MEnvSC / Department of Ecology and Environmental Science

Em razão dos problemas causados pelos insetos nos armazéns, no Brasil tem-se usado principalmente substâncias químicas para controlar essas pragas, porém, com relativo sucesso na solução do problema. Em fase disso, alguns aspectos sobre a proteção de grãos armazenados foram estudados, entre eles: 1) o efeito do volume de pulverização no teor de umidade dos grãos, 2) a estabilidade de inseticidas durante o processamento de amostras, 3) a influência da formulação concentrado emulsionável nas propriedades físicas do líquido, características do spray e depósito de inseticidas, 4) os resíduos de inseticidas nos grãos e em alguns de seus produtos processados e 5) a eficácia de inseticidas no controle de Sitophilus oryzae (L.), Sitophilus zeamais Motsch. e Rhyzopertha dominica (Fabr.). Para o tratamento dos grãos foi utilizado o bico hidráulico de jato duplo, modelo, TJ-60 8002EVS. O sistema de aplicação foi calibrado de modo a se obter tratamento dos grãos na concentração teórica de 10 e/ou 0,5 mg.kg-1 de fenitrotion e esfenvalerato, respectivamente. Os experimentos de eficácia foram instalados aos 15 dias, 30 dias e, a partir desta em intervalos mensais até 12 meses após o tratamento dos grãos, ou até que não houvesse mais mortalidade nas parcelas. Nos experimentos de resíduo, amostras de grãos foram coletadas ao zero, 15, 30, 60, 90, 120, 180, 240 e 360 dias após o tratamento e as amostras dos produtos processados foram obtidos na mesma data. A umidade dos grãos após a pulverização dos volumes de água estudados teve pequeno acréscimo quando comparado com o teor inicial. Os grãos processados junto com gelo seco proporcionaram a maior porcentagem de recuperação dos inseticidas. Houve grande influência da formulação concentrado emulsinável nas propriedades físicas do líquido, características do spray e depósito dos inseticidas nos grãos. Os resíduos de fenitrotion e esfenvalerato em grãos de trigo concentraram-se preferencialmente no farelo e em menores quantidades na farinha. A taxa de degradação do esfenvalerato foi independente do tipo de grão. O resíduo de fenitrotion no período de carência de 120 dias foi superior ao Limite Máximo de Resíduo (LMR) permitido pela legislação brasileira, e esta se mostra inadequada na regulamentação de ambos os inseticidas. O esfenvalerato foi ineficiente no controle das espécies estudadas; já o fenitrotion foi eficiente no controle de S. oryzae e S. zeamais. A linhagem SR de R. dominica foi mais resistente do que a linhagem CG aos inseticidas testados. As CL50s estimadas concordaram parcialmente com os resultados obtidos nos testes de eficácia. De forma geral, a mortalidade de S. oryzae decresceu mais rapidamente do que a mortalidade de S. zeamais. Houve maior mortalidade das duas espécies de Sitophilus em grãos de trigo do que em grãos de milho. Foi constatado efeito sinergista da mistura fenitrotion + esfenvalerato no controle das duas linhagens de R. dominica. Na ausência de inseticidas S. oryzae apresentou progênie mais numerosa no trigo em relação àquela no milho; já S. zeamais apresentou número similar de progênie nos dois tipos de grãos. Na testemunha, a linhagem CG de R. dominica apresentou progênies mais elevadas do que a linhagem SR, sendo que ocorreu o inverso quando da aplicação dos inseticidas. / Because of the problems caused by insects in storehouses, chemicals have been chiefly used in Brazil to control these pests. However, success in solving problems with pests has been limited. In view of this, some aspects about stored grain protection were studied, among which are the following: 1) spray volume effect on grain moisture content, 2) stability of insecticides during sample processing, 3) influence of the emulsifiable concentrate formulation on the physical properties of the liquid, spray characteristics, and deposition of insecticides, 4) insecticide residues in grain and some of their processed products, and 5) effectiveness of insecticides in the control of Sitophilus oryzae (L.), Sitophilus zeamaiszeamais Motsch., and Rhyzopertha dominica (Fabr.). Grain treatment was accomplished by using a twin jet, model TJ-60 8002EVS hydraulic nozzle. The application system was calibrated to provide grain treatment at theoretical concentrations of 10 and/or 0.5 mg kg-1 fenitrothion and esfenvalerate, respectively. The effectiveness experiments were installed at 15 days, 30 days and at monthly intervals thereafter