Genetically modified (GM) maize was developed mainly to control lepidopteran pests such as the maize stem borer (Busseola fusca) (Lepidoptera: Noctuidae). Since the first commercialization of GM crops with insecticidal properties, possible non-target effects such as the effect at the third trophic level on important predators for example lacewing species (Chrysoperla spp.) have been of concern. Contradicting results were reported in previous studies with regard to the effect of Cry 1Ab protein produced by Bt maize on the performance of lacewings. Some studies found that Bt proteins had no effect while others reported that C. carnea performed poorly if they consumed prey that consumed Cry 1Ab protein. In South Africa one of the most common chrysopid species in maize ecosystems is Chrysoperla pudica (Navás) (Neuroptera: Chrysopidae). Evolution of Bt resistant pests, such as B. fusca in South Africa facilitates a new pathway for exposure of predators to healthy prey that consumes Cry 1Ab proteins. The aims of this study was to determine the effect of the Cry 1Ab protein expressed in Bt maize on a non-target organism‟s (C. pudica) biology via indirect exposure, and to determine the concentration of Cry 1Ab protein in the plant, prey and predator. Chrysoperla pudica larvae were indirectly exposed to the Bt-toxin through healthy Bt-maize feeding prey (B. fusca larvae) in two feeding experiments and lacewing survival and life history parameters recorded. Bt had a limited effect on some parameters that were evaluated. The larval and pupal periods of C. pudica larvae that were exposed to the Bt-toxin had a significant difference from that of the control treatment. The Bt-toxin had a significant effect on fecundity, fertility and malformation after emergence of C. pudica adults of which larvae fed only on Bt resistant B. fusca larvae, but not on the mortality rate. Cry 1Ab concentration was the highest in the plant, followed by the prey and lacewing larvae. This study showed that the Cry 1Ab protein had a slight adverse effect only on certain life parameters of C. pudica, and that Cry 1Ab protein was hardly detectable in C. pudica larvae. However, since this study represented a worst-case scenario where diverse prey was not available, insignificant effects is expected under field conditions where prey is diverse. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

A tolerância pode ser considerada um dos pilares da imunologia. Sabe-se que a tolerância a um antígeno pode gerar tolerância a outro antígeno não relacionado, fenômeno conhecido como tolerância cruzada. Neste trabalho caracterizamos a tolerância cruzada utilizando a OVA como tolerógeno e extrato de Blomia tropicalis (Bt) ou Hemocianina de Keyhole limpet (KLH) como alérgenos. Verificamos que é possível reproduzir o fenômeno da tolerância cruzada neste modelo de inflamação alérgica induzida tanto pelo KLH quanto pela Bt, com diminuição do infiltrado inflamatório no pulmão, eosinófilos, IgE total e produção de muco. Ainda, a estratégia de utilizar a tolerância cruzada terapeuticamente, i.é., após a sensibilização com KLH, a indução de tolerância cruzada não foi capaz de prevenir a resposta alérgica. Em conjunto, nossos dados mostram que a tolerância à OVA modifica as respostas alérgicas tanto à Bt quanto ao KLH no modelo de inflamação pulmonar experimental de forma profilática, mas que a tolerância cruzada não é eficiente em animais já sensibilizados. / Tolerance is among the Immunology pillars. Experimental data indicate that tolerance towards an antigen can promote tolerance to an unrelated antigen, a phenomenon known as cross-tolerance. Here we sought to characterize cross tolerance using OVA as a tolerogen and Blomia tropicalis (Bt) extract or Keyhole limpet Hemocianina (KLH) as allergens. We found that cross tolerance can be reproduced in the model of allergic lung disease induced by KLH or Bt, with less inflammatory infiltrate in the lung, eosinophils, total IgE and mucus production. Using cross tolerance therapeutically, i.e., after KLH sensitization, was not effective, since the allergic lung response was not modulated. Altogether, our data shows that OVA tolerance modulate allergic lung disease induced either by Bt or KLH when used as prophylactic model, however cross tolerance is ineffective in sensitized animals.

