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Vers une meilleure compréhension des bases moléculaires de la résistance des moustiques au Bti (Bacillus thuringiensis subsp. israelensis) / Toward a better understanding of the molecular basis of Bti (bacillus thuringiensis var. israelensis) resistance in mosquitoesStalinski, Renaud 10 December 2015 (has links)
Le Bti est un bioinsecticide mondialement utilisé dans la lutte contre les moustiques. La toxicité du Bti est due à un cristal, constitué de quatre toxines principales, produit par la bactérie Bacillus thuringiensis subsp. israelensis. Le Bti représente une bonne alternative aux insecticides chimiques car il est peu persistant dans l’environnement et spécifique des insectes ciblés. La sélection d’une souche de moustique (Aedes aegypti) de laboratoire au Bti a entrainé une résistance modérée au Bti (3.5 fois) mais plus élevée aux toxines testées séparément (jusqu’à 60 fois). Ce résultat suggère que l’adaptation des moustiques au Bti en populations naturelles peut être corrélée à une résistance accrue à chacune des toxines. Pour pouvoir détecter une adaptation au Bti, il est donc nécessaire de mieux caractériser la résistance à chaque toxine qui compose le Bti.Cette thèse se structure en trois axes. 1. L’analyse du transcriptome par RNA-seq des souches résistantes à chaque toxine et au Bti a permis de mettre en évidence plusieurs gènes candidats potentiellement impliqués dans la résistance. La résistance développée au laboratoire est complexe, combinant des mécanismes de résistances spécifiques de chaque toxine et généralistes. 2. L’analyse du transcriptome d’une souche sensible exposée au Bti a permis d’identifier des gènes impliqués dans la réponse à l’intoxication, notamment les phosphatases alcalines. De plus, l’exposition à chaque toxine et au Bti induit une modification de l’expression de ces gènes bien plus importante chez les souches résistantes que chez la souche sensible. 3. L’implication de la réponse immunitaire dans la résistance et dans la réponse à une exposition au Bti est discutée. L’exposition au Bti accroit la compétence vectorielle d’A. aegypti pour deux maladies tropicales (Dengue et Chikungunya), différemment selon si la souche est sensible ou résistante au Bti. Cette thèse permet de mieux comprendre les mécanismes de résistances au Bti et à ses toxines ; ces connaissances sont nécessaires pour mieux anticiper la résistance sur le terrain. / Bti is a bioinsecticide used worldwide for mosquito control. Bti toxicity is due to a crystal, composed of four main toxins, produced by the bacteria Bacillus thuringiensis subsp. israelensis. Bti represents a good alternative to chemical insecticides because it is known to have a low persistence in the environment and to be specific to the targeted insects. The selection of a laboratory mosquito (Aedes aegypti) strain with a persistent form of Bti led to moderate resistance to Bti (3.5-fold) but to higher resistance (up to 60-fold) to Bti toxins tested separately. This result suggests that the adaptation of mosquitoes to Bti in the field may be correlated to a higher resistance to each toxin. In order to detect an adaptation to Bti, it is thus necessary to characterize better the resistance against each Bti toxin.This thesis is organized in three parts. 1. The transcritpome analysis of strains resistant to each toxin and to Bti using RNA-seq technique enabled to highlight genes and mechanisms potentially involved in the resistance. The resistance developed in the laboratory is complex, combining generalist and specific mechanisms to resist to each toxin. 2. The transcriptome analysis of a susceptible strain exposed to Bti enabled identifying genes involved in the response to the intoxication, especially alkaline phosphatases. Furthermore, exposure to Bti and to each toxin resulted in gene expression modification being far higher in resistant strains than in susceptible strain. 3. The role of immunity in the resistance and in the response to Bti exposure is discussed. The Bti exposure increases the vector competence of A. aegypti for two tropical diseases (Dengue and Chikungunya) differently, depending on the strain being susceptible or resistant to Bti. This thesis enables better understanding of resistance mechanisms to Bti and its toxins; this knowledge is necessary for anticipating field resistance development.
