Estudio de las bases de la resistencia a las proteínas insecticidas de bacillus thuringiensis en ostrinia nubilalis

Ostrinia nubilalis (Hübner) es una de las plagas más devastadoras de los cultivos de maíz de Europa y Norte América, y a nivel económico la obtención de un control eficaz de esta plaga es un logro fundamental. En 1996, se permitió la comercialización de las plantas transgénicas que llevan insertado en el genoma un gen procedente de Bacillus thuringiensis (Berliner) (Bt) y que codifica para una proteína insecticida de las que esta bacteria produce en forma de cristales paraesporales (proteínas Cry). A partir de esta fecha, la adopción de los cultivos de maíz Bt que expresan en concreto la toxina Cry1Ab ha ido aumentando progresivamente en todo el mundo, con el resultado de que las pérdidas económicas producidas por O. nubilalis se han reducido de forma considerable. Recientemente, un nuevo evento de maíz transgénico que expresa la proteína Cry1F, también tóxica frente O. nubilalis, ha salido a la venta, aumentando el abanico de plantas Bt empleadas para el control de esta plaga. Las ventajas derivadas del empleo de las plantas Bt son muchas pero esta tecnología no está libre de amenazas: el mayor peligro reside en el potencial de los insectos diana para desarrollar resistencia a las toxinas insecticidas empleadas contra ellos. La posible aparición de resistencia se puede producir por mutaciones en cualquiera de los genes involucrados en cada uno de los pasos del mecanismo de acción de las proteínas Cry. Por esta razón, el conocimiento de las bases bioquímicas por las cuales actúan estas toxinas, así como de las bases genéticas, son esenciales para preservar a largo plazo las tecnologías fundamentadas en B. thuringiensis. Para la realización de este trabajo de investigación se planteó abordar tanto la caracterización de poblaciones españolas de O. nubilalis, estudiando el potencial de desarrollo de resistencia a la toxina Cry1Ab, como el estudio de las moléculas del epitelio intestinal que pueden estar implicadas en el mecanismo de toxicidad de las proteínas Cry en este insecto plaga. En la primera parte de esta tesis, comparamos la susceptibilidad de una población de campo procedente del sur-este de España con la de una colonia de laboratorio a cuatro proteínas Cry ensayadas tanto en forma de toxina activada como de protoxina, así como al producto estándar HD-1-S2005. La característica principal de la población de campo escogida fue su procedencia: invernaderos cultivados con pimientos, donde se utilizaban formulados Bt para el control de las plagas de lepidópteros durante toda la temporada vegetativa. Los resultados de los bioensayos de mortalidad indicaron que las proteínas más toxicas para O. nubilalis son las toxinas de la clase 1 mientras que la toxina Cry2Aa ejerce un efecto tóxico menor. Con este tipo de ensayos, encontramos diferencias significativas entre la susceptibilidad de la colonia de campo y de la colonia de laboratorio, siendo esta última más susceptible a todas las toxinas de la clase 1. Sin embargo, el conjunto de todos los resultados obtenidos en este trabajo, que incluyen también el estudio de la mortalidad funcional y un ensayo de selección en laboratorio de resistencia a la proteína Cry1Ab, apunta a que las diferencias encontradas se debían más probablemente a variación intraespecífica que a un proceso de selección de resistencia producido en el invernadero. En el segundo apartado de esta tesis, la tolerancia de O. nubilalis a la proteína Cry1Ab se estudió bajo un punto de vista genético cuantitativo. Se fundaron isolíneas derivadas del apareamiento de adultos procedentes de dos diferentes localidades españolas, y las progenies se sometieron a cribado con la proteína Cry1Ab, a una concentración que causó una mortalidad media entre todas las isolíneas del 87% (± 17% SD). Solamente aquellos individuos que pertenecían a las isolíneas que tuvieron una supervivencia mayor del 40% se utilizaron para obtener la generación siguiente, que se sometió a cribado con la misma concentración de proteína Cry1Ab. Pudimos observar una clara reducción en la mortalidad, ya que la mortalidad media entre todas las isolíneas en la generación F2 fue de 62% (± 17% SD). El límite superior