Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2011. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2011-05-23T14:12:46Z No. of bitstreams: 1 2011_EricoAugustoRosasVasconcelos.pdf: 4770212 bytes, checksum: 5d478696a64090851b80de519f4fb674 (MD5) / Approved for entry into archive by Raquel Viana(tempestade_b@hotmail.com) on 2011-05-24T11:33:57Z (GMT) No. of bitstreams: 1 2011_EricoAugustoRosasVasconcelos.pdf: 4770212 bytes, checksum: 5d478696a64090851b80de519f4fb674 (MD5) / Made available in DSpace on 2011-05-24T11:33:57Z (GMT). No. of bitstreams: 1 2011_EricoAugustoRosasVasconcelos.pdf: 4770212 bytes, checksum: 5d478696a64090851b80de519f4fb674 (MD5) / Uma busca por novas proteínas entomotóxicas de Bacillus thuringiensis S811 indicou que a atividade enzimática quitinolítica contribuía para a toxicidade dessa cepa contra o bicudo-do-algodoeiro (Anthonomus grandis). Durante os experimentos para a construção de uma molécula recombinante efetiva contra A. grandis e que fosse formada pela fusão de uma quitinase com uma toxina Cry, uma nova classe de Proteínas Inibidoras de Xilanases (XIP) foi descoberta dentro da família 8 de Proteínas Relacionadas a Patogênese (PR-8) de Café (Coffea arabica), a qual compreende as quitinases de classe III de plantas. Tal proteína foi denominada CaclXIP (Coffea arabica Chitinase-Like Xylanase Inhibitor Protein). O gene codificador da proteína inibidora de xilanase paráloga a quitinases de classe III de café (CaclXIP) foi isolado a partir de folhas e subclonado no vetor pGAPZα-B para expressão em Pichia pastoris. Sua seqüência de aminoácidos, que prediz uma topologia do tipo barril (β/α)8, comum a quitinases de classe III de plantas (família 18 das glicosil hidrolases (GH18)), partilha alta similaridade com outros membros da família GH18, embora ela careça do ácido glutâmico catalítico, que encontra-se substituído por uma glutamina. Ensaios de atividade enzimática com a proteína recombinante purificada mostraram que CaclXIP não apresenta atividade quitinolítica, contudo esta molécula mostrou-se capaz de inibir em 57% a atividade de xilanases de Acrophialophora nainiana em uma proporção de 12:1 (enzima:inibidor). Adicionalmente, quando testada a 1,5 μg/μL, CaclXIP foi capaz de inibir em 45% a germinação de esporos de Phakopsora pachyrhizi, o agente causador da Ferrugem asiática, uma doença de difícil controle que afeta severamente os campos de soja no Brasil. Estes resultados indicam que CaclXIP pertence a uma classe de proteínas sem atividade enzimática que evoluiu dentro da família das quitinase de classe III de plantas. Assim como as quitinases, essas novas proteínas também atuam na defesa vegetal, porém como inibidores de xilanases. Seu papel na interferência da germinação de esporos de P. pachyrhizi faz dela uma molécula candidata em potencial para programas biotecnológicos que visem o controle da Ferrugem asiática. ______________________________________________________________________________ ABSTRACT / Looking for a new entomotoxins from Bacillus thuringiensis S811, It has been realized that chitinolytic activity of those bacterial protein extracts could contribute to the strain toxicity towards the cotton boll-weevil (Anthonomus grandis). During experiments to construct one recombinant molecule lethal to A. grandis, and formed by a fusion between a chitinase and a Cry toxin, a new class of Xylanase Inhibitor Proteins (XIP) was discovered into Pathogenesis Related Proteins family 8 (PR-8) from Coffee (Coffea arabica), comprehending plants Class III Chitinases. This molecule was named CaclXIP (Coffea arabica Chitinase-Like Xylanase Inhibitor Protein). The gene for a xylanase inhibitor protein (XIP), which is paralogous to class III chitinase from coffee (CaclXIP), has been isolated from leaves and subcloned into pGAPZα-B vector to be expressed in Pichia pastoris. Its amino acid sequence, that predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins (GH18)), share high similarity with other GH18 members although it lack the catalytic glutamic acid, which is replaced by a glutamine amino acid residue. Enzymatic assay using purified recombinant CaclXIP showed no chitinolytic activity. On the other hand, it has been capable to inhibit xylanases from Acrophialophora nainiana in a 57% rate when assayed at a 12:1 (enzyme:inhibitor) ratio. Additionally, when tested at 1.5 μg/μL, CaclXIP was able to inhibit about 45% the germination of spores of Phakopsora pachyrhizi, the agent of Soybean Asian rust, a harsh disease difficult to control in Brazilian soybean fields. These achievements indicate that CaclXIP belongs to a class of proteins without enzymatic activity, which evolved from plants class III quitinase family. Like chitinases, theses new proteins works on plant defence too, but as xylanase inhibitors. Moreover, its role in arresting P. pachyrhizi spores germination makes it an eligible candidate to biotechnological programs aiming the asian rust control.