for 12 months after treating the grains, or until mortality ceased in the plots. In the residue experiments, grain samples were collected at zero, 15, 30, 60, 90, 120, 180, 240, and 360 days after treatment, and processed product samples were obtained on the same dates. After spraying the water volumes planned in the study, only a small increase in grain moisture was observed when compared with the initial content. Grains processed together with dry ice provided the greatest insecticide recovery percentages. The emulsifiable concentrate formulation had a great influence on the physical properties of the liquid, spray characteristics, and deposition of insecticides on the grain. The fenitrothion and esfenvalerate residues in wheat grain were concentrated preferentially in the meal and at smaller amounts in the flour. The rate of esfenvalerate degradation did not depend on grain type. During a withholding period of 120 days, the fenitrothion residue was higher than the Maximum Residue Limit (MRL) allowed by Brazilian law, which has been demonstrated to be unsuitable for the regulation of both insecticides. Esfenvalerate was ineffective in controlling the species studied; fenitrothion, however, was effective for the control of S. oryzae and S. zeamais. The SR line of R. dominica was more resistant to the insecticides tested than the CG line. The estimated LC50 values agreed in part with the results obtained in the effectiveness tests. In general, S. oryzae mortality decreased more rapidly than S. zeamais mortality. There was greater mortality of both Sitophilus species on wheat grain than on corn grain. A synergistic effect was verified for the fenitrothion + esfenvalerate mixture in controlling both R. dominica lines. In the absence of insecticides, S. oryzae showed a more numerous offspring on wheat in relation to corn; S. zeamais. however, showed a similar number of offspring on both types of grain. In the control, the R. dominica CG line showed higher numbers of offspring than the SR line, and the contrary was verified when insecticides were applied.

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

Em razão dos problemas causados pelos insetos nos armazéns, no Brasil tem-se usado principalmente substâncias químicas para controlar essas pragas, porém, com relativo sucesso na solução do problema. Em fase disso, alguns aspectos sobre a proteção de grãos armazenados foram estudados, entre eles: 1) o efeito do volume de pulverização no teor de umidade dos grãos, 2) a estabilidade de inseticidas durante o processamento de amostras, 3) a influência da formulação concentrado emulsionável nas propriedades físicas do líquido, características do spray e depósito de inseticidas, 4) os resíduos de inseticidas nos grãos e em alguns de seus produtos processados e 5) a eficácia de inseticidas no controle de Sitophilus oryzae (L.), Sitophilus zeamais Motsch. e Rhyzopertha dominica (Fabr.). Para o tratamento dos grãos foi utilizado o bico hidráulico de jato duplo, modelo, TJ-60 8002EVS. O sistema de aplicação foi calibrado de modo a se obter tratamento dos grãos na concentração teórica de 10 e/ou 0,5 mg.kg-1 de fenitrotion e esfenvalerato, respectivamente. Os experimentos de eficácia foram instalados aos 15 dias, 30 dias e, a partir desta em intervalos mensais até 12 meses após o tratamento dos grãos, ou até que não houvesse mais mortalidade nas parcelas. Nos experimentos de resíduo, amostras de grãos foram coletadas ao zero, 15, 30, 60, 90, 120, 180, 240 e 360 dias após o tratamento e as amostras dos produtos processados foram obtidos na mesma data. A umidade dos grãos após a pulverização dos volumes de água estudados teve pequeno acréscimo quando comparado com o teor inicial. Os grãos processados junto com gelo seco proporcionaram a maior porcentagem de recuperação dos inseticidas. Houve grande influência da formulação concentrado emulsinável nas propriedades físicas do líquido, características do spray e depósito dos inseticidas nos grãos. Os resíduos de fenitrotion e esfenvalerato em grãos de trigo concentraram-se preferencialmente no farelo e em menores quantidades na farinha. A taxa de degradação do esfenvalerato foi independente do tipo de grão. O resíduo de fenitrotion no período de carência de 120 dias foi superior ao Limite Máximo de Resíduo (LMR) permitido pela legislação brasileira, e esta se mostra inadequada na regulamentação de ambos os inseticidas. O esfenvalerato foi ineficiente no controle das