasciatus. Neste estudo, o alelo r cqm1REC, selecionado e identificado anteriormente na colônia R2362, foi detectado por PCR alelo-específica em quatro populações de Recife com frequências entre 0,001 e 0,017. Em duas populações foram identificados novos alelos r, o cqm1REC-D16 e o cqm1REC- D25, com frequência entre 0,002-0,006. Estes alelos são caracterizados por deleções de 16 e 25-nt, respectivamente, as quais geram códon de terminação da tradução prematuro (CTTP) e não codificam proteínas com âncora GPI. Um segundo alelo r (cqm1REC- 2) foi identificado na colônia R2362 e possui uma mutação nonsense (G1292A) que também gera um CTTP, impedindo a localização de receptores Cqm1 no epitélio. O alelo cqm1REC-2 foi co-selecionado com o cqm1REC na colônia R2362 e uma análise da competição entre eles mostrou que o cqm1REC-2 predomina sob pressão de seleção com Bsp, enquanto que o cqm1REC é majoritário na ausência de Bsp. A expressão relativa dos alelos cqm1REC e cqm1REC-2, avaliada por PCR em tempo real, mostrou que ambos possuem uma expressão significativamente menor em relação ao cqm1. Amostras de microvilli intestinal de larvas homozigotas para cada alelo apresentaram uma baixa capacidade de interação com a toxina Bin, corroborando os dados de expressão gênica e o fenótipo de resistência. Este estudo mostrou a detecção e caracterização de novos alelos de C. quinquefasciatus que conferem resistência a Bsp e estes dados são fundamentais para o diagnóstico e manejo da resistência em programas de controle.

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

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.

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

Bacillus sphaericus binary toxin action on Culex quinquefasciatus larvae relies on the binding to Cqm1alpha-glucosidases, which act as midgut receptors. Resistance of two laboratory-selected colonies is associated with the allele cqm1(REC) that prevents Cqm1 expression as membrane-bound molecules. This study evaluated stability of resistance after the interruption of selection pressure and introduction of susceptible individuals in these colonies. Bioassays showed that frequency of resistant larvae did not decrease throughout 11 generations, under these conditions, and it was associated to a similar frequency of larvae lacking the Cqm1alpha-glucosidase receptor, detected by in gel enzymatic assays. Direct screening of the cqm1(REC) allele, by specific PCR, showed that its frequency remained stable throughout 11 generations. Parental resistant colony did not display biological costs regarding fecundity, fertility and pupal weight and data from susceptibility assays, enzymatic assays and PCR screening showed that cqm1(REC) was not disfavored in competition with the susceptible allele and persisted in the progenies, in the lack of selection pressure. Characterization of molecular basis of resistance is essential for developing diagnostic tools and data have relevant implication for the establishment of strategies for resistance management.

Detection of an allele conferring resistance to Bacillus sphaericus binary toxin in Culex quinquefasciatus populations by molecular screening.

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 alpha-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 alpha-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.

Diagnóstico da resistência do vetor Culex quinqufasciatus ao biolarvicida Bacillus sphaericus / Resistance diagnostic of the vector Culex quinquefasciatus to the bioinsecticide Bacillus sphaericus

A eficácia e seletividade do Bacillus sphaericus (Bsp) para larvas do vetor Culex quinquefasciatus é devida à ligação específica da toxina binária (Bin), principal proteína inseticida do Bsp, a um receptor presente no epitélio intestinal das larvas, a glicosidase Cqm1. Um mecanismo de resistência descrito para este culicídeo é devido a uma deleção de 19 nucleotídeos (d19) no gene cqm1 (cqm1-d19) que, em homozigose, impede a expressão do receptor no epitélio. O objetivo deste trabalho foi determinar a freqüência do alelo cqm1-d19 em populações naturais de Cx. quinquefasciatus, não tratadas e tratadas com o Bsp, além de avaliar polimorfismos no gene cqm1. Uma análise in vivo da susceptibilidade ao Bsp em larvas de Cx. quinquefasciatus mostrou que as populações não tratadas de Varadouro, Peixinhos e Fazenda Nova apresentaram valores de razão de resistência (RR) de 1,3 a 4,0 vezes, enquanto que para a população tratada de Água Fria a RR variou de 2,7 a 8,6 vezes, em relação à população susceptível usada como controle. Os valores de RR inferiores a 10 indicaram que não houve alteração significativa na susceptibilidade. Os alelos cqm1 e cqm1- d19 foram identificados através de método diagnóstico de PCR utilizando primers que flanqueiam a d19 e as amostras das populações analisadas variaram entre 162 e 353 larvas. O alelo cqm1-d19 foi detectado pela primeira vez em populações naturais e sua freqüência em áreas não tratadas da Região Metropolitana do Recife (RMR) foi de 1,16 x 10-2 em Varadouro e de 7,8 x 10-3 em Peixinhos. Na população de Fazenda Nova, isolada geograficamente da RMR, o alelo não foi detectado. Em Água Fria, tratada com o Bsp desde 2003, as freqüências foram bem mais elevadas em relação às populações não tratadas: 6,94 e 5,55 x 10-2 após 13 e 24 tratamentos, respectivamente. Em paralelo à detecção do alelo foi feita a avaliação da freqüência de expressão da -glicosidase Cqm1, através de ensaios enzimáticos in gel, e os resultados corroboraram aqueles de freqüência alélica em todas as populações. A partir da análise de polimorfismos no gene cqm1 em 15 indivíduos de Água Fria, foram identificados 13 alelos, baseados em 13 alterações na seqüência de aminoácidos, a maioria na região C terminal. Dentre os alelos identificados, dois ocorreram em maior freqüência e os demais foram variações destes principais. Os resultados deste estudo mostraram que a freqüência do alelo cqm1-d19 é alta em populações naturais, e mais elevada naquelas que estão sob pressão de seleção, além de demonstrarem que o gene cqm1 é altamente polimórfico. As abordagens moleculares utilizadas neste estudo são mais sensíveis que os bioensaios, visto que foi possível detectar indivíduos heterozigotos e homozigotos resistentes, e são adequadas para avaliar a susceptibilidade de populações de Cx. quinquefasciatus ao Bsp. Estes dados reforçam a adoção de medidas visando o uso sustentável do biolarvicida Bsp em programas de controle de vetores.