Made available in DSpace on 2017-07-25T19:29:58Z (GMT). No. of bitstreams: 1 Elderson Ruthes.pdf: 4240882 bytes, checksum: 070ecb98a882a5dd0c10730ab8030e55 (MD5) Previous issue date: 2012-08-27 / Bt corn is characterized as a technology for pest management that has been used in Brazil since the 2008/2009 season. Bt corn hybrids currently available on the market resulted from five transgenic events for the control of caterpillars, MON 810, Bt11, TC 1507, MON 89034, and MIR 162, which express Cry1Ab, Cry1Ab, Cry1F, Cry1A.105 + Cry2Ab2, and Vip3A toxins, respectively. The increased adoption of no-tillage farming has a major influence on insect pests, including caterpillars. Black oat is the main winter cover crop preceding soybeans and corn crops in the summer, and it is grown in large areas in the states of Rio Grande do Sul, Santa Catarina, and Paraná.Direct sowing of maize on desiccated black oat may favor the occurrence of insects with potential for damage, especially during germination and seedling stage. Therefore, the aim of this study was to evaluate the effectiveness of Bt corn associated with the following management practices: a) interval between black oat desiccation and maize sowing, with and without application of the insecticide lufenurom; b) seed treatment with the insecticides thiamethoxan + fipronil and thiodicarb + imidacloprid to reduce the damage caused by Pseudaletia sequax and Spodoptera frugiperda caterpillars in the beginning of the development of corn plants.Four experiments were conducted under field conditions and no-tillage farming on desiccated black oat, two with natural infestation of insect pests and two with artificial infestation of S. frugiperda using cages. The four experiments were conducted in Itaberá, SP during the seasons 2009/2010 and 2010/2011. The 40-day interval between black oat desiccation and maize sowing proved to be effective in reducing damage caused by P. sequax in Bt corn 30F53H (Cry1F), regardless of insecticide application for desiccation or seed treatment. When black oat desiccation was carried out 15 days before maize sowing, a higher occurrence of damage caused by P.sequax in 30F53H Bt corn was observed. In this case, the seed treatment with thiodicarb + imidacloprid proved to be more effective in reducing damage. Corn hybrid 30F53H was effective in reducing damage caused by S. frugiperda, regardless of desiccation interval or use of insecticides. Corn hybrids DKB 240YG (Cry1Ab),DKB 240H (Cry1F), 30F53H (Cry1F), DKB 390PRO (Cry1A.105 + Cry2Ab2), and Garra Viptera (Vip3A) were effective in reducing the number of plants cut by S. frugiperda third instar caterpillars compared with non-Bt corn, regardless of the seed treatment used. Corn hybrids DKB 240H, DKB 390PRO, and Garra Viptera were effective in reducing damage caused by S. frugiperda, such as scraped plants and damaged whorls, compared with non-Bt corn, regardless of the seed treatment used. Corn hybrid DKB 240YG presented reduction in damaged whorls only when associated with seed treatment with the insecticide thiodicarb + imidacloprid. The use of Bt corn with other management practices significantly contributed to reduction of damage caused by P. sequax and S. frugiperda in the beginning of the development of the crop. Therefore, the use of Bt corn should be considered as one more practice in corn integrated pest management. / O milho Bt caracteriza-se como uma tecnologia para o manejo de pragas que vem sendo utilizada no Brasil desde a safra 2008/2009. Os híbridos de milho Bt disponíveis atualmente no mercado resultaram de cinco eventos transgênicos para o controle de lagartas, MON 810, Bt11, TC 1507, MON 89034 e MIR 162, os quais expressam as toxinas Cry1Ab, Cry1Ab, Cry1F, Cry1A.105 + Cry2Ab2 e Vip3A,respectivamente. O aumento na adoção do sistema de plantio direto na palha apresenta grande influência sobre os insetos-praga, inclusive as lagartas. A aveiapreta é a principal cultura de cobertura que antecede o cultivo de soja e milho no verão, ocupando extensas áreas nos estados do Rio Grande do Sul, Santa Catarina e Paraná. A semeadura do milho sobre plantas dessecadas de aveia-preta pode favorecer a ocorrência de insetos com potencial de danos, principalmente durante a germinação e a fase de plântula. Assim, o objetivo deste estudo foi avaliar a eficácia do milho Bt associada às seguintes práticas de manejo: a) intervalo entre a dessecação da aveia-preta e a semeadura do milho, com e sem aplicação do inseticida lufenurom em pulverização; b) tratamento de sementes com os inseticidas tiametoxam + fipronil e tiodicarbe + imidacloprido para redução dos danos ocasionados pelas lagartas Pseudaletia sequax e Spodoptera frugiperda no início do desenvolvimento da cultura do milho. Foram realizados quatro experimentos em condições de campo sob o sistema de plantio direto na palha após a cultura da aveia-preta, dois deles com infestação natural dos insetos-praga e dois com infestação artificial de S. frugiperda utilizando-se gaiolas. Os quatro experimentos foram conduzidos no município de Itaberá, SP durante as safras 2009/2010 e 2010/2011. O intervalo de 40 dias entre a dessecação da aveia-preta e a semeadura do milho se mostrou eficaz na redução dos danos ocasionados por P. sequax no híbrido de milho Bt 30F53H (Cry1F), independentemente da aplicação de inseticidas na dessecação ou via tratamento de sementes. Quando a dessecação da aveiapreta foi realizada 15 dias antes da semeadura do milho, houve maior ocorrência de danos ocasionados por P. sequax no híbrido Bt 30F53H. Nesse caso, o tratamento de sementes com tiodicarbe + imidacloprido se mostrou eficiente na redução dos danos. O híbrido 30F53H foi eficaz na redução dos danos ocasionados por S.frugiperda, independentemente do intervalo de dessecação e da utilização de inseticidas. Os híbridos DKB 240YG (Cry1Ab), DKB 240H (Cry1F), 30F53H (Cry1F),DKB 390PRO (Cry1A.105 + Cry2Ab2) e Garra Viptera (Vip3A) foram eficazes na redução do número de plantas cortadas por lagartas de S. frugiperda de terceiro ínstar em relação ao híbrido não Bt, independentemente do tratamento de sementes utilizado. Os híbridos DKB 240H, DKB 390PRO e Garra Viptera foram eficazes na redução dos danos ocasionados por S. frugiperda, tais como plantas raspadas e cartuchos danificados, em relação ao híbrido não Bt, independentemente do tratamento de sementes utilizado. O híbrido DKB 240YG apresentou redução de cartuchos danificados somente quando associado ao tratamento de sementes com o inseticida tiodicarbe + imidacloprido. Pode-se afirmar que a utilização desses híbridos Bt associada a outras práticas de manejo contribuiu significativamente na redução dos danos ocasionados por P. sequax e S. frugiperda no início do desenvolvimento da cultura. Assim, a utilização do milho Bt deve ser considerada como mais uma prática no manejo integrado de pragas na cultura do milho.

Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica encontrada em várias partes do mundo e tem a capacidade de produzir cristais durante a fase estacionária do crescimento. Esses cristais são toxinas compostas por proteínas ativas contra uma ampla variedade de fases imaturas de insetos. As toxinas do Bt têm sido usadas como bioinseticidas por décadas no controle de insetos das ordens Coleoptera, Lepidoptera e Diptera, e algumas toxinas já foram selecionadas como efetivas contra ácaros, nematoides e insetos sugadores de floema da ordem Hemiptera, como os afídeos. Recentemente, foi comprovada a patogenicidade de três isolados de Bt a outro inseto dessa ordem, o psilídeo Diaphorina citri. Esse psilídeo é vetor das bactérias Candidatus Liberibacter spp., agentes causais da principal doença dos citros: o Huanglongbing ou HLB. A descoberta da capacidade endofítica de Bt em plantas de diferentes espécies vegetais abriu perspectivas para novos estudos usando o Bt para controle de insetos sugadores, como a D. citri. O objetivo deste trabalho foi avaliar o efeito patogênico de cada toxina Cry ou Cyt presente nestes três isolados através de isolados de Bt recombinantes expressando as toxinas individualmente. Foram montados bioensaios para confirmar a taxa de mortalidade causada por isolados de Bt contra ninfas de D. citri. Os bioensaios foram feitos usando cinco plantas de laranja doce (Citrus sinensis (L.) Osbeck) e dez ninfas de 3º ínstar de D. citri por planta, além dos controles (sem Bt). A mortalidade das ninfas foi avaliada diariamente durante cinco dias. Durante a avaliação, as ninfas mortas foram coletadas para o isolamento do Bt além da detecção do gene da toxina por PCR. No final do experimento, as folhas jovens onde as ninfas se alimentavam também foram avaliadas quanto à presença do Bt. Nossos resultados confirmaram que os isolados de Bt previamente identificados como patogênicos a D. citri causaram elevada mortalidade (em média 68-93%) em ninfas de 3º ínstar. Dentre os isolados recombinantes testados, um deles apresentou grande destaque, causando a mortalidade de 68-81% das ninfas em 48 horas e 83-93% em 120 horas. Esse isolado possui grande potencial de uso no controle biológico de D. citri através da produção de um bioinseticida ou produção de plantas de citros transgênicas. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium found in several locations around the world and has the ability to produce crystals during the stationary phase of growth. These crystals are composed of protein toxins that are active against a wide variety of insect larvae. The Bt toxins have been used for decades as biopesticides to control insects of the orders Coleoptera, Lepidoptera, and Diptera. Some toxins have been selected as effective for mites, nematodes and phloem-sucking insects of the order Hemipera, such as aphids. The pathogenicity of strains of Bt to Diaphorina citri, an hemipteran known as Asian citrus psyllid, has been recently demonstrated. This psyllid is the vector of the bacteria Candidatus Liberibacter spp., the causal agents of the main disease of citrus: Huanglongbing or HLB. The discovery of the ability of Bt strains to endophytically colonize plants of different species has opened new perspectives for studies. The aim of this work was to evaluate the pathogenic effect of each Cry or Cyt toxin present in these three isolates through the use of recombinant Bt isolates expressing each toxin individually. A series of bioassays were done in order to confirm the mortality caused by Bt strains against nymphs of D. citri. Bioassays were performed in five seedlings of Citrus sinensis (L.) Osbeck (five replicates/ bioassay) and using ten D. citri 3rd instar nymphs to each assay plant. A suspension containing Bt spores was inoculated via drench in each seedling, and five seedlings were maintained as negative control (without Bt). Mortality was assessed daily for five days. During the bioassays, dead nymphs were collected for further Bt isolation and detection by PCR. At the end of the experiment, young leaves where nymphs feed were also collected to Bt isolation and detection. Our results confirmed that the previously identified Bt isolates pathogenic to D. citri cause high mortality to 3rd instar nymphs. Among the ten recombinant isolates tested, one of them stood out, causing 68-81 % and 83-93% mortality after 48 and 120 hours of inoculation, respectively. The isolation of Bt from dead nymphs and young leaves, and PCR performed with specific primers confirmed the presence of the Bt isolates in psyllids and plants, and their involvement in psyllid mortality. This recombinant Bt with the higher mortality rate found in our results has the potential to be used as a bioinsecticide to control D. citri and the toxin gene can be used to genetic engineering of citrus.