espécies estudadas; já o fenitrotion foi eficiente no controle de S. oryzae e S. zeamais. A linhagem SR de R. dominica foi mais resistente do que a linhagem CG aos inseticidas testados. As CL50s estimadas concordaram parcialmente com os resultados obtidos nos testes de eficácia. De forma geral, a mortalidade de S. oryzae decresceu mais rapidamente do que a mortalidade de S. zeamais. Houve maior mortalidade das duas espécies de Sitophilus em grãos de trigo do que em grãos de milho. Foi constatado efeito sinergista da mistura fenitrotion + esfenvalerato no controle das duas linhagens de R. dominica. Na ausência de inseticidas S. oryzae apresentou progênie mais numerosa no trigo em relação àquela no milho; já S. zeamais apresentou número similar de progênie nos dois tipos de grãos. Na testemunha, a linhagem CG de R. dominica apresentou progênies mais elevadas do que a linhagem SR, sendo que ocorreu o inverso quando da aplicação dos inseticidas. / Because of the problems caused by insects in storehouses, chemicals have been chiefly used in Brazil to control these pests. However, success in solving problems with pests has been limited. In view of this, some aspects about stored grain protection were studied, among which are the following: 1) spray volume effect on grain moisture content, 2) stability of insecticides during sample processing, 3) influence of the emulsifiable concentrate formulation on the physical properties of the liquid, spray characteristics, and deposition of insecticides, 4) insecticide residues in grain and some of their processed products, and 5) effectiveness of insecticides in the control of Sitophilus oryzae (L.), Sitophilus zeamaiszeamais Motsch., and Rhyzopertha dominica (Fabr.). Grain treatment was accomplished by using a twin jet, model TJ-60 8002EVS hydraulic nozzle. The application system was calibrated to provide grain treatment at theoretical concentrations of 10 and/or 0.5 mg kg-1 fenitrothion and esfenvalerate, respectively. The effectiveness experiments were installed at 15 days, 30 days and at monthly intervals thereafter for 12 months after treating the grains, or until mortality ceased in the plots. In the residue experiments, grain samples were collected at zero, 15, 30, 60, 90, 120, 180, 240, and 360 days after treatment, and processed product samples were obtained on the same dates. After spraying the water volumes planned in the study, only a small increase in grain moisture was observed when compared with the initial content. Grains processed together with dry ice provided the greatest insecticide recovery percentages. The emulsifiable concentrate formulation had a great influence on the physical properties of the liquid, spray characteristics, and deposition of insecticides on the grain. The fenitrothion and esfenvalerate residues in wheat grain were concentrated preferentially in the meal and at smaller amounts in the flour. The rate of esfenvalerate degradation did not depend on grain type. During a withholding period of 120 days, the fenitrothion residue was higher than the Maximum Residue Limit (MRL) allowed by Brazilian law, which has been demonstrated to be unsuitable for the regulation of both insecticides. Esfenvalerate was ineffective in controlling the species studied; fenitrothion, however, was effective for the control of S. oryzae and S. zeamais. The SR line of R. dominica was more resistant to the insecticides tested than the CG line. The estimated LC50 values agreed in part with the results obtained in the effectiveness tests. In general, S. oryzae mortality decreased more rapidly than S. zeamais mortality. There was greater mortality of both Sitophilus species on wheat grain than on corn grain. A synergistic effect was verified for the fenitrothion + esfenvalerate mixture in controlling both R. dominica lines. In the absence of insecticides, S. oryzae showed a more numerous offspring on wheat in relation to corn; S. zeamais. however, showed a similar number of offspring on both types of grain. In the control, the R. dominica CG line showed higher numbers of offspring than the SR line, and the contrary was verified when insecticides were applied.