Les propriétés insecticides du Bacillus thuringiensis, découvert par ShigentaneIshiwatari, ont été utilisées pendant des décennies comme biopesticides et cette utilisation a augmenté rapidement en raison de préoccupations au sujet des effets environnementaux négatifs des pesticides chimiques. Actuellement, la toxine Bt dans la forme de biopesticides et des plantes transgéniques Bt peut compléter ou remplacer les pesticides chimiques. Il y a peu d’indication que la toxine Bt a un effet nocif pour l'environnement ou la santé humaine. Néanmoins, il ya des préoccupations que les cultures transgéniques commerciales peuvent avoir des effets néfastes sur l'environnement. Après son introduction dans le sol l'exsudation racinaire et la dégradation des résidus végétaux, la toxine Bt interagit avec les particules de sol. Les interactions de la toxine Bt avec des particules de sol influencent sa mobilité, sa biodisponibilité, sa persistance et sa toxicité.Dans cette étude, nous visons à établir l'importance relative des facteurs biologiques et physico-chimiques dans la détermination de la dynamique des protéines Cry détectables dans les sols, de clarifier si la protéine adsorbée conserve ses propriétés insecticides et d'identifier les propriétés du sol qui déterminent le devenir des protéines Cry dans le sol. Les résultats montrent que les protéines Cry ont une forte affinité sur la surface du sol. Cependant, il y avait peu de relation entre l'affinité pour le sol ou le rendement d'extraction et les propriétés du sol, y compris la teneur en argile, teneur en carbone organique et le pH du sol. Il y avait peu de rapport entre l'affinité et le rendement d'extraction. Les protéines diffèrent à la fois dans leur affinité pour les sols et leurs rendements d'extraction.Une évaluation du rôle du sol et des facteurs environnementaux dans le sort des protéines Cry de la formulation de biopesticides commerciale a montré un déclin rapide de la protéine Cry détectable soumise aux rayons du soleil sous la condition de laboratoire, alors que peu d'effet a été observé dans des conditions de terrain. La demi-vie des protéines dans le sol dans des conditions naturelles était d'environ 1 semaine. Des effets de la température forts ont été observés, mais ils diffèrent pour les biopesticides et la protéine purifiée, indiquant différentes étapes limitantes. Pour le biopesticide, la baisse observée était ralenties par des facteurs biologiques, y compris éventuellement sporulation. En revanche pour des protéines purifiées, augmentation de la température améliorée des changements conformationnels de la protéine adsorbée du sol, conduisant à une fixation et, par conséquent diminué efficacité d'extraction qui a diminué avec le temps. En outre, l'étude de la persistance de diverses protéines Cry dans les sols contrastés a été réalisée par immuno-détection et dosage biologique a montré que la toxine extractible diminue avec incubation allant jusqu'à quatre semaines. L'activité insecticide était toujours maintenue à l'état adsorbé, mais a disparue après deux semaines d'incubation à 25°C. La baisse de la protéine extractible et la toxicité était beaucoup plus faible à 4°C à 25°C. La stérilisation du sol n'a pas eu d'effet significatif sur la persistance de la toxine Cry indiquant que le déclin observé était provoqué par la fixation en fonction du temps de la protéine adsorbée ce qui diminue la quantité de toxine Cry extractable, la dégradation de la protéine par l’activité microbienne jouant un rôle plus mineur.L’exposition des insectes aux protéines Cry sous la forme adsorbé pourrait avoir un impact significatif sur les insectes cibles et même les insectes non cibles, et devrait être plus étudiée afin de déterminer son impact potentiel. / The insecticidal properties of Bacillus thuringiensis, discovered by Shigentane Ishiwatari, have been used for decades as biopesticides and this use has been increasing rapidly because of concerns about the negative environmental effects of chemical pesticides. Currently, Bt toxin in the form of both biopesticides and Bt transgenic plantsmay supplement or replace chemical pesticide. There is little evidence to demonstrate that Bt toxin has any harmful effect to the environment or to human health. Nevertheless, there are concerns that commercial transgenic crops may have harmful impacts on the environment. After release into soil via root exudation and breakdown of plant residues, Bt toxin