Orientador: Gil Eduardo Serra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-21T09:37:52Z (GMT). No. of bitstreams: 1 CesareVidaurre_TeresaJacoba_D.pdf: 4896751 bytes, checksum: 36b2186bfac3b85207eac90811a5d618 (MD5) Previous issue date: 1996 / Resumo: O presente trabalho teve como objetivo o desenvolvimento e a otimização da produção de bioinseticida bacteriano a partir do isolamento e seleção de uma linhagem de Bacillus thuringiensis que apresentasse uma diferenciada e alta atividade inseticida contra larvas de mosquitos de importância na saúde pública. A partir de amostras de solo, isolou-se uma bactéria entomopatogênica, denominada SP-36. Este isolado apresentou uma atividade de 30.767 ITU/mg, contra larvas de Aedes aegypti quase duas vezes mais alta que a linhagem padrão B. thuringiensis israelensis IPS-82, e foi classificado taxonomicamente como sendo um Bacillus thuringiensis. Na sorotipagem do isolado, este foi classificado como uma cepa autoglutinante de B.t.. O perfil electroforético das proteínas que compõem os cristais do isolado (110, 68, 38 e 25 kDa) apresentou significativa diferenças com os polipeptídios produzidos pela linhagem padrão. A otimização das condições fermentativas foram planejadas através da Metodologia por Superfícies de Resposta (MSR) com um delineamento experimental 34. Os resultados experimentais demonstraram uma resposta de 1,37 x 108 para contagem de esporos, completa lise celular e atividade de 35.000 ITU/mg, utilizando-se meio basal em condições de 30°C, 350 rpm, 5 % de inóculo e 7,2 de pH inicial. Para seleção dos componentes de meio de cultura à base de sub-produtos agro-industriais, foram utilizados fatoriais fraccionários, permitindo a seleção das fontes de carbono, nitrogênio e suplementos minerais, assim como o ajuste preliminar das concentrações dos componentes. foram selecionados os seguintes componentes: melaço, água de maceração de milho, água de levedura e fosfato de potássio. Para a otimização da concentração dos componentes selecionados utilizou.:se de delineamento central composto 54 da metodologia por superfícies de resposta, obtendo-se contagem de 1,28 x 108 esporos/ml, em 24 horas com um meio otimizado: 3,5g/l de melaço, 7,O g/l de água de maceração de milho, 16g/l de água de levedura e 6,O g/l de fosfato de potássio. Foram realizados testes de escalamento, com 30 e 50 % de oxigênio dissolvido, obtendo-se velocidades específicas de crescimento de 0,25 e.0,51 h-1, e contagem de esporos de 1,76 e 1,96 x 109 esp/ml respectivamente, em 24 e 18 horas / Abstract: The purpose of this work was to develop and improve the production of bacterial bioinsecticide from a microbiological screening and selection of a Bacillus thuringiensis strain which presents a high and differentiated insecticide activity against mosquitoes larvae of public heath importance. The entomopathogenic bacteria B.t. strain SP-36, has been isolated from soil samples, according to serotyping it as an auto-agglutinated B.t. strain. This isolate presented an activity of 30.767 ITU/mg against Aedes aegypti larvae, almost twice as high as the B. thuringiensis israelensis IPS-82 standard strain. The electrophoretic pattern of the proteins, which constitute the isolate crystals (110, 68, 38 and 25 kDa), has presented significant differences as to the polypeptides produced by the standard strain. The improvement of the fermentative conditions has been planned through the Response Surfaces Methodology (MSR) with an experimental design 34. The experimental results have shown a response of 1,37 x 108 for the spore count, complete cellular lysis and an activity of 35.000 ITU/mg, using a basal medium at 30°C, 350 rpm, 5% inoculum and initial pH 7,2. In order to select the culture medium based on agroindustrial by-products, fractional fatorials have been used, so that the sources of carbon, nitrogen and mineral suplements could be selected, as well as the preliminary setting of the components concentration. The following components have been selected: sugarcane molasses, com-steep liquor, yeast liquor and potassium phosphate. In order to improve the selected components concentration a central composite design 54 of the response surfaces methodology was used, obtaining a count, of 1,28 x 108 spores/ml, in 24 hours, in an optimized medium: 3,5g/l of molasses, 7,0g/l of com-steep liquor, 16g/l of yeast liquor and 6,0g/l of potassium phosphate. Scale-up tests have been performed, with 30 and 50% dissolved oxygen, obtaining specific growth rate of 0,25 and 0,51 h-1 and spores count of 1,76 and 1,96 x 109 spores/ml, in 24 hours, respectively / Doutorado / Doutor em Tecnologia de Alimentos

