Entomopathogenic fungi are highly susceptible to the damaging effects of solar radiation. Attempts to protect entomopathogenic fungi from solar radiation have been, for the most part, unsuccessful. A new strategy for formulating entomopathogenic fungi for protection from solar radiation and desiccation has been developed tested using the acridid entomopathogen Metarhizium anisopliae var acridum (IMI 330189). This strategy involves coating spores with water-soluble materials that provide protection from solar radiation and enhance spore survival during drying. Development of this formulation involved the following: 1) production of an infective spore-type in liquid culture that could survive drying; 2) coating spores during an air-drying process; 3) reducing formulation particle size for oil suspension; 4) testing the effects spore coating on spore-tolerance simulated sunlight; and 6) testing the effects of spore coating infectivity to Schistocerca americana. Aerial conidia, submerged conidia, and blastospores produced in a high-osmolality liquid medium all had high desiccation tolerance relative to blastospores produced in Adamek's media. Blastospores produced in high osmolality medium were the most infective to S. americana in an aqueous 20% molasses solution followed by submerged conidia and aerial conidia, with LT50 values (95% C.I.) at 1 x 106 spores/insect of 7.8 d (6.7 to 9.0 d), 10.5 d (9.5 to 11.6 d), 14.6 d (11.9 to 18.0 d), respectively. Comparisons were made among cell-wall characteristics of these spore-types, including cell-wall thickness, lectin-binding, charge, and hydrophobicity. An optimal spore-coating formulation was selected on the basis of spore survival and germination over time after air-drying, particle-size reduction, and storage at 28 Ë C. This spore-coating formulation, consisting of skim milk, Kraft lignin (Curan 10®) and glycerol, greatly improved the tolerance of aerial conidia and submerged conidia to simulated sunlight, increasing the LT50 (95% C.I.) of aerial conidia from 4.0 hr (3.1-5.1) to 17.0 hr (12.5-23.0). The spore coating formulation decreased the infectivity of spores in oil to adult S. americana; reducing the LT50 values of aerial conidia at a dose of 1 x 105 spores / insect from 5.8 d (4.9-6.9 d) to 8.2 d (7.3-9.3 d). / Ph. D.

Notre étude avait pour objectifs d'évaluer les effets de la technique d'étuvage traditionnelle pratiquée en Guinée et ceux des huiles essentielles extraites des feuilles d'Ocimum basilicum L. (Ob), d'Ocimum gratissimum L. (Og) et de Cymbopogon citratus Stapf (Cc) sur deux ravageurs importants (Sitophilus oryzae L. et Tribolium castaneum Herbst) fréquemment rencontrés ensembles dans les stocks de riz des régions chaudes et humides d'Afrique. Nos hypothèses de recherche s'appuyaient sur le fait que ces techniques pourraient empêcher le développement de ces ravageurs dans le système de stockage. Pour vérification des hypothèses, nous avons mesuré, dans cinq milieux alimentaires, la survie, le nombre de trous de ponte et le nombre d'adultes émergés survivants en fonction du temps dans le cas de l'étuvage. Les milieux alimentaires ont été préparés en collaboration avec la population paysanne. Dans le cas des huiles essentielles, les évaluations ont été effectuées sous la forme de poudre d'argile aromatisée appliquée dans les milieux alimentaires, sous la forme d'huile pure par fumigation pendant 6 jours et l'application des huiles à différentes concentrations dans les milieux alimentaires. L'extraction des huiles essentielles a été effectuée par entraînement à la vapeur d'eau à partir des feuilles des trois plantes et le meilleur rendement moyen a été obtenu avec les feuilles d'Og. La poudre d'argile a été choisie parmi trois types, dont deux à l'état naturel (argiles rouge et blanche) et une purifiée. Ces trois argiles ont été soumises à des mesures de la conductivité, du pH et de la diffraction des rayons X pour l'identification des éléments minéraux. L'argile blanche naturelle a été retenue à cause de la stabilité de sa conductivité et de son pH d'une part et d'autre part, à cause de la grande quantité de smectite, support d'adsorption pour les molécules d'intérêt agronomique qu'elle contient et de son abondance dans le pays. Dans le cas de la technique traditionnelle d'étuvage deux expériences ont été réalisées pour contrôler la survie et la dynamique des populations des insectes ravageurs dans les différents milieux alimentaires. Pour la première expérience, les milieux ont été infestés seulement par S. oryzae. Les deux ravageurs (S. oryzae et T. castaneum) ont été associés pour la deuxième expérience. Les résultats ont mis en évidence l'effet de l'étuvage sur la survie, sur la ponte et sur l'émergence des adultes de S. oryzae sur une période d'au moins trois mois. Le nombre d'adultes survivants et le nombre de trous de ponte étaient significativement réduits dans les riz étuvés que dans les autres milieux alimentaires pour les deux expériences. Le dénombrement