Thèses : « Bacillus thuringiensis sérotype israelensis »

1

Tokcaer, Zeynep. « Response Surface Optimization Of Bacillus Thuringiensis Israelensis Fermentation ». Master's thesis, METU, 2003. http://etd.lib.metu.edu.tr/upload/3/1109602/index.pdf.

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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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Purcell, Mark Douglas. « The mosquitocidal activity of Bacillus thuringiensis ssp. israelensis ». Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624705.

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Clark, Burton David. « Characterization of plasmids from Bacillus thuringiensis var. israelensis / ». The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487330761220416.

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Houston, J. « Control of sewage filter flies using Bacillus thuringiensis var israelensis ». Thesis, Cardiff University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377875.

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5

Boonserm, Panadda. « Structure-function studies of the Bacillus thuringiensis subsp. Israelensis mosquitocidal toxins ». Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620578.

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6

Vollmann, Ketlyn. « Análise ecotoxicológica de diferentes formulações do bioinseticida Bacillus thuringiensis var. israelensis ». reponame:Repositório Institucional da UFSC, 2012. http://repositorio.ufsc.br/xmlui/handle/123456789/95896.

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Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2011
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O Bacillus thuringiensis var. israelensis (Bti) é uma bactéria entomopatogênica, Gram-positiva, aeróbica facultativa, formadora de esporos que, durante a esporulação, sintetiza um cristal protéico parasporal (?-endotoxina) em adição ao endósporo, tóxico a uma grande variedade de insetos que são economicamente importantes como pestes. Sua ação depende da ingestão dos cristais, que são solubilizados no intestino de larvas susceptíveis, onde as protoxinas são liberadas, formando poros na membrana, causando a morte da larva. Ele tem sido utilizado em Joinville e região para o controle dos mosquitos Aedes aegypti e Simulium pertinax, cujos ataques causam sérios impactos e inconveniência à população ribeirinha e ao turismo local. O objetivo do presente estudo é testar a metodologia de cultivo semi-contínuo para a produção de Bacillus thuringiensis var. israelensis proposta por Silva (2007); desenvolver uma formulação bioinseticida contendo o Bti obtido e verificar sua eficácia no controle populacional de larvas de A. albopictus em comparação com os bioinseticidas comerciais Vectobac AS e Teknar HP-D , bem como sua segurança em testes de toxicidade aguda utilizando, como bioindicadores, organismos não-alvo de diferentes níveis tróficos: o flagelado Euglena gracilis, representando os produtores primários, o microcrustáceo Daphnia similis, representanto os consumidores secundários e o peixe Danio rerio como representante dos consumidores terciários. Sob as condições experimentais utilizadas, a formulação bioinseticida produzida demonstrou ser eficiente para o controle de larvas de A. albopictus, com valores de DL50 de 3,97 mg/L, enquanto que os produtos comerciais formulados Vectobac AS e Teknar HP-D , tiveram valores de DL50 de 0,019 e 0,013 mg/L respectivamente. O bioinseticida Bti-Univille formulado demonstrou ser mais seguro ao organismo não-alvo Daphnia similis (DL50= 130 mg/L) que os produtos comerciais Vectobac AS® (DL50= 50 mg/L) e Teknar HP-D® (DL50= 32 mg/L), requerendo doses maiores para causar a mesma letalidade. Para todas as formulações bioinseticidas testadas, os valores de DL50 à D. similis foram superiores aos de DL50 estimados para o inseto-alvo; frente ao peixe Danio rerio, nenhuma das amostras bioinseticida testadas demonstrou toxicidade em doses até 100 mg/L. Os resultados obtidos nos ensaios de toxicidade aguda com Euglena gracilis foram inconclusivos, sendo necessária a realização de novos testes sob as mesmas condições experimentais para estimar a toxicidade do bioinseticida produzido a este organismo não-alvo.

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Beltrão, Henrique de Barros Moreira. « Interação das toxinas Cry do Bacillus thuringiensis svar. israelensis com o mesêntero de larvas do vetor Aedes aegypti (Diptera : Culicidae) ». reponame:Repositório Institucional da FIOCRUZ, 2006. https://www.arca.fiocruz.br/handle/icict/3931.

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O Bacillus thuringiensis svar. israelensis (Bti) é um importante entomopatógeno utilizado na produção de larvicidas para o controle do Aedes aegypti, vetor da dengue. A toxicidade do Bti está baseada no cristal, produzido durante a esporulação, que contém quatro protoxinas Cry11Aa (70 kDa), Cry4Aa (125 kDa), Cry4Ba (130 kDa) e Cyt1A (28 kDa). Sua ação ocorre através da ingestão dos cristais que são solubilizados no mesêntero, onde as protoxinas são liberadas e clivadas por serina-proteases em toxinas ativas que agem em sinergia no epitélio intestinal e provocam a morte das larvas. Apesar da alta seletividade do Bti, ainda não foi completamente elucidado como as toxinas Cry interagem com os receptores específicos presentes no epitélio das larvas. O objetivo principal do trabalho foi caracterizar, através de ensaios in vitro de natureza quantitativa, a capacidade de ligação de cada toxina Cry (4Aa, 4Ba e 11Aa) às preparações de microvilli intestinal (BBMF) de larvas de Ae. aegypti. Para tal, cada componente Cry foi produzido a partir de cepas recombinantes, Bt cepa 4Q2-81, para produção de biomassas. A atividade inseticida das biomassas para larvas do 3o/4o estádios foi determinada através de bioensaios e, outra parte da biomassa foi utilizada para a obtenção dos cristais. Os cristais contendo cada protoxina foram processados in vitro e uma amostra de cada uma delas foi marcada com iodo (I125). Para realizar os estudos de ligação foram feitas preparações BBMF, a partir de larvas do 3o/4o estádios. Os estudos da capacidade de ligação da toxina foram realizados através de ensaios de competição, de saturação e de cinética, através de incubações entre a toxina- I125 e preparações de BBMF, na ausência ou na presença de um competidor. (...) Os resultados obtidos mostraram que as toxinas Cry competem pelos mesmos sítios e partilham receptores presentes na BBMF. Em todos os casos estudados, a afinidade do complexo toxinareceptor não foi elevada, e não foi detectada sinergia entre as toxinas Cry para a ligação à BBMF. A ligação entre as toxinas-I125 e a BBMF é irreversível, e observou-se uma forte tendência à oligomerização nos três casos. Os resultados obtidos nesse trabalho sugerem que a toxicidade das toxinas Cry para larvas de Aedes está relacionada à etapa irreversível de ligação com os receptores, e não é caracterizada por um padrão elevado de afinidade do complexo toxina-receptor ...

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Abdoarrahem, Mostafa Mohamed Omar. « Factors influencing the activity of mosquito control agent (Bacillus thuringiensis subsp. israelensis) ». Thesis, Cardiff University, 2010. http://orca.cf.ac.uk/54112/.

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For toxicity, B. thuringiensis must be taken into the larval midgut, where a community of other bacteria is already present. The culturable flora from the Aedes aegypti mosquito midgut was analysed and its role in larval growth and insect mortality was determined. In contrast to published reports concerning B. thuringiensis subsp. kurstaki, subsp. israelensis caused toxicity and larval death even in the absence of other bacteria. The pBtoxis plasmid of B. thuringiensis subsp. israelensis encodes all the mosquitocidal toxins and a number of other coding sequences. The potential effects of selected genes on host phenotype was assessed. No evidence was found for antibiotic production from putative antibiotic synthesis genes. The plasmid also carries potential germination genes organised in a single ger operon. Comparison of the germination responses of spores from strains with and without pBtoxis revealed that this plasmid could promote activation of the spores under alkaline conditions but not following heat treatment. Introduction of the ger operon on a recombinant plasmid to the plasmidless strain established this operon as the first with an identified role in alkaline activation. Mosquito midgets provide an alkaline environment and in which enhanced germination may occur. Co-feeding experiments showed that in competition to colonise intoxicated A. aegypti larvae, B. thuringiensis carrying pBtoxis, are able to outgrow the plasmid-cured strain. This indicates a selective advantage for the presence of pBtoxis. The strain carrying the recombinant ger genes also outgrew its plasmidless parent, indicating that the ger genes may be responsible for this effect, perhaps by allowing strains a head-start by germinating more rapidly in the insect gut.

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Angsuthanasombat, Chanan. « Mechanism of action of Bacillus thuringiensis subsp. israelensis mosquito-larvicidal #delta#-endotoxins ». Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318348.

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10

Ward, Elizabeth Sally. « Molecular genetics of an insectidal delta-endotoxin from Bacillus thuringiensis var israelensis ». Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377842.

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11

Armstrong, Graham. « Directed mutagenesis of the CytA #delta#-endotoxin of Bacillus thuringiensis ssp. israelensis ». Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259545.