Submitted by Amauri Alves (amauri.alves@ufv.br) on 2015-11-03T15:06:33Z No. of bitstreams: 1 texto completo.pdf: 1310681 bytes, checksum: ae3d027db5649c35ff8ef366ea4689e0 (MD5) / Made available in DSpace on 2015-11-03T15:06:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1310681 bytes, checksum: ae3d027db5649c35ff8ef366ea4689e0 (MD5) Previous issue date: 2015-02-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bacillus thuringiensis é uma espécie comumente associada a solos, insetos e plantas. Diversas linhagens são importantes para diferentes formas de controle biológico na agricultura e em outras áreas. Neste trabalho, foi sequenciado e anotado o genoma de B. thuringiensis BAC3151 endofítico do feijoeiro comum (Phaseolus vulgaris), um isolado que tem demonstrado atividade antimicrobiana contra bactérias patogênicas, incluindo bactérias fitopatogênicas do feijoeiro, possuindo, portanto, potencial para biocontrole de doenças nessa leguminosa. O genoma de BAC3151 e o de outros B. thuringiensis publicados forneceu a oportunidade para realizarmos análises de genômica comparativa entre diferentes linhagens dessa espécie dinâmica. A análise filogenômica utilizando os genes core das linhagens avaliadas agrupou BAC3151 com B. thuringiensis MC28 isolado de solo. Também foram identificados rearranjos cromossomais de grandes segmentos em BAC3151 e em outras linhagens, sugerindo que rearranjos podem contribuir para diferenças funcionais dessas linhagens. O genoma core das linhagens estudadas consistiu em 3413 sequências codificadoras de proteínas (CDSs) e incluiu as CDSs com funções fisiológicas gerais e de sobrevivência da espécie, enquanto as CDSs do genoma acessório foi associada a plasmídeos e a regiões cromossômicas específicas. Além disso, foram encontrados múltiplos genes potencialmente envolvidos na ação antagonista de BAC3151 contra outros micro-organismos e insetos, incluindo genes inseticidas, policetídeos sintases, bacteriocinas, peptídeos não-ribossomais sintetases, sideróforos, quitinases, N-acil homoserina lactonase e outros. Este estudo mostrou diversas variações entre linhagens de B. thuringiensis em nível genômico e também que B. thuringiensis BAC3151 endofítico do feijoeiro é potencialmente útil para o desenvolvimento de estratégias para o controle biológico de doenças nessa importante leguminosa. / Bacillus thuringiensis is a species commonly associated with soil, insects and plants. Several strains are important for different types of biological control in agriculture and other areas. In this work, was sequenced and annotated the genome of endophytic B. thuringiensis BAC3151 of common bean (Phaseolus vulgaris), an isolate that has demonstrated antimicrobial activity against pathogenic bacteria, including plant pathogenic bacteria of the common bean, having, in this way, potential for biocontrol of diseases in this legume. The genome of BAC3151 and the other B. thuringiensis published provided the opportunity to accomplish comparative genomic analysis between different strains of this dynamic species. The phylogenomic analysis using the core genes of the evaluated strains grouped BAC3151 with B. thuringiensis MC28 isolated from soil. Also, large chromosomal rearrangements were identified in BAC3151 and other strains, suggesting that rearrangements may contribute to functional differences of these strains. The core genome of the strains studied consisted of 3413 sequences encoding proteins (CDSs) and included the CDSs with general physiological functions and species survival, while the accessory genome CDSs were associated with plasmids and specific chromosomal regions. In addition, were found multiple potential genes involved in the antagonistic action of BAC3151 against other microorganisms and insects, including insecticidal genes, polyketide synthases, bacteriocins, nonribosomal peptide synthetases, siderophores, chitinases, N-acyl homoserine lactonase and others. This study showed several variations among strains of B. thuringiensis in genomic level and also that endophytic B. thuringiensis BAC3151 of common bean is potentially useful for the development of strategies for biological control in this important legume.