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-02T11:16:10Z No. of bitstreams: 1 Marcileyne Pessoa Leite de Lima.pdf: 517075 bytes, checksum: b7ddab703563527f0c2b8e77f710122c (MD5) / Made available in DSpace on 2016-12-02T11:16:11Z (GMT). No. of bitstreams: 1 Marcileyne Pessoa Leite de Lima.pdf: 517075 bytes, checksum: b7ddab703563527f0c2b8e77f710122c (MD5) Previous issue date: 2008-02-04 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The synthetic insecticide is the main method used to control Spodoptera frugiperda (J.E. Smith). However, the inadequate application of the insecticide has contributed to increase costs, reduction of the natural enemies, and selection of resistant populations, among other problems. Attempting to obtain alternative control of S. frugiperda more ecologically acceptable, the present work investigated the effects of neem (Azadirachta indica A. Juss) and Bacillus thuringiensis subsp. aizawai (Bta) on S. frugiperda under laboratory, greenhouse and field conditions. In the field, the commercial formulations of Natuneem® (10 mL/L in 2005 and 15 mL/L in 2006) and Neemseto® (5 and 10 mL/L in 2005, and 10 and 15 mL/L in 2006) and the biological insecticide Xentari® (10 g/L in 2005 and 2006) were applied at 15, 30 and 45 and at 15, 25 e 35 days after plant emergency in 2005 and 2006, respectively. Thee evaluations consisted of using a scale of foliar injury from 0 to 5 prior each spray. In 2005, only Neemseto® 10 mL/L was efficient at 30 days. In 2006, Neemseto® 15 mL/L and Xentari® with average score of 0.93 and 0.98, respectively, were different from the control, which produced a score of 2.38 at 35 days after plant emergency. The greenhouse assay consisted of treatment of plants 40-days old with the insecticides Natuneem®, Neemseto® and Xentari® followed by artificial infestation with newly-hatched larvae of S. frugiperda. After 10 days, it was verified that the treatment were equally efficient, differing from the control, regarding to the injury score, as well as, the number of surviving larvae. In the last bioassays, in the laboratory, 10-days old S. frugiperda larvae were fed with corn leaves treated with the insecticides Natuneem®, Neemseto® and Xentari® at 2.5; 5.0; 7.5; 10 mL or g/mL and control (water). The effect of the insecticides increased as function of increasing concentration, causing higher larval mortality, longer period and lower larval weight. In some cases, reduction of pupa weight and viability and longevity of adults were also verified. Larvae 0-24 h old were more susceptible to the insecticides, as compared with 10-days old larvae. / A utilização de inseticidas sintéticos tem sido o principal método de controle de Spodoptera frugiperda (J.E. Smith), mas o uso inadequado tem contribuindo para o aumento do custo de produção, redução de inimigos naturais, seleção de populações resistentes, entre outros problemas. Desta forma, buscando alternativas de controle mais ecológicas, estudou-se no presente trabalho os efeitos de nim (Azadirachta indica A. Juss) e Bacillus thuringiensis subsp. aizawai (Bta) sobre S. frugiperda em experimentos de laboratório, casa de vegetação e campo. Foram testadas em campo formulações comerciais de inseticidas botânicos Natuneem® (10 mL/L em 2005 e 15 mL/L em 2006) e Neemseto® (5 e 10 mL/L em 2005, e 10 e 15 mL/L em 2006) e do inseticida biológico Bta (Xentari® 10 g/L em 2005 e 2006), aplicados aos 15, 30 e 45 e aos 15, 25 e 35 dias após a emergência das plantas em 2005 e 2006, respectivamente. Antes de cada pulverização foram atribuídas notas de 0 a 5 para as injúrias foliares. Em 2005, somente Neemseto® 10 mL/L foi eficiente aos 30 dias. Em 2006, Neemseto® 15 mL/L e Bta 10 g/L, com notas médias de 0,93 e 0,98, diferiram da testemunha, com nota 2,38, na avaliação realizada aos 35 dias. Plantas de milho, com cerca de 40 dias de idade, foram pulverizadas em casa de vegetação com os inseticidas Natuneem®, Neemseto e Bta, e posteriormente infestadas,artificialmente, com lagartas de S. frugiperda recém-eclodidas. Após 10 dias verificou-se que os tratamentos foram igualmente eficientes, diferindo da testemunha, tanto em relação às notas, quando ao número de lagartas sobreviventes. Nos últimos bioensaios lagartas de S. frugiperda com 10 dias de idade foram alimentadas com folhas de milho submersas na calda inseticida nas concentrações 2,5; 5,0; 7,5; 10 mL ou g/L e testemunha (água). Os efeitos dos inseticidas dependeram da concentração utilizada, pois provocaram mortalidade crescente das lagartas, alongaram o período e reduziram o peso larval. Em alguns casos, também, reduziram o peso e a viabilidade pupal e a longevidade de adultos. Lagartas com 0-24 h de idade foram mais susceptíveis aos inseticidas, em relação às com 10 dias de idade.