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12

Epp, Liam Jonathan. « Assessing the Effect of Bacillus Thuringiensis Var. Israelensis on Nontarget Chironomidae Emergence ». Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41118.

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Bacillus-derived larvicides, which selectively target mosquito (Diptera: Nematocera: Culicidae) populations to reduce nuisance and health risks, were applied in the South March Highlands Conservation Forest near residential neighbourhoods in Ottawa, Ontario. The objective was to assess effects of application on the nontarget mosquito relative, Chironomidae (Diptera: Nematocera: Chironomidae), and other nontarget aquatic taxa captured using emergence traps. A secondary objective was to assess physicochemical variables that influence Chironomidae emergence. Study ponds received an application of Bacillus thuringiensis var. israeliensis, a subset also received an application of Bacillus sphaericus, and a group of control ponds were left untreated over 3 years (2016-2018). Weekly sampling included trap collections and measurements of water temperature, pH, water depth, conductivity, dissolved oxygen, ammonia, nitrate, and sulphate. Drought in 2016, high precipitation throughout 2017, and seasonal precipitation in 2018 influenced variable physicochemical conditions. Principal component analyses identified differences between sampling groups and between years. Redundancy analyses correlated insect emergence with pond pH, average water depth and water temperature and indicated a strong relationship between Chironomidae emergence and average water depth. Although significantly less Chironomidae annual emergence was observed at treated sites in 2017 and 2018, zero-inflated negative binomial generalized linear mixed modelling failed to detect a significant Bti treatment effect when controlling for within group variation. Rather, variations in pH, mean water depth and water temperature were identified as drivers of Chironomidae emergence. Culicidae emergence was reduced to zero briefly following treatment in 2017 and 2018. The model detected a marginal negative treatment effect on Culicidae in 2017 only, and a positive treatment effect in 2018 at the onset of a secondary hydroperiod, in the absence of treatment. Variations in pH and water temperature were also identified to be drivers of Culicidae emergence. Modelling failed to detect treatment effects on any of the nontarget taxa abundance, including Diptera, Lepidoptera, Ephemeroptera, Odonata, Coleoptera, Hymenoptera, and Arachnida. An inverse relationship between insectivore and prey taxa abundance was observed. In 2018, taxa richness increased between years and trended higher at treated sites and a positive relationship between insectivore and prey taxa richness was observed. In 2017, Shannon-Weiner index and Simpson’s index of diversity were higher at untreated sites, and in 2018 diversity indices were higher at treated sites, with taxa richness increasing between years and higher evenness trending at treated sites. Our data suggest that treatment effects were potentially shrouded by natural variability of physicochemical variables, especially due to the varying hydroperiod observed over the three years of sampling. Additional work is needed to capture average conditions and separate confounding variables from treatment effects. This study provides an inventory of the current wetland insect community in the South March Highlands Conservation Forest landscape that offers a reference for ongoing mosquito management.

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Ernandes, Samara [UNESP]. « Obtenção de bioinseticida a partir de Bacillus thuringiensis var. israelensis e aplicação de bioensaios em culicídeos ». Universidade Estadual Paulista (UNESP), 2004. http://hdl.handle.net/11449/100750.

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A bactéria Bacillus thuringiensis var. israelensis (Bti) gera certas toxinas com ação inseticida que podem ser usadas no controle de doenças transmissíveis através de culicídeos, especialmente o Aedes aegypti, vetor da dengue. Este bioinseticida tem sido produzido através de fermentação submersa e, no Brasil, esta produção tem sido realizada através de pequenos centros de investigação e, mais recentemente, por uma pequena empresa. Para a implementação de um programa de controle de vetores viável através de bioinseticidas, alguns estudos sobre meio de cultura são essenciais para unir eficiência e baixos custos. Assim, tem-se utilizado resíduos ou subprodutos agroindustriais como fontes de nutrientes em meio de cultura. Neste estudo, alguns resíduos e subprodutos tais como água de maceração de milho (“milhocina”), um subproduto do processamento industrial do milho, “manipueira”, um subproduto do processamento da farinha de mandioca e sangue bovino oriundo de abatedouros foram avaliados com o intuito de se verificar a eficiência em processo fermentativo para obtenção de bioinseticidas. O crescimento celular foi avaliado por densidade óptica a 620 nm, a produção de esporo por plaqueamento em ágar nutriente e a CL 50 (Concentração letal média), por bioensaios contra larvas de 4º instar de Ae. aegypti. Foi verificado, em estudos com “manipueira”, que quando este subproduto é utilizado em baixa concentração, há o mesmo ou um melhor efeito do que quando se utiliza em concentração alta. A esterilização parece ser necessária ao processo fermentativo, pois quando se comparam ambos os meios de “manipueira”, um submetido à esterilização e outro sem este processo, o primeiro produziu mais esporos e uma melhor eficiência em bioensaios. Da mesma forma, quando o sangue de bovinos é acrescentado ao meio de “manipueira”, há um aumento...
The bacterium Bacillus thuringiensis var. israelensis (Bti) generates certain toxins with pesticid action, which can be used on the control of transmissible diseases by culicides, specially Aedes aegypti, the dengue´s vector. This biopesticide has been produced by submerged fermentation and, in Brazil, this production has been made by very little research centers and, more recently, by a unique small enterprise. For the implementation of a viable vectors control program through bioinsecticides, some studies about culture media are essential in order to join efficiency and low costs. So, agroindustrial wastes or by-products have been utilized as nutrient source for the culture media production. In this study, some wastes and by-products like corn steep liquor (“milhocina”), a corn industrial processing byproduct, “manipueira”, a by-product from cassava flour processing and cattle blood proceeding from slaughterhouses were utilized as substrate in order to verify the efficiency on fermentative process to obtain bioinsecticides. Cellular growth was evaluated by optical density at 620 nm, spore production by pour plate count and LC50 by bioassays against 4th instar larvae. It was verified, in studies with “manipueira”, that a low concentration of this byproduct has the same or better effect compared with high concentrations. The sterilization was a requisite to the fermentative process because when compared both sterilized and no sterilized “manipueira” media, the first yielded more spores and better efficiency in bioassays. Likewise, when cattle blood is added to “manipueira” media, there is an increase on the spores number and effectiveness against Ae. aegypti larvae. It was compared a traditional nitrogen source, peptone, with “manipueira”, with and without salts and glucose, tested separately and joined. In both media, salts had an importance, but the glucose didn´t seem necessary.

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Kouassi, Koffi Marcel. « Comportement et activité larvicide de Bacillus thuringiensis sérovariéte israelensis dans un cours d'eau ». Thèse, Université du Québec à Trois-Rivières, 1987. http://depot-e.uqtr.ca/5757/1/000569504.pdf.

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Huang, Fang. « The purification, characterization and mode of action of Bacillus thuringiensis subspecies israelensis proteins ». Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/6541.

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The gram-positive, spore forming bacteria Bacillus thuringiensis synthesize cytoplasmic crystalline inclusions, the Bt proteins, which are toxic to insect larvae. Although Bt proteins have been used as bioinsecticides for many years and a considerable number of studies have been carried out to investigate their mode of action, their precise mechanism of action still remains unclear. A 27 kDa protein from Bt subsp. israelensis (cytA27) is known for its broad cytolytic activity against a wide range of invertebrate and vertebrate cells in vitro as well as mosquitocidal activity in vivo. Given the interest in the mechanism of the membrane interaction of cytA27 and its proteolytic product, a 24 kDa protein (cytA24), we have undertaken a study of their interactions with model phospholipid membranes. The cytA27 and cytA24 proteins were purified by selective solubilization and ion exchange HPLC and characterized by SDS-PAGE, N-terminal sequencing and mass spectrometry. Fluorescence spectroscopy was used to characterize the cytA-induced release of fluorescence markers from vesicles and the cytA-vesicle affinity and stoichiometry. The results indicate that cytA proteins bind to lipids and affect the integrity of phospholipid vesicles. The binding is non-specific in that it does not require a receptor. CytA27 and cy8tA24 interact with PC-LUV with apparent binding constants of $\rm (0.34\pm0.02)\times10\sp5M\sp{-1}$ and $\rm (1.54\pm0.10)\times10\sp5M\sp{-1}$ respectively. Binding isotherm data indicate that a critical number of cytA molecules must associate with the membrane in order to induce vesicle leakage. Approximately 324 of cytA27 and 157 of cytA24 molecules are required to bind to one PC-LUV before the latter starts to release its contents. CytA-induced vesicle leakage follows an all-or-none mechanism, i.e. each LUV either releases all of its contents or remains intact. (Abstract shortened by UMI.)

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Ernandes, Samara. « Obtenção de bioinseticida a partir de Bacillus thuringiensis var. israelensis e aplicação de bioensaios em culicídeos / ». Araraquara : [s.n.], 2004. http://hdl.handle.net/11449/100750.