para la heredabilidad (h2) de la tolerancia a la proteína Cry1Ab se calculó mediante dos métodos y se situó entre 0.82 y 0.90, indicando que buena parte de la variación fenotípica en la tolerancia a la proteína Cry1Ab dependía de diferencias genéticas. La aplicación de la ecuación de Lande confirmó que la tolerancia a Cry1Ab tenía una base poligénica, ya que era debida a, como mínimo, dos loci. La contribución del gen de la caderina (candidato por estar ligado a la resistencia en otros lepidópteros) a la tolerancia a la proteína Cry1Ab se comprobó utilizando marcadores moleculares EPIC-PCR; el estudio de la segregación alélica en las isolíneas más tolerantes indicó su posible contribución en una de las cinco isolíneas examinadas. La unión de las proteínas Cry al epitelio intestinal de lepidópteros como O. nubilalis es un mecanismo fundamental para la toxicidad. Abordamos el estudio de las diferencias entre la unión de dos proteínas de la clase 1 (Cry1Ab y Cry1F), ambas expresadas en el maíz transgénico resistente a O. nubilalis, mediante ensayos de preincubación del epitelio intestinal con las mismas toxinas, lectinas, y anticuerpo anticaderina. La unión de las proteínas Cry1Ab y Cry1F (marcadas con biotina) resultó verse afectada de forma diferente: mientras que ningún tipo de preincubación pareció afectar la unión de la toxina Cry1Ab, la unión de la toxina Cry1F disminuyó tras preincubación con las toxinas Cry1Ab y Cry1F sin marcar y con el ácido síalico. Estas diferencias apuntan a la existencia de diferentes receptores (además de unos compartidos) para estas dos toxinas. Ensayos de inmunodetección de la unión de las proteínas Cry1Ab y Cry1F indicaron que éstas unen a proteínas del epitelio intestinal de O. nubilalis de un tamaño de 150 y 75 kDa, y que solamente Cry1Ab une a una proteína de alrededor de 250 kDa. Mientras que no obtuvimos ningún resultado en la identificación de las proteínas que componían la banda de 250 kDa, las proteínas más abundantes que componían las dos bandas de 75 y 150 kDa fueron identificadas como varias isoformas de aminopeptidasas N (APN). Estas son enzimas intestinales propuestos como receptores para la unión de las proteínas Cry al epitelio intestinal en varios lepidópteros. Finalmente, las APN expresadas en el intestino de O. nubilalis se caracterizaron a nivel genético, funcional y en lo que respecta a su interacción con las proteínas Cry1Ab y Cry1F. Las secuencias codificantes para seis isoformas de APN más una aminopeptidasa sensible a puromicina (PSA) se clonaron a partir de ADNc de intestino, y se caracterizaron buscando la presencia de motivos funcionales y analizando sus relaciones filogenéticas. El patrón de expresión espacial y temporal indicó que todos los genes están expresados en el intestino de las larvas de O. nubilalis durante todo el desarrollo larvario, y que solo un transcrito (onapn8) está expresado también en los túbulos de Malpighio. El gen psa es el único en tener una expresión ubicua en todos los tejidos analizados. Cinco APN se expresaron con éxito en las células Sf21 utilizando el sistema Bac-to-Bac® basado en baculovirus. La caracterización de la actividad enzimática de cada isoforma expresada en las células Sf21 demostró que cada una de ellas es funcionalmente diferente, tanto en la afinidad a varios sustratos como por la sensibilidad a diferentes inhibidores. La interacción de las APN expresadas ex vivo con las proteínas Cry se abordó mediante ensayos de inmunohistoquímica. Este tipo de aproximación nos permitió visualizar la interacción diferencial de las APN con las toxinas Cry1Ab y Cry1F: la isoforma OnAPN1 es capaz de unir a ambas toxinas mientras que las isoformas OnAPN3a y OnAPN8 solamente unen a Cry1F. Como conclusión, este trabajo aporta resultados que se añaden a los publicados previamente y que son necesarios para evaluar el potencial de desarrollo de resistencia a las proteínas Cry en O. nubilalis. En primer lugar, la tolerancia a las proteínas Cry1Ab parece ser un rasgo común en poblaciones de campo españolas. En segundo lugar, este rasgo parece derivar del efecto conjunto de varios genes, según hemos concluido tras realizar estudios de genética cuantitativa con isolíneas fundadas con individuos de zonas geográficamente distantes