interacts with soil particles. The interactions of Bt toxin with soil particles influence its mobility, its bioavailability, its persistence and its toxicity. In this study, we aim to establish the relative importance of biological and physicochemical factors in the determination of the dynamics of detectable Cry proteins in soils, to clarify if adsorbed protein maintains its insecticidal properties and to identify the soil properties that determine the fate of Cry proteins in soil. The results show that Cry proteins have strong affinity on soil surface. However, there was little relationship between affinity for soil or the extraction yield and soil properties including clay content, organic carbon content and soil pH. There was little relationship between the affinity and the extraction yield. The proteins differ in both their affinity for soil and their extraction yields.An assessment of role of soil and environmental factors in the fate of Cry protein from commercial biopesticide formulation showed a rapid decline of detectable Cry protein subjected to direct sunlight under the laboratory condition, whereas, little effect was observed under field conditions. The half-life of proteins in soil under natural conditions was about one week. Strong temperature effects were observed, but theydiffered for biopesticide and purified protein, indicating different limiting steps. For biopesticide, the observed decline was due to biological factors, possibly including sporulation. In contrast for purified proteins, increased temperature enhanced conformationalchanges of the soil-adsorbed protein, leading to fixation and hence extraction efficiency decreased that decreased with time. Moreover, the study of persistence of various Cry proteins in contrasting soils was carried out by immuno-detection and bioassay showed that extractable toxin decreased with incubation of up to four weeks. Insecticidal activity was still retained in the adsorbed state, but lost after two weeks of incubation at 25°C. The decline in extractable protein and toxicity was much lower at 4°C than 25°C. There was no significant effect of soil sterilization to persistence of Cry toxin indicating that decrease in detectable Cry toxin in soil may be time-dependent fixation of adsorbed protein as well as decreasing solubilization in larva midgut, but not microbial breakdown.Exposition to Cry in the adsorbed form could have a significant impact on target and even non target insects and should be investigation to determine the potential impact.

Os custos adaptativos associados à resistência de insetos a inseticidas podem ser explorados mediante a integração com outras estratégias de controle de pragas em programas de Manejo Integrado de Pragas (MIP). No presente estudo, objetivou-se verificar custos adaptativos associados à resistência de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) ao inseticida piretroide lambda-cyhalothrin e suas implicações na utilização do hospedeiro pelo parasitoide larval Campoletis aff. flavicincta (Hymenoptera: Ichneumonidae) e as interações com milho geneticamente modificado (MON810) que expressa a proteína Cry1Ab de Bacillus thuringiensis Berliner (milho Bt) e na resposta imunológica ao parasitismo por Campoletis aff. flavicincta. Foram verificados presença de custos adaptativos associados à resistência de S. frugiperda a lambda-cyhalothrin, dado o prolongamento no desenvolvimento larval e duração pupal, redução do peso de pupas fêmeas e longevidade das fêmeas, razão sexual, taxa líquida de reprodução (Ro), taxa intrínseca de aumento (rm) e taxa finita de aumento () de insetos resistentes ao inseticida. Não foi verificada diferença na aceitação de lagartas de S. frugiperda suscetível e resistente a lambda-cyhalothrin por Campoletis aff. flavicincta. Entretanto, o parasitismo de lagartas resistentes foi maior que de suscetíveis em estudos de gaiolas com milho Bt e não-Bt. Posteriormente, foram conduzidos estudos para avaliar, por PCR em tempo real, a expressão diferencial de genes associados ao metabolismo (proteína rica em metionina), resposta imunológica (calreticulina, lisozima, colágeno IV-2, hemócito protease-3, serina protease, imunolectina, receptor scavenger classe C) e detoxificação de xenobióticos (glutationa-S-transferase 145 e as monoxigenases P450 Cyp9A31 e Cyp333B2) expressos em diferentes tecidos (tecido adiposo, hemócitos e/ou mesêntero), na ausência e presença de