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Orientador: Iracema de Oliveira Moraes
Coorientador: Vanildo Luiz Del Bianchi
Resumo: A bactéria Bacillus thuringiensis var. israelensis (Bti) gera certas toxinas com ação inseticida que podem ser usadas no controle de doenças transmissíveis através de culicídeos, especialmente o Aedes aegypti, vetor da dengue. Este bioinseticida tem sido produzido através de fermentação submersa e, no Brasil, esta produção tem sido realizada através de pequenos centros de investigação e, mais recentemente, por uma pequena empresa. Para a implementação de um programa de controle de vetores viável através de bioinseticidas, alguns estudos sobre meio de cultura são essenciais para unir eficiência e baixos custos. Assim, tem-se utilizado resíduos ou subprodutos agroindustriais como fontes de nutrientes em meio de cultura. Neste estudo, alguns resíduos e subprodutos tais como água de maceração de milho ("milhocina"), um subproduto do processamento industrial do milho, "manipueira", um subproduto do processamento da farinha de mandioca e sangue bovino oriundo de abatedouros foram avaliados com o intuito de se verificar a eficiência em processo fermentativo para obtenção de bioinseticidas. O crescimento celular foi avaliado por densidade óptica a 620 nm, a produção de esporo por plaqueamento em ágar nutriente e a CL 50 (Concentração letal média), por bioensaios contra larvas de 4º instar de Ae. aegypti. Foi verificado, em estudos com "manipueira", que quando este subproduto é utilizado em baixa concentração, há o mesmo ou um melhor efeito do que quando se utiliza em concentração alta. A esterilização parece ser necessária ao processo fermentativo, pois quando se comparam ambos os meios de "manipueira", um submetido à esterilização e outro sem este processo, o primeiro produziu mais esporos e uma melhor eficiência em bioensaios. Da mesma forma, quando o sangue de bovinos é acrescentado ao meio de "manipueira", há um aumento... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The bacterium Bacillus thuringiensis var. israelensis (Bti) generates certain toxins with pesticid action, which can be used on the control of transmissible diseases by culicides, specially Aedes aegypti, the dengue's vector. This biopesticide has been produced by submerged fermentation and, in Brazil, this production has been made by very little research centers and, more recently, by a unique small enterprise. For the implementation of a viable vectors control program through bioinsecticides, some studies about culture media are essential in order to join efficiency and low costs. So, agroindustrial wastes or by-products have been utilized as nutrient source for the culture media production. In this study, some wastes and by-products like corn steep liquor ("milhocina"), a corn industrial processing byproduct, "manipueira", a by-product from cassava flour processing and cattle blood proceeding from slaughterhouses were utilized as substrate in order to verify the efficiency on fermentative process to obtain bioinsecticides. Cellular growth was evaluated by optical density at 620 nm, spore production by pour plate count and LC50 by bioassays against 4th instar larvae. It was verified, in studies with "manipueira", that a low concentration of this byproduct has the same or better effect compared with high concentrations. The sterilization was a requisite to the fermentative process because when compared both sterilized and no sterilized "manipueira" media, the first yielded more spores and better efficiency in bioassays. Likewise, when cattle blood is added to "manipueira" media, there is an increase on the spores number and effectiveness against Ae. aegypti larvae. It was compared a traditional nitrogen source, peptone, with "manipueira", with and without salts and glucose, tested separately and joined. In both media, salts had an importance, but the glucose didn't seem necessary.
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Roberts, Gillian Mary. « The combination of Bacillus thuringiensis var. israelensis and surface active monolayers for mosquito control ». Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328501.

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Boisvert, Mario. « Étude de la persistance d'un insecticide biologique, Bacillus thuringiensis serovariete israelensis dans le milieu aquatique ». Thèse, Université du Québec à Trois-Rivières, 1988. http://depot-e.uqtr.ca/5657/1/000569502.pdf.

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Corrêa, Roberto Franco Teixeira. « Avaliação da Toxicidade de proteínas Cry e Cyt de Bacillus thuringiensis subsp. israelensis para diferentes linhagens de células de inseto e de mamífero ». reponame:Repositório Institucional da UnB, 2012. http://repositorio.unb.br/handle/10482/10935.

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Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2012.
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Bacillus thuringiensis é uma bactéria Gram-positiva que produz proteínas formadoras de cristal ou δ-endotoxinas que compreendem as toxinas Cry, com atividade inseticida específica, e Cyt, com atividade citolítica inespecífica. Diferentes δ-endotoxinas mostram-se específicas para diferentes insetos-alvos. Esta especificidade deve-se a receptores de membrana distintos nas células do intestino dos insetos e à presença de diferentes proteases próprias dos insetos, necessárias para ativação proteolítica da prótoxina produzida na fase de esporulação do B. thuringiensis. Os genes cry4Aa, cry11A e cyt2Ba foram amplificados por PCR a partir das estirpes de Bacillus thuringiensis subsp. israelensis, S-1989 ou S-1806. Os produtos das PCR foram clonados em um vetor de expressão em B. thuringiensis, pSVP27A. O gene cyt2Ba foi também clonado em vetores de transferência para construção de baculovírus recombinantes para expressão da proteína Cyt2Ba nativa ou fusionada à proteína Poliedrina. Estirpes de B. thuringiensis acristalíferas foram usadas para expressão individual das proteínas. Após purificação e solubilização das proteínas heterólogas, as memsmas foram ativadas com tripsina ou suco gástrico de Spodoptera frugiperda, para a realização de ensaios de atividade em culturas de células de Lepidoptera, Diptera e mamífero. Foram usados, nos ensaios de citotoxicidade, 20 μg/mL de cada toxina Cry individual ou em combinações e 20 μg/mL ou 5 μg/mL da toxina Cyt2Ba. Atividades tóxicas das toxinas heterólogas ativadas por tripsina foram detectadas para todas as linhagens de células de insetos testadas, fato que não ocorreu após ativação das mesmas toxinas com suco gástrico de S. frugiperda. Para células humanas, apenas Cyt2Ba mostrou-se tóxica. Entretanto, a combinação entre Cyt2Ba e as toxinas Cry4Aa e Cry11A apresentou citotóxicidade maior que Cyt2Ba isoladamente, o que pode sugerir que Cyt2Ba funcione como receptor para Cry4Aa e Cry11A em células humanas. _________________________________________________________________________________ ABSTRACT
Bacillus thuringiensis is a Gram-positive bacterium that produces crystal forming proteins, δ-endotoxins, which consist of Cry toxins harboring specific insecticidal activity, and Cyt toxins that contain non-specific cytolytic activity. Different δ-endotoxins are specific to different target insects. This specificity is due to distinct membrane receptors on the surface of the insect´s midgut cells and to the presence of different host´s proteases, which are necessary to proteolytic activation of the protoxin expressed during B. thuringiensis sporulation phase. The cry4Aa, Cry11A and cyt2Ba genes were amplified from Bacillus thuringiensis subsp. israelensis Brazilian strains, S-1989 or S-1806 by PCR. The PCR products were cloned into the B. thuringiensis expression vector, pSVP27A. The cyt2Ba gene was also cloned into transfer vectors to allow construction of recombinant baculoviruses, in order to express the native Cyt2Ba protein, or Cyt2Ba fusioned to the Polyhedrin protein. Acrystaliferous B. thuringiensis strains were used for the expression of the proteins individually. After purification and solubilization of the heterologous proteins, toxin activation by either trypsin or Spodoptera frugiperda´s gastric juice was carried out to perform citotoxicity assays using Lepidopteran, Dipteran and human cultured cells. Each Cry toxin was used at the concentration of 20 μg/mL, while Cyt2Ba was used at two different concentrations: 20 μg/mL or 5 μg/mL. Cytotoxic activities of the trypsin activated heterologous proteins were detected to all insect cell lines tested, but when the same proteins were digested by S. frugiperda´s gastric juice, no toxic activities were detected. Only Cyt2Ba was shown to be toxic to the human cells tested. Nevertheless, the combination of Cyt2Ba and both Cry4Aa and Cry11A toxins yelded a higher toxicity than that produced by Cyt2Ba isolatedly, which could suggest that Cyt2Ba could be functioning as a receptor for Cry4Aa and Cry11A on the surface of human cells.

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Bourgouin, Catherine. « Les Toxines de Bacillus thuringiensis israelensis et de Bacillus sphaericus leur utilisation en lutte biologique contre les vecteurs de maladies tropicales / ». Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb376121877.

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Charles, Jean-François. « Bacillus thuringiensis sérotype H 14 et bacillus sphaericus : sporulation, biogenèse des cristaux larvicides et cytopathologie sur larves de moustiques (diptères ; culicidae) ». Paris 6, 1987. http://www.theses.fr/1987PA066303.