mensuel des adultes survivants a montré également une réduction significative des adultes dans les riz étuvés. Avec T. castaneum, l'effet de l'étuvage a été marqué par un ralentissement du cycle de développement des larves qui sont restées tout le temps de l'expérience au premier stade. Pour ce qui concerne l'évaluation des poudres d'argile aromatisées avec les huiles essentielles, deux expériences ont été aussi réalisées. Pour la première expérience, les milieux alimentaires ont été d'abord infestés par les insectes puis conservés pendant 15 jours avant l'ajout des poudres aromatisées. Pour la seconde expérience, les milieux alimentaires ont été mélangés aux poudres puis, infestés immédiatement par les insectes. Les résultats ont montré des effets insecticides variant en fonction des milieux alimentaires, des expériences, de la nature des huiles essentielles et les espèces d'insectes, Pour l'expérience 1, les poudres d'Og et d'Ob ont réduit significativement le nombre d'adultes de S. oryzae dans les milieux. Les nombres moyens d'adultes (82,93) et (97,66) obtenus après trois mois étaient les plus petits nombres significatifs par rapport à celui du témoin riz avec argile (123,2) et celui de Cc (118,16) non différents puis, de celui du riz seul (154,73) différent des deux derniers. Quant à l'expérience 2, toutes les trois huiles avaient réduit considérablement le nombre d'adultes émergés. Les nombres moyens étaient 7,26 pour Cc, 5,03 pour Ob et 2,13 pour Og dans le milieu RNEB. Dans le cas des milieux RNEP, REB, les huiles ont inhibé totalement la survie et la prolifération de S. oryzae. Ceci met en évidence dans les deux expériences, l'effet combiné de l'argile et des huiles essentielles. Des fluctuations observées avec les paddy pourraient être attribuées soit à l'effet des huiles essentielles soit à la capacité des insectes de se cacher dans les grains ce qui rendait leur dénombrement difficile. Un ralentissement du cycle de développement a été constaté chez T. castaneum sous l'effet des poudres avec la présence d'un grand nombre de larves de premier stade également. S. oryzae a été plus sensible aux poudres aromatisées que T. castaneum et la méthode utilisée pour l'expérience 2 a été la plus efficace. Avec la fumigation, deux expériences ont été aussi réalisées. La première a porté sur les volumes de 10 à 100 µl et la deuxième, de 2 à 100 µl. Les huiles ont montré des activités insecticides en fonction de leur nature, des volumes utilisées et du temps d'exposition. Les huiles d'Ob et de Og, avaient peu d'effet durant les 3 premiers jours d'exposition pour les volumes de 2 à 80 µl. C'est à partir du jour 4 que les effets étaient manifestes. Les CL50 et CL90 obtenues pour l'huile d'Ob, du jour 4 au jour 6 variaient respectivement de 71,42 à 5,93 µ I/insecte pour S. oryzae. Avec Og, elles variaient de 26,58 à 7,59 µl/insecte pour S. oryzae et de 35,11 à 8,73 µ/insecte pour T. castaneum, S. oryzae a été plus sensible aux effets d'Ob et d'Og alors que l'huile de Cc n'a pas manifesté d'effet insecticide. L'huile d'Og pourrait ainsi être retenue comme la plus toxique à cause de ses effets sur les deux ravageurs et des faibles valeurs de CL50et de CL90 obtenues. Enfin, les huiles essentielles ont été appliquées directement dans les milieux alimentaires en deux expériences. La première expérience concernait l'évaluation des effets des volumes de 1 à 5 µl et la deuxième ceux des volumes de 10 à 50 µl. Les résultats de l'expérience 1 ont montré une diminution graduelle du nombre d'adultes survivants de S. oryzae en fonction des volumes, des milieux ainsi qu'une réduction de leur survie dans les riz étuvés. Ceci indique les effets de l'étuvage et des huiles essentielles. L'huile de Cc a été moins active par rapport aux autres huiles. Les résultats similaires ont été observés chez T. castaneum. Dans le cas de l'expérience 2, aucun adulte survivant n'a été dénombré sauf dans les milieux témoins. Ceci met en évidence l'efficacité des trois huiles dans les grains dans le contexte de ces deux expériences. En général, les hypothèses ont été toutes confirmées. Nos travaux ont permis d'obtenir de nouvelles informations dans des domaines ayant peu de publications scientifiques et permettraient de valider les techniques traditionnelles étudiées comme outil de lutte écologiquement et économiquement viables pour la protection des denrées en post-récolte particulièrement le riz. Elles pourraient éventuellement servir de bases pour la sensibilisation des populations paysannes. La technique traditionnelle d'étuvage et l'utilisation des huiles essentielles pourraient conduire à l'amélioration de la sécurité alimentaire dans les pays en développement. Le défi est de répandre cette approche traditionnelle et durable parmi les paysans. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Riz étuvé, Sitophilus oryzae, Tribolium castaneum, Biopesticide, Huile essentielle, Ocimum basilicum, Ocimum gratissimum, Cymbopogon citratus, Argile, Fumigation.