parasitismo de lagartas das duas linhagens de S. frugiperda por Campoletis aff. flavicincta. No geral, a expressão gênica em lagartas suscetíveis foi superior àquela de lagartas resistentes a lambda-cyhalothrin, independente do período de desenvolvimento, do tecido avaliado e da presença ou não do parasitismo por Campoletis aff. flavicincta. E por fim, foram conduzidos estudos para avaliar o efeito da resistência de S. frugiperda a lambda-cyhalothrin nas respostas imunológica celular (contagem total de hemócitos) e humoral (atividade das fenoloxidases, lisozimática e antimicrobiana e concentração de óxido nítrico) de lagartas, tanto na ausência como na presença do parasitismo por Campoletis aff. flavicincta. A resistência de S. frugiperda a lambda-cyhalothrin induziu somente a pequenas alterações no sistema imunológico do hospedeiro (aumento do número total de hemócitos, redução da atividade antimicrobiana e aumento da atividade lisozimática), as quais não interferem a ponto de resultar em custos adaptativos que leve à maior exploração de lagartas resistentes na presença do parasitismo por Campoletis aff. flavicincta. Portanto, o manejo de S. frugiperda mediante o emprego da tecnologia de milho Bt e do controle biológico via parasitoide Campoletis aff. flavicincta pode favorecer o restabelecimento da suscetibilidade de S. frugiperda a lambda-cyhalothrin. / Fitness costs of insect resistance to insecticides can be exploited by integrating other pest control strategies in Integrated Pest Management (IPM) programs. The objective of this research was to evaluate the existence of fitness costs associated with the resistance of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) to the pyrethroid insecticide lambda-cyhalothrin and their implications for host use by the larval parasitoid Campoletis aff. flavicincta (Hymenoptera: Ichneumonidae) and interactions with the genetically modified maize (MON810) that expresses Cry1Ab toxin of Bacillus thuringiensis Berliner (Bt maize) and in the immune response to parasitization by Campoletis aff. flavicincta. Fitness costs associated to resistance of S. frugiperda to lambda-cyhalothrin were detected by the delay in larval and pupal development, reduction in the pupal weight and longevity of females, sex ratio, net reproductive rate (Ro), intrinsic rate of natural increase (rm) and the finite rate of increase (). No differences were detected in host acceptance and survival of Campoletis aff. flavicincta in susceptible and lambda-cyhalothrin-resistant larvae of S. frugiperda. However, larval parasitization was higher on the resistant than on the susceptible strain of S. frugiperda in cage studies with Bt and non-Bt maize plants. Then, studies were conducted by using Real time-PCR to evaluate the differential expression of genes associated with metabolism (methionine-rich protein), immune response (calreticulin, lysozyme, collagen IV-2, protease-3 hemocyte, serine protease, immunolectin, scavenger receptor class C) and xenobiotic detoxification (glutathione-S-transferase 145 and P450 monooxygenases Cyp9A31 and Cyp333B2) expressed in different tissues (fat body, hemocytes and/or midgut), in the absence and presence of larval parasitization of both strains of S. frugiperda by Campoletis aff. flavicincta. Overall, gene expression in susceptible larvae was higher than that of lambdacyhalothrin- resistant larvae, regardless of the period of development, tissue evaluated and presence or not of parasitization by Campoletis aff. flavicincta. Finally, studies were conducted to evaluate the effect of the resistance of S. frugiperda to lambda-cyhalothrin on cellular (total hemocyte count) and humoral (phenoloxidases, lysozyme and antimicrobial activities and nitric oxide concentration) immune responses in the absence or presence of parasitization by Campoletis aff. flavicincta. The resistance of S. frugiperda to lambdacyhalothrin conferred only minor changes in the host immune system (increased total hemocyte count, reduced antimicrobial activity and increased lysozyme activity), which may not interfere with fitness costs leading to higher exploitation of resistant larvae in the presence of parasitization by Campoletis aff. flavicincta. Therefore, the management of S. frugiperda by using the Bt maize technology and the biological control via parasitoid Campoletis aff. flavicincta can favor the resetting to susceptibility of S. frugiperda to lambda-cyhalothrin.