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Devenir du bioinsecticide Bti dans l'environnement et impact sur le développement de résistances chez le moustique / Fate of the bioinsecticide Bti in the environment and impact on the development of resistance in mosquito speciesTetreau, Guillaume 30 May 2012 (has links)
Le Bti est un bioinsecticide mondialement utilisé pour contrôler les populations de moustiques. La toxicité du Bti est due à un cristal constitué de plusieurs toxines, produit par la bactérie éponyme Bacillus thuringiensis var. israelensis. Qualifié de peu persistant dans l'environnement et sans résistance connue en populations naturelles, le Bti représente une alternative « propre » aux insecticides chimiques. Cependant, une litière végétale prélevée plusieurs mois après un traitement insecticide a montré une forte toxicité contre les larves de moustiques, due à la présence de Bti. La sélection d'une souche de moustique de laboratoire Aedes aegypti avec cette litière (souche LiTOX) a permis d'obtenir une résistance modérée au Bti (3,5 fois) mais plus élevée aux toxines testées séparément (jusqu'à 60 fois). La présente thèse se décompose en trois axes. 1. Un test permettant le suivi du Bti dans l'environnement a été développé et breveté. Il a permis de mettre en évidence un recyclage du Bti dans les litières toxiques et une persistance différentielle des toxines. 2. La résistance au Bti de la souche LiTOX a été étudiée par des approches globales et fonctionnelles, permettant de mieux caractériser les mécanismes de résistance multigéniques impliqués. 3. La résistance a été recherchée en populations naturelles par des bioessais aux toxines du Bti testées séparément, outil plus sensible que les bioessais « classiques » au Bti, et a permis de mettre en évidence une tolérance accrue de certaines populations. Cette thèse apporte les connaissances et les outils nécessaires pour mieux caractériser les paramètres favorisant la prolifération du Bti en milieu naturel et les mécanismes de résistance potentiellement sélectionnés chez les moustiques. / Bti is a bioinsecticide used worldwide for mosquito control. Bti toxicity is due to a toxins-containing crystal, produced by the bacteria Bacillus thuringiensis var. israelensis. Bti is known to have a low persistence in the environment and no firm resistance has been found yet in the field, therefore representing a “safe” alternative to chemical insecticides. Nevertheless, leaf litters collected several months after a treatment exhibited high toxicity against mosquito larvae, due to persistent Bti. The selection of a laboratory mosquito strain of Aedes aegypti with this litter (LiTOX strain) led to a moderate 3.5-fold resistance to Bti but to higher resistance (until 60-fold) to Bti toxins tested separately. This thesis is structured in three main axes. 1. A test has been developed and patented to follow the fate of Bti in the environment. We showed that Bti has recycled in the collected toxic leaf litters and that the different Bti toxins persist differently. 2. The Bti-resistance of the LiTOX strain has been studied by global and functional approaches, leading to a better understanding of the multigenic mechanisms implicated in the resistance phenotype. 3. Cryptic Bti-resistance has been investigated performing bioassays with separate Bti Cry toxins, showing that some populations exhibit increased Cry tolerance. This thesis gives the knowledge and new tools necessary to better understand how Bti can persist and recycle in the environment and how mosquitoes can develop resistance.
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Avaliação do glicerol proveniente da fabricação do biodiesel como substrato para produção de endotoxinas por Bacillus thuringiensis var. israelensis / Evaluation of glycerol derived from biodiesel production as substrate for endotoxins production by Bacillus thuringiensis var. israelensis.Barbosa, Claudia Rodrigues 19 June 2009 (has links)
A utilização do glicerol proveniente da fabricação do biodiesel como substrato para a obtenção de produtos biotecnológicos é uma alternativa promissora como forma de disposição adequada deste subproduto. O bioinseticida formulado com toxinas de Bacillus thuringiensis tem sido utilizado para o controle de insetos veiculadores de doenças, como a dengue, que atingem milhões de pessoas em todo o mundo. Para que este bioinseticida seja competitivo com os inseticidas químicos é necessário que o custo de sua produção seja diminuído, o que pode ser feito com a utilização de fontes de carbono alternativas, de forma a diminuir o custo do meio de fermentação. Neste trabalho, empregou-se o glicerol proveniente da fabricação do biodiesel de sebo bovino como componente do meio de fermentação para a produção de bioinseticida por Bacillus thuringiensis var. israelensis. Foram avaliados diferentes tratamentos do resíduo contendo glicerol, baseados em acidificação, decantação e aquecimento, para remoção de impurezas. O ajuste do pH até 7 pela adição de ácido fosfórico, seguido de decantação, aquecimento para remoção de metanol e nova decantação, foi a forma de tratamento escolhida, uma vez que proporcionou a maior atividade tóxica do meio fermentado contra larvas de Aedes aegypti. O meio de fermentação foi formulado determinando-se as concentrações de glicerol, extrato de levedura, sulfato de amônio e cloreto de cálcio que