Submitted by Kian Eghrari Moraes null (kianem@gmail.com) on 2017-03-14T19:44:37Z No. of bitstreams: 1 Dissertação_Kian_Eghrari_Moraes.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-21T19:54:29Z (GMT) No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) / Made available in DSpace on 2017-03-21T19:54:29Z (GMT). No. of bitstreams: 1 moraes_ke_me_jabo.pdf: 1379647 bytes, checksum: 5a9daad8a0289711efcdab3175a3dd1e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os híbridos de milho transgênicos, em geral, apresentam o locus transgênico em hemizigose. O objetivo do trabalho foi avaliar o efeito do número de alelos transgênicos em híbridos de milho em relação à expressão de Cry1F nas folhas, ataque de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) e a produtividade de grãos, utilizando cinco híbridos isogênicos nas versões transgênicas homozigota e hemizigota, além da versão convencional de um dos híbridos. O nível de expressão da proteína Cry1F (PRYF) foi quantificado pela técnica de ELISA. Nos experimentos de campo, conduzidos na primeira e segunda safras do ano agrícola 2015/2016, avaliou-se o ataque de S. frugiperda em campo (ALC), por infestação natural, e a produtividade de grãos (PG). Dois bioensaios foram realizados em laboratório para avaliar a sobrevivência larval de S. frugiperda de 1º instar (SL) alimentadas com as folhas dos híbridos. Os híbridos transgênicos, homozigotos e hemizigotos, não apresentaram silenciamento gênico. Os híbridos homozigotos apresentaram maior concentração de proteína Cry1F. Quando houve elevado ALC, na primeira safra, os híbridos transgênicos foram superiores à testemunha convencional na PG, entretanto, não houve diferença entre os híbridos homozigotos e hemizigotos. Os híbridos transgênicos também foram superiores à testemunha convencional nos bioensaios, sendo que os homozigotos apresentaram as menores médias de SL. A presença de um alelo transgênico a mais nos híbridos homozigotos propiciou comportamento genético aditivo para a expressão de Cry1F e controle de S. frugiperda, diminuindo ALC e SL, sem diminuir a capacidade produtiva das plantas. Diante do exposto, não há limitações para a utilização de híbridos de milho homozigotos para o transgene, pois apresentaram melhor controle de S. frugiperda em comparação com os híbridos hemizigotos. / Genetically modified (GM) maize hybrids, in general, possess the transgenic locus in a hemizygous state. The aim of the study was to assess the effect of the number of transgenic alleles in maize hybrids regarding the Cry1F leaf expression, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) attack and grain yield, through the evaluation of five isogenic hybrids in the homozygous and hemizygous transgenic versions and a non-GM hybrid. Cry1F protein expression levels (PRYF) were quantified by ELISA. In field experiments conducted during the summer and autumn seasons of 2015/2016, we assessed the leaf-feeding injury of S. frugiperda in the field (LFI) by natural infestation and grain yield (GY). Two bioassays were carried out in the laboratory to evaluate the survival of first-instar S. frugiperda larvae (SL) fed with the maize hybrids. Transgenic hybrids did not present gene silencing. Homozygous hybrids presented higher Cry1F expression levels than hemizygous hybrids. With high LFI during the summer season, transgenic hybrids were superior to the non-GM for GY, however, there was no difference between homozygous and hemizygous hybrids. The transgenic hybrids were superior to the non-GM hybrid in the bioassays, and the homozygotes caused the highest mortality of S. frugiperda. The addition of one transgenic allele in the homozygous hybrids provided an additive genetic effect, increasing PRYF and S. frugiperda control, whereas GY was not affected. In conclusion, there are no limitations to the use of transgenic homozygous maize hybrids, which presented better S. frugiperda control comparing to hemizygous hybrids.

Die vorliegende Arbeit untersucht kooperative und hierarchische Erscheinungsformen des institutionellen Wandels am Beispiel des Anbaus von gentechnisch verändertem Bt-Mais MON810 in Deutschland. In den Jahren 2005 bis 2008 war der kommerzielle Anbau dieser Pflanze in Deutschland möglich, und überwiegend Landwirte in den ostdeutschen Bundesländern machten von dieser Möglichkeit der Bekämpfung des Maisschädlings Ostrinia nubilalis Hübn. (Maiszünsler) Gebrauch. Unter Zuhilfenahme des Analyserahmens „Institutionen der Nachhaltigkeit“ werden in vier empirischen Kapiteln die Aspekte 1) Risikowahrnehmung und institutionelle Präferenz, 2) Möglichkeiten der Bereitstellung des Gutes „Gentechnikfreie Atmosphäre, 3) regionale Faktoren für Adaptionsunterschiede und 4) Adaption von Bt-Mais als Verhandlungsspiel zwischen Akteuren des ländlichen Raums beleuchtet. Hierbei wird deutlich, dass eine verstärkte Risikowahrnehmung beim Anbau von Bt-Mais MON810 eine Präferenz für eine Verschärfung des gegenwärtigen gesetzlichen Rahmens zum Umgang mit gentechnisch veränderten Pflanzen bedingt. Ebenso bevorzugen Akteure mit einer entsprechenden Risikowahrnehmung eine hierarchische Steuerung zur Sicherstellung der Gentechnikfreiheit, beispielsweise in Form von staatlich ausgewiesenen Gentechnikfreien Regionen oder Anbauverboten. Bei der Adaption von Bt-Mais kommt der Maisfläche je Betrieb eine große Bedeutung zu. Durch den rechtlichen Rahmen in Deutschland, die jeweilige Agrarstruktur und das soziale Umfeld wird der Anbau von Bt-Mais erschwert. Die unterschiedlichen Akteure wenden daher Maßnahmen an, um ihre eigene Verhandlungsposition zu verbessern oder die des Mitspielers zu verschlechtern. Hierzu zählen beispielsweise die Reduktion von Mindestabständen durch nachbarschaftliche Absprachen oder auch die Ausübung von öffentlichem Druck auf Bt-Mais anbauende Landwirte sowie Feldzerstörungen. / This work aims at analysing co-operative and hierarchical forms of institutional change in the case of Bt-maize cultivation in Germany. Genetically modified Bt-maize expresses a toxin which is lethal for the larvae of a main pest in maize production, the European Corn Borer. From 2005 to 2008, the cultivation of this transgenic crop was legally possible in Germany and adoption took mainly place in the former eastern German federal states. In four empirical chapters, the aspects of 1) risk perception and institutional preferences, 2) hierarchical or co-operative provision of the good “GMO-free atmosphere”, 3) regional adoption patterns of Bt-maize and 4) the adoption as a bargaining game will be analysed. It becomes apparent that actors with a strong risk perception prefer stricter institutions when dealing with the cultivation of GM crops and also favour hierarchical governance forms instead of co-operative solutions. The adoption of Bt-maize at the federal state level in Germany is significantly influenced by the factor maize area per farm. When conceptualizing Bt-maize adoption as a game under a strict regulatory framework and different agricultural and social structures, several measures can be identified which are employed by the actors to improve one’s own bargaining position, such as private agreements on the reduction of isolation distances or impair the one of the opposite player through public pressure or field destructions.