Acompanha memorial / Tese (livre-docencia) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-16T08:07:42Z (GMT). No. of bitstreams: 1 Habib_MohamedEzzEl-DinMostafa_LD.pdf: 6621704 bytes, checksum: f55463be23eb86b925f2eacb9517e66e (MD5) Previous issue date: 1982 / Resumo: A patogenicidade de duas variedades de Bacillus tnuringiensis Berliner foi investigada em larvas de Lepi-doptera e Diptera. Através do enfoque patológico no estudo, tentou-se esclarecer alguns aspectos importantes nas áreas de pesquisa, industrialização de patógenos e aplicação de produtos à base de B. thuringiensis no campo. Os sintomas externos pré-mortais foram descritos em larvas de Lepidoptera infectadas por B. thuringiensis var. kurstaki e em larvas de Diptera infectadas por B. thuringien-sis var. israelensis. Algumas alterações histológicas foram detectadas em alguns tecidos de tais larvas, principalmente , no intestino médio, sistema nervoso e musculatura. As alterações observadas nesses tecidos foram relacionadas com o desencadeamento dos sintomas externos da bacteriose. A variedade kurstaki teve ação fatal lenta (2 a 3 dias) nas larvas de Lepidoptera, quando comparada com a ação da variedade israelensis nas larvas aquáticas de Diptera,onde a morte ocorre em poucas horas após a infecção. Nenhum dos lepidópteros estudados pertence o tipo I da classicificação de Heimpel & Angus (1959); pois não ocorreu paralisia geral ou aumento no pH da hemolinfa nas larvas doentes. Entre as larvas dessas espécies, as de Brasso-lis sophorae revelaram-se mais susceptiveis em relação às demais. Por outro lado, as larvas de Spodoptera latifascia e-ram as mais resistentes à mesma variedade kurstaki ( sorotipo H-3a:3b). A origem do patógeno, o hospedeiro do qual este foi isolado e o processamento da produção comercial, além da variedade do bacilo e da espécie do inseto infectado, eram fatores responsáveis pela variação nas respostas das larvas infectadas. A expressão da quantidade do patógeno em termos de Unidades Internacionais de virulência / unidade de peso do inseto tratado revelou-se altamente precisa para comparações de susceptibilidade de insetos e virulência de produtos. 0 uso de critérios de DL50 e CL50 _ mostrou-se muito adequado, e até pode ser recomendado, para estudos com populações de insetos geneticamente menos variáveis. A resposta dos indivíduos, no caso, seria diretamente relacionada com e em função da dose ou a concentração aplicada. 0 uso de TL50 , por outro lado, revelou-se mais funcional e, também, pode ser recomendado para investigações com populações de maior variabilidade genética. Os níveis de susceptibilidade revelados para cada uma das espécies estudadas, indicaram a alta possibilidade de obter resultados satisfatórios no campo, quando produtos comerciais à base da variedade kurstaki forem aplicados contra larvas de B. sophorae , Alabama argillacea e Plodia inter-punctella em jardins e áreas urbanas, lavoura e armazéns respectivamente. A morte iniciou-se, nas larvas aquáticas dos dípteros estudados, apenas poucas horas após a infecção pela variedade israelensis. As larvas de Culex declarator (Culicidae) mostraram-se mais susceptíveis a essa variedade do que as lar vas de simulídeos, quando infectadas com este patógeno. Entre as duas espécies de simulídeos, as larvas de Simulium goeldii eram 4 vezes mais susceptíveis do que as de S. rorotaense. As larvas do último estádio de C. declarator revelaram-se mais susceptíveis ao patógeno do que as dos estádios iniciais. No mesmo tempo, as larvas no início do último estádio (42) eram mais susceptíveis do que no final do mesmo estádio. Este último aspecto, juntamente com a morte rápida causada pelo patógeno ( 2 a 3 horas), devem ser considerados nas recomendações de alguns órgãos oficiais no exterior. Tais órgãos, exigem que o 42 estádio larval de Aedes aegypti seja usado nos bioensaios de padronização de produtos à base da variedade israelensis ; porém, sem estabelecer uma fase desse estádio. A especificidade e a alta virulência da variedade israelensis para larvas aquáticas de Diptera, revelam o seu grande valor como agente promissor no controle microbiano de larvas de Culicidae e Simuliidae. Os sintomas externos observados nas larvas de. C. declarator, as alterações nistológicas e a morte rápida foram suficientemente convenientes para mostrar a ação da S-endo toxina produzida pelo patógeno. Tal toxina deve ter afetado drasticamente o sistema nervoso e a musculatura da larva, dificultando assim a manutenção desta na superfície da água e conseqüentemente a sua respiração do ar atmosférico. Assim , a larva afundada morreu por asfixia. Esses estudos revelaram a presença de um novo campo para investigações de patologia de insetos e controle microbiano para os dípteros aquáticos neotropicais e subtropicais . Tais investigações terão o seu alto valor do ponto de vista humano, desde que várias dessas espécies tem a sua importância como insetos vetores de doenças humanas / Abstract: The pathogenicity of two varieties of Bacillus thuringiensis Berliner was investigated using some lepi-dopterous and dipterous larvae. The aim of the present study was to clarify certain aspects related to the application of insect pathology, such as susceptibility and virulence évalua tions, standardisation