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Schmeisser, Glen A. « Location of the insect binding specificity domain of the bacillus thuringiensis subsp. israelensis 128 kDa toxin ». Virtual Press, 1994. http://liblink.bsu.edu/uhtbin/catkey/897503.

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The ultimate goal of this research was to perform a domain exchange between a computer identified insect specificity region of the mosquito larvicidal protein Cry IVB and a previously identified domain in a related protein toxin which targets lepidopteran insect larvae. If the insect specificity domain has been correctly identified, an exchange of DNA in this manner transfers the toxicity of one peptide to another by an exchange of the insect specificity domains. New, chimeric peptides may be designed which will target a larger spectrum of insect larvae.In previous research a domain exchange was performed between the two genes carried on plasmid vectors in E. coli and low levels of toxicity to mosquito larvae were observed. Initial efforts of this research attempted to identify these recombinants. However, stability was not achieved by sequential colony screens. Furthermore, a recently published three-dimensional structural model for all the B. thuringiensis crystalline toxins became available and it was quickly determined that the first exchanges excluded most of the f3-sheet domain that is responsible for insect cell receptor binding, the feature that gives the toxins their specificity. Therefore, it was decided that a larger, more inclusive region of Cry IVB DNA must be exchanged between the two toxins.Extensive computer analyses of the Cry IVB sequence and retroactive comparison of these sequences to the three-dimensional model yielded a fragment of DNA that encoded more than 60% of the putative insect specificity domain. Oligonucleotide primers were subsequently designed to flank this region so that the polymerase chain reaction could be employed to amplify the region. Additionally, the primers were engineered to contain terminal restriction endonuclease sites to ease in the exchange of the domain encoding region into Cry IA(c). The region of Cry IVB DNA flanked by the oligonucleotide primers was successfully amplified by the PCR and cloned into the plasmid vector pUC 19 as a reservoir for a future domain exchange.
Department of Biology

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Stalinski, Renaud. « Vers une meilleure compréhension des bases moléculaires de la résistance des moustiques au Bti (Bacillus thuringiensis subsp. israelensis) ». Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV054/document.

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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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Helvering, Leah M. « Cloning of genes encoding larvicidal proteins from Bacillus thuringiensis subsp. israelensis into the cyanobacterial hybrid vector, pTNTV ». Virtual Press, 1989. http://liblink.bsu.edu/uhtbin/catkey/562782.

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Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus.
Department of Biology

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Panarotto, Cíntia. « Influência de parâmetros operacionais, fontes protéicas e substratos energéticos sobre o cultivo de Bacillus thuringiensis var. israelensis ». reponame:Repositório Institucional da UCS, 2006. https://repositorio.ucs.br/handle/11338/169.

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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.
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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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Larget-Thiery, Isabelle. « Évaluation et contrôle au laboratoire du pouvoir entomopathogène de Bacillus thuringiensis var. Israelensis et de Bacillus sphaericus sur larves de Culicidae (Diptères, Nématocères) ». Paris 11, 1988. http://www.theses.fr/1988PA112119.

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Bacillus thuringiensis var, israelensis (B. T. I. ) et Bacillus sphaericus sont des bactéries synthétisant, pendant leur sporulation, des inclusions parasporales ou « cristaux » toxiques sur larves de moustiques. B. T. I. Est très toxique pour les larves de moustiques et de simulies tandis que B. Sphaericus est plus spécifique des genres Culex et Anopheles. La sélection, au laboratoire, de souches entomopathogènes puis de formulations expérimentales est réalisée par essais biologiques. Des méthodes standardisées d'élevage de masse et de titrages biologiques contre des poudres étalons de référence ont été mises au point. L’aptitude au stockage des formulations sélectionnées à des températures (de 0 à 50°C) a été évaluée pendant plusieurs mois. La persistance de leur activité larvicide est tributaire, après traitement, de divers facteurs étudiés au laboratoire. Après ingestion par les larves, les cristaux sont dissous par les enzymes intestinales, dans le tube digestif, libérant plusieurs polypeptides toxiques. L'étude de la toxicité des polypeptides, issus des cristaux de B. T. I et de B. T. Morrisoni (souche PG14 : très similaire aux souches de B. T. I. De par sa toxicité et la composition biochimique de ses cristaux), montre que l'activité larvicide nécessite l'association des protéines de 28 kD et de 68 kD. L'innocuité de B. T. I. Et de B. Sphaericus sur la faune non-cible aquatique et sur mammifères a permis l'expérimentation à grande échelle et la commercialisation très rapide de leurs formulations
Bacillus thuringiensis var. Israelensis and Bacillus sphaericus synthetize during sporulation proteic parasporal bodies called "crystals" which are toxic towards mosquito larvae. B. T. I. Is mostly efficient on Culicidae and against blackflies while B. Spaericus is specific to Culicidae, mainly to Culex and Anopheles genera. To select, in the laboratory, entomopathogenic strains and experimental formulations, we used bioassays. Standardized methods of mosquito mass-rearing and of biological titration against reference standard bacterial powders were designed. These formulations can be stored several months at temperatures ranging from 0 to 50°C without significant loss of toxicity. After treatment, persistence of larvicidal activity in water depends of factors which were studied in the laboratory. When ingested by larvae, crystals were dissolved by gut enzymes, releasing toxic polypeptides within the gut. Toxicity study of crystals from B. T. I. And B. T. Morrisoni (PG14 strain: which presents similar toxicity level and biochemical crystal composition) showed that larvicidal activity is related to the association of 28 kD and 68 kD proteins. Innocuity of B. T. I. And B. Sphaericus on non-target aquatic organisms and mammals has enabled large-scale field trials end rapid commercialization of industrial formulations

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Angelo, Elisangela Andrade. « Otimização de meio de cultivo para a produção de toxinas por Bacillus thuringiensis SUBSP. israelensis empregando resíduos agroindustriais ». Universidade Estadual de Londrina. Centro de Ciências Agrárias. Programa de Pós-Graduação em Ciência de Alimentos, 2009. http://www.bibliotecadigital.uel.br/document/?code=vtls000148696.

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O controle de insetos é realizado, em sua maioria, por produtos químicos; cujos efeitos cumulativos ocasionam grandes prejuízos ambientais e à saúde humana, destacando-se ainda a rápida seleção de insetos resistentes. O controle biológico por entomopatógenos é uma alternativa eficiente, principalmente devido a sua alta especificidade, menor freqüência de resistência nos insetos alvos e baixo efeito residual no ambiente. Bacillus thuringiensis é uma bactéria Gram-positiva esporulante, produtora de cristais protéicos com atividade inseticida. Apesar do amplo uso B. thuringiensis no controle biológico, há poucos trabalhos publicados quanto a sua produção, visto que muitas informações são segredos industriais. Além disso, a diversidade de subprodutos da agroindústria são fontes adequadas de carbono e nitrogênio para o cultivo de microrganismos e podem contribuir significativamente para a redução de custos na produção de biomoléculas. Portanto, o presente trabalho teve por objetivo otimizar um meio de cultura, empregando resíduos agroindústrias, para produção de toxinas por B. thuringiensis subsp. israelensis, utilizando a metodologia de superfície de resposta. Foi estudada também a cinética de crescimento associada à produção de toxina pela bactéria, bem como a toxicidade da cultura pós-fermentação, por um período de 28 dias. As fontes de nitrogênio orgânico estudadas foram: farinha de soja, farinha de crisálida, peptona bacteriológica e uréia agroindustrial. As fontes de carbono estudadas foram sacarose, glicose e fécula de mandioca. Como fonte de nitrogênio inorgânico foi estudada sulfato de amônia. O parâmetro analisado para a otimização do meio foi toxicidade, por meio de bioensaio contra Aedes aegypti. Foram estudados também o percentual de esporulação e o crescimento (por meio da contagem de Unidades Formadoras de Colônia). A farinha de crisálida e a glicose foram as fontes mais adequadas de nitrogênio orgânico e carbono, respectivamente, para a produção de toxinas. A superfície de resposta mostrou-se um método eficaz para a otimização, resultando em um meio com as seguintes faixas de concentrações das fontes de carbono e nitrogênio: glicose 0,3 a 0,55g/L; farinha de crisálida 55 a 58g/L e sulfato de amônia 0,6 a 1,1g/L. O meio otimizado resultou em uma toxicidade de 0,703ppm (v/v), valor superior a de meios comumente utilizados para B. thuringiensis israelensis. A toxicidade da cultura aumentou com o tempo de cultivo, sendo máxima com 96 horas. A cultura manteve-se tóxica durante os 28 dias de análise, sendo que o armazenamento sem refrigeração mostrou-se mais eficaz. Esses resultados contribuem para o desenvolvimento de uma produção local e para o aproveitamento de resíduos agroindustriais da região.
Insect control is realized, in its majority, for chemical products, whose cumulative effects become problematic due to the environment and to human's healthy and also the selection of resistant insects. Biological control by entomopatogen is an efficient alternative, mainly due to its own specification, lower resistance in the target insects and low residual effect on the environment. Bacillus thuringiensis is a Gram-positive spore-forming bacterium that produces a parasporal crystal protein toxic for many insects. Besides this bacteria is used widely worldwide in controlling insects, there aren't many studies about its production, because many information are industrial secrecy. Besides, the diversity of agro industrial byproducts are appropriate sources of carbon and nitrogen for the growth of microorganism and signficatively contribute for the reduction of cost of biomolecule. Therefore, the object of this study was to optimize the culture medium using agro industrial byproducts for producing toxins by B. thuringiensis subsp israelensis, using the Response Surface Analyses (RSA) methodology. We also studied the kinetics of growth and production of the toxin by the bacteria and its post-fermentation toxicity for 28 days. The organic nitrogen sources were: soybean meal, Bombix mori pupae meal (BMP), bacterial peptone and agricultural urea. The source of carbon studied were glucose, sucrose and cassava starch. The inorganic nitrogen source studied was ammonium sulfate. The parameter used for the medium optimization was toxicity, evaluated by bioassays again Aedes aegypti. It was also studied the percentage of the bacteria sporulation and growth (Colony Forming Units, CFU,). The BMP and glucose were the most appropriate sources of organic nitrogen and carbon, respectively, for toxins production. The RSA, proved to be an effective method for medium optimization, resulting in a medium composition for maximal response with the following ranges of carbon and nitrogen concentrations: glucose 0,3 to 0,55gL-1; BMP 55 to 58gL-1 and ammonium sulfate 0,6 to 1,1gL-1. The optimized medium resulted in a toxicity of 0,703ppm (v/v), which is above the mediums toxicity commonly obtained used B. thuringiensis israelensis. The culture medium toxicity increased with cultivation time until 96 hours. The culture medium toxicity remains stable under refrigeration temperatures during 28 days and when it was kept without refrigeration, the response was more effective. These results contribute to the development of a local technology and to promote the use of agro industrial byproducts produced in the region.