proporcionaram a maior atividade larvicida do meio fermentado, de acordo com planejamento fatorial 24. A toxicidade foi avaliada pela CL50 (concentração do complexo esporo-cristal necessária para matar 50% da população de larvas). O melhor valor de CL50 foi obtido com a utilização de glicerol a 10 g/L, extrato de levedura a 12 g/L, cloreto de cálcio a 0,24 g/L e sem adição de sulfato de amônio. A análise estatística dos dados confirmou a significância das variáveis glicerol, extrato de levedura e cloreto de cálcio sobre a atividade larvicida do meio fermentado. Cloreto de cálcio não influenciou significantemente a produção de esporos pela bactéria, e a taxa de esporulação não foi influenciada por cloreto de cálcio e sulfato de amônio nas concentrações estudadas. Para a otimização da produção de endotoxinas pela bactéria, foi realizado um planejamento fatorial 22, variando-se apenas as concentrações de extrato de levedura e de cloreto de cálcio, fixando-se a concentração de glicerol em 10 g/L e excluindo-se o sulfato de amônio do meio de fermentação. O melhor valor de CL50 (0,295 mg/mL) foi obtido no ensaio com 15 g/L de extrato de levedura e 0,4 g/L de cloreto de cálcio. / The use of glycerol derived from biodiesel production as a substrate for the acquisition of biotechnology products is a promising alternative as an adequate disposal mean of this by-product. The bioinsecticide formulated with toxins from Bacillus thuringiensis has been used to control vectors insects of diseases, such as dengue, which affect millions of people around the world. For this bioinsecticide be competitive with chemical insecticides is necessary a lower cost of its production, which can be achieved by the use of alternative carbon sources in order to reduce the fermentation medium cost. In this work, it was used glycerol from beef tallow biodiesel production as a component of the fermentation medium for the production of bioinsecticide by Bacillus thuringiensis var. israelensis. Different treatments of the residue containing glycerol, based on acidification, sedimentation and heating, to remove impurities, were evaluated. The adjustment of pH to 7 by addition of phosphoric acid, followed by decantation, then heating for removal of methanol, and a new decantation, was the treatment selected, since it provided the highest toxic activity of the fermented broth against Aedes aegypti larvae. The fermentation medium was formulated by determining the concentration of glycerol, yeast extract, ammonium sulfate and calcium chloride that provided the greatest larvicidal activity of the fermented broth according to 24 factorial design. The toxicity was measured by the LC50 (concentration of the spore-crystal complex required to kill 50% of the larvae). The best value of LC50 was obtained using glycerol at 10 g/L, yeast extract at 12 g/L, calcium chloride at 0.24 g/L and without addition of ammonium sulfate. Statistical analysis confirmed the significance of the variables glycerol, yeast extract and calcium chloride on the larvicidal activity of fermented broth. Calcium chloride did not influence significantly the production of spores by the bacteria and the sporulation ratio was not affected by calcium chloride and ammonium sulfate at the studied concentrations. For the endotoxins production optimization, a 22 factorial design was carried out, varying only the concentrations of yeast extract and calcium chloride, maintaining the concentration of glycerol at 10 g/L and excluding ammonium sulfate of the fermentation medium. The best value of LC50 (0,295 mg/mL) was obtained in the assay with 15 g/L of yeast extract and 0.4 g/L of calcium chloride.
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Avaliação do glicerol proveniente da fabricação do biodiesel como substrato para produção de endotoxinas por Bacillus thuringiensis var. israelensis / Evaluation of glycerol derived from biodiesel production as substrate for endotoxins production by Bacillus thuringiensis var. israelensis.Claudia Rodrigues Barbosa 19 June 2009 (has links)
A utilização do glicerol proveniente da fabricação do biodiesel como substrato para a obtenção de produtos biotecnológicos é uma alternativa promissora como forma de disposição adequada deste subproduto. O bioinseticida formulado com toxinas de Bacillus thuringiensis tem sido utilizado para o controle de insetos veiculadores de doenças, como a dengue, que atingem milhões de pessoas em todo o mundo. Para que este bioinseticida seja competitivo com os inseticidas químicos é necessário que o custo de sua produção seja diminuído, o que pode ser feito com a utilização de fontes de carbono alternativas, de forma a diminuir o custo do meio de fermentação. Neste trabalho, empregou-se o glicerol proveniente da fabricação do biodiesel de sebo bovino como componente do meio de fermentação para a produção de bioinseticida por Bacillus thuringiensis var. israelensis. Foram avaliados diferentes tratamentos do resíduo contendo glicerol, baseados em acidificação, decantação e aquecimento, para remoção de impurezas. O ajuste do pH até 7 pela adição de ácido fosfórico, seguido de decantação, aquecimento para remoção de metanol e nova decantação, foi a forma de tratamento escolhida, uma vez que proporcionou a maior atividade tóxica do meio fermentado contra larvas de Aedes aegypti. O meio de fermentação foi formulado determinando-se as concentrações de