L'objectif de ce projet était de déterminer les effets létaux et sub-létaux d'un savon insecticide sur le puceron vert du pêcher (Myzus persicae (Sulzer)) et une guêpe parasitoïde du puceron, Aphidius colemani (Viereck). Dans cette étude, les hypothèses suivantes ont été vérifiées: 1) la mortalité des insectes augmente en fonction de la concentration du savon, 2) les doses sub-létales du savon insecticide ont des effets sur la valeur adaptative du puceron (mesurée par la longévité, le taux de développement et la fécondité) et sur la valeur adaptative (estimée par la longueur de tibias et le nombre d'oeufs) et le comportement du parasitoïde (contact antennaire, attaque et ponte). La concentration de savon causant 100% de mortalité 24h après le traitement chez tous les stades du puceron était de 37.5 g/L. La CL₅₀ était de 1.50, 3.25 et de 5.50 g/L pour les larves de stades 1 et 2, 3 et 4, et les adultes respectivement. La longévité des pucerons qui ont survécu à la CL₅₀ était significativement différente de celle des témoins. Par contre, les larves exposées au savon n'ont pas eu un taux de développement significativement différent des témoins. La fécondité des adultes n'était pas significativement différente des témoins. Ce savon insecticide pourrait être une bonne façon de contrôler les pucerons. La concentration de savon causant 100% de mortalité 24h après le traitement chez les adultes de la guêpe était de 17.5 g/L. La CLs₅₀ était de 2.75 g/L. La survie, la longueur des tibias, le nombre d'oeufs matures par femelle émergeant de pucerons parasités et traités au savon, ainsi que le comportement des guêpes mises en contact avec des pucerons traités n'ont pas été affectés. Par contre, les guêpes ont significativement moins pondu dans les pucerons traités ayant survécu au savon. Il semble évident que les programmes de lutte biologique peuvent être améliorés par des applications de savon insecticide si ces dernières sont faites une journée avant le relâcher de parasitoïdes en serre. Ceci éviterait le contact des parasitoïdes avec le savon et permettrait aux pucerons traités de muer. Les parasitoïdes pourront ainsi pondre dans les pucerons qui auront survécu au savon ce qui augmentera le contrôle du puceron.