Dans un contexte où l’utilisation de substance chimique en agriculture est de plus en plus décriée, il est nécessaire de trouver de nouveaux moyens de protections des cultures, tout en maintenant une agriculture économiquement performante. Ainsi, un peptide extrait de graines de pois nommée PA1b (Pea Albumin 1 sous-unité b), présentant une forte activité insecticide a été découvert au laboratoire. PA1b provoque chez l’insecte modèle du laboratoire, le charançon des céréales Sitophilus sp., 100% de mortalité. L’action de PA1b passe par la liaison à un récepteur présent chez les charançons sensibles, et cette liaison est absente chez les charançons résistants ; ce récepteur est une pompe à protons nommée V-ATPase pour Vacuolar ATPase. Elle est composée de 14 sous-unités organisées en deux complexes protéiques nommés V1 (intracellulaire) et V0 (membranaire). PA1b agissant à l’extérieur des cellules seul le complexe V0 composé des sous - unités a, c, d et e pouvait être le récepteur de notre toxine. Mon premier objectif de thèse a été de comprendre le mode d’action de PA1b, en identifiant d’abord la ou les sous-unités réceptrices de PA1b, puis en recherchant par quel mécanisme la liaison de PA1b induit la mort de l’insecte. Nous avons cloné chez le charançon tous les gènes du complexe Vo, puis j’ai complémenté des levures déficientes pour ces gènes. Ce travail, mais également celui réalisé en collaboration avec d’autres équipes, nous a permis de proposer un modèle de perception de PA1b qui implique les sous-unités c et e de la V-ATPase, et permet également de proposer des hypothèses pour les différentes résistances au peptide. Par des méthodes d’immunohistologie et de biochimie, j’ai ensuite montré de manière concordante que la liaison de PA1b à la V-ATPase déclenche un phénomène d’apoptose qui conduit à la mort cellulaire, puis à la mort de l’insecte. Le second objectif de ma thèse était la mise en place d’un système de production hétérologue de PA1b. Grâce à l’expression hétérologue par infiltration de feuille de tabac (Nicotiana benthamiana) nous avons mis en place une technique de production efficace de la protéine PA1b. Après avoir déterminé les parties du gène codant PA1b nécessaires à la production de la protéine fonctionnelle, le système de production a ensuite été simplifié par la construction d’une cassette d’expression. Ainsi six isoformes de PA1b présents chez le pois, dont l’activité individuelle restait inconnue, ont été produits et testés, permettant de montrer que le caractère amphiphile de PA1b était primordial pour son activité. Par cette technique nous avons mis en place un système de production rapide et efficace permettant de tester la toxicité de nombreux isoformes de PA1b. Ce travail sera une aide précieuse pour le projet, dont l’un des objectifs majeurs est l’optimisation de PA1b, c’est-à-dire de déterminer la séquence peptidique la plus toxique. / In a context where chemical pesticides are increasingly criticized, new crops protection strategies that do not affect agriculture efficiency and productivity, must be found. A new peptide extracted from pea (Pisum sativum) seeds, named PA1b (Pea albumin 1 subunit b), and showing an important insecticide activity, was discovered in our laboratory. PA1b induces 100% mortality in our insect model, the cereal weevil, Sitophilus sp. PA1b acts by interacting with a receptor, this interaction is present in sensitive weevil, but not present in resistant weevil. The PA1b receptor is the vacuolar H+ -ATPase (V-ATPase), a multi-subunit proton pump. The V-ATPase is composed of two functional protein complexes named V1 (in the cytoplasm) and V0 (in the membrane). As PA1b is known to act only in the extracellular space, thus only the V0 complex, composed on the subunits a, c, d and e, can be the toxin receptor. The first aim of this thesis is to understand the PA1b mode of action: (i) identifying the subunit(s) acting as the receptor(s), (ii) understanding how the binding mechanism of PA1b on the receptor lead to the insect death. The weevil V0 complex genes were cloned and we used them for a functional complementation tests in yeasts strains deleted for these genes. Our data, together with those obtained through collaboration, lead to the proposal of model for the PA1b perception signaling which would involve subunits c and e of the V-ATPase. The identification of the PA1b receptor allows us to propose a hypothetical model explaining resistance mechanism to the peptide. Using immunohistology and biochemistry methods, we showed that PA1b-receptor interaction induced cells death triggered by apoptosis thus leading to insect death. The second aim of this thesis was the development of a PA1b heterologous production system. Through Agrobacterium tumefaciens mediated transient transformation by infiltration in tobacco leaves (Nicotiana benthamiana) an efficient system for PA1b production was developed. After identification of the essential parts of the complex PA1 gene necessary for efficient PA1b production, we created an expression cassette to simplify our heterologous production system. We used the system to produce six pea PA1b-isoformes with unknown individual toxic activity. The isoforms toxicity was experimentally determined, and our data showed that the amphiphilic properties of PA1b are essential for the maintenance of its toxic activity. For the first time, we implemented a quick and efficient production system, which allows to produce and to test many naturals or synthetics PA1b isoforms. This work will be useful to achieve one of the most important objectives of the research on this molecule, that is the identification.