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Hicks, Teri Ann. « Expression of the bacillus thuringiensis var. israelensis 130kDa delta-endotoxin and the firely luciferase reporter gene in escherichia coli ». Virtual Press, 1991. http://liblink.bsu.edu/uhtbin/catkey/770955.

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The use of the larvacidal delta-endotoxin of the sporeforming bacterium Bacillus thuringiensis var. israelensis has been examined as a promising means to control insects that carry diseases such as malaria. An ultimate goal of this project was to genetically engineer both E. coli and the cyanobacterium Synechococcus PCC 7942 to express high levels of this delta-endotoxin and to construct the recombinant to carry a gene which would allow for monitoring of recombinants in the field. Previous research performed by a member of our laboratory involved cloning the gene fragment encoding the delta-endotoxin into a hybrid plasmid yielding recombinant E. coli clones which were toxic to mosquito larvae. Unfortunately, upon further examination of these recombinants using agarose gel electrophoresis and mosquitocidal assays, the clones were found to be unstable and lost their toxin encoding genes readily. Furthermore, cloning of the stabilizing parB locus into one of the recombinant plasmids did not enhance segregational stability as had been shown with some plasmids in E. coli. In another approach oligonucleotide primers were constructed which flanked the 130 kDa toxin gene but excluded a transposon-likesequence postulated to contribute to instability. These primers were used in the polymerase chain reaction in order to amplify this smaller DNA fragment for cloning experiments. Only a small quantity of primers were made and amplification of the DNA was not achieved prior to depletion of the primers. Future work will involve synthesizing new primers to be used for amplification and cloning of the B.t.i. toxin gene.In order to construct a traceable recombinant, the luciferase reporter gene (Luc) had been previously cloned into a hybrid plasmid that was capable of transforming both E. coli and the cyanobacterium Synechococcus PCC 7942. The new construction was then transformed into E. coli, to yield a pool of uncharacterized recombinants. In the present work, I determined that the luciferase enzyme was being expressed in the E. coli recombinants in the presence of the substrate luciferin. Initially, bioluminescence of these E. coli clones was detected by using OG-1 film which fogs in the presence of light. In order to quantify expression of the clones, lysates of the E. coli recombinants were also examined using a luminometer. Comparisons of bioluminescence were made between lysates with the parent E. coli plasmid harboring the luciferase gene and recombinants in which the Luc gene was placed downstream of the powerful rightward lambda promoter. Luminometer readings indicated that luciferase expression was enhanced six fold (from 2.0 X 10-6 to 3.0 X 10-5 by units/cell) in the recombinant plasmid. Plasmid DNA was isolated from the two luciferase expressing E. coli clones. Recombinants were obtained as determined by agarose gel electrophoresis examination of the plasmid DNA. This recombinant DNA was used to transform Synechococcus PCC 7942. However, because enzyme releasing methods were unsuccessful for the more rigid Synechococcus PCC 7942, the level of expression of the Luc gene could not be determined by either method mentioned above. Apparently, the methods used either failed to lyse the cells or they were too harsh and inactivated the enzyme. Future endeavors will involve the use of a French press to more gently lyse the cells so that the level of expression can be determined.
Department of Biology

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Barbosa, Claudia Rodrigues. « Avaliação do glicerol proveniente da fabricação do biodiesel como substrato para produção de endotoxinas por Bacillus thuringiensis var. israelensis ». Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/97/97131/tde-26092012-155413/.

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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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Taha, Awad Khalafalla. « Ecology of Aedes cantans (Meigen) larvae and effects of Bacillus thuringiensis var. israelensis on mosquito larvae and non-target organisms ». Thesis, University of Newcastle Upon Tyne, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357080.

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Alves, Giselly Batista. « Análises de genomas de Bacillus thuringiensis subsp. israelensis isolados em Tocantins com toxicidade para mosquitos de interesse em saúde pública ». Universidade Federal do Tocantins, 2017. http://hdl.handle.net/11612/586.

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Bacillus thuringiensis subsp. israelensis é uma bactéria gram positiva, amplamente utilizada no controle de insetos Diptera, família Culicidae, tais como Aedes aegypti e Culex quinquefasciatus. O sequenciamento de genomas completos de estirpes de B. thuringiensis tem permitido a identificação e caracterização de proteínas inseticidas, assim como a realização de estudos de genômica comparativa, permitindo evidenciar diferenças funcionais e filogenéticas. Neste contexto, o presente estudo teve como objetivo realizar análises genômicas de quatro isolados de B. thuringiensis subsp. israelensis que apresentam diferentes toxicidades para larvas de A. aegypti e C. quinquefasciatus. O sequenciamento dos genomas dos isolados T0124, T0131, T0137 e T0139 foi realizado a partir da plataforma MiSeq-Ilumina, e a montagem e anotação foram feitas utilizando ferramentas do programa de bioinformática Geneious. As análises resultaram em cromossomos com tamanhos aproximados de 5.414 Kb, conteúdo G+C de 35,2% e 5.358 regiões codificantes. Com relação ao conteúdo extracromossomal, foram montados três plasmídeos menores de 5,4; 6,8 e 7,6 Kb, e três plasmídeos maiores de 127, 235 e 359 Kb para todos os isolados, e estes foram enumerados de 1 a 6. A anotação revelou que apenas os plasmídeos de número 4, que correspondem ao pBtoxis, possuem proteínas inseticidas, sendo elas Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba e Cyt1Ca. A análise comparativa entre os genomas demonstra que os cromossomos T0124, T0131, T0137 e T0139 apresentam sequências colineares e com 99% de identidade, e seus respectivos plasmídeos 1, 2, 3, 4, 5 e 6 compartilham todas as suas regiões codificantes, incluindo aquelas relacionadas a δ-endotoxinas. A comparação de sequências de enterotoxinas, metaloproteases e fosfolipases demonstra que estes também são conservados entre os genomas. Na análise filogenética, os cromossomos T0124, T0131, T0137 eT0139 agruparam-se juntamente com os genomas dos isolados AM65-52 e HD-789 disponíveis no GenBank, sugerindo uma estreita relação genética entre estirpes de B. thuringiensis subsp. israelensis. Os isolados T0124, T0131, T0137 e T0139 apresentam diferentes concentrações letais para larvas de A. aegypti e C. quinquefasciatus, e a adição de dados proteômicos aos dados genômicos, aqui obtidos, poderão auxiliar na compreensão destas diferenças de toxicidade. Contudo as análises comparativas entre os genomas dos isolados T0124, T0131, T0137 e T0139 mostram alto grau de conservação genética a nível de nucleotídeos de B. thuringiensis subsp. israelensis.
Bacillus thuringiensis subsp. israelensis is a gram-positive bacterium widely used to control insects of the order Diptera, such as Aedes aegypti (Diptera: Culicidae) and Culex quinquefasciatus (Diptera: Culicidae). The sequencing of complete genomes of B. thuringiensis strains has allowed the identification and characterization of insecticidal proteins, as well as the comparative genomic studies, allowing the identification of functional and phylogenetic differences. In this context, the present study aimed to perform genomic analysis of four isolates of B. thuringiensis subsp. israelensis having different toxicities for larvae of A. aegypti and C. quinquefasciatus larvae. Sequencing of genomes of the isolates T0124, T0131, T0137 and T0139 was performed from the MiSeq-Ilumina platform, and assembly and annotation were done using tools from the Geneious bioinformatics program. The analyzes resulted in chromosomes with approximate sizes of 5,414 Kb, G + C content of 35.2% and 5,358 coding regions. Regarding extrachromosomal content, three smaller plasmids of 5.4, 6.8 and 7.6 Kb, and three larger plasmids of 127, 235 and 359 Kb were assembled for all the isolates, and these were enumerated from 1 to 6. The annotation revealed that only the plasmids of number 4, corresponding to pBtoxis, possess insecticidal proteins, being Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba and Cyt1Ca. Comparative analysis between the genomes demonstrates that chromosomes T0124, T0131, T0137 and T0139 have collinear sequences and 99% identity, and their respective plasmids 1, 2, 3, 4, 5 and 6 share all of their coding regions, including those related to δ-endotoxins. Comparison of enterotoxin, metalloprotease and phospholipase sequences shows that these are conserved among the genomes. In the phylogenetic analysis, chromosomes T0124, T0131, T0137 and T0139 were grouped together with the genomes of the isolates AM65-52 and HD-789 available in GenBank, suggesting a close genetic relation between strains of B. thuringiensis subsp. israelensis. The isolates T0124, T0131, T0137 and T0139 present different lethal concentrations to larvae of A. aegypti and C. quinquefasciatus, and the addition of proteomic data to the genomic data obtained here may help to understand these differences in toxicity. However, the comparative analyzes between the genomes of isolates T0124, T0131, T0137 and T0139 show a high degree of genetic conservation at the nucleotide level of B. thuringiensis subsp. israelensis.