Orientador: Daniela Carvalho dos Santos / Resumo: Devido ao uso indiscriminado de inseticidas de largo espectro na produção agrícola e a vulnerabilidade dos inimigos naturais a este tipo de exposição, efeitos colaterais podem afetar a função biológica destes inimigos, tornando sua utilização no controle biológico impraticáveis. A busca por pesticidas mais seletivos sobre esses organismos benéficos é necessária, e entre as alternativas, enfatizamos o aumento do uso dos biopesticidas. Um dos biopesticidas amplamente utilizados é o óleo de nim (Azadirachta indica), e sua segurança e compatibilidade com os inimigos naturais têm sido mais esclarecidos através de estudos ecotoxicológicos que verificam os efeitos da exposição indireta através da ingestão de presas contaminadas. Assim, este estudo analisou a resposta das células epiteliais do mesêntero de adultos do crisopídeo Ceraeochrysa claveri quando expostos por ingestão de presa contaminada com óleo de nim durante a fase larval e a ocorrência de efeitos letais e subletais em seu desenvolvimento. Larvas de C. claveri foram alimentadas com ovos de Diatraea saccharalis tratados com óleo de nim nas concentrações de 0,5%, 1% e 2% durante todo período larval. Fêmeas adultas obtidas a partir destes tratamentos foram utilizadas em análises morfológicas e ultraestruturais da resposta celular do mesêntero. Além disso, parâmetros biológicos também foram avaliados durante o desenvolvimento pós-embrionário. O óleo de nim provoca efeitos citotóxicos no mesêntero através de pronunciadas alte... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the indiscriminate use of broad-spectrum insecticides in agricultural production and the vulnerability of natural enemies to this type of pesticide exposure, side effects can affect the biological function of these enemies, making their use for biological control unfeasible. A search for more selective pesticides on these beneficial organisms is necessary, and among the alternatives, we emphasize the increased use of biopesticides. One of the biopesticides widely used is the neem oil (Azadirachta indica), and its safety and compatibility with natural enemies have been more clarified through ecotoxicological studies which check the effects of indirect exposure by intake of poisoned preys. Thus, this study analyzed the response of midgut epithelial cells of adult of lacewing Ceraeochrysa claveri when exposed by intake of contaminated prey with neem oil during the larval stage and the occurrence of lethal and sublethal effects on its development. Larvae of C. claveri were fed on eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% and 2% throughout the larval stage. Adults female obtained from these treatments were used in the morphological and ultrastructural analyses of cellular response of midgut. Moreover, biological parameters were also measured during the post embryonic development. Neem oil causes cytotoxic effects on the midgut through pronounced alterations as cell swollen, loss of integrity of cell cortex, dilated cisternae of the ro... (Complete abstract click electronic access below) / Doutor

L'utilisation de pesticides conventionnels, en particulier les pesticides chimiques de synthèse, a considérablement réduit les pertes de récoltes et a connu un succès commercial. Cependant, l'utilisation excessive de pesticides chimiques qui manquent de toxicité spécifique a provoqué une série de problèmes environnementaux et de santé publique. L'intérêt de la recherche vers de nouveaux biopesticides naturels avec de nouveaux modes d'actions vise à un meilleur équilibre entre l'efficacité des pesticides et la réduction des méfaits possibles pour l'environnement et les humains. Les plantes sont une source importante de biopesticides. Les acides chlorogéniques (CQA), isolés à partir de diverses plantes et présentent in vivo et in vitro un large spectre d'activités biologiques, ont attiré l'attention avec un potentiel comme biopesticides basé sur la toxicité brevetée de l'acide 3,5-di-O-caféoylquinique contre les larves de Myzus persicae. L'étude des propriété insecticides notamment la mode d'action et l'étude de certaines relations structure-activité pourrait bénéficier de la synthèse de différents dérivés et analogues de CQA. Des analogues des acides 4-désoxy-3,5-dicinnamoy quiniques et 3,4- et 4,5-dicinnamoylquiniques naturels ont été synthétisés. Sept analogues dans la série 4-désoxy ont été soumis à des essais insecticides et deux composés présentent une activité insecticide plus élevée que l'acide 3,5-dicaféoylquinique naturel. Comme perspectives à ce travail, confirmer l'activité des composés synthétisés sur d'autres espèces de pucerons d'importance agronomique pourrait être réalisé. De plus, d’autres collaborations avec des biologistes pourraient être