criteria, and field applications of microbial products to the control of some harmful insects. The sequence of external symptoms was described in lepidopterous larvae infected with the B. thuringiensis var. kurstaki (syrotype K-3a:3b) and in dipterous larvae infected with the variety israelensis (syrotype H-14)-The histological alterations, in these infected larvae, were described, principally those in the mid-gut, nervous system, and musculature These alterations were found to be directly associated with the different external symptoms of the disease. The progress of the disease up to the death in the lepidopterous larvae lasted longer time (2-3 days) than in dipterous ones (2-3 hours). Type I of Heimpel & Angus (1959) classification did not occur among the lepidopterous species studied, since nei_ ther general paralysis nor pH alterations in the hemolymph were detected. Brassolis sophorae larvae were found to "be the most susceptible to syrotype H-3a:3b, when compared with the other lepidopterous species. On the other hand, Spodoptera lati-fascia larvae were the most resistant. Origin, natural host, and mass production criteria of the pathogen, in addition to its variety and the tested insect species, were the most important factors responsible for the variation in responses of the infected larvae. Therefore, these aspects should be considered to choice the more adequate product for each insect species to be combatted. The quantity of the pathogen expressed in International Units of toxicity per unit of body weight, showed to be highly precise for the comparison of insect susceptibility and pathogen virulence. The LD^Q and LC^0 criteria were found to be suitable and, therefore, can be recommended for investigations where the populations of insects are of low genetic variability. On the other hand, for genetically variable populations, the LT^q was found to be more adequate. B. sophorae, Alabama argillacea and Flodia inter-punctella were found to be highly susceptible to the syrotype H-3a:3b. Therefore, this pathogen can be recommended for their control. The aquatic dipteran larvae, studied in the present work, died some hours after infection with syrotype H-14.Culex declarator (Culicidae) larvae were more susceptible than those .132 . of the simulid species. Within Simuliidae, Simulium goeldii larvae were 4 times more susceptible than the S_. rorotaense ones. The last instar (4th.) larvae of C. declarator were more susceptible than those of the first instars. Moreover, within the same instar, these larvae showed different levels of susceptibility. In the initial phase of the last instar they were more susceptible than in the final phase. These informations, in addition to the rapid fatal action of the toxin in dipteran aquatic larvae, should be considered by the governamental agencies, in establishing standardization criteria for future commercial products based on this variety.At the present, these agencies establish the 4th instar larvae of Aedes aegypti as a test insect for stadardization bio-assays, ignoring the difference in susceptibility within the same instar. Because of the high specificity and virulence of B. thuringiensis var. israelensis for dipteran aquatic larvae, it can be considered as a promissing agent for microbial control of mosquitos and black flies larvae. Considering the mode of action of syrotype H-14 in mosquito larvae, the results led us to beleive that the £-endotoxin liberated in the high alkaline mid-gut affected the ventral nervous system and consequently the musculature , provoking disfunction of the latter.The effect on the muscles made the larva lose its capacity to maintain itself at the water surface. Consequently, the larva submerged and died by asphixiation. The observed sequence of the external symptoms, as well as the histological alterations sustained this hypothesis. The results obtained in the present study reveal the possibility to utilize the syrotype H-14 in the control of vectors of some human diseases, such as malaria and filaria This application, however, requires additional extensive research work to complete urgently needed informations / Tese (livre-docencia) - Univer / Livre-Docente em Ciencias Biologicas

The bollworm, Helicoverpa zea (Boddie), is an economically important pest of cotton and is becoming more difficult to control in the United States. This project was designed to develop a better understanding of the effects of resistance development and plant structures on the efficacy of Bt cotton and foliar insecticides. Experiments examined larval behavior and damage in current cotton varieties expressing proteins from Bacillus thuringiensis. Other experiments examined the effects of floral structures on the control of bollworm using foliar insecticides. Results suggest that larval feeding and avoidance behavior is dependent on point of eclosion. Fruiting form damage increased in two-gene cotton compared to earlier research but was low in cotton expressing the Vip3A protein. Increases in damage can lead to more frequent applications of foliar insecticides. Floral structures can hinder insecticide efficacy by reducing exposure. Results from these experiments will be important for refining management recommendations for bollworm in Bt cotton.