de España. Adicionalmente, se ha analizado la interacción de las proteínas Cry1Ab y Cry1F con el epitelio intestinal de O. nubilalis, encontrando diferencias de especificidad en la unión de estas proteínas e identificando a las APN como posibles receptores. Se han caracterizado las secuencias de los transcritos de seis isoformas de APN, y cinco han sido expresados en cultivo celular para determinar su actividad biológica y su interacción con las proteínas Cry. Los resultados indican que, de las APN estudiadas, las que podrían jugar un papel en la unión de las proteínas Cry al epitelio intestinal son tres. Además, según hemos evidenciado, estas proteínas parecen interactuar de forma diferencial con las toxinas Cry1Ab y Cry1F. Las resultados obtenidos a lo largo de esta tesis complementan los datos existentes en la literatura y esencialmente son útiles para el diseño de estrategias eficaces de manejo de la resistencia a las tecnologías Bt utilizadas a día de hoy en el control de O. nubilalis.Ostrinia nubilalis (Hübner) is one of the key pests of maize cultivations both in Europe and North America. Its effective control is one the most important goals of the pest management, since this lepidopteran species causes economically relevant losses. Commercialization of transgenic plants expressing insecticidal Cry proteins from Bacillus thuringiensis (Berliner) (Bt) started in 1996 and since then cultivation of Bt maize expressing Cry1Ab has been steadily increasing, reaching an effective control of O. nubilalis. Recently, a second Bt maize event expressing Cry1F has been commercialized. The effectiveness of the technologies based on Bt is seriously threatened by the possible development of the resistance in the target insects. Resistance may be caused by mutations that occur in one of the multiple steps of the mode of action of the Cry toxins in Lepidoptera. The knowledge of the biochemical basis that undergone the toxicity process produced by the Cry proteins, as well as the genetics of the resistance, are fundamental to design strategies aimed to preserve the efficacy of the Bt technologies as long as possible. The work presented here had two main objectives: first, the evaluation of the potential in Spanish populations of O. nubilalis to develop resistance to Cry toxins; second, the characterization of the Cry1Ab and Cry1F interactions with the midgut epithelium of O. nubilalis. To these purposes, we analyzed the susceptibility of a greenhouse population collected from cultivations regularly sprayed with Bt formulations. Also, we applied quantitative genetic techniques to investigate the tolerance to Cry1Ab in field populations and we developed a molecular marker to assess the contribution of the candidate resistance gene cadherin to the tolerance. Moreover, we characterized the O. nubilalis aminopeptidases (APNs) that have been previously described as candidate receptor for Cry proteins. The baseline susceptibility of the Spanish greenhouse population of O. nubilalis to four individual Cry protoxins, activated toxins and to the standard product HD-1-S-2005, was compared with the one obtained for a laboratory strain never exposed to B. thuringiensis. The greenhouse population was sampled from greenhouses routinely sprayed with Bt products during the whole growing season, to control lepidopteran pest infestations. Results from mortality assays indicated that toxins from class Cry1 are the most effective against O. nubilalis while Cry2Aa exerts a minor toxic effect. We found significant differences in susceptibility between the greenhouse and the laboratory strains to all the individual Cry1 protoxins and toxins tested. However, the overall results that included functional mortality bioassays and laboratory resistance selection with Cry1Ab protein, suggested that the susceptibility differences probably reflected intraspecific variation instead to be due to a selection process occurred in the greenhouse. Tolerance to Cry1Ab protein in Spanish O. nubilalis populations was studied as a quantitative genetic trait, using isolines established from field-derived insects. F1 offspring was tested for susceptibility to trypsin-activated Cry1Ab using a concentration that caused a mean larval mortality of 87% (± 