L’utilisation du chlordécone (CLD) pour éradiquer les populations de charançon noir dans les bananeraies des Antilles françaises (Guadeloupe et Martinique) entre 1972 et 1993 a conduit à la contamination des sols et de l’environnement. Cet insecticide organochloré très hydrophobe persiste dans les sols d’où il transfère lentement vers les ressources en eau et vers les biotes terrestre et aquatique (plantes, animaux, poissons, crustacées). Réputé « indégradable », le CLD résiste à la photolyse, à l’hydrolyse et à la biodégradation. A ce jour, il n’existe pas de méthode pour remédier les 20 000 hectares de sols pollués avec cet insecticide. Compte-tenu de l’étendue de la pollution avec le CLD, les procédés biologiques de décontamination paraissent appropriés au contexte antillais. Les objectifs de mon travail de thèse étaient d’explorer les possibilités de transformation microbienne du CLD et l’impact écotoxicologique du CLD sur la communauté microbienne des sols. Mes travaux reposent sur l’hypothèse selon laquelle des populations microbiennes exposées de manière chronique au CLD se seraient adaptées à sa dégradation pour détoxifier leur environnement ou éventuellement pour l’utiliser comme source d’énergie pour leur croissance. Pour cela, j’ai développé une méthode d’analyse du CLD dans les sols et les cultures microbiennes basée sur l’isotopie stable. J’ai conduit des expériences d’enrichissement à partir de sols de Guadeloupe pollués avec le CLD. Une centaine de souches fongiques et près de 200 souches bactériennes ont été isolées. Aucunes souches bactériennes dégradantes n’ont pu être mises en évidence bien que certaines formaient un halo de dissolution du CLD sur milieu gélosé. Parmi les isolats fongiques, seul F. oxysporum sp. MIAE01197 se développait sur un milieu minéral contenant le CLD comme seul source de carbone et dissipait 40% du CLD. Cet isolat était deux fois plus tolérant au CLD qu’un isolat de référence jamais exposé au CLD. Cet isolat minéralisait très peu le 14C-CLD, formait très peu de 14C-métabolites, mais le 14C-CLD s’adsorbait sur les parois fongiques, suggérant que l’adsorption était le principal mécanisme impliqué dans la dissipation du CLD. L’analyse de trois autres isolats appartenant au genre Aspergillus a confirmé que l’exposition au CLD était un des paramètres améliorant la tolérance des souches fongiques au CLD et que la biomasse fongique était capable d’adsorber le CLD dans des proportions proches de celles obtenues avec du charbon actif utilisé pour traiter l’eau potable aux Antilles. L’évaluation de l’impact écotoxicologique du CLD sur la communauté microbienne et les fonctions qu’elle supporte a été menée sur deux sols aux propriétés physicochimiques contrastées n’ayant jamais été exposés au CLD. L’analyse de la structure globale (évaluée par RISA), de l’abondance et de l’activité de la communauté microbienne du sol argilo-limoneux n’étaient pas affectées par le CLD. En revanche, la composition taxonomique (qPCR) et l’activité respiratoire de la communauté microbienne étaient affectées par le CLD dans le sol sableux. Ces résultats montrent que la toxicité du CLD pour la communauté microbienne dépend des propriétés physicochimiques du sol qui conditionne sa biodisponibilité. Des études complémentaires devront être menées pour évaluer la toxicité possible du CLD sur des fonctions écosystémiques des sols des Antilles. / The use of chlordecone (CLD) to eradicate the weevil populations in the banana plantations in the French West Indies (Guadeloupe and Martinique) between 1972 and 1993 led to the contamination of the soil and the environment. This very hydrophobic organochlorine insecticide persists in the soil where it slowly transfers not only to the water resources but also to terrestrial and aquatic biota (plants, animals, fishes, shellfishes). Deemed “non-degradable”, CLD is resistant to photolysis, hydrolysis and biodegradation. To date, there is no method to remediate the 20,000 hectares of polluted soil with this insecticide. Given the extent of CLD pollution, biological decontamination processes appear appropriate to the Caribbean context. The objectives of my thesis were to explore the possibilities of microbial transformation of CLD and to assess the ecotoxicological impact of CLD on the soil microbial community. My work is based on the hypothesis that microbial populations chronically exposed to CLD would be adapted to its degradation to detoxify their environment or possibly for use as an energy source for growth.To do so, I developed an analysis method in soils and microbial cultures based on the use of stable isotope to trace CLD. I conducted enrichment experiments with CLD polluted soils from Guadeloupe yielding in the isolation of one hundred fungal strains and nearly two hundred bacterial strains. No degrading bacterial strains have been identified although few of them formed dissolution halo of CLD on solid media. Among the fungal isolates, only F. oxysporum sp. MIAE01197 grew on a mineral medium containing CLD as sole carbon source and dissipated 40% of the CLD. This isolate was twice more tolerant than the reference isolate which had never been exposed to CLD. This isolate