établies afin d’évaluer d'autres activités des composés synthétisés ou les utiliser comme outils pour étudier des mécanismes de biosynthése. / The use of conventional pesticides, especially the synthetic chemical pesticides, has greatly reduced the crop losses and gained a commercial success. However, the excessive use of pesticides lacking toxic specificity has caused a series of environmental and public health problems. The research interest toward new naturally-occurring biopesticides with novel modes of actions aims at a better balance between the efficiency of pesticide and reducing possible harms to environment and humans. Botanicals are an important source of biopesticides. Cinnamoyl quinic acids (CQA), isolated from various plants and shown to exhibit in vivo and in vitro a wide spectrum of biological activities, have attracted the attention with potential as biopesticides based on the patented toxicity of 3,5-di-O-caffeoylquinic acid against the larvae of Myzus persicae. The investigation of the insecticidal activity of CQA including their mode of action and the study of some structure-activity relationships could benefit from the synthesis of different CQA derivatives and analogs. A series of natural CQA derivatives natural 3,4- and 4,5-dicinnmamoylquinic acid derivatives but also analogs like 4-deoxy-3,5-dicinnamoylquinic acids were so synthesized. Seven targeted 4-deoxy CQA analogs were subjected to insecticidal assays, and two compounds were found to exhibit higher insecticidal activities than natural 3,5-dicaffeoylquinic acid. As perspectives to this work, confirming the activity of the synthesized compounds on other aphid species of agronomic importance could be performed. Furthermore, other collaborations could be established with biologists dedicated to measure other bioactivities of the synthesized compounds or use them as tools to investigate various biological pathways.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-10-02T21:48:26Z No. of bitstreams: 1 DISERTA??O_FINAL_DAIANE (REVISADA) (1).pdf: 2567387 bytes, checksum: 15b7a9c355bab95c6ac54d2f36eed753 (MD5) / Made available in DSpace on 2017-10-02T21:48:26Z (GMT). No. of bitstreams: 1 DISERTA??O_FINAL_DAIANE (REVISADA) (1).pdf: 2567387 bytes, checksum: 15b7a9c355bab95c6ac54d2f36eed753 (MD5) Previous issue date: 2017-03-31 / The exploitation potential of new biopesticides sources is very broad in Brazil and worldwide. In the past few years, the search for these products has been intensified due to serious health and environmental issues caused by the use of synthetic insecticides. In this context, essential oils and extracts of vegetal species have been considered as an alternative to agriculture. Thus, the aim of this work was to test the insecticidal potential of species belonging to the genus Lippia Lippia insignis Mondenk 1976, Lippia lasiocalycina Schauer, 1832 e Lippia thymoides Martius & Schauer, 1832 no controle de insetos-praga que provocam danos econ?micos (Callosobruchus maculatus Fabricius, 1775 e Nasutitermes corniger Motschulsky, 1855) in the control of insect-pest responsible for economic damage (Callosobruchus. maculatus and Nasutitermes corniger). The samples were collected in the experimental area in the Horto Florestal (UEFS) and after the material was dried, the essential oils were extracted and the extracts were prepared. In vitro trials were performed in order to verify the effects of essential oils and extracts against C. maculatus and N. corniger. The methodology of exposition to contaminated surface, testing an initial concentration of aqueous and methanolic extract and essential oils was adopted to determine the insecticidal activity of extracts and oils. The mortality was verified 24 and 48 hours after treatment application, and the product with major potential on the insect mortality was diluted in different concentrations to determine the medium lethal concentration (LC50). The repellent activity against C. maculatus and N. corniger was evaluated. Moreover, specific tests with C. maculatus investigated the product efficiency in two methods of application, as well as the quantification and identification of chemical constituents present in the studied species oils using GC/FID and GC/MS techniques. The results obtained revealed, occurrence highlighted of ?-mirceno and E -ocimenona in the sample of L. lasiocalycina, in L. insignis were prominent ?