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Widespread anthelmintic resistance in trichostrongyle nematodes of ruminants has created an urgent need for alternatives to commercial anthelmintics. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins during sporulation, which can be lethal to insects in multiple orders when ingested. One protein, Cry5B, has demonstrated effectiveness against multiple parasitic nematodes. We hypothesized that Cry5B would be effective against Haemonchus contortus, a highly pathogenic parasite, in sheep. Two experiments tested efficacy of Cry5B in sheep experimentally infected with H. contortus. In the first, a live genetically modified, asporogenous strain of B. thuringiensis expressing cytosolic Cry5B protein (BaCC) was administered orally daily for four days (~40mg/kg Cry5B/day). The mean fecal egg count (FEC) of treated animals was reduced by 94% three days after treatment, and at necropsy the female worm burden was significantly reduced by 98%. In the second experiment inactivated, asporogenous Bt expressing cytosolic Cry5B (IBaCC) was used. Treated animals received 60mg/kg Cry5B, administered daily for three days. By 72 hours after the first treatment FEC was reduced by 91%. Mean total worm burden of treated sheep at necropsy was significantly reduced, with female worms reduced by 95%. A third study tested the effect of BaCC and IBaCC on development of eggs to infective larvae in feces under laboratory and outdoor environmental conditions. Cry5B (15mg) added to feces (10g) reduced numbers of infective larvae by 99% in both environments within 12 days. Cry5B appears to have potential for controlling H. contortus in sheep. All protocols approved by VT IACUC and IBC. / Master of Science / Many animals and humans can be infected with roundworm, also called nematode, parasites. Infection of animals and humans by parasitic nematodes can result in disease. Some animals like ruminants (cows, sheep, and goats) can be infected with multiple species at once with few effects on the host. However, certain species can cause major disease, and even kill their ruminant host. Younger animals like lambs can easily become overwhelmed by these parasites. Anthelmintics are the type of drug used to treat those infected with these parasitic worms. However, just like bacteria are becoming resistant to antibiotics, these worms are also becoming resistant to anthelmintics. Because of this, researchers are looking for new compounds and materials that are lethal to the parasite and can be used to treat infected animals. One species of bacterium, Bacillus thuringiensis, is usually found in the soil. This bacterium can produce a large crystal structure that is made up of proteins. These crystal (Cry) proteins can be lethal to pest insects like beetles, caterpillars, and mosquitos. When the insect eats the protein, it binds to cells in the insect intestine, creating holes in the insect gut. These proteins can be lethal to nematodes as well when they are eaten by the worms. Because of this, these proteins are being investigated as potential alternative treatments for parasitic nematodes. One type of protein, Cry5B, has been tested in hamsters, mice, and pigs. We hypothesized that Cry5B would also be effective against a sheep stomach worm called Haemonchus contortus. We tested the Cry5B in two different formulations and found that the protein was effective against both the adult worm in the stomach, and the young worms in the feces of the host. This protein could potentially be used to treat parasitic nematodes that have become resistant to anthelmintics.

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.