mineralizes 14C-CLD very lowly, formed very few 14C-metabolites, but the 14C-CLD was adsorbed on the fungal cell walls, suggesting that the adsorption was the main mechanism involved in the dissipation of the CLD. Analysis of three other isolates belonging to the genus Aspergillus confirmed that exposure to CLD was one of the parameters improving the tolerance of fungal strains to CLD and fungal biomass was capable of adsorbing the CLD in proportions close to those obtained with activated carbon used to treat drinking water in the French West Indies.The assessment of the CLD ecotoxicological impact on the microbial community and functions it supports was carried out on two soils never exposed to CLD showing contrasting physicochemical properties. The analysis of the overall structure (evaluated by RISA), the abundance and the activity of the microbial community of the silty-clay soil were not affected by the CLD. However, the taxonomic composition (evaluated by group specific qPCR) and respiratory activity of the microbial community were affected by the CLD in the sandy soil. These results showed that the toxicity of CLD for microbial community depends on the physicochemical properties of the soil which may determine its bioavailability. Further studies are needed to evaluate the possible toxicity of the CLD on Caribbean soil ecosystemic functions.

<p>  </p> <p>Vector-borne diseases (VBDs) represent a significant health burden worldwide, threatening approximately 80% of the global population. Insecticide-based vector control is the most effective method to manage many VBDs, but its efficacy has been declining due to high levels of resistance in vector populations to the main insecticide classes which operate via limited modes of action. Therefore, the discovery of new chemistries from non-conventional chemical classes and with novel modes of action is a priority for the control of vectors and VBDs. Natural products (NPs) are diverse in chemical structures and, potentially, modes of action. They have been used as insecticides for many decades and have inspired the development of multiple synthetic insecticides, suggesting the discovery of novel NPs could lead to the development of highly effective insecticides. </p> <p><br></p> <p>In this thesis, I report two studies with a main goal to identify novel mosquito-active insecticide leads that operate via modes of action distinct from existing insecticides. First, I tested the hypothesis that new mosquito-active insecticide leads with novel chemical structures, possibly operating via novel modes of action, can be identified by high-content larval phenotypic screening against a natural product collection and using novel phenotypic endpoints in addition to mortality endpoints. Here, I performed a high-content larval phenotypic screen using first instar (L1) larvae of <em>Aedes aegypti</em> (Linnaeus, 1762) against 3,680 compounds from the AnalytiCon MEGx Natural Product Libraries and a screening platform developed by Murgia et al., (2022). Compounds were screened in a 384-well plate format using the Perkin Elmer Opera Phenix and larvae were scored for lethal and novel phenotypic endpoints. Screening revealed five chemistries that caused larval mortality, including rotenone and a new NP chemistry, NP-4. The identification of rotenone confirmed the ability of the screen to detect mosquito-active NP chemistries. NP-4 caused high levels of larval mortality in the screen, and toxicity was confirmed in a subsequent concentration-response assay against third instar (L3) larvae of <em>Ae. aegypti</em>. 140 chemistries that caused atypical larval phenotypes, including cuticular pigmentation and morphometric changes relative to negative controls, were also identified by the screen. Some of these chemistries may operate by disruption of pathways regulating melanization, growth and development, and novel targets in the insect nervous systems, thus representing potential leads for further insecticide toxicity and mode of action studies. To facilitate quantitative analyses of atypical phenotypes, an attempt was made to assess the morphometrics of the thorax in larvae exposed to test chemistry, relative to control larvae. However, assessment was limited by the number of larvae images of suitable quality for measurements. </p> <p><br></p> <p>In the second study, I tested the hypothesis that metergoline (Murgia et al., 2022) and NP-4 (this study), two chemistries identified by the HTP phenotypic screen described in this project, operate via disruption of targets in the insect nervous systems that are distinct from the current insecticidal modes of products used in mosquito control programs. Specifically, I explored the hypothesis that metergoline operates via one or more insect orthologs of the mammalian G protein-coupled serotonin and dopamine receptors. An electrophysiology study was performed using the suction electrode technique and ganglia of the German cockroach, <em>Blattella germanica </em>(Linnaeus, 1767). To facilitate the investigation of metergoline agonism/antagonism and disruption of invertebrate GPCR signaling, 5-hydroxytryptamine (5-HT; serotonin) was included as a chemical probe. Electrophysiological recordings showed 5-HT (10µM and 1mM) and metergoline (10µM) caused no significant neurological activity at the tested concentrations in comparison to the saline control. However, a consistent neuro-inhibitory trend was observed, suggesting possible agonism of a 5-HT1-like receptor ortholog and antagonism of a putative 5-HT7-like receptor ortholog in the cockroach, respectively.  