-mirceno e o limoneno. For L. thymoides the main constituents were: E-cariofileno e o ?xido de cariofileno.for C. maculatus, the essential oil of L. insignis stood out as insecticidal activity with CL50 of 3,11 ?l/ml. Although both methods of application showed efficiency in the control of this insect-pest, there was no repellent activity verified in the oil of L. insignis (5?l/ml). For N. corniger, the essential oils of all tested species, as well as the methanolic extract of L. insignis exhibited promising results in the control of this insect-pest. It was also verified that L. lasiocalycina possessed lower LC50 (0,47 ?l/ml) compared to L. insignis (0,88 ?l/ml) and L. thymoides (3,64 ?l/ml), while the LC50 of L. insignis methanolic extract was 27,84 mg/ml. Furthermore, essential oils and methanolic extracts of Lippia studied species revealed repellent activity in the N. corniger in vitro trials. / O potencial de explora??o de novas fontes de biopesticidas ? bastante amplo no Brasil e no mundo. Nos ?ltimos anos, a busca por esses produtos tem se intensificado em virtude dos graves problemas de sa?de e ambientais advindos do uso de inseticidas sint?ticos. Neste contexto, os ?leos essenciais e extratos de esp?cies vegetais tem se mostrado uma boa alternativa para a agricultura. Deste modo, o objetivo deste trabalho foi testar o potencial inseticida de esp?cies pertencentes ao g?nero Lippia (Lippia insignis Mondenk 1976, Lippia lasiocalycina Schauer, 1832 e Lippia thymoides Martius & Schauer, 1832 no controle de insetos-praga que provocam danos econ?micos (Callosobruchus maculatus Fabricius,1775 e Nasutitermes corniger Motschulsky, 1855). A coleta das plantas foi realizada na ?rea experimental do Horto Florestal da UEFS e o material coletado, ap?s a secagem, foi destinado a extra??o de ?leos essenciais e preparo dos extratos. Foram realizados ensaios in vitro para verifica??o dos efeitos dos ?leos essenciais e extratos contra C. maculatus e N. corniger. Para a determina??o inicial da atividade inseticida dos extratos e ?leos foi utilizada a metodologia de exposi??o ? superf?cie contaminada, testando-se uma concentra??o inicial do extrato aquoso, extrato metan?lico e ?leos essenciais. A mortalidade foi verificada 24 e 48 hs ap?s a aplica??o dos tratamentos. O produto com maior potencial sobre a mortalidade dos insetos testados foi dilu?do em diferentes concentra??es para determina??o da concentra??o letal mediana (CL50). A atividade repelente contra C. maculatus e N. corniger foi avaliada. Al?m disso, testes espec?ficos com C. maculatus investigaram a efici?ncia do produto em dois m?todos de aplica??o, bem como a quantifica??o e identifica??o de constituintes qu?micos presentes nos ?leos de esp?cies estudados utilizando t?cnicas GC/ FID e GC / MS. Os resultados demonstraram, a ocorr?ncia destacada do ?-mirceno e E-ocimenona nas amostras de L. lasiocalycina, em L. insignis destacaram-se, ?-mirceno e limoneno. Para L. thymoides os constituintes principais foram: E-cariofileno e o ?xido de cariofileno. Para C. maculatus, que o ?leo essencial da esp?cie L. insignis destacou-se quanto ? atividade inseticida com CL50 de 3,11 ?l/ml. Embora ambos os m?todos de aplica??o tenham se mostrado eficientes no controle deste inseto-praga, n?o foi verificada atividade repelente do ?leo de L. insignis (5?l/ml). Para N. corniger, os ?leos essenciais de todas as esp?cies testadas, al?m do extrato metan?lico da esp?cie L. insignis mostraram-se promissores no controle a este inseto-praga. Foi verificado que a esp?cie L. lasiocalycina apresentou menor CL50 (0,47 ?l/ml) comparada a L. insignis (0,88 ?l/ml) e L. thymoides (3,64 ?l/ml), j? o extrato metan?lico de L. insignis obteve CL50 de (27,84 mg/ml). Os ?leos essenciais e extrato metan?lico das esp?cies de Lippia estudadas demonstram atividade repelente a N. corniger nos ensaios in vitro.

Visando explorar o potencial bioativo de plantas da família Solanaceae como fonte alternativa para o manejo de Spodoptera frugiperda (Lepidoptera: Noctuidae), a lagarta-do-cartucho-do-milho, realizou-se, primeiramente, uma triagem de extratos etanólicos preparados de diferentes partes de 25 espécies vegetais. Após sete dias de exposição das lagartas em dietas tratadas, constatou-se que os extratos etanólicos de folhas de Acnistus arborscens e de Datura stramonium, na concentração de 4.000 mg kg -1, foram os mais promissores causando inibição do ganho de peso de 97,85 e 95,72%, respectivamente. A seguir, foi realizado um fracionamento biomonitorado, a partir do qual, a fração diclorometânica foi selecionada como a mais ativa para essas duas espécies, causando efeito subletal (inibição do ganho do peso) e letal (mortalidade), porém este inferior ao causado pela formulação comercial Azamax® 1,2 EC (controle positivo). Em sequencia, foi possível estimar as concentrações letais para as frações diclorometânicas durante o período de 168 horas. Em relação ao efeito sobre o desenvolvimento do inseto, os extratos etanólicos foram testados na concentração de 4.000 mg kg -1 e as frações diclorometânicas na concentração letal mediana (4.088 e 3.694 mg kg -1 para D. stramonium e A. arborescens, respectivamente). Na