glicerol, extrato de levedura, sulfato de amônio e cloreto de cálcio que proporcionaram a maior atividade larvicida do meio fermentado, de acordo com planejamento fatorial 24. A toxicidade foi avaliada pela CL50 (concentração do complexo esporo-cristal necessária para matar 50% da população de larvas). O melhor valor de CL50 foi obtido com a utilização de glicerol a 10 g/L, extrato de levedura a 12 g/L, cloreto de cálcio a 0,24 g/L e sem adição de sulfato de amônio. A análise estatística dos dados confirmou a significância das variáveis glicerol, extrato de levedura e cloreto de cálcio sobre a atividade larvicida do meio fermentado. Cloreto de cálcio não influenciou significantemente a produção de esporos pela bactéria, e a taxa de esporulação não foi influenciada por cloreto de cálcio e sulfato de amônio nas concentrações estudadas. Para a otimização da produção de endotoxinas pela bactéria, foi realizado um planejamento fatorial 22, variando-se apenas as concentrações de extrato de levedura e de cloreto de cálcio, fixando-se a concentração de glicerol em 10 g/L e excluindo-se o sulfato de amônio do meio de fermentação. O melhor valor de CL50 (0,295 mg/mL) foi obtido no ensaio com 15 g/L de extrato de levedura e 0,4 g/L de cloreto de cálcio. / The use of glycerol derived from biodiesel production as a substrate for the acquisition of biotechnology products is a promising alternative as an adequate disposal mean of this by-product. The bioinsecticide formulated with toxins from Bacillus thuringiensis has been used to control vectors insects of diseases, such as dengue, which affect millions of people around the world. For this bioinsecticide be competitive with chemical insecticides is necessary a lower cost of its production, which can be achieved by the use of alternative carbon sources in order to reduce the fermentation medium cost. In this work, it was used glycerol from beef tallow biodiesel production as a component of the fermentation medium for the production of bioinsecticide by Bacillus thuringiensis var. israelensis. Different treatments of the residue containing glycerol, based on acidification, sedimentation and heating, to remove impurities, were evaluated. The adjustment of pH to 7 by addition of phosphoric acid, followed by decantation, then heating for removal of methanol, and a new decantation, was the treatment selected, since it provided the highest toxic activity of the fermented broth against Aedes aegypti larvae. The fermentation medium was formulated by determining the concentration of glycerol, yeast extract, ammonium sulfate and calcium chloride that provided the greatest larvicidal activity of the fermented broth according to 24 factorial design. The toxicity was measured by the LC50 (concentration of the spore-crystal complex required to kill 50% of the larvae). The best value of LC50 was obtained using glycerol at 10 g/L, yeast extract at 12 g/L, calcium chloride at 0.24 g/L and without addition of ammonium sulfate. Statistical analysis confirmed the significance of the variables glycerol, yeast extract and calcium chloride on the larvicidal activity of fermented broth. Calcium chloride did not influence significantly the production of spores by the bacteria and the sporulation ratio was not affected by calcium chloride and ammonium sulfate at the studied concentrations. For the endotoxins production optimization, a 22 factorial design was carried out, varying only the concentrations of yeast extract and calcium chloride, maintaining the concentration of glycerol at 10 g/L and excluding ammonium sulfate of the fermentation medium. The best value of LC50 (0,295 mg/mL) was obtained in the assay with 15 g/L of yeast extract and 0.4 g/L of calcium chloride.
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Response Surface Optimization Of Bacillus Thuringiensis Israelensis FermentationTokcaer, Zeynep 01 December 2003 (has links) (PDF)
The control of pest populations by using insect pathogens has been an attractive alternative to the application of chemical pesticides employed for the same purpose. As these chemicals not only damage the environment, but also trigger development of resistance by the pests and can harm other organisms together with the target pest,
biological control is preferable and Bacillus thuringiensis (Bt) subspecies have been the most widely used bioinsecticides in forestry, agriculture and mosquito/ black fly control.
The most important property of Bt subspecies is the synthesis of protoxins named as delta-endotoxins (crystal proteins). In this study, response surface optimization of Bt subsp. israelensis HD500 batch fermentation for high level production of its toxin proteins Cry4Ba and Cry11Aa was performed. As the interaction of the medium components as well as cultivation conditions are expected to influence the production of the toxin proteins, an experimental chart was prepared by accepting the previously reported optimal values for the most important parameters as zero points: [Mn], 10-6 M / [K2HPO4], 50 mM / C:N ratio, 20:1 and incubation temperature / 30° / C. When the combinations of these variables at different levels were studied at 30 batch cultures and analysed for the optimum toxin protein concentrations, temperature: 28.3& / #61616 / C, [Mn]: 3.3x10-7M, C:N ratio: 22.2 and [K2HPO4]: 66.1mM yielded the highest concentrations of both Cry4Ba and Cry11Aa toxin proteins.