Made available in DSpace on 2016-12-23T14:37:39Z (GMT). No. of bitstreams: 1 Carolina Oliveira Bernardes.pdf: 507333 bytes, checksum: f1bdcef3de907c50ccb23b8a69a35e0a (MD5) Previous issue date: 2009-12-10 / A soja é considerada um dos produtos de maior importância para a economia brasileira e dentre as pragas mais importantes, que limitam a sua produção, destaca-se Anticarsia gemmatalis. Visando reduzir a utilização de inseticidas no controle de pragas, têm-se buscado alternativas de controle, dentre as quais a utilização do controle biológico. Entre os agentes de controle biológico se destacam a bactéria entomopatogênica Bacillus thuringiensis (Bt) e os parasitoides de ovos do gênero Trichogramma. Este estudo avalia a interação desses dois agentes de controle biológico utilizados no controle de A. gemmatalis. Inicialmente, foram feitos testes de seleção com onze linhagens de Trichogramma, sendo a linhagem T. pretiosum 12, a que apresentou melhor desempenho para esta praga. Testes de patogenicidade e virulência foram realizados com 23 isolados de Bt, além do produto comercial Dipel® (B. thuringiensis var. kurstaki), para selecionar os mais agressivos para o inseto. Entretanto, os isolados 80, 997, 1054, 716 e 633 e o Dipel® foram os mais promissores. Por fim, foram realizados dois experimentos de interação entre o T. pretiosum 12, os isolados 80, 997, 1054, 716 e 633 e Dipel®. Foi observado que para o experimento em que os isolados foram misturados ao alimento houve uma interação positiva, em que os isolados 633 e 1054 causaram aumento no parasitismo. Com relação ao segundo experimento, foi observado que a imersão das cartelas com ovos de A. gemmatalis nas suspensões, contendo os diferentes isolados de Bt 80, 633, 716, 993, 1054 e o Dipel® reduziu o número total de ovos parasitados devido à repelência do T. pretiosum 12 aos ovos imersos em suspensão com Bt. Estudos histopatológicos da interação do Bt com as células do intestino médio de Trichogramma são necessários para elucidar como o Bt atua no inseto adulto. Esses estudos, aliados à caracterização molecular das toxinas presentes nos isolados, contribuirão para elucidar a interação parasitoide x entomopatógeno x hospedeiro / Soybean is considered one of the most important products to the brazilian economy and among the most important pests, which make its production limited, Anticarsia gemmatalis is detached. Aiming to reduce the use of insecticides on the pests control, alternatives of control have been searched, among these the use of biological control. Among the biological control agents the entomopathogenic bacterium Bacillus thuringiensis (Bt) and the egg parasitoids from the genus Trichogramma are detached. This study evaluates the interaction between these two biological control agents used on A. gemmatalis control. Initially selection tests were made with eleven Trichogramma strains and the strain T. pretiosum 12 was the one which presented best performance for this pest. Patogenicity and virulence tests were carried out with 23 Bt isolates and the commercial product Dipel® (B. thuringiensis var. kurstaki) to select the most virulent ones for the insect. This way the isolates 80, 997, 1054, 716, 633 and Dipel® were the most promising. With these results, two interaction experiments were performed among T. pretiosum 12, the isolates 80, 997, 1054, 716, 633 and Dipel®. It was observed that for the experiment in which the isolates were mixed to the food there was a positive interaction, where the isolates 633 and 1054 caused an increase on parasitism. Concerning the second experiment it was observed that the immersion of the cards containing A. gemmatalis eggs in the different Bt isolates 80, 633, 716, 993, 1054 and Dipel® reduced the total number of parasitized eggs due to repellence of T. pretiosum to the immersed eggs in Bt suspensions. Histopathologic studies of Bt interaction with T. pretiosum midgut cells are necessary to elucidate how Bt acts on the adult insect. These studies allied to molecular characterization of the toxins presented on the isolates will contribute to elucidate the interaction parasitoid x entomopathogen x host

Le groupe Bacillus cereus est composé de huit espèces de bactéries à Gram positif sporulantes qui peuvent coloniser plusieurs niches écologiques. Les espèces les plus importantes sont B. cereus, une bactérie ubiquitaire du sol et un pathogène opportuniste; B. thuringiensis, un entomopathogène très utilisé comme biopesticide; et B. anthracis l’agent de la maladie du charbon. Bien que ces espèces présentent différents phénotypes, elles sont étroitement liées génétiquement et leurs facteurs de virulences principaux sont portés par des plasmides. Le cycle infectieux de B. thuringiensis dans la larve d’insecte est régulé par l’activation séquentielle de systèmes de quorum sensing de la famille RNPP. Parmi eux, les systèmes Rap-Phr, caractérisés chez B. subtilis, ont très peu été étudiés dans le groupe B. cereus. Ces systèmes régulent divers processus bactériens importants dont la sporulation. L’objectif de cette