Neste trabalho foi feito um estudo para obtenção de formulações multiparticuladas, formadas por micropartículas de polímeros naturais, solúveis em água, não tóxicos e biodegradáveis. Os polímeros utilizados foram: Caseína, Hidroxietilcelulose (HEC), Hidroxipropilmetilcelulose (HPMC), Alginato de sódio e Quitosana. As técnicas utilizadas para obtenção das micropartículas foram o spray drying e a atomização de alginato de sódio em uma solução de CaCl2, para gelificação das gotículas formadas, com uma complexação ou não com quitosana. O estudo de micropartículas secas de alginato de cálcio e alginato de cálcio recoberta com uma membrana de quitosana revelou que diferentes procedimentos e variáveis de processo influenciavam nas características das micropartículas obtidas. O diâmetro médio das micropartículas de alginato e alginato-quitosana variou de 60 a 553 µm. A superfície de micropartículas com quitosana se mostrou mais rugosa, com uma grande quantidade de poros menores que 1 µm. As micropartículas com diâmetro médio de 60 µm apresentaram uma boa esfericidade e uma distribuição de tamanho de partícula uniforme. O teor de cálcio apresentou variações, diminuindo em processos com quitosana. A maioria das micropartículas de alginato e alginato-quitosana eram estáveis em água, mas instáveis em soro fisiológico e tampão fosfato 0,1M. Após a realização do estudo das características destas micropartículas foram incorporados a elas materiais com atividade bioinseticida (Bacillus thuringiensis var. krusfaki (Btk) e de Baculovirus (Bv)). As micropartículas obtidas com alginato de cálcio formaram pós contendo sistemas matriciais capazes de microencapsular e reter microrganismos entomopatogênicos, inclusive após redispersão em água. Os processos de microencapsulação de desenvolvidos e avaliados demonstraram ser adequados para a manutenção da viabilidade de Btk e da integridade dos poliedros de Bv, assim como, a conservação da capacidade bioinseticida destes microrganismos. As micropartículas obtidas com as misturas poliméricas Caseína/HEC e Caseína/HPMC pelo processo de spray drying revelaram-se inadequadas na medida que se dissolviam rapidamente depois de dispersas em água e não poderiam assim proteger o material bioinseticida no meio ambiente. / The aim of this work was to develop powder formulations containing microparticles, to be used as multiparticulate delivery systems. Two different methods were investigated : 1) the preparation of microparticles by spray drying using casein, hydroxyethylcellulose (HEC) and hydroxypropilmetilcellulose (HPMC) and 2) the preparation of calcium alginate and chitosan-alginate microparticles by using an atomizer device. Different experimental procedures to prepare calcium alginate and chitosanalginate microparticles were evaluated and variables beHeved to be important for the membrane formation were examined. The mean particles diarneter ranged from 60 to 553 µm. When a comparison was made between the surface morphology of calcium alginate and alginate-chitosan microparticles, remarkable roughness and more porous structure was observed in the chitosan-alginate microparticles. Some properties of the microparticles depended on the method and the procedure conditions of forming the chitosan-alginate complex. Calcium alginate and chitosan-alginate microparticles containing two different bioinsecticides were also prepared: 1) a spore/δ-endotoxinscomplex of Bacillus thuringiensis var.kurstaki (Btk) and 2) polyhedra of Baculovirus anticarsia (Bv), a viral insecticide. The results shown that the encapsulation of suspensions of Btk containing spore/toxin complex or polyhedra of B. anticarcia in calcium alginate and chitosan-alginate microparticles did not decrease the larvicidal activity of these biopesticides against lepidopterous pests. The formulations developed in this study remained unchanged, did not swell, did not release the spores (Btk) or the polyhedra (Bv), when dispersed in water and could be useful to be applied by aqueous spray as bioinsecticides in agriculture. On the other hand, casein, HEC and HPMC microparticles prepared by spray drying were not suitable to encapsulate bioinsecticides because they dissolved fastly after dispersion in water.