17% SD). The progeny of the most tolerant isolines (that had shown mortalities lower than 60%) was crossed to obtain the F2 generation that was exposed to the same Cry1Ab concentration. A clear reduction in mortality (62% ± 17% SD) was observed. The upper limit for heritability was estimated to range between 0.82 and 0.90, suggesting that a high part of phenotypic variation in tolerance to Cry1Ab was attributable to genetic differences. An estimate of the minimum number of segregating factors indicated that the loci involved in tolerance to Cry1Ab were at least two. The role of the cadherin, which is a B. thuringiensis resistance gene in some lepidopteran species, was assessed in the most tolerant isolines by using an EPIC-PCR marker specifically developed for this study. Association between cadherin and tolerance was obtained in one tolerant isoline; however it could be not confirmed by segregation analysis in the F2 progeny because F2 offspring was not viable. Our results indicate that the tolerance trait is common in Spanish field populations of O. nubilalis. Binding of Cry proteins to the midgut epithelium of susceptible lepidopteran larvae is an essential step in the toxicity process. We try to discriminate the interaction of two Cry proteins from class 1 (Cry1Ab and Cry1F), both of them nowadays expressed in the transgenic Bt maize, to the O. nubilalis midgut epithelium. We assayed the effect of preincubations of the component of the binding assays (biotinilated toxins or brush border membrane vesicles) with Cry1Ab or Cry1F toxins, sugars, lectins or anticadherin antibody. Results showed that Cry1Ab protein bind to BBMV in a different way than Cry1F: binding signal of Cry1Ab did not decrease after any preincubation while Cry1F binding signal decreased after preincubations with Cry1F, Cry1Ab and sialic acid. These differences pointed out the existence of a midgut receptor not shared by the two Cry proteins, in addition to one or more common receptors. Ligand blot assays showed that Cry1Ab and Cry1F toxins bind to 75 and 150 kDa proteins from midgut epithelium. Cry1Ab toxin also binds to a band of 250 kDa. Identification of the protein present in the 250 kDa band did not render any result, whereas the most abundant proteins composing bands of 75 and 150 kDa were identified as aminopeptidasas N (APNs). These proteins are midgut enzymes that had been previously described as candidate receptor for Cry proteins in several lepidopteran species. APNs from O. nubilalis midgut were sequenced, characterized for functional motifs and expression pattern, and expressed ex vivo to evaluate their biological activity and their interaction with Cry1Ab and Cry1F proteins. In this work, seven transcripts expressed in O. nubilalis midgut and codifying for aminopeptidases were sequenced. Six out the seven sequences analyzed were classified as OnAPN while one sequence was identified as puromycin-sensible aminopeptidase (OnPSA). Expression of all the transcripts was steadily detected in the midgut of the larvae throughout the whole larval stage. However, only one OnAPN transcript (onapn8) was expressed also in Malpighian tubules whereas onpsa gen showed a ubiquitous expression. Five OnAPN were successfully expressed as recombinant proteins in Sf21 cells using the Bac-to-Bac® system. Characterization of the enzymatic activity displayed differences among the cell-expressed OnAPNs in substrate affinity and sensitivity to inhibitors. Interaction of the cell-expressed OnAPNs with the Cry1Ab and Cry1F proteins was assessed by using fluorescent antibodies. The results showed differential binding specificity of Cry proteins to the OnAPN isoforms: OnAPN1 binds to both Cry1Ab and Cry1F toxins while OnAPN3a and OnAPN8 only bind to Cry1F protein. In summary, this work provides data on the susceptibility and the potential for resistance appearance to Bt products in O. nubilalis populations. Also, data presented here are useful to understand the mode of action of two Cry proteins, Cry1Ab and Cry1F, nowadays expressed in Bt maize resistant to O. nubilalis attacks. This information complement data reported in literature, helping to design effective resistance management strategies for the Bt technologies used in the O. nubilalis control