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Influência de parâmetros operacionais, fontes protéicas e substratos energéticos sobre o cultivo de Bacillus thuringiensis var. israelensisPanarotto, Cíntia 20 October 2006 (has links)
Bacillus thuringiensis var. israelensis é uma bactéria entomopatogênica capaz de sintetizar um cristal protéico composto por endotoxinas. Estas endotoxinas são utilizadas em formulações de biolarvicidas, sendo efetivas contra dípteros que representam um problema para a saúde humana. Neste trabalho, estudou-se a influência de fontes protéicas, de substratos energéticos, da temperatura e do pH sobre o crescimento celular, a esporulação e a expressão de endotoxinas de B. thuringiensis var. israelensis, em frascos agitados e em fermentador de bancada. Foram estudadas fontes protéicas de menor custo em substituição ao extrato de levedura de laboratório. Melhores resultados, com relação ao crescimento celular, à esporulação e à mortalidade de larvas de Culex quinquefasciatus, foram obtidos com 12 g/L de extrato de levedura bruto Prodex (ELB) ou 20 g/L de farelo de soja (FS) em biorreator de bancada, com valores máximos de demanda de oxigênio pelo cultivo (OUR) de 58,1 e 53,3 mmolO2/L/h, máxima concentração celular (X) de 12 g/L, esporulação de 3.1012 e 6.1012 esporos/mL e concentração letal mediana (CL50) de 0,21 e 0,45 ppm, respectivamente. Entre estas alternativas, o ELB, com um custo muito inferior ao do extrato de levedura de laboratório, foi considerado como uma fonte de proteína mais adequada que o farelo de soja por ser completamente solúvel o que representaria uma vantagem em processos em larga escala. Com relação aos substratos energéticos estudados (glicose, sacarose e lactose), a glicose, nas concentrações de 10 e 20 g/L, apresentou os resultados mais expressivos em termos de respiração (OUR máximo apoximadamente 60 mmolO2/L/h, em ambos os cultivos), de crescimento (X máxima = 8,2 e 12 g/L, respectivamente) e esporulação (1012 esporos/mL em 26 e 28 horas, respectivamente). A expressão de endotoxinas foi maior em meios com glicose ou sacarose, observando-se em eletroforese as bandas de 26, 70 e 128 kDa. Apesar do crescimento celular inferior, a sacarose (10 g/L), um carboidrato de custo relativamente baixo no Brasil, pode ser utilizada como substrato para Bti uma vez que permite a expressão das endotoxinas em estudo. A temperatura mostrou grande influência sobre o cultivo de Bti, sendo 27 e 30oC as que proporcionaram as mais altas OUR (88,1 e 66,1 mmolO2/L/h), X (13 g/L, em ambos os cultivos) e esporulação (8 e 7.1010 esporos/mL), assim como maior expressão das endotoxinas de 70 e 26 kDa em meios com ELB (12 g/L) e glicose ( 20 g/L). Entre as duas, a temperatura de 27oC, porém, foi considerada mais adequada por facilitar a transferência de oxigênio em cultivo de Bti. Os baixos valores de pH, observados quando o cultivo foi realizado sem controle e quando foi controlado em 5,5, influenciaram negativamente a produção de biomassa que atingiu 9,5 g/L. Com o pH controlado em 6,2 e 7,0 ou quando variou entre 7,0 e 5,5, o maior valor de X foi de cerca de 12 g/L. Apesar disso, a contagem de esporos foi semelhante em todos os ensaios: aproximadamente 1012 esporos/mL, em cerca de 30 horas de cultivo. Uma melhor expressão das endotoxinas de 26, 70 e 128 kDa foi evidenciada quando o pH foi mantido próximo da neutralidade. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-13T17:26:06Z
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dissertacao Cintia Panarotto.pdf: 1176937 bytes, checksum: b1ab2e0e4c43ddf42a0f132a88cf7c77 (MD5) / Made available in DSpace on 2014-05-13T17:26:06Z (GMT). No. of bitstreams: 1
dissertacao Cintia Panarotto.pdf: 1176937 bytes, checksum: b1ab2e0e4c43ddf42a0f132a88cf7c77 (MD5) / The entomopathogenic bacterium Bacillus thuringiensis var. israelensis produces a crystal protein formed by endotoxins. These endotoxins are used in the formulation of biolarvicides effective for the control of diptera, which represent a problem for human health. In this work, the influence of nitrogen sources, energetic substrates, temperature, and pH on the cell growth, spore formation, and the expression of endotoxins by B. thuringiensis var. israelensis was studied in shake-flask and bioreactor experiments. Low-cost nitrogen sources were studied for the substitution of laboratory yeast extract. The best results for cell growth, spore formation and lethality of Culex quinquefasciatus larvae were obtained in bench bioreactor with medium containing 12 g/L of the bulk yeast extract Prodex (BYE) or 20 g/L of soy bran (SB), with maximum oxygen uptake rate values (OUR) of 58.1 and 53.3 mmolO2/L/h, maximum cell concentration of 12 g/L, spore counting of 3.1012 and 6.1012 spores/mL, and medium lethal concentration (LC50) of 0.21 and 0.45 ppm, respectively. In this comparison, BYE, which is much cheaper than laboratory yeast extract, was preferred as a protein source than soy bran because it is completely soluble, which could be an advantage in large-scale processing. With respect to the energetic substrates evaluated (glucose, sucrose and lactose), glucose, in concentrations of 10 and 20 g/L, led to the most significant results for respiration (maximum OUR approximately 60 mmolO2/L/h for both conditions), cell growth (maximum X = 8.2 and 12 g/L, respectively), and spore counting (1012 spores/mL in 26 and 28 hours, approximately). In media containing glucose or sucrose, the presence of toxins of 26, 70, and 128 kDa was observed. Despite the inferior cell growth observed, sucrose (10 g/L), a relatively low-cost carbohydrate in Brazil, could be used as a substrate for Bti cultivation since the endotoxins studied were expressed. With lactose, no significant result was found. The temperature of the medium exerts a major influence on the cultivation of Bti. At 27 and 30ºC, the higher values for the maximum OUR (88.1 and 66.1 mmolO2/L/h), X (13 g/L for both conditions) and spore counting (8 and 7.1010 spores/mL) were measured. The more intense expression for proteins of 70 and 26 kDa, in mediu containing BYE (12 g/L) and glucose (20 g/L), were also observed at these temperatures. The temperature of 27ºC can be considered as a better temperature for this process since oxygen supply would be facilitated at this condition. The low pH observed when the cultivation was performed without pH control or with pH kept constant at 5.5 led to decreasing values of biomass concentration which reached 9.5 g/L. When the pH was controlled in 6.2 and 7.0 or varied from 7.0 to 5.5, the maximum X was measured as 12 g/L. Despite such a difference, spore counting was similar in all experiments: 1012 spores/mL in approximately 30 hours of cultivation. A better expression of endotoxins of 26, 70, and 128 kDa was detected when the pH was kept close to neutral values.