Quatro linhagens capazes de crescer com acefato foram isoladas a partir de solos com históricos de aplicação de deste composto. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. e Pandoreae sp. foram identificadas através do rDNA 16S e perfil de ácidos graxos de membrana. Rhodococcus sp. foi o isolado mais eficiente na degradação de acefato, removendo 99.24% deste composto em meio de cultura com acefato como única fonte de carbono. Quando avaliado com acefato como fonte combinada de carbono e nitrogênio, este organismo degradou 19% de acefato com formação de metamidofós (17%). Staphylococcus sp. apresentou 21% de degradação de acefato utilizando-o como fonte de carbono e nitrogênio, mas não manteve o crescimento com este composto como fonte de carbono. Stenotrophomonas maltophilia e Pandoreae sp. não mantiveram crescimento com acefato como única fonte de carbono isoladamente. Estas linhagens apresentaram crescimento em acefato como fonte de nitrogênio e enxofre, porém, as análises de GC/MS demonstraram que não houve degradação nestas condições. / Four strains of microorganisms capable of growth on acephate were isolated from soil samples with a history of acephate application. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. and Pandoreae sp. were identified based on 16S rRNA gene sequencing and fatty acid profiling. Rhodococcus sp. was the most efficient acephate degrader of the isolates, it removed 99.24% of acephate from defined growth media when the compound was provided as sole carbon source. When provided as a combined carbon and nitrogen source, the organisms degraded 19% of acephate with formation of methamidophos (17%). Staphylococcus sp. degraded 21% of acephate when provided as sole nitrogen and carbon source but did not grow on the compound as a sole source of carbon. Pandoreae sp. and Stenotrophomonas maltophilia failed to grow on acephate as sole source of carbon in defined medium. These strains grew in media where the pesticide was provided as a combined nitrogen and carbon source, but no acephate biodegradation could be demonstrated in these instances.

Made available in DSpace on 2017-07-10T19:24:35Z (GMT). No. of bitstreams: 1 Patricia Stadler Rosa Lucca.pdf: 1596078 bytes, checksum: 26545cacc279bf8104c7bd2edea4d008 (MD5) Previous issue date: 2010-06-10 / The use of natural substances for pest management in agriculture is, economically, a viable option and has benefits for both human-beings and environment, due to its low persistence and toxicity. Thus, this trial aimed at determining the insecticidal potential of extracts and essential oils of fennel (Foeniculum vulgare), sweet herb (Pimpinella anisum), clove (Caryophillus aromaticus) and of homeopathic preparations from CH 05 and CH 06 aphid on the control of Brevicoryne brassicae in cabbage (Brassica oleracea var. acephala). The treatments were: fennel extracts, sweet herb and cloves at 10%; fennel oils, sweet herb and cloves at 1%; homeopathic preparations with CH 05 and CH 06 aphid; control with distilled water. The mortality tests were done concerning nymphs and adults of aphids in laboratory, with three replications, each one consisting of a cabbage leaf kept inside a Petri dish containing moistened cotton. On each plate, ten nymphs were released added with a 1 mL spray of the respective solutions. The plates were covered with a perforated plastic film and incubated with a 12 hour photoperiod at 25 ° C. Evaluations were performed after 1, 12, 24, 48 and 72 hours. After