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Rossi, Juliana Regina [UNESP]. « Bacillus thuringiensis var. israelensis SPS1 : caracterização da região promotora de genes cry e efeito em larvas de Aedes aegypti (L.) (Diptera : Culicidae) ». Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/92678.

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Dentre os microrganismos empregados no controle de mosquitos, a bactéria Bacillus thuringiensis var. israelensis (Bti) se destaca por apresentar atividade tóxica contra insetos da ordem Diptera, na qual produz inclusões cristalinas compostas pelas proteínas Cry, que são codificadas por genes cry presentes em um único plasmídeo. A produção dessas proteínas em grande escala está relacionada a mecanismos transcricionais, como por exemplo, a expressão de um gene sob o controle de um promotor forte. Tendo em vista que a bactéria Bti SPS1 (Patente PI0200228-0) foi isolada do território brasileiro e que possui potencial para o controle de vetores por produzir uma maior quantidade de esporos/cristais em menor tempo, este trabalho teve por finalidade a caracterização da região promotora dos genes cry4Aa, cry4Ba e cry11Aa de Bti SPS1 pelas técnicas de PCR e “Southern blotting”. Em associação a estas técnicas, foi realizado bioensaio para a verificação da mortalidade de larvas de Aedes aegypti. Os resultados das análises moleculares indicaram homologia no perfil de hibridização da região promotora da linhagem padrão Bti T14-001 e do isolado Bti SPS1. A quantificação das suspensões em espectrofotômetro (DO600nm) e a leitura de esporos em câmara de Neubauer, revelaram que o isolado Bti SPS1 produz uma maior quantidade de esporos/cristais em relação à linhagem padrão Bti T14-001. O bioensaio apresentou elevados índices de mortalidade. Estes resultados tornam a bactéria Bti SPS1 uma fonte promissora para novas formulações visando o controle de vetores.
Among the microorganisms used for mosquitoes’ control the bacterium Bacillus thuringiensis var. israelensis (Bti) is frequently considered since it presents toxic activities’ against Diptera, producing crystal inclusions including Cry proteins, which are coded by a sole plasmid borne set of genes. The production of these proteins in large scale is related to transcriptional mechanisms, as an example, a particular gene expression controlled by a strong promoter. Since the Bti bacterial isolate SPS1 (Patent PI0200228-0) was isolated within the Brazilian territory and exhibits potential for the control of vector insects due to a higher crystal production ability in shorter time period, this work had as objective the characterization of the promotion region for the genes cry4Aa, cry4Ba e cry11Aa using PCR and “Southern blotting” techniques. Also some bioassays using Aedes aegypti larvae were carried out. The results from the molecular analysis have indicated homology for the hybridization profile from the promoter region from the type strain of Bti T14-001 and that of the SPS1 isolate. Spectrofotometric (OD600nm) and Neubauer chamber measures have revealed that the SPS1 isolate produces a higher amount of spore/crystal as compared to the Bti T14-001 strain. The bioassay presented higher mortality levels. These results seem to indicate that the isolate SPS1 is a promising bacterial strain to be used on formulations able to control insect vector pests.

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Joeressen, Hermann-Josef. « In vivo- und In vitro-Untersuchungen, [delta]-Endotoxin-Analysen und molekulare Struktur der Parasporalkörper eines neuen Bacillus thuringiensis-Isolates (K24) im Vergleich mit B. thuringiensis ssp. israelensis / ». [S.l.] : [s.n.], 1993. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10449.

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Rossi, Juliana Regina. « Bacillus thuringiensis var. israelensis SPS1 : caracterização da região promotora de genes cry e efeito em larvas de Aedes aegypti (L.) (Diptera : Culicidae) / ». Jaboticabal : [s.n.], 2007. http://hdl.handle.net/11449/92678.

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Orientador: Manoel Victor Franco Lemos
Banca: Cristina Lacerda Soares Petrarolha Silva
Banca: Sérgio Antonio de Bortoli
Resumo: Dentre os microrganismos empregados no controle de mosquitos, a bactéria Bacillus thuringiensis var. israelensis (Bti) se destaca por apresentar atividade tóxica contra insetos da ordem Diptera, na qual produz inclusões cristalinas compostas pelas proteínas Cry, que são codificadas por genes cry presentes em um único plasmídeo. A produção dessas proteínas em grande escala está relacionada a mecanismos transcricionais, como por exemplo, a expressão de um gene sob o controle de um promotor forte. Tendo em vista que a bactéria Bti SPS1 (Patente PI0200228-0) foi isolada do território brasileiro e que possui potencial para o controle de vetores por produzir uma maior quantidade de esporos/cristais em menor tempo, este trabalho teve por finalidade a caracterização da região promotora dos genes cry4Aa, cry4Ba e cry11Aa de Bti SPS1 pelas técnicas de PCR e "Southern blotting". Em associação a estas técnicas, foi realizado bioensaio para a verificação da mortalidade de larvas de Aedes aegypti. Os resultados das análises moleculares indicaram homologia no perfil de hibridização da região promotora da linhagem padrão Bti T14-001 e do isolado Bti SPS1. A quantificação das suspensões em espectrofotômetro (DO600nm) e a leitura de esporos em câmara de Neubauer, revelaram que o isolado Bti SPS1 produz uma maior quantidade de esporos/cristais em relação à linhagem padrão Bti T14-001. O bioensaio apresentou elevados índices de mortalidade. Estes resultados tornam a bactéria Bti SPS1 uma fonte promissora para novas formulações visando o controle de vetores.
Abstract: Among the microorganisms used for mosquitoes' control the bacterium Bacillus thuringiensis var. israelensis (Bti) is frequently considered since it presents toxic activities' against Diptera, producing crystal inclusions including Cry proteins, which are coded by a sole plasmid borne set of genes. The production of these proteins in large scale is related to transcriptional mechanisms, as an example, a particular gene expression controlled by a strong promoter. Since the Bti bacterial isolate SPS1 (Patent PI0200228-0) was isolated within the Brazilian territory and exhibits potential for the control of vector insects due to a higher crystal production ability in shorter time period, this work had as objective the characterization of the promotion region for the genes cry4Aa, cry4Ba e cry11Aa using PCR and "Southern blotting" techniques. Also some bioassays using Aedes aegypti larvae were carried out. The results from the molecular analysis have indicated homology for the hybridization profile from the promoter region from the type strain of Bti T14-001 and that of the SPS1 isolate. Spectrofotometric (OD600nm) and Neubauer chamber measures have revealed that the SPS1 isolate produces a higher amount of spore/crystal as compared to the Bti T14-001 strain. The bioassay presented higher mortality levels. These results seem to indicate that the isolate SPS1 is a promising bacterial strain to be used on formulations able to control insect vector pests.
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Paula, de Araújo Ana. « Avaliaçao de um biolarvicida à base de Bacillus thuringiensis sorovar. israelensis, desenvolvido no Brasil, para o controle do Aedes aegypti (Diptera:Culicidae) ». Universidade Federal de Pernambuco, 2006. https://repositorio.ufpe.br/handle/123456789/648.