Genetically modified (GM) maize was developed mainly to control lepidopteran pests such as the maize stem borer (Busseola fusca) (Lepidoptera: Noctuidae). Since the first commercialization of GM crops with insecticidal properties, possible non-target effects such as the effect at the third trophic level on important predators for example lacewing species (Chrysoperla spp.) have been of concern. Contradicting results were reported in previous studies with regard to the effect of Cry 1Ab protein produced by Bt maize on the performance of lacewings. Some studies found that Bt proteins had no effect while others reported that C. carnea performed poorly if they consumed prey that consumed Cry 1Ab protein. In South Africa one of the most common chrysopid species in maize ecosystems is Chrysoperla pudica (Navás) (Neuroptera: Chrysopidae). Evolution of Bt resistant pests, such as B. fusca in South Africa facilitates a new pathway for exposure of predators to healthy prey that consumes Cry 1Ab proteins. The aims of this study was to determine the effect of the Cry 1Ab protein expressed in Bt maize on a non-target organism‟s (C. pudica) biology via indirect exposure, and to determine the concentration of Cry 1Ab protein in the plant, prey and predator. Chrysoperla pudica larvae were indirectly exposed to the Bt-toxin through healthy Bt-maize feeding prey (B. fusca larvae) in two feeding experiments and lacewing survival and life history parameters recorded. Bt had a limited effect on some parameters that were evaluated. The larval and pupal periods of C. pudica larvae that were exposed to the Bt-toxin had a significant difference from that of the control treatment. The Bt-toxin had a significant effect on fecundity, fertility and malformation after emergence of C. pudica adults of which larvae fed only on Bt resistant B. fusca larvae, but not on the mortality rate. Cry 1Ab concentration was the highest in the plant, followed by the prey and lacewing larvae. This study showed that the Cry 1Ab protein had a slight adverse effect only on certain life parameters of C. pudica, and that Cry 1Ab protein was hardly detectable in C. pudica larvae. However, since this study represented a worst-case scenario where diverse prey was not available, insignificant effects is expected under field conditions where prey is diverse. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

The fall armyworm, Spodoptera frugiperda (J. E. Smith 1797) (Lepidoptera: Noctuidae), is considered the main insect-pest of corn in Brazil. Its control, in recent years, is mainly accomplished through the use of Bt plants and spray insecticides. Aiming to evaluate the efficiency of Bt proteins expressed by maize commercially available, as well as their interaction with the use of insecticides for the control of S. frugiperda to subsidize IPM and IRM programs in Bt maize, experiments were carried under field condition during two crop seasons in the 2015-16. Eight technologies of Bt maize and one non-Bt hybrid were evaluated, as well as seven insecticide application programs. Damage assessments were performed every five days, counting the number of damaged plants and assigning a score according to the Davis Scale (1992). For the application of the insecticides, a control level was established in which the spraying was carried out whenever 10% of the plants showed a damage score > 3. In the 1st season 2015/16, Agrisure TL (Cry1Ab), Herculex (Cry1F) and Optimum Intrasect (Cry1Ab/ Cry1F) and non-Bt maize required 1 to 3 sprays of insecticides to complement the control of S. frugiperda. In this season, all tested insecticides were efficient in control. In the case of YieldGard VT PRO (Cry1A.105 / Cry2Ab2), YieldGard VT PRO 3 (Cry1A.105 / Cry2Ab2 / Cry3Bb1), PowerCore (Cry1A.105 / Cry2Ab2 / Cry1F), Agrisure Viptera (Vip3Aa20) and Agrisure Viptera 3 (Vip3Aa20 / Cry1Ab) the use of insecticides was not necessary. In the second crop year 2015/16, with the exception of Agrisure Viptera and Agrisure Viptera 3, all other hybrids of Bt and non-Bt maize required 1 to 4 sprays of insecticides. The results obtained in the present work demonstrate that some Bt maize technologies (expressing genes that encode Cry1 proteins) present low control efficiency, thus making insecticides indispensable to complement the control of S. frugiperda. In contrast, the Vip3Aa20 protein shows high toxicity to S. frugiperda, showing insignificant damage to the culture. Among the insecticides tested in the work, spinetoram (12 g of a.i./ha), in general presented the highest control efficiency for fall armyworm. / A lagarta-do-cartucho, Spodoptera frugiperda (J. E. Smith 1797) (Lepidoptera: Noctuidae), é considerada o principal inseto-praga da cultura do milho no