analysis in the laboratory, some tests were done with the culture, in pots, only with the Indian cloves extracts at 10% and fennel oil at 1% treatments, since they were significant. There was, moreover, the sensorial analysis of cabbage, treated with clove extract at 10% and fennel oil at 1%. Data analysis was performed by analysis of variance and averages comparison by Tukey, at 5% of significance. In lab tests, it was found out that fennel oil at 1% showed the best rate of mortality on aphid nymphs, which means 70%, at 72 h, followed by clove extract at 10% with 37% of mortality. It was registered for survival of adults aphids that the fennel oil at 1%, fennel extract at 10% and clove extract at 10% had the lowest survival rates, as: 46%, 53% and 53 %, respectively. A mortality test was carried out in the culture and it was registered the effectiveness of clove extract at 10% and fennel oil at 1%, and they had 6% and 8% of survival in adults aphids, respectively. The sensorial analysis of cabbage did not indicate any significant difference among them, at 5% significance. / O uso de substâncias naturais para manejo de pragas da agricultura é uma opção economicamente viável e traz benefícios tanto ao homem como ao ambiente, devido a sua baixa persistência e toxicidade. Dessa forma, o objetivo do trabalho em questão foi verificar o potencial inseticida de extratos e óleos essenciais de funcho (Foeniculum vulgare), erva doce (Pimpinella anisum), cravo-da-Índia (Caryophillus aromaticus) e dos preparados homeopáticos para o controle de Brevicoryne brassicae em couve (Brassica oleracea var. acephala). Os tratamentos utilizados foram os extratos de funcho, erva-doce e cravo-da-Índia a 10%; óleos de funcho, erva-doce e cravo-daÍndia a 1%; preparados homeopáticos de pulgão CH 05 e CH 06 e controle com água destilada. Realizaram-se testes de mortalidade sobre ninfas e adultos de pulgão em condições de laboratório, com três repetições, cada uma constituída por uma folha de couve mantida no interior de uma placa de Petri contendo algodão umedecido. Em cada placa, foram liberadas dez ninfas e pulverizado 1 mL das respectivas caldas. As placas foram cobertas com filme plástico perfurado e incubadas com fotoperíodo de 12 horas a 25 °C. Avaliações foram realizadas após 1, 12, 24, 48 e 72 h. Após análise em condições de laboratório, realizaram-se testes na cultura, em vasos, somente com os tratamentos extrato de cravo-da-Índia a 10% e óleo de funcho a 1%, pois os mesmos apresentaram-se significativos. Realizou-se, ainda, análise sensorial das couves tratadas com o extrato de cravo a 10% e óleo de funcho a 1%. A análise dos dados ocorreu por análise de variância e comparação de médias por Tukey, a 5% de significância. Nos testes realizados em laboratório, verificou-se que, o óleo de funcho a 1% apresentou a melhor mortalidade sobre as ninfas de pulgão, ou seja, 70%, às 72 h, seguido do extrato de cravo a 10%, com 37% de mortalidade. Quanto à sobrevivência de adultos de pulgão, verificou-se que o óleo de funcho a 1%, extrato de funcho a 10% e o extrato de cravo a 10% apresentaram os menores índices de sobrevivência, ou seja, 46%, 53% e 53%, respectivamente. No teste de mortalidade realizado na cultura, observou-se a efetividade do extrato de cravo a 10% e do óleo de funcho a 1%, em que os mesmos obtiveram 6% e 8% de sobrevivência de adultos de pulgões, respectivamente. A análise sensorial das couves não indicou diferença significativa entre as mesmas, a 5% de significância.