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Influência de parâmetros operacionais, fontes protéicas e substratos energéticos sobre o cultivo de Bacillus thuringiensis var. israelensisPanarotto, Cíntia 20 October 2006 (has links)
Bacillus thuringiensis var. israelensis é uma bactéria entomopatogênica capaz de sintetizar um cristal protéico composto por endotoxinas. Estas endotoxinas são utilizadas em formulações de biolarvicidas, sendo efetivas contra dípteros que representam um problema para a saúde humana. Neste trabalho, estudou-se a influência de fontes protéicas, de substratos energéticos, da temperatura e do pH sobre o crescimento celular, a esporulação e a expressão de endotoxinas de B. thuringiensis var. israelensis, em frascos agitados e em fermentador de bancada. Foram estudadas fontes protéicas de menor custo em substituição ao extrato de levedura de laboratório. Melhores resultados, com relação ao crescimento celular, à esporulação e à mortalidade de larvas de Culex quinquefasciatus, foram obtidos com 12 g/L de extrato de levedura bruto Prodex (ELB) ou 20 g/L de farelo de soja (FS) em biorreator de bancada, com valores máximos de demanda de oxigênio pelo cultivo (OUR) de 58,1 e 53,3 mmolO2/L/h, máxima concentração celular (X) de 12 g/L, esporulação de 3.1012 e 6.1012 esporos/mL e concentração letal mediana (CL50) de 0,21 e 0,45 ppm, respectivamente. Entre estas alternativas, o ELB, com um custo muito inferior ao do extrato de levedura de laboratório, foi considerado como uma fonte de proteína mais adequada que o farelo de soja por ser completamente solúvel o que representaria uma vantagem em processos em larga escala. Com relação aos substratos energéticos estudados (glicose, sacarose e lactose), a glicose, nas concentrações de 10 e 20 g/L, apresentou os resultados mais expressivos em termos de respiração (OUR máximo apoximadamente 60 mmolO2/L/h, em ambos os cultivos), de crescimento (X máxima = 8,2 e 12 g/L, respectivamente) e esporulação (1012 esporos/mL em 26 e 28 horas, respectivamente). A expressão de endotoxinas foi maior em meios com glicose ou sacarose, observando-se em eletroforese as bandas de 26, 70 e 128 kDa. Apesar do crescimento celular inferior, a sacarose (10 g/L), um carboidrato de custo relativamente baixo no Brasil, pode ser utilizada como substrato para Bti uma vez que permite a expressão das endotoxinas em estudo. A temperatura mostrou grande influência sobre o cultivo de Bti, sendo 27 e 30oC as que proporcionaram as mais altas OUR (88,1 e 66,1 mmolO2/L/h), X (13 g/L, em ambos os cultivos) e esporulação (8 e 7.1010 esporos/mL), assim como maior expressão das endotoxinas de 70 e 26 kDa em meios com ELB (12 g/L) e glicose ( 20 g/L). Entre as duas, a temperatura de 27oC, porém, foi considerada mais adequada por facilitar a transferência de oxigênio em cultivo de Bti. Os baixos valores de pH, observados quando o cultivo foi realizado sem controle e quando foi controlado em 5,5, influenciaram negativamente a produção de biomassa que atingiu 9,5 g/L. Com o pH controlado em 6,2 e 7,0 ou quando variou entre 7,0 e 5,5, o maior valor de X foi de cerca de 12 g/L. Apesar disso, a contagem de esporos foi semelhante em todos os ensaios: aproximadamente 1012 esporos/mL, em cerca de 30 horas de cultivo. Uma melhor expressão das endotoxinas de 26, 70 e 128 kDa foi evidenciada quando o pH foi mantido próximo da neutralidade. / The entomopathogenic bacterium Bacillus thuringiensis var. israelensis produces a crystal protein formed by endotoxins. These endotoxins are used in the formulation of biolarvicides effective for the control of diptera, which represent a problem for human health. In this work, the influence of nitrogen sources, energetic substrates, temperature, and pH on the cell growth, spore formation, and the expression of endotoxins by B. thuringiensis var. israelensis was studied in shake-flask and bioreactor experiments. Low-cost nitrogen sources were studied for the substitution of laboratory yeast extract. The best results for cell growth, spore formation and lethality of Culex quinquefasciatus larvae were obtained in bench bioreactor with medium containing 12 g/L of the bulk yeast extract Prodex (BYE) or 20 g/L of soy bran (SB), with maximum oxygen uptake rate values (OUR) of 58.1 and 53.3 mmolO2/L/h, maximum cell concentration of 12 g/L, spore counting of 3.1012 and 6.1012 spores/mL, and medium lethal concentration (LC50) of 0.21 and 0.45 ppm, respectively. In this comparison, BYE, which is much cheaper than laboratory yeast extract, was preferred as a protein source than soy bran because it is completely soluble, which could be an advantage in large-scale processing. With respect to the energetic substrates evaluated (glucose, sucrose and lactose), glucose, in concentrations of 10 and 20 g/L, led to the most significant results for respiration (maximum OUR approximately 60 mmolO2/L/h for both conditions), cell growth (maximum X = 8.2 and 12 g/L, respectively), and spore counting (1012 spores/mL in 26 and 28 hours, approximately). In