Transcriptome profiling reveals differential gene expression of detoxification enzymes in a hemimetabolous tobacco pest after feeding on jasmonate-silenced Nicotiana attenuata plants

Putatively unique transcripts (PUTs) used in real-time qPCR (RT-qPCR) expression analysis. (XLSX 12 kb

Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

Among the butterflies of the genus Papilio (Lepidoptera: Papilionidae), Papilio hospiton (Gene) has a geographical distribution limited to the Mediterranean islands of Sardinia (Italy) and Corsica (France). This is mainly due to the host range that includes only a few plant species of Apiaceae and Rutaceae growing on these islands. In a previous electrophysiological investigation conducted on the maxillary gustatory system of larvae of P. hospiton and its closely phylogenetically related species Papilio machaon, a significantly higher spike activity was shown for the gustatory neurons of lateral and medial styloconic sensilla in P. hospiton when bitter compounds were tested. This effect was possibly correlated to the limited host choice range for P. hospiton. To shed light on the molecular aspects of this phenomenon, we investigated the expression pattern of sensory-related sequences by conducting a transcriptomic analysis from total RNA isolates of P. hospiton larval maxillae. We identified several transcripts that may be involved in taste (one gustatory receptor, one divergent ionotropic receptor, and several transient receptor potential channels, TRPs) as well as transcripts supporting an olfactory function for this appendage, including odorant receptors (ORs), antennal ionotropic receptors (A-IRs), sensory neuron membrane proteins (SNMPs), and odorant-binding proteins (OBPs). We used Human Embryonic Kidney (HEK293A) cells to heterologously express two of the identified receptors, PhospOR1 and PhospPain, together with their orthologs from P. machaon, for functional characterization. While our data suggest no activation of these two receptors by the ligands known so far to activate the electrophysiological response in larval maxillary neurons of Papilio species, nor temperature activation of both Papilio TRPA-channel Painless, they represent the first attempt in connecting neuronal activity with their molecular bases to unravel diet specialization between closely related Papilio species

Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

Among the butterflies of the genus Papilio (Lepidoptera: Papilionidae), Papilio hospiton (Géné) has a geographical distribution limited to the Mediterranean islands of Sardinia (Italy) and Corsica (France). This is mainly due to the host range that includes only a few plant species of Apiaceae and Rutaceae growing on these islands. In a previous electrophysiological investigation conducted on the maxillary gustatory system of larvae of P. hospiton and its closely phylogenetically related species Papilio machaon, a significantly higher spike activity was shown for the gustatory neurons of lateral and medial styloconic sensilla in P. hospiton when bitter compounds were tested. This effect was possibly correlated to the limited host choice range for P. hospiton. To shed light on the molecular aspects of this phenomenon, we investigated the expression pattern of sensory-related sequences by conducting a transcriptomic analysis from total RNA isolates of P. hospiton larval maxillae. We identified several transcripts that may be involved in taste (one gustatory receptor, one divergent ionotropic receptor, and several transient receptor potential channels, TRPs) as well as transcripts supporting an olfactory function for this appendage, including odorant receptors (ORs), antennal ionotropic receptors (A-IRs), sensory neuron membrane proteins (SNMPs), and odorant-binding proteins (OBPs). We used Human Embryonic Kidney (HEK293A) cells to heterologously express two of the identified receptors, PhospOR1 and PhospPain, together with their orthologs from P. machaon, for functional characterization. While our data suggest no activation of these two receptors by the ligands known so far to activate the electrophysiological response in larval maxillary neurons of Papilio species, nor temperature activation of both Papilio TRPA-channel Painless, they represent the first attempt in connecting neuronal activity with their molecular bases to unravel diet specialization between closely related Papilio species

Quantitative genetic analysis of Cry1Ab tolerance in Ostrinia nubilalis Spanish populations 2