Made available in DSpace on 2017-05-12T14:47:57Z (GMT). No. of bitstreams: 1 Patricia Stadler Rosa Lucca.pdf: 1596078 bytes, checksum: 26545cacc279bf8104c7bd2edea4d008 (MD5) Previous issue date: 2010-06-10 / The use of natural substances for pest management in agriculture is, economically, a viable option and has benefits for both human-beings and environment, due to its low persistence and toxicity. Thus, this trial aimed at determining the insecticidal potential of extracts and essential oils of fennel (Foeniculum vulgare), sweet herb (Pimpinella anisum), clove (Caryophillus aromaticus) and of homeopathic preparations from CH 05 and CH 06 aphid on the control of Brevicoryne brassicae in cabbage (Brassica oleracea var. acephala). The treatments were: fennel extracts, sweet herb and cloves at 10%; fennel oils, sweet herb and cloves at 1%; homeopathic preparations with CH 05 and CH 06 aphid; control with distilled water. The mortality tests were done concerning nymphs and adults of aphids in laboratory, with three replications, each one consisting of a cabbage leaf kept inside a Petri dish containing moistened cotton. On each plate, ten nymphs were released added with a 1 mL spray of the respective solutions. The plates were covered with a perforated plastic film and incubated with a 12 hour photoperiod at 25 ° C. Evaluations were performed after 1, 12, 24, 48 and 72 hours. After analysis in the laboratory, some tests were done with the culture, in pots, only with the Indian cloves extracts at 10% and fennel oil at 1% treatments, since they were significant. There was, moreover, the sensorial analysis of cabbage, treated with clove extract at 10% and fennel oil at 1%. Data analysis was performed by analysis of variance and averages comparison by Tukey, at 5% of significance. In lab tests, it was found out that fennel oil at 1% showed the best rate of mortality on aphid nymphs, which means 70%, at 72 h, followed by clove extract at 10% with 37% of mortality. It was registered for survival of adults aphids that the fennel oil at 1%, fennel extract at 10% and clove extract at 10% had the lowest survival rates, as: 46%, 53% and 53 %, respectively. A mortality test was carried out in the culture and it was registered the effectiveness of clove extract at 10% and fennel oil at 1%, and they had 6% and 8% of survival in adults aphids, respectively. The sensorial analysis of cabbage did not indicate any significant difference among them, at 5% significance. / O uso de substâncias naturais para manejo de pragas da agricultura é uma opção economicamente viável e traz benefícios tanto ao homem como ao ambiente, devido a sua baixa persistência e toxicidade. Dessa forma, o objetivo do trabalho em questão foi verificar o potencial inseticida de extratos e óleos essenciais de funcho (Foeniculum vulgare), erva doce (Pimpinella anisum), cravo-da-Índia (Caryophillus aromaticus) e dos preparados homeopáticos para o controle de Brevicoryne brassicae em couve (Brassica oleracea var. acephala). Os tratamentos utilizados foram os extratos de funcho, erva-doce e cravo-da-Índia a 10%; óleos de funcho, erva-doce e cravo-daÍndia a 1%; preparados homeopáticos de pulgão CH 05 e CH 06 e controle com água destilada. Realizaram-se testes de mortalidade sobre ninfas e adultos de pulgão em condições de laboratório, com três repetições, cada uma constituída por uma folha de couve mantida no interior de uma placa de Petri contendo algodão umedecido. Em cada placa, foram liberadas dez ninfas e pulverizado 1 mL das respectivas caldas. As placas foram cobertas com filme plástico perfurado e incubadas com fotoperíodo de 12 horas a 25 °C. Avaliações foram realizadas após 1, 12, 24, 48 e 72 h. Após análise em condições de laboratório, realizaram-se testes na cultura, em vasos, somente com os tratamentos extrato de cravo-da-Índia a 10% e óleo de funcho a 1%, pois os mesmos apresentaram-se significativos. Realizou-se, ainda, análise sensorial das couves tratadas com o extrato de cravo a 10% e óleo de funcho a 1%. A análise dos dados ocorreu por análise de variância e comparação de médias por Tukey, a 5% de significância. Nos testes realizados em laboratório, verificou-se que, o óleo de funcho a 1% apresentou a melhor mortalidade sobre as ninfas de pulgão, ou seja, 70%, às 72 h, seguido do extrato de cravo a 10%, com 37% de mortalidade. Quanto à sobrevivência de adultos de pulgão, verificou-se que o óleo de funcho a 1%, extrato de funcho a 10% e o extrato de cravo a 10% apresentaram os menores índices de sobrevivência, ou seja, 46%, 53% e 53%, respectivamente. No teste de mortalidade realizado na cultura, observou-se a efetividade do extrato de cravo a 10% e do óleo de funcho a 1%, em que os mesmos obtiveram 6% e 8% de sobrevivência de adultos de pulgões, respectivamente. A análise sensorial das couves não indicou diferença significativa entre as mesmas, a 5% de significância.