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Neste trabalho foram determinados a atividade tóxica e o desempenho em campo simulado de um larvicida experimental à base de Bacillus thuringiensis israelensis contra larvas de Aedes aegypti. Diferentes lotes de produção de pó técnico (PT) pré-formulado, foram avaliados em bioensaios, para definição das concentrações que eliminam 50% (CL50) das larvas e definição da potência do produto. Um dos lotes foi submetido a diferentes doses de radiação gama para a inativação dos esporos, sendo avaliado quanto à viabilidade microbiológica e toxicidade. As apresentações em comprimido (C) contendo 15% de princípio ativo, em PT e em pó técnico irradiado (PTI) foram testadas nas concentrações de 250 mg/50L de água, em recipientes plásticos, em condições simuladas de campo (TCS). A eficácia inicial do produto foi estimada pela mortalidade de 50 L4 de Ae. aegypti, após 48 h de exposição, e a persistência, mortalidade observada ao longo do tempo, pela introdução periódica de 50 L1 e recuperação de pupas. Os recipientes foram submetidos às seguintes variáveis: exposição solar ou sombra, renovação de 20% ou 60% do volume da água periodicamente, e a diferentes freqüências de colonização. Amostras de água foram coletadas para a verificação de esporos viáveis (UFC/ml) nos recipientes tratados. Os resultados demonstraram que existem diferentes níveis de toxicidade entre os lotes avaliados em laboratório, mas que estas diferenças não comprometem a atividade larvicida do produto em TCS. A CL50 média foi estimada em 0,26 ± 0,1 mg/L, com potência de 750 UTI/mg. A dose de 20 KGy de radiação gama inativou o maior percentual de esporos (99,9%) com menor perda da toxicidade. Em TCS, PTI, PT e C promoveram de 90 a 100% de mortalidade inicial e controle total durante 6 meses, na sombra. Não houve diferenças entre recipientes que sofreram ou não reposição de água, nem entre aqueles colonizados com diferentes números de larvas. A densidade de larvas também não influenciou a concentração de esporos ao longo do experimento. Nos recipientes tratados com PTI as concentrações de esporos viáveis foram inferiores às observadas para o PT em todas as coletas. A exposição solar foi o único fator que limitou o tempo de persistência do produto nos recipientes. Concluímos que o produto avaliado apresenta boa qualidade de produção com níveis altos de toxicidade e excelente persistência

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Lara, Ana Paula de Souza Stori de. « Expressão heteróloga da toxina Cry 11Aa de Bacillus thuringiensis (Berliner, 1919) var. israelensis em Escherichia coli (Escherich, 1885), visando o controle biológico ». Universidade Federal de Pelotas, 2013. http://repositorio.ufpel.edu.br/handle/ri/2322.

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Bacillus thuringiensis (Bt) is a Gram-positive bacteria, ubiquitous, facultative anaerobic, and form spores. During sporulation produce a parasporal crystals inclusion. Within these inclusions there are δ-endotoxin proteins well known for its insecticides proprieties. Among them, the Cry (crystal) is wide employed for biological control of plagues. The δ-endotoxin has an advantage of been more specific than chemical insecticides, thus been consider more favorable for the environment. The aim of this study was to obtain the Cry 11Aa recombinant protein of Bacillus thuringiensis var. israelensis in Escherichia coli, active for use in biocontrol. Two expression E. coli strains were tested: BL 21 (DE3) C41 and BL 21 (DE3) Ril. The protein Cry 11Aa was expressed and secreted in a soluble form by the two strains. The expression was demonstrated by Western Blot using anti-histidin monoclonal antibody. The strain BL 21 (DE3) C41 express the protein Cry 11Aa ~3.6 times more than the strain Rill, and showed a biologic efficiency of 95% of mortality for Culex quinquefaciatus larvae. The data obtained in this study suggest that the protein recombinant Cry 11Aa expressed in E. coli has a potential to be used in biological control.
Bacillus thuringiensis (Bt) é uma bactéria Gram-positiva, de ocorrência ubíqua, anaeróbica facultativa, formadora de esporos. Produz cristais, como inclusões parasporal durante a esporulação. Estas inclusões contêm proteínas chamadas de δ-endotoxinas, que são bem conhecidas pelas suas propriedades inseticidas. Dentre elas as toxinas Cry (crystal) são largamente empregadas no controle biológico de pragas. As δ-endotoxinas têm a vantagem de serem mais específicos do que os inseticidas químicos sintéticos, portanto, são considerados como agentes de controles favoráveis ao meio ambiente. O objetivo deste estudo foi a obtenção da proteína Cry 11Aa recombinante de Bacillus thuringiensis var. israelensis em Escherichia coli, ativa, para utilização no controle biológico.Duas cepas de E. coli de expressão foram testadas: BL 21 (DE3) C41 e BL 21 (DE3) Ril. A proteína Cry 11Aa foi expressa e secretada na forma solúvel pelas duas cepas. A expressão foi demonstrada por Western blot utilizando-se anticorpo monoclonal anti-histidina. A cepa BL 21 (DE3) C41 expressou a proteína Cry 11Aa ~3.6 vezes mais que a cepa BL 21 (DE3) Ril, e apresentou, em teste biológico, uma eficácia de 95% de mortalidade sobre larvas de Culex quinquefaciatus. Com os dados obtidos neste trabalho podemos sugerir que a proteína recombinante Cry 11Aa expressa em E. coli é um potencial candidato para ser utilizado no controle biológico.

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Silvia, Antone G. « Life history and production of the dominant Chironomidae in the New River, with emphasis on the effects of Bacillus thuringiensis var. Israelensis ». Thesis, This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-05092009-040337/.

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Villeneuve, Stéphane. « Impact de conditions variables sur la croissance et la sensibilité au Bacillus thuringiensis Serovar. israelensis de larves de moustiques Aedes triseriatus (Say) / ». Thèse, Trois-Rivières, Université du Québec à Trois-Rivières, 2001. http://www.uqtr.ca/biblio/notice/resume/03-2224109R.html.

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DELECLUSE, ARMELLE. « Caracterisation des genes codant pour les toxines de bacillus thuringiensis serovar israelensis, actif sur les larves de dipteres, vecteurs de maladies tropicales ». Paris 7, 1991. http://www.theses.fr/1991PA077027.

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Bacillus thuringiensis israelensis (bti) est une bacterie a gram (+), qui a la particularite de produire au cours de la sporulation des inclusions cristallines, toxiques pour les larves de certains dipteres vecteurs de maladies tropicales. Les cristaux de bti sont constitues de quatre polypeptides majeurs de 28 kda, 68 kda, 125 kda et 135 kda. Les genes codant pour les proteines de 28, 125 et 135 kda et pour un polypeptide mineur (orf1) ont ete clones. Les proteines de 125 kda et 135 kda et le produit d'orf1 appartiennent a une famille de proteines de type 130 kda et possedent une moitie carboxy-terminale similaire, la partie amino-terminale qui constitue le motif toxique etant variable. L'etude de l'activite des produits des genes clones a mis en evidence que la proteine de 28 kda n'est pas essentielle pour l'activite larvicide; les proteines de 125 kda et 135 kda, ainsi que le produit d'orf1 sont en revanche impliques, seuls ou en association, dans la toxicite des cristaux de bti. Chacune de ces proteines possede une specificite d'action differente, mais aucune ne presente le niveau de toxicite des cristaux de la souche de bti. La toxicite importante des cristaux de bti serait le resultats d'un phenomene de synergie entre plusieurs constituants du cristal. La presence de sequences d'insertion, designees is240, a ete detectee, au voisinage du gene codant pour la proteine de 125 kda. Un essai d'amelioration de potentialites de bti, par introduction dans cette bacterie de genes codant pour des toxines actives sur larves de moustiques, provenant d'une autre bacterie entomopathogene (bacillus sphaericus) est egalement presente

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Villeneuve, Stéphane. « Impact de conditions variables sur la croissance et la sensibilité au Bacillus thuringiensis Serovar. israelensis de larves de moustiques Aedes triseriatus (Say) ». Thèse, Université du Québec à Trois-Rivières, 2001. http://depot-e.uqtr.ca/3104/1/000677378.pdf.

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Robinson, Mary J. « Cloning a mosquitocidal fragment of Bacillus thuringiensis subsp. israelensis and location of the insect binding specificity domain of the 130 kDa toxin gene ». Virtual Press, 1991. http://liblink.bsu.edu/uhtbin/catkey/774740.