Brasil. Seu controle, nos últimos anos, é realizado principalmente por meio do uso de plantas Bt e pulverização de inseticidas. Visando avaliar a eficiência das proteínas Bt expressas pelos milhos disponíveis no mercado, bem como sua interação com o uso de inseticidas para o controle de S. frugiperda para subsidiar programas de MIP e MRI em milho Bt, foram realizados experimentos, em condição de campo, durante duas épocas de cultivo na safra 2015-16. Foram avaliadas oito tecnologias de milho Bt e um híbrido não-Bt, bem com sete programas de aplicação de inseticidas. A cada cinco dias, foram realizadas avaliações de dano, contabilizando o número de plantas danificadas e atribuindo uma nota de acordo com a Escala de Davis (1992). Para a aplicação dos inseticidas, foi estabelecido um nível de controle em que era realizada a pulverização sempre que 10% das plantas apresentassem nota de dano >3. Na 1a safra 2015/16, Agrisure TL (Cry1Ab), Herculex (Cry1F) e Optimum Intrasect (Cry1Ab/Cry1F) e milho não-Bt necessitaram de 1 a 3 pulverizações de inseticidas para complementar o controle de S. frugiperda. Nesta safra, todos os inseticidas testados foram eficientes no controle. Já em milho YieldGard VT PRO (Cry1A.105/Cry2Ab2), YieldGard VT PRO 3 (Cry1A.105/Cry2Ab2/Cry3Bb1), PowerCore (Cry1A.105/Cry2Ab2/ Cry1F), Agrisure Viptera (Vip3Aa20) e Agrisure Viptera 3 (Vip3Aa20/Cry1Ab) não foi necessário o uso de inseticidas. Na 2a safra 2015/16, com exceção de Agrisure Viptera e Agrisure Viptera 3, todos as demais híbridos de milho Bt e não-Bt necessitaram de 1 a 4 pulverizações de inseticidas. Os resultados obtidos no presente trabalho demonstram que algumas tecnologias de milho Bt (expressando genes que codificam proteínas Cry1) apresentam baixa eficiência de controle, tornando-se assim indispensável o uso de inseticidas para complementar o controle de S. frugiperda. Em contraste, a proteína Vip3Aa20 apresenta alta toxicidade para S. frugiperda, apresentando danos insignificantes à cultura. Dentre os inseticidas testados no trabalho, spinetoram (12 g de i.a./ha), de modo geral apresentou a maior eficiência de controle para lagarta-do-cartucho.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Atualmente sÃo liberadas para comercializaÃÃo no mercado agrobiotecnolÃgico brasileiro, pelo menos, 12 variedades de algodÃo expressando proteÃnas Cry de Bacillus thruringiensis (Bt) para conferir resistÃncia ao ataque de insetos. Contudo, essas proteÃnas sÃo ativas contra lepidÃpteros, enquanto que a principal praga da cotonicultura brasileira à um coleÃptero, o bicudo-do-algodoeiro (Anthonomus grandis). Neste contexto, tÃm-se feito um grande esforÃo na busca por novas molÃculas Cry ativas contra coleÃpteros. Utilizando tÃcnicas de evoluÃÃo molecular dirigida in vitro, foi desenvolvida a proteÃna mutante Cry8Ka5, que à vÃrias vezes mais tÃxica contra o bicudo do que a proteÃna original, Cry8Ka1, inicialmente descoberta. Dada a sua atividade promissora contra coleÃpteros, Cry8Ka5 està sendo utilizada para o desenvolvimento de um novo algodÃo Bt, bem como de outras culturas Bt de importÃncia econÃmica. No entanto, como parte das exigÃncias legais para a liberaÃÃo comercial de uma planta transgÃnica, essa proteÃna recombinante deve ser testada quanto a sua biosseguranÃa alimentar (BA) a fim de detectar potenciais efeitos alergÃnicos, tÃxicos e/ou antimetabÃlicos. Assim, este trabalho objetivou realizar uma ampla avaliaÃÃo de BA da proteÃna mutante Cry8Ka5 e da proteÃna Cry1Ac, jà incorporada em vÃrias culturas Bt comercializadas. A Ãltima foi utilizada como proteÃna controle ou teste dependendo do ineditismo da abordagem empregada em cada seÃÃo do trabalho. Nesse Ãmbito, a avaliaÃÃo de BA proposta cobriu desde as exigÃncias legais atà a execuÃÃo de metodologias, atà entÃo, nunca propostas para avaliaÃÃo de seguranÃa de consumo de produtos de Bt. Para tanto, este trabalho estruturou-se em quatro capÃtulos. O CapÃtulo 1 objetivou realizar uma revisÃo da literatura acerca do estado da arte das plantas Bt e proteÃnas Cry, bem como fornecer um entendimento geral sobre os conceitos e a legislaÃÃo vigente em BA de plantas GM. Jà o CapÃtulo 2 objetivou avaliar a BA da entomotoxina mutante Cry8Ka5 atravÃs do mÃtodo de duas etapas baseado em pesos de evidÃncia que, por sua vez, atende a todas as recomendaÃÃes da CTNBio. Neste caso, a proteÃna Cry1Ac foi utilizada como controle experimental, pois abordagens similares jà foram realizadas com essa molÃcula. Baseando-se nos mÃtodos oficiais, à possÃvel concluir que nÃo à esperado qualquer risco associado ao consumo da proteÃna Cry8Ka5 devido aos seguintes resultados encontrados: 1. Produtos Bt mostraram ter longo histÃrico de uso seguro; 2. A proteÃna Cry8Ka5 nÃo mostrou