Abstract 2 Tolerance to Bacillus thuringiensis Cry1Ab toxin in Spanish Ostrinia nubilalis 3 populations was analyzed by quantitative genetic techniques, using isolines established 4 from field-derived insects. F1 offspring was tested for susceptibility to trypsin activated 5 Cry1Ab using a concentration that caused a mean larval mortality of 87% (± 17% SD). 6 The progeny of the most tolerant isolines (that had shown mortalities lower than 60%) 7 was crossed to obtain the F2 generation that was exposed to the same Cry1Ab 8 concentration. A clear reduction in mortality (62% ± 17% SD) was observed. The upper 9 limit for heritability was estimated to range between 0.82 and 0.90, suggesting that a 1

The evolution of olfactory gene families in Drosophila and the genomic basis of chemical-ecological adaptation in Drosophila suzukii

How the evolution of olfactory genes correlates with adaption to new ecological niches is still a debated topic. We explored this issue in Drosophila suzukii, an emergin gmodel that reproduces on fresh fruit rather than in fermenting substrates likemost other Drosophila. We first annotated the repertoire of odorant receptors (ORs), odorant binding proteins (OBPs), and antennal ionotropic receptors (aIRs) in the genomes of two strains of D. suzukii and of its close relative Drosophila biarmipes. We then analyzed these genes on the phylogeny of 14 Drosophila species: whereas ORs and OBPs are characterized by higher turnover rates in some lineages including D. suzukii, aIRs are conserved throughout the genus. Drosophila suzukii is further characterized by a non-random distribution of OR turnover on the gene phylogeny, consistent with a change in selective pressures. In D. suzukii, we found duplications and signs of positive selection in ORs with affinity for short-chain esters, and loss of function of ORs with affinity for volatiles produced during fermentation. These receptors-Or85a and Or22a-are characterized by divergent alleles in the European and American genomes, and we hypothesize that they may have been replaced by some of the duplicated ORs in corresponding neurons, a hypothesis reciprocally confirmed by electrophysiological recordings. Our study quantifies the evolution of olfactory genes in Drosophila and reveals an array of genomic events that can be associated with the ecological adaptations of D. suzukii

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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Quantitative genetic analysis of Cry1Ab tolerance in Ostrinia nubilalis Spanish populations

30 p.-2 fig.-3 tab.Tolerance to Bacillus thuringiensis Cry1Ab toxin in Spanish Ostrinia nubilalis populations was analyzed by quantitative genetic techniques, using isolines established from field-derived insects. F1 offspring was tested for susceptibility to trypsin activated Cry1Ab using a concentration that caused a mean larval mortality of 87% (±17% SD). The progeny of the most tolerant isolines (that had shown mortalities lower than 60%) was crossed to obtain the F2 generation that was exposed to the same Cry1Ab concentration. A clear reduction in mortality (62 ± 17% SD) was observed. The upper limit for heritability was estimated to range between 0.82 and 0.90, suggesting that a high part of phenotypic variation in tolerance to Cry1Ab was attributable to genetic differences. An estimate of the minimum number of segregating factors indicated that the loci involved in tolerance to Cry1Ab were at least two. The role of the cadherin gene, which is a B. thuringiensis resistance gene in Lepidoptera, was assessed in the most tolerant isolines by using an EPIC-PCR marker specifically developed for this study. Association between cadherin and tolerance was obtained in one tolerant isoline; however it could be not confirmed by segregation analysis in the F2 progeny because F2 offspring was not viable. Our results indicate that the tolerance trait is common in Spanish field populations. Quantitative genetic techniques may be helpful for estimating the influence of genetic factors to Cry1Ab tolerance in O. nubilalis.Support for this project was provided by the EU Fifth Framework Project ‘‘ProBenBt’’ (Ref. QLK3-CT-2002-01969), by the Spanish Ministerio de Educación y Ciencia (project Ref. at the University of Valencia AGL2006-11914) and by the Spanish Ministerio de Ciencia e Innovación (project Ref. at the Centro de Investigaciones Biológicas of Madrid AGL 2009-08813). Cristina Crava was funded by a V Segles grant from the University of Valencia (Ref. UV-BVS- 07-2083).Peer reviewe