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Various strains of Bacillus thuringiensis Mt.) produce crystalline endotoxins specific for larvae of different insect classes. Two strains, B.t. subspp. israelensis and kurstaki produce similar 130 kDa toxins encoded by the CryIVB gene (toxic to Diptera) and the CryIA gene (toxic to Lepidoptera), respectively. The N-terminal region of the CryIVB gene was cloned into the Escherichia coli expression vector pKX223-3. A mosquitocidal transformant was obtained as determined by mosquito bioassays. The gene fragment, if stable, can be cloned into cyanobacteria to achieve biological control of mosquito-borne diseases. A second goal was to identify the binding specificity domain of the CryIVB gene which encodes the portion of the protein toxin that binds the insect midgut causing cell lysis and death. Two potential insect binding specificity domains identified by computer analyses were switched with a known binding specificity region of the CryIA gene. The polymerase chain reaction was utilized to obtain gene fragments of the CryIVB gene which replaced the CryIA gene binding specificity domain. The resulting recombinant clones carrying the CryIA gene containing the .000nd proposed insect binding specificity domain of the CryIVB gene were fotsd to be mosquitocidal.
Department of Biology

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Litz, Sara Leandra. « Construction of a library of the plasmids of Bacillus thuringiensis subsp. israelensis and identification of a lameda clone encoding the 135 kDa mosquitocida polypeptide ». Virtual Press, 1990. http://liblink.bsu.edu/uhtbin/catkey/722428.

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Bacillus thuringiensis subsp. israelensis (B.t.i.) produces a plasmid encoded parasporal crystalline protein which is larvacidal to mosquitoes carrying parasites for malaria and other infectious diseases. The purpose of this study was to construct a library of random fragments from the nine plasmids of wild type B.t.i. strain 402. The library was to be utilized in order to clone a 135kDa mosquitocidal polypeptide carried on a 108 kb B.t.i. plasmid.The library construction involved isolation of plasmid DNA by equilibrium density centrifugation, generation of random fragments of the nine plasmids by a partial Sau3A restriction digest, and ligation of these fragments into XbaI-BamHI restricted Lambda GEM-11 vector. Escherichia coli strain LE392 was infected by the packaged recombinant lambda and over 1000plaques were pooled to comprise the library. In order to verify construction of the library, both plaque screens of the library and Southern Analysis of restricted clones subjected to agarose gel electrophoresis were performed with labeled probes. The labeled probes were included: 1) radioactive end-labeled oligonucleotides constructed from published sequences of the B.t.i. 135 kDa toxic protein, 2) radioactive end-labeled random fragments from all nine plasmids of B.t.i., 3) radiolabeled entire plasmids of all nine plasmids of B.t.i., and 4) dioxigenin-labeled oligonucleotides. No homology between the lambda library digested DNA and the B.t.i. plasmid was observed. The results suggested that no lambda library of B.t.i. was constructed and, therefore, a lambda clone encoding the 135 kDa mosquitocidal polypeptide was not isolated.
Department of Biology

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Dupont, Claude. « Étude des modifications histopathologiques des structures intestinales de larves de mouches noires (Diptere : Simuliidae) à la suite d'une ingestion de Bacillus thuringiensis serovariete israelensis ». Thèse, Université du Québec à Trois-Rivières, 1986. http://depot-e.uqtr.ca/5825/1/000559850.pdf.

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Ozcelik, Hayriye. « Productivity Analyses In Fermentations With Three Different Biolarvacides ». Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12604988/index.pdf.

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The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

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Allgeier, Stefanie [Verfasser], Carsten A. [Akademischer Betreuer] Brühl et Ralf [Akademischer Betreuer] Schulz. « Mosquito control based on Bacillus thuringiensis israelensis (Bti) - Ecological effects on wetland food chains and public acceptance of control alternatives / Stefanie Allgeier ; Carsten A. Brühl, Ralf Schulz ». Landau : Universität Koblenz-Landau, Campus Landau, 2019. http://d-nb.info/120227384X/34.

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Tousignant, Mario. « Étude de la dispersion et de la perte du larvicide biologique particulaire, Bacillus thuringiensis serovar. israelensis, sur les substrats benthiques et dans la zone hyporhéique des cours d'eau ». Thèse, Université du Québec à Trois-Rivières, 1991. http://depot-e.uqtr.ca/5581/1/000592856.pdf.

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Paris, Margot. « Evolution de la résistance au bactério-insecticide Bti chez les moustiques ». Phd thesis, Université de Grenoble, 2010. http://tel.archives-ouvertes.fr/tel-00629116.

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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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Bernard, James-Christopher. « Étude in vitro des changements physiologiques des cellules épithéliales du moustique Aedes aegypti en réponse à une exposition aux toxines du bacille de Thuringe ». Thèse, 2016. http://hdl.handle.net/1866/19160.

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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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Shiau, B. C., et 蕭伯昌. « Laboratory and evaluations of Bacillus thuringiensis israelensis o larvae ». Thesis, 1994. http://ndltd.ncl.edu.tw/handle/85654760545871202853.

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碩士
東海大學
生物學研究所
82
The commerical development of Bacillus thuringiensis israelensisollow its discovery in 1976. Its efficacy against several speciesitoes is documented in various habitats worldwide. Since Bti wasin Taiwan in 1994 and was not yet use in the field, it isalute its efficacy in the laboratory and in various habitats inhe other hand, the impact of this bacterial insecticide on theof mosquito larvae and nontarget organisms was also studied in the presentformulations of Bacillus thuringiensis israelensis were evaluatedry against five species of mosquito larvae. All formulationsd high levels of activity against Armigeres subalbatus larvae.ion formulation showed better persistent activity than that offormulations. Antagonism was found when Bti was mixed withantibiotics but not with herbicides. Under natural field conditions, all formulationod inital control of Cx. tritaediorhynchus, Cx. quinquefasciatustus larvae. In control persisted up to 7 days posttreament. Alld no noticeable adverse effect on Gambusia affinis and prevailingnau except chironomid larvae in the laboratory and field studies.

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Tsai, Pei-Jung, et 蔡佩蓉. « Simulation and Field Studies of Bacillus thuringiensis subsp. israelensis against Dengue Vectors ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/22918760368753580920.

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碩士
國立高雄大學
運動健康與休閒學系碩士班
98
Because Kaohsiung area of Taiwan carries out chemistry control against dengue fever for a long time, these vectors have been resulted in resistance of pyrethrum. Bacillus thuringiensis subsp. israelensis (Bti) is a biological insecticides of safety and environmental protection. This study was designed to the effectiveness and residual efficacy of Bti against dengue vectors. Aedes aegypti susceptible strain (Bora-bora), Ae. aegypti and Ae. albopictus wild strains of Kaohsiung was released to containers filled with 60 L dechlorinated tap water indoor and outdoor. Each set of experiment was performed in five times. The usage of Bti water dispersible granular formulation VectoBac (WDG) was dispersed each dosage of 4 g/1000 L and 8 g/1000 L, respectively. The control groups were treated without the dosage. Lethal time 50% (LT50) and 24 h and 48 h mortality were determined to assess the killing effectiveness. In intervals of 7 days, the dengue vectors were added to determine LT50 and 24 h and 48 h mortality for the assessment of residual effects (water was filled 60 L). LT50 were calculated according to Finney Probit Analysis and mortalities were corrected with the Abbott’s formula. Outdoor field trials selected apartment type residential area and the similarity of the two neighboring blocks as the experimental area and control area to assess the ovitrap index of before treatment, during the application and after treatment. During the treatment spraying once a week for 6 times to water and non-water area. Indoor field trials selected the basement of 30 water places, spraying or putting Bti to control Aedes aegypti larvae, and observed 24 hours larvae mortality after Bti treatment. To assess killing effect, observed mosquito breeding situation weekly. Bti against larvae of dengue vectors to both outdoor or indoor simulation tests are less than half the time half an hour death, for the immediate killing effect; outdoor simulation results of the residual effect of control only to a week, 100% mortality. Control laboratory experiment of the effectiveness of the residual effect of 8 weeks can be to 100% mortality. Outdoor field trials of the effectiveness of prevention, application of Bti once a week, compared with the control experiment area area ovitrap indices decreased significantly, one week after cessation of spraying the experimental area ovitrap index higher than the control area. Indoor field tests of control effectiveness, the killing effect of 24-hour mortality was 100%; the residual effect of at least 8 weeks or more 24-hour mortality rate was 100%. Bti can be used as quickly kill dengue vectors of biological pesticides. Outdoor field control should be effective once a week with Bti application. The residual effects indoor were longer than outdoor. It suggested that a better Bti application in the interior to reach good residual effects. Bti in the drinking water is safety (WHO, 1999). The Bti formulation evaluated in this study should be suitable for application to large-scale spraying, especially in stagnant water bodies.

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