similaridade significativa de sua sequÃncia de aminoÃcidos primÃria com proteÃnas alergÃnicas, tÃxicas e/ou antinutricionais; 3. As proteÃnas Cry mostraram possuir modo de aÃÃo bastante compreendido e elevada especificidade contra insetos; 4. Cry8Ka5 foi altamente susceptÃvel à digestÃo em fluÃdo gÃstrico simulado; 5. A proteÃna Cry8Ka5 nÃo causou efeitos adversos relevantes em camundongos (5.000 mg de proteÃna/Kg de peso corpÃreo, via oral, dose Ãnica). Por sua vez, o CapÃtulo 3 objetivou avaliar a proteÃna Cry8Ka5 e a Cry1Ac quanto aos seus efeitos cito- e genotÃxicos em uma sÃrie de testes clÃssicos e alternativos, alÃm de pesquisar seus efeitos antimicrobianos. As proteÃnas nÃo causaram efeitos cito- ou genotÃxicos em linfÃcitos humanos (ambas com CI50 > 1.000 Âg/mL em todos os testes), nÃo promoveram hemÃlise em suspensÃes de eritrÃcitos de humanos (tipos A, B, AB e O), de coelho e rato (ambas com CI50 > 1.000 Âg/mL para todas as espÃcies), enquanto somente a Cry8Ka5 causou alteraÃÃes na topografia de membrana celular de eritrÃcitos humanos do tipo O, na concentraÃÃo de 1.000 Âg/mL, detectadas via microscopia de forÃa atÃmica. AlÃm disso, as proteÃnas Cry8Ka5 e Cry1Ac nÃo apresentaram citotoxicidade elevada contra naÃplios de Artemia sp. (CL50 > 700 e > 1000 Âg/mL, respectivamente) e nÃo inibiram o crescimento de quatro cepas de bactÃrias e quatro de leveduras (ambas com CIM > 1.000 Âg/mL para todos os microrganismos testados). Por fim, o CapÃtulo 4 objetivou avaliar os efeitos das proteÃnas Cry8Ka5 e Cry1Ac e de um âpoolâ de peptÃdeos de cada uma (Cry8Ka5-pep e Cry1Ac-pep, respectivamente), obtidos por digestÃo sequencial in vitro, sobre o perfil de expressÃo gÃnica de cÃlulas de carcinoma de mama MCF-7 e de carcinoma de cÃlon Caco-2, ambas de origem humana. Nenhuma das amostras testadas causou alteraÃÃes no perfil de expressÃo gÃnica das cÃlulas Caco-2 diferenciadas (utilizadas como um modelo de epitÃlio intestinal humano), mesmo quando testadas em alta concentraÃÃo (100 Âg/mL). Nos testes de exposiÃÃo com cÃlulas MCF-7 indiferenciadas, apenas a proteÃna Cry1Ac causou alteraÃÃes significativas no padrÃo de expressÃo gÃnica. Contudo, esse efeito nÃo foi detectado apÃs tratamento dessas cÃlulas com a amostra digerida de Cry1Ac (Cry1Ac-pep), que, por sua vez, mimetiza melhor as condiÃÃes reais de exposiÃÃo Ãs molÃculas Cry. As abordagens experimentais utilizadas nos trÃs Ãltimos capÃtulos mostraram-se bastante Ãteis e, apÃs tudo que foi exposto, foi possÃvel confirmar o carÃter inÃcuo da proteÃna Cry1Ac, bem como concluir que a proteÃna Cry8Ka5 à uma ferramenta biotecnolÃgica segura para o desenvolvimento de plantas de algodÃo Bt para conferir resistÃncia ao ataque do bicudo-do-algodoeiro. / Currently at least 12 varieties of cotton expressing Cry proteins from Bacillus thruringiensis (Bt) are released for sale in Brazil to confer resistance to insect attack. However, these proteins are active against lepidopteran, whereas the major pest of Brazilian cotton is a beetle, the cotton boll weevil (Anthonomus grandis). In this context, great efforts have been dedicated in the search for new Cry molecules active against coleopteran. Using techniques of in vitro directed molecular evolution, the Cry8Ka5 mutant protein was developed, which is several times more toxic against the boll weevil than the original protein, Cry8Ka1, initially discovered. Given its promising activity against coleopteran, Cry8Ka5 is being used for the development of a new Bt cotton, as well as of other Bt crops of economic importance. However, as part of legal requirements for the commercial release of a transgenic plant, the recombinant products should be tested for their food safety (FS) in order to detect potential allergenic, toxic and antimetabolic effects. This study aimed to perform a comprehensive FS assessment of the Cry8Ka5 mutant protein and of the Cry1Ac protein, already incorporated in several commercialized Bt crops. The latter was used as a protein control or test depending on the originality of the approach used in each section of this work. The FS evaluation performed covered not only the legal requirements but also the implementation of methodologies hitherto never proposed to assess the safety of consumption of Bt. For that, this study was structured in four chapters. Chapter 1 aimed to conduct a literature review on the state of the art of Bt plants and Cry proteins, as well as provide a general understanding of the concepts and current legislation in GM plants FS. Chapter 2 aimed to evaluate the FS of Cry8Ka5 mutant entomotoxin through the two-step method based on weights of evidence that, in turn, meets the recommendations of CTNBio. In this section, the Cry1Ac protein was used as