media containing glucose or sucrose, the presence of toxins of 26, 70, and 128 kDa was observed. Despite the inferior cell growth observed, sucrose (10 g/L), a relatively low-cost carbohydrate in Brazil, could be used as a substrate for Bti cultivation since the endotoxins studied were expressed. With lactose, no significant result was found. The temperature of the medium exerts a major influence on the cultivation of Bti. At 27 and 30ºC, the higher values for the maximum OUR (88.1 and 66.1 mmolO2/L/h), X (13 g/L for both conditions) and spore counting (8 and 7.1010 spores/mL) were measured. The more intense expression for proteins of 70 and 26 kDa, in mediu containing BYE (12 g/L) and glucose (20 g/L), were also observed at these temperatures. The temperature of 27ºC can be considered as a better temperature for this process since oxygen supply would be facilitated at this condition. The low pH observed when the cultivation was performed without pH control or with pH kept constant at 5.5 led to decreasing values of biomass concentration which reached 9.5 g/L. When the pH was controlled in 6.2 and 7.0 or varied from 7.0 to 5.5, the maximum X was measured as 12 g/L. Despite such a difference, spore counting was similar in all experiments: 1012 spores/mL in approximately 30 hours of cultivation. A better expression of endotoxins of 26, 70, and 128 kDa was detected when the pH was kept close to neutral values.
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Étude in vitro des changements physiologiques des cellules épithéliales du moustique Aedes aegypti en réponse à une exposition aux toxines du bacille de ThuringeBernard, James-Christopher 12 1900 (has links)
Bacillus thuringiensis sérotype israelensis (Bti) produit quatre toxines entomocides utilisées à grande échelle pour le biocontrôle des populations de diptères nuisibles et vecteurs de maladies : Cry4Aa, Cry4Ba, Cry11Aa et Cyt1Aa. Chacune de ces toxines présente un effet létal sur différents insectes mais, lorsqu’elles sont combinées, on observe un effet synergique et l’absence de résistance. Bien que cette synergie soit bien documentée par des tests de toxicité, il existe très peu d’information sur son mécanisme aux niveaux cellulaire et moléculaire. À l’aide d’intestins isolés des larves du moustique Aedes aegypti, le principal vecteur du paludisme, et de microélectrodes, nous avons observé une dépolarisation membranaire en présence de Cyt1Aa et de Cry4Aa individuellement. Cette dépolarisation se produit cependant plus rapidement lorsque la Cyt1Aa est utilisée en même temps que la Cry4Aa. D’autre part, des expériences réalisées avec la sonde calcique Fura-2 sur une lignée cellulaire provenant d’Anopheles gambiae (Ag55), ont révélé une forte activité lytique de la Cyt1Aa, mais très peu d’effets des autres Cry, et ce même en combinaison. Nous avons dissocié les cellules de l’épithélium intestinal isolé du moustique pour des expériences de Fura2. Nos résultats, quoique préliminaires, montrent les effets variables de ces toxines lorsqu’elles sont administrées seules sur les cellules dissociées : une augmentation du calcium intracellulaire, ou une fuite de la sonde se traduisant par une perte du signal fluorescent, ou la lyse cellulaire. On observe également en présence de Cyt1Aa et de Cry4Ba, que les effets sont presque instantanés. / Bacillus thuringiensis var israelensis (Bti) produces four insecticidal toxins used around the world to control disease-borne and harmful dipterans populations: Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. They each present their lethal effect on different dipterans, but combined, they generate a synergistic activity and a reduced resistance is observed. Though these synergies are well documented and supported by toxicity bioassays, little is known regarding the cellular and molecular mechanisms of these synergies. Here, by using freshly isolated midguts from the mosquito Aedes aegypti, an important malaria vector, and glass microelectrodes, we measured the electrical potential of the apical membrane when exposed to these toxins alone or in combination. We observed a depolarisation when treated with Cyt1Aa and Cry4Aa. Toxin mixture assays only revealed a faster depolarisation of the membrane when the above two toxins were combined together, and a variety of responses with other toxin mixtures. Microspectrofluometry using the calcium probe Fura-2 on an immortal cell line from Anopheles gambiae (Ag55) showed massive effect of Cyt1Aa, but very little effect of the Cry toxins alone or in mixture. Microspectrofluometry experiments were also conducted on freshly dissociated cells from Aedes aegypti. Though these experiments are innovative and the results preliminary, it was observed that some cells responded differently to Cyt1Aa and Cry4Ba, showing the various ways these toxins affect cells, by inducing either intracellular calcium change, or by entirely losing the probe, or by cell lysis. The mixture of these toxins is very efficient and almost instantaneous.