étude est d’analyser les systèmes Rap-Phr dans le groupe B. cereus, pour connaitre leur distribution, leur localisation et leur diversité afin d’obtenir une vue globale de ces systèmes chez ces bactéries. De plus, leur possible implication dans la régulation du processus de sporulation a été prédite sur la base de données structurales décrites chez RapH de B. subtilis. Les gènes rap, toujours associés à un gène phr, sont présents dans toutes les souches étudiées avec une moyenne de six gènes rap-phr par souche et avec 30% de ces systèmes qui sont portés par des plasmides. Les souches de B. thuringiensis portent six fois plus de systèmes Rap-Phr plasmidiques que les souches de B. cereus. Par ailleurs, les souches phylogénétiquement proches possèdent un profil de gènes rap-phr similaire. Un tiers des protéines Rap sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Cette prédiction a été partiellement validée par des tests de sporulation suggérant que les résidus impliqués dans cette activité chez B. subtilis sont conservés mais insuffisants pour prédire cette fonction. Le système Rap63-Phr63 porté par le plasmide pAW63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. La protéine Rap63 a un effet modéré sur la sporulation et retarde l’expression des gènes régulés par Spo0A. La Rap63 est inhibée par son peptide Phr63, dont la forme mature correspond à l’extrémité C-terminale du pro-peptide. Les résultats de sporulation dans l’insecte suggèrent une activité synergique des systèmes Rap63-Phr63 et Rap8-Phr8 (porté par le pHT8_1) dans la régulation de la sporulation. Malgré la similarité entre les Phr63 et Phr8 aucun cross-talk n’a pu être mis en évidence, ce qui confirme la spécificité de ces systèmes de communication cellulaire. L’ensemble de ces résultats démontre la grande diversité des systèmes Rap-Phr dans le groupe B. cereus et souligne l’impact des systèmes plasmidiques dans le développement de ces bactéries. Par conséquent, les plasmides sont des éléments importants pour l’adaptation et la survie de ces bactéries et particulièrement pour B. thuringiensis. / The Bacillus cereus group of Gram positive spore forming bacteria is comprised by eight species that are able to colonize several ecological niches. The most important species are B. cereus, a ubiquitous soil bacterium and an opportunistic pathogen; B. thuringiensis, an entomopathogen widely used as biopesticide; and B. anthracis, the causative agent of anthrax. Even if they present different phenotypes, they are genetic closely related and their main virulence factors are encoded on plasmids. The infectious cycle of B. thuringiensis in the insect larvae is regulated by the sequential activation of quorum sensing systems from the RNPP family. Among them, the Rap-Phr was extensively studied in B. subtilis but just punctually in B. cereus group species. The Rap-Phr systems were shown to regulate various bacterial processes, including the sporulation. The objective of this study was to analyze the Rap-Phr systems in the B. cereus group, regarding their distribution, location and diversity to achieve an overview of these systems in these bacteria. Moreover, their possible involvement in the control of the sporulation process was predicted based on structural data described for RapH in B. subtilis. The rap genes, always associated with a phr gene, were present in all 49 studied strains with an average of six rap-phr genes per strain and 30% were located on plasmids. Comparison among B. cereus and B. thuringiensis strains revealed that the last one harbors six-fold more plasmid rap-phr system then the former. Moreover, phylogenetic closer strains possess a similar profile of rap-phr genes. Interestingly, 32% of the Rap proteins were predicted to inhibit sporulation and these proteins were preferentially located on plasmids and therefore in B. thuringiensis strains. This prediction was partially validated by sporulation efficiency assays suggesting that residues identified in B. subtilis as involved in the phosphatase activity are conserved but not sufficient to predict the sporulation function. Then, the plasmid-borne Rap63-Phr63 system from pAW63 plasmid of B. thuringiensis HD73 strain was further studied. The Rap63 protein moderately inhibits the sporulation and delays the expression of Spo0A-regulated genes. Rap63 is counteracted by its cognate Phr63 peptide, which mature form corresponds to the C-terminal end of the pro-peptide. Sporulation assays in insect larvae suggest a synergistic activity of Rap63-Phr63 and Rap8-Phr8 (from pHT8_1 of B. thuringiensis HD73 strain) systems on sporulation efficiency. Despite the similarities of Phr63 and Phr8 no cross-talk was found between these two systems, confirming their specificity. Altogether, these results reveal the high diversity of the Rap-Phr systems in the B. cereus group and highlight the relevance of the plasmid-borne systems to cell development. Therefore, the results demonstrated the importance of the plasmids in the adaptation and the survival of these bacteria, especially for B. thuringiensis.