In this thesis, we examined the effects of the exposures to anthropogenic pollutants on the fish, primarily juvenile chinook salmon, immune system using newly and recently developed immune assays. In addition, we developed a new assay for measuring immunocompetence of fish. In the first chapter, the Alamar Blue assay was developed to quantify the proliferation of chinook salmon (Oncorhynchus tshawytscha) leukocytes. Isolated splenic and pronephric leukocytes were stimulated with different concentration of mitogens (LPS, PWM, and ConA) for various incubation times. Optimum cell culture conditions (cell density, mitogen concentration, and incubation time) for the Alamar Blue assay were evaluated by comparison with flow cytometric analysis. The Alamar Blue dye was non-toxic for leukocytes, and the assay proved to be able to quantify the mitogenic responses using LPS, but PWM and ConA. In the second chapter, we determined the effects and mechanisms by which p,p'- DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha). Isolated salmon splenic and pronephric leucocytes were incubated with different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'- DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leucocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leucocytes appeared to vary between developmental stages or season. The mitogenic response of leukocytes of chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a significantly lower percentage of Ig+ blasting cells than controls. Our results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression. In the third chapter, we evaluated the direct effects of in vitro exposures to tributyltin (TBT), widely used biocide, on the cell mediated immune system of chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile chinook salmon were exposed for 6, 24, or 96 hr to a concentration range of 0.03 0.1 mg TBT 1����� in cell cultures. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in the viability in cell cultures following the induction of apoptosis. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon LPS stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg 1����� of TBT in the cell cultures. Flow cytometric assay with the fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cell in the chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications. / Graduation date: 2004

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Le DEET (N, N-diéthyl-m-toluamide), est connu comme le répulsif le plus utilisé au monde. Bien qu'il soit efficace contre un large groupe d'arthropodes, son mode d'action exact et sa cible moléculaire ne sont pas encore connus précisément. Grâce à l'utilisation des techniques d'électrophysiologie (patch-clamp et oil-gap), d'imagerie calcique et biochimique, nous avons étudié le mode d'action du DEET sur des cellules neurosécrétrices identifiées, les DUM neurones de la blatte Periplaneta americana. Le DEET, à forte concentration, inhibe l'activité de l'acétylcholinestérase (AChE) au niveau du DUM neurone. A faible concentration, il induit une augmentation de la concentration en calcium intracellulaire via l'activation des récepteurs cholinergiques de type muscariniques (mAChRs). Dans un deuxième temps, les interactions synergiques entre le DEET et le propoxur, un carbamate connu pour inhiber l'AChE, ont été étudiées. Les résultats ont révélé que les mAChRs, correspondent bien à une nouvelle cible potentielle pour le DEET et qu'ils sont impliqués dans l'effet synergique. Le DEET, à faible et à forte concentration, agit sur des sites allostériques positifs et négatifs des mAChRs respectivement. L'action du DEET sur le site allostérique positif des mAChRs est responsable de l'effet synergique via une augmentation de la concentration en calcium intracellulaire qui potentialise l'effet anti-AChE du propoxur. L'utilisation d'outils pharmacologiques sélectifs a permis l'identification de la voie de signalisation intracellulaire (PLC, PI-PLC, CaMKinase II, récepteurs IP3) impliquée dans l'effet synergique du propoxur. Les résultats présentés dans ce mémoire vont contribuer au développement de nouvelles stratégies basées sur l'utilisation de combinaisons d'insecticides de familles chimiques différentes afin de réduire les doses des traitements tout en augmentant l'efficacité.