fase larval, os extratos etanólicos de D. stramonium e A. arborescens ocasionaram 77,50 e 62,50% de mortalidade, respectivamente, e, além disso, ambos os tratamentos causaram alongamento dessa fase, e mortalidade, deformidade e redução do peso pupal. A fração diclorometânica de A. arborescens proporcionou 100% de mortalidade das lagartas expostas, enquanto a mortalidade em D. stramonium foi 83,19% e, as lagartas necessitaram de 41,75 dias para atingir a fase pupa, que apresentou baixo peso (158,60 mg) e altas deformidades (40%) em comparação ao controle (255,90 mg e 8,26%, respectivamente). Além disso, as frações diclorometânicas ocasionaram atividade fagodeterrente nas CL25, CL50 e CL90. Novamente, utilizando-se a fração diclorometânica, foi realizado novo fracionamento, e após sete dias de exposição das lagartas neonatas, todas as subfrações ocasionaram efeitos subletais. Com isso, tornaram-se necessárias novas investigações químicas a fim de se compreender o (s) composto (os) responsável (is) pela bioatividade, porém a complexidade de algumas subfrações não permitiu uma caracterização química. Ainda assim, foi constatada nas subfrações acetato de etila e metanólica de D. stramonium a presença de compostos pertencentes à classe dos vitanolidos. Dessa forma, os derivados de Solanaceae apresentam potencial bioativo passíveis de serem estudados mais profundamente visando ao emprego dos mesmos no manejo de S. frugiperda. / Aiming to explore the bioactive potential of Solanaceae plants as alternative resources for the management of Spodoptera frugiperda (Lepitoptera: Noctuidae), the fall armyworm, it was performed, firstly, a screening of ethanolic extracts prepared from different parts of 25 plant species. After seven days of exposure of caterpillars to treated diets, it was verified that ethanolic extracts of Acnistus arborscens and Datura stramonium leaves in the concentration of 4,000 mg kg-1 were the most promising, causing an inhibition of 97.85% and 95.72%, respectively, in weight gain. After this, a bioguided fractionation was performed, from which the dichloromethane fraction was selected as the most active in both species, causing sublethal (inhibition of weight gain) and lethal (mortality) effects, although both were less effective than the Azamax® 1,2 EC commercial formulation (positive control). Following the previous step, it was possible to estimate the lethal concentrations for dichloromethane fraction during the period of 168 hours. In relation to the effect on the insect development, the ethanolic extracts were tested in the concentration of 4,000 mg kg-1 and the dichloromethane fractions in their median lethal concentrations (4,088 and 3,694 mg kg-1 for D. stramonium and A. arborescens, respectively). In the larval phase, ethanolic extracts of D. stramonium and A. arborescens lead to 77.50% and 62.50% of mortality, respectively, and both treatments lead to the elongation of this phase, mortality, deformity and pupal weight reduction. The dichloromethane fraction of A. arborescens lead to 100% of mortality of the exposed caterpillars, while mortality of D. stramonium was 83.19%, and the caterpillars needed 41.75 days to reach the pupal stage, which presented low weight (158.60 mg) and were highly deformed (40%) in comparison with the negative control (255.90 mg and 8.26%, respectively). Furthermore, the dichloromethane lead to antifeedant activity in CL25, CL50 and CL90. Once again, using the dichloromethane fraction, another fractionation was performed, and after seven days of exposure of neonates caterpillars, all subfractions lead to sublethal effects. Therefore, new chemical evaluations are necessary to understand the compounds responsible for bioactivity, however, the complexity of some subfractions did not permit chemical characterization. Nevertheless, it was verified in the ethyl acetate and methanolic subfractions of D. stramonium the presence of withanolides compounds. Therefore, derivatives of Solanaceae present bioactive potential that may be liable for further evaluation aiming their employment in the management of S. frugiperda.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