NP-4 caused significant neuro-inhibition at the tested concentration of 20µM, in comparison to the negative saline control. Given the demonstration of rapid contact toxicity to <em>Ae. aegypti</em> larvae and neurological inhibition in <em>B. germanica</em>, we propose NP-4 may act at one or more conserved targets in the insect nervous systems, which remain to be elucidated. </p> <p><br></p> <p>The significance of the present study is three-fold. First, this study reports the first high-content phenotypic screen of mosquito larvae against a NP collection and identification of 145 mosquito-active chemistries associated with lethal and phenotypic endpoints. These data confirm that the screening platform provided an innovative and effective system to rapidly identify mosquito-active small molecules with potential novel modes of action. Second, metergoline and NP-4 represent potential novel chemical leads for the development of new insecticides that can be incorporated into vector control programs targeting insecticide-resistant populations. Lastly, the study describes the first electrophysiology study of 5-HT, metergoline, and NP-4 via the suction electrode technique in an insect system and contributes new knowledge to the study of the insect serotonergic system, which represents an expanding area of vector biology research given its roles in feeding regulation.  </p> <p><br></p> <p>Future studies resulting from this thesis might include: (1) development of a set of morphometric criteria for quantitative analyses of atypical larval phenotypes, (2) incorporation of new phenotypic endpoints to expand the capacity of the screen to identify novel mode of action chemistries for insecticide discovery, and (3) identification of chemistry candidates suitable for further development from the 140 chemistries associated with atypical larval phenotypes in the primary screen using chemo-informatic and toxicological studies. In addition, studies using reverse transcription-polymerase chain reaction (RT-PCR), cell-based expression systems, mutant/insecticide resistant strains, and patch clamp electrophysiology could be pursued to further investigate the molecular mode of action of metergoline and NP-4, and potential for vector control.</p>

No description available.

Master of Science / Department of Entomology / Franklin Arthur / Kun Yan Zhu / Tribolium spp. are major pests in structures used for the processing and storage of grain-based products (e.g., flourmills, warehouses, retail stores). Consumers and regulators have little tolerance for insect-damaged or contaminated food products. The direction and breadth of pest-control strategies in the food industry have changed significantly over the past few years, creating the need to optimize insecticides through improved integrated pest management (IPM) techniques, specifically through the identification of new control agents that are low in mammalian toxicity, as well as any factors that might affect susceptibility to these agents. There is currently renewed interest in developing reduced-risk, low toxicity chemicals that can be effectively utilized in a setting in which grain and other food commodities are vulnerable to insect infestation, as a means of replacing outdated, and at times, less effective methods of insect control. Over the past decade, developed countries have made significant progress toward alternative insect control strategies by employing a variety of applied insecticides. Two classes of insecticide include natural pyrethrum and insect growth regulators (IGRs), which are substances that mimic insect hormones essential to normal development and reproduction. Pyrethrin is a highly efficient, broad spectrum, botanical insecticide that causes a rapid knockdown in exposed insects. Synergists are used to extend the economic usage of natural pyrethrins and because pyrethrum is rapidly metabolized, it is often mixed with a synergist. Methoprene, a juvenile hormone analog, is labeled as an aerosol and surface treatment inside mills, warehouses and other food storage facilities. There is little recent research with large-scale aerosol applications in stored-food facilities; furthermore, there are few published references regarding the efficacy of using methoprene in combination with synergized pyrethrin, in aerosol form. Therefore, the purpose of this research was to evaluate the use of aerosol applications of two aerosol concentrations on flour and finished stored-product packaging materials for the control of Tribolium spp. Results of this research show that T. castaneum are effectively controlled with 1% aerosol application, while the 3% formulation is required to effectively control T. confusum. With regards to the various packaging material surfaces, few differences between the surfaces emerged.