experimental control, since similar approaches have already been performed with this molecule. Based on official methods, we conclude that it is not expected any risk associated with consumption of protein Cry8Ka5 due to the following results found: 1. Bt products were shown to have a long history of safe use, 2. The protein showed no significant similarity in its primary amino acid sequence with known allergenic, toxic and/or antinutritional proteins; 3. The mode of action and high specificity against insects of Cry proteins are well known; 4. Cry8Ka5 was highly susceptible to digestion in simulated gastric fluid; 5. The protein Cry8Ka5 caused no significant adverse effects in mice (5,000 mg protein/kg body weight, orally, single dose). Chapter 3 aimed to evaluate the Cry8Ka5 and Cry1Ac proteins as to their cyto- and genotoxicity in a serie of conventional and alternative tests as well as antimicrobial effects. The proteins did not cause cyto- or genotoxic effects in human lymphocytes (both with IC50 > 1,000 Âg/mL in all tests), did not cause hemolysis in human (types A, B, AB and O), rabbit and rat (both with IC50 > 1,000 Âg/mL for all species) rythrocytes, but only Cry8Ka5 caused changes in the topography of the cell membrane of human O type erythrocytes at a concentration of 1,000 Âg/mL, detected by atomic force microscopy. Furthermore, the Cry8Ka5 and Cry1Ac proteins showed no cytotoxicity against Artemia sp. nauplii (LC50 > 700 and >1000 Âg/mL, respectively), and did not inhibit the growth of four strains of bacteria and four of yeasts (both with MIC > 1,000 Âg/mL for all microrganisms tested). Finally, Chapter 4 aimed to evaluate the effects of Cry8Ka5 and Cry1Ac proteins and of a "pool" of peptides of each one (Cry8Ka5-pep and Cry1Ac-pep, respectively), obtained by in vitro sequential digestion, on the gene expression profile of MCF-7 breast carcinoma cells and Caco-2 colon carcinoma cells, both human. No sample caused changes in the gene expression profile of differentiated Caco-2 cells (used as a model for human intestinal epithelium), even when tested at high concentration (100 Âg/mL). In the exposure tests with undifferentiated MCF-7 cells, only Cry1Ac (at 100 Âg/mL) caused significant changes in the gene expression pattern. However, this effect was not detected after treatment of these cells with the sample digested of Cry1Ac Cry1Ac-pep), which, in turn, better mimic actual conditions of exposure to Cry molecules. The experimental approaches used in the last three chapters were quite useful and, after all the above, it was possible to confirm the harmless nature of the Cry1Ac protein and conclude that the Cry8Ka5 protein is a safe biotechnological tool for the development of Bt cotton plants to confer resistance against the cotton boll weevil attack.

A tolerância pode ser considerada um dos pilares da imunologia. Sabe-se que a tolerância a um antígeno pode gerar tolerância a outro antígeno não relacionado, fenômeno conhecido como tolerância cruzada. Neste trabalho caracterizamos a tolerância cruzada utilizando a OVA como tolerógeno e extrato de Blomia tropicalis (Bt) ou Hemocianina de Keyhole limpet (KLH) como alérgenos. Verificamos que é possível reproduzir o fenômeno da tolerância cruzada neste modelo de inflamação alérgica induzida tanto pelo KLH quanto pela Bt, com diminuição do infiltrado inflamatório no pulmão, eosinófilos, IgE total e produção de muco. Ainda, a estratégia de utilizar a tolerância cruzada terapeuticamente, i.é., após a sensibilização com KLH, a indução de tolerância cruzada não foi capaz de prevenir a resposta alérgica. Em conjunto, nossos dados mostram que a tolerância à OVA modifica as respostas alérgicas tanto à Bt quanto ao KLH no modelo de inflamação pulmonar experimental de forma profilática, mas que a tolerância cruzada não é eficiente em animais já sensibilizados. / Tolerance is among the Immunology pillars. Experimental data indicate that tolerance towards an antigen can promote tolerance to an unrelated antigen, a phenomenon known as cross-tolerance. Here we sought to characterize cross tolerance using OVA as a tolerogen and Blomia tropicalis (Bt) extract or Keyhole limpet Hemocianina (KLH) as allergens. We found that cross tolerance can be reproduced in the model of allergic lung disease induced by KLH or Bt, with less inflammatory infiltrate in the lung, eosinophils, total IgE and mucus production. Using cross tolerance therapeutically, i.e., after KLH sensitization, was not effective, since the allergic lung response was not modulated. Altogether, our data shows that OVA tolerance modulate allergic lung disease induced either by Bt or KLH when used as prophylactic model, however cross tolerance is ineffective in sensitized animals.