Visando explorar o potencial bioativo de plantas da família Solanaceae como fonte alternativa para o manejo de Spodoptera frugiperda (Lepidoptera: Noctuidae), a lagarta-do-cartucho-do-milho, realizou-se, primeiramente, uma triagem de extratos etanólicos preparados de diferentes partes de 25 espécies vegetais. Após sete dias de exposição das lagartas em dietas tratadas, constatou-se que os extratos etanólicos de folhas de Acnistus arborscens e de Datura stramonium, na concentração de 4.000 mg kg -1, foram os mais promissores causando inibição do ganho de peso de 97,85 e 95,72%, respectivamente. A seguir, foi realizado um fracionamento biomonitorado, a partir do qual, a fração diclorometânica foi selecionada como a mais ativa para essas duas espécies, causando efeito subletal (inibição do ganho do peso) e letal (mortalidade), porém este inferior ao causado pela formulação comercial Azamax® 1,2 EC (controle positivo). Em sequencia, foi possível estimar as concentrações letais para as frações diclorometânicas durante o período de 168 horas. Em relação ao efeito sobre o desenvolvimento do inseto, os extratos etanólicos foram testados na concentração de 4.000 mg kg -1 e as frações diclorometânicas na concentração letal mediana (4.088 e 3.694 mg kg -1 para D. stramonium e A. arborescens, respectivamente). Na fase larval, os extratos etanólicos de D. stramonium e A. arborescens ocasionaram 77,50 e 62,50% de mortalidade, respectivamente, e, além disso, ambos os tratamentos causaram alongamento dessa fase, e mortalidade, deformidade e redução do peso pupal. A fração diclorometânica de A. arborescens proporcionou 100% de mortalidade das lagartas expostas, enquanto a mortalidade em D. stramonium foi 83,19% e, as lagartas necessitaram de 41,75 dias para atingir a fase pupa, que apresentou baixo peso (158,60 mg) e altas deformidades (40%) em comparação ao controle (255,90 mg e 8,26%, respectivamente). Além disso, as frações diclorometânicas ocasionaram atividade fagodeterrente nas CL25, CL50 e CL90. Novamente, utilizando-se a fração diclorometânica, foi realizado novo fracionamento, e após sete dias de exposição das lagartas neonatas, todas as subfrações ocasionaram efeitos subletais. Com isso, tornaram-se necessárias novas investigações químicas a fim de se compreender o (s) composto (os) responsável (is) pela bioatividade, porém a complexidade de algumas subfrações não permitiu uma caracterização química. Ainda assim, foi constatada nas subfrações acetato de etila e metanólica de D. stramonium a presença de compostos pertencentes à classe dos vitanolidos. Dessa forma, os derivados de Solanaceae apresentam potencial bioativo passíveis de serem estudados mais profundamente visando ao emprego dos mesmos no manejo de S. frugiperda. / Aiming to explore the bioactive potential of Solanaceae plants as alternative resources for the management of Spodoptera frugiperda (Lepitoptera: Noctuidae), the fall armyworm, it was performed, firstly, a screening of ethanolic extracts prepared from different parts of 25 plant species. After seven days of exposure of caterpillars to treated diets, it was verified that ethanolic extracts of Acnistus arborscens and Datura stramonium leaves in the concentration of 4,000 mg kg-1 were the most promising, causing an inhibition of 97.85% and 95.72%, respectively, in weight gain. After this, a bioguided fractionation was performed, from which the dichloromethane fraction was selected as the most active in both species, causing sublethal (inhibition of weight gain) and lethal (mortality) effects, although both were less effective than the Azamax® 1,2 EC commercial formulation (positive control). Following the previous step, it was possible to estimate the lethal concentrations for dichloromethane fraction during the period of 168 hours. In relation to the effect on the insect development, the ethanolic extracts were tested in the concentration of 4,000 mg kg-1 and the dichloromethane fractions in their median lethal concentrations (4,088 and 3,694 mg kg-1 for D. stramonium and A. arborescens, respectively). In the larval phase, ethanolic extracts of D. stramonium and A. arborescens lead to 77.50% and 62.50% of mortality, respectively, and both treatments lead to the elongation of this phase, mortality, deformity and pupal weight reduction. The dichloromethane fraction of A. arborescens lead to 100% of mortality of the exposed caterpillars, while mortality of D. stramonium was 83.19%, and the caterpillars needed 41.75 days to reach the pupal stage, which presented low weight (158.60 mg) and were highly deformed (40%) in comparison with the negative control (255.90 mg and 8.26%, respectively). Furthermore, the dichloromethane lead to antifeedant activity in CL25, CL50 and CL90. Once again, using the dichloromethane fraction, another fractionation was performed, and after seven days of exposure of neonates caterpillars, all subfractions lead to sublethal effects. Therefore, new chemical evaluations are necessary to understand the compounds responsible for bioactivity, however, the complexity of some subfractions did not permit chemical characterization. Nevertheless, it was verified in the ethyl acetate and methanolic subfractions of D. stramonium the presence of withanolides compounds. Therefore, derivatives of Solanaceae present bioactive potential that may be liable for further evaluation aiming their employment in the management of S. frugiperda.