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Evolution de la résistance au bactério-insecticide Bti chez les moustiquesParis, Margot 01 February 2010 (has links) (PDF)
La résistance aux insecticides chez les moustiques pose des problèmes de santé publique car ils sont vecteurs de nombreuses maladies. Une alternative aux insecticides chimiques est l'utilisation du bactério-insecticide Bacillus thuringiensis subsp israelensis (Bti) qui a l'avantage de produire un mélange de six toxines spécifiques des Diptères. Cependant le Bti commercial peut proliférer et s'accumuler, entrainant une forte toxicité dans les litières végétales de certains gîtes à moustiques. Afin d'étudier l'évolution de la résistance au Bti chez les moustiques, j'ai sélectionné en laboratoire une souche d'Aedes aegypti avec des litières végétales contenant des toxines de Bti. Une résistance multigénique aux toxines Cry du Bti est apparue en seulement quelques générations chez la souche sélectionnée. Plusieurs approches ont été utilisées pour rechercher les bases génétiques de la résistance au Bti chez la souche d'Ae. aegypti résistante. Deux "scans génomiques" ont permis de déterminer plusieurs régions du génome présentant des signatures de sélection. Ensuite, les niveaux de transcription de plus de 6000 gènes ont été étudiés par séquençage haut débit. La combinaison de ces résultats avec une approche "gènes candidats" a permis d'obtenir une liste de gènes potentiellement liés à la résistance au Bti. Parmi les gènes identifiés, un gène codant pour une cadhérine présente des signatures de sélection chez la souche résistante et semble donc impliqué dans la résistance au Bti. De plus, une étude de génomique des populations de l'espèce de terrain Aedes rusticus traitée depuis 20 ans au Bti a mis en évidence des signatures de sélection liées au traitement et des flux de gènes importants chez cette espèce dans la région Rhône-Alpes. La caractérisation de facteurs génétiques liés à la résistance et de facteurs biologiques liés aux espèces traitées peut aider à la mise en place de stratégies de gestion limitant l'évolution de la résistance au Bti dans ces populations.
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Devenir du bioinsecticide Bti dans l'environnement et impact sur le développement de résistances chez le moustiqueTetreau, Guillaume 30 May 2012 (has links) (PDF)
Le Bti est un bioinsecticide mondialement utilisé pour contrôler les populations de moustiques. La toxicité du Bti est due à un cristal constitué de plusieurs toxines, produit par la bactérie éponyme Bacillus thuringiensis var. israelensis. Qualifié de peu persistant dans l'environnement et sans résistance connue en populations naturelles, le Bti représente une alternative " propre " aux insecticides chimiques. Cependant, une litière végétale prélevée plusieurs mois après un traitement insecticide a montré une forte toxicité contre les larves de moustiques, due à la présence de Bti. La sélection d'une souche de moustique de laboratoire Aedes aegypti avec cette litière (souche LiTOX) a permis d'obtenir une résistance modérée au Bti (3,5 fois) mais plus élevée aux toxines testées séparément (jusqu'à 60 fois). La présente thèse se décompose en trois axes. 1. Un test permettant le suivi du Bti dans l'environnement a été développé et breveté. Il a permis de mettre en évidence un recyclage du Bti dans les litières toxiques et une persistance différentielle des toxines. 2. La résistance au Bti de la souche LiTOX a été étudiée par des approches globales et fonctionnelles, permettant de mieux caractériser les mécanismes de résistance multigéniques impliqués. 3. La résistance a été recherchée en populations naturelles par des bioessais aux toxines du Bti testées séparément, outil plus sensible que les bioessais " classiques " au Bti, et a permis de mettre en évidence une tolérance accrue de certaines populations. Cette thèse apporte les connaissances et les outils nécessaires pour mieux caractériser les paramètres favorisant la prolifération du Bti en milieu naturel et les mécanismes de résistance potentiellement sélectionnés chez les moustiques.
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