To contribute to the knowledge of the chemical species P. klotzschianum, P. hispidum and P. arboreum, analyzes of volatile obtained by hydrodistillation-(HD), extracts obtained by supercritical fluid extraction-(SFE) and identification of isolated compounds were performed using techniques such as GC/MS/FID, NMR (1D, 2D) and IV. Sesquiterpenes present in P. klotzschianum, P. hispidum and P. arboreum as the (E)-caryophyllene, bicyclogermacrene and germacrene D observed only in P.hispidum and P. klotzschianum were the main substances responsible for the high values obtained sesquiterpenes identified by HD. For the monoterpenes £]-pinene (27.19¡Ó0.27%) and £-pinene (7.17¡Ó0.50%) identified in the chemical composition of the stems of P. klotzschianum and Ô-3-carene (17.39¡Ó0.15%, 19.13¡Ó0.48%) respectively present in fresh and dry leaves of P. hispidum also deserved highlighted. The highest yield of extract using supercritical carbon dioxide-(SC-CO2) was 1.36% (80 ¢XC/220 bar) P. klotzschianum and 1.92% (80 ¢XC/200 bar) for P. hispidum, already with the addition of modifiers 2.18% for P. klotzschianum and 3.62% P. hispidum both using 5% methanol at 80 ¢XC/220 bar. By analysis of variance and F test the cosolvent was the variable that most influenced the yields of extracts for F = 288.95 (P. klotzschianum) and F = 409.59 (P.hispidum). The major compounds identified by GC/MS/FID of P. hispidum using SC-CO2 and SC-CO2 + co-solvents were germacrene D (21.08%-80 ¢XC/200 bar-isopropanol 1%) and squalene (15.18%-80 ¢X C/200 bar). Four compounds not initially identified by GC/MS/FID were isolated from extracts of SC-CO2 by column-chromatography (CC) followed by preparative layer chromatography-(CCDP): N-[3-(6-methoxy-3´, 4´-methylenedioxyphenyl)-2(Z)-propenoyl]-pyrrolidine (PHC7_1), N-[3-(6´-methoxy-3´, 4´-methylenedioxyphenyl)-2 (E)-propenoyl]-pyrrolidine (PHC7_2), N-[7 (3´, 4´ methylenedioxyphenyl)-2(Z), 4(E)-heptadienoil]-pyrrolidine (PHC6_2) and N-[7 (3´, 4´ methylenedioxyphenyl)-2(E), 4(Z)-heptadienoil]-pyrrolidine (PHC9), among these only PHC7_1 was cited in the literature on P. hispidum, the others were first reported in the species, with an PHC6_2 PHC9 unpublished in the literature. In chemical composition of the extracts of P. klotzschianum, germacrene D (25.00¡Ó0.11%-40 ¢XC/220 bar/isopropanol 3%), bicyclogermacrene (15.41¡Ó0.23%-40 ¢XC/220 bar/isopropanol 3%), 14-oxy-muuroleno-£ (21.23¡Ó0.10%-80 ¢XC/180 bar/ethanol 3%) pipercalosidina (22.06¡Ó0.01%-40 ¢XC/180 bar/methanol 1%) and (E)-caryophyllene (11.66¡Ó0.05%-40 ¢XC/220 bar/isopropanol 3%) are the major constituents present in the extracts obtained with SC-CO2 or addition modifier. Among the samples tested in front of IC50 HepG2 and HL-60 cells, the samples P. hispidum stems oil (94.77¡Ó1.93%-HepG2), P. klotzschianum fruit oil (90.53¡Ó1.10%-HepG2, 76.64¡Ó7.44%-HL-60) and P. klotzschianum oil fresh leaves (93.24¡Ó0.70%-HepG2) had cytotoxic activity. The oil obtained from the dried leaves of P. klotzschianum (122.372¡Ó1.247 £gg mL-1), dried leaves of P. hispidum (141.876¡Ó2.333 £gg mL-1), fresh leaves of P. arboreum (187.901¡Ó2.106 £gg mL-1) and fresh leaves P. klotzschianum (223.051¡Ó1.253 £gg mL-1) showed larvicidal activity against the mosquito Aedes aegypti. / Visando contribuir para o conhecimento quimico das especies P. klotzschianum, P. hispidum e P. arboreum, analises dos volateis obtidos por hidrodestilacao-(HD), extratos obtidos por extracao com fluido supercritico-(EFS) e identificacao de compostos isolados foram realizadas por tecnicas como CG/EM/DIC, RMN (1D, 2D) e IV. Sesquiterpenos presentes na P. klotzschianum, P. hispidum e P. arboreum como o (E)-cariofileno, biciclogermacreno e o germacreno D observados somente na P. klotzschianum e P. hispidum foram as principais substancias responsaveis pelos altos percentuais de sesquiterpenos identificados obtidos por HD. Para os monoterpenos o £]-pineno (27,19¡Ó0,27%) e £-pineno (7,17¡Ó0,50%) identificados na composicao quimica dos caules de P. klotzschianum e Ô-3-careno (17,39¡Ó0,15%-19,13¡Ó0,48%) respectivamente presente nas folhas frescas e folhas secas da P. hispidum mereceram destaque. O maior rendimento de extrato utilizando dioxido de carbono supercritico-(SC-CO2) foi 1,36% (80 ¢XC/220 bar) P. klotzschianum e 1,92% (80 ¢XC/200 bar) para a P. hispidum, ja com adicao de modificadores 2,18% para a P. klotzschianum e 3,62% para a P. hispidum ambas utilizando 5% de metanol a 80 ¢XC/220 bar. Pela analise de variancia e teste F o co-solvente foi a variavel que maior influenciou para os rendimentos dos extratos F=288,95 (P. klotzschianum) e F=409,59 (P.hispidum). Os constituintes majoritarios identificados por CG/EM/DIC para a P. hispidum utilizando SC-CO2 e SC-CO2 + co-solventes foram o germacreno D (21,08%-80 ¢XC/200 bar- isopropanol 1%) e o esqualeno (15,18%-80 ¢XC/200 bar). Quatro substancias inicialmente nao identificadas por CG/EM/DIC foram isoladas dos extratos de SC-CO2 por meio de cromatografia em coluna-(CC) seguida de cromatografia em camada preparativa-(CCDP) N-[3-(6¡¦-metoxi-3¡¦,4¡¦-metilenodioxifenil)-2(Z)-propenoil]-pirrolidina (PHC7_1), N-[3-(6¡¦-metoxi-3¡¦,4¡¦-metilenodioxifenil)-2(E)-propenoil]-pirrolidina (PHC7_2), N-[7(3¡¦,4¡¦ metilenodioxifenil)-2(Z),4(E)-heptadienoil]-pirrolidina (PHC6_2) e N-[7(3¡¦,4¡¦ metilenodioxifenil)-2(E),4(Z)-heptadienoil]-pirrolidina (PHC9), dentre estas apenas a PHC7_1 foi citada na literatura sobre P. hispidum, as demais foram relatadas pela primeira vez na especie, sendo a PHC6_2 e PHC9 ineditas na literatura. Na composicao quimica dos extratos da P. klotzschianum o germacreno D (25,00¡Ó0,11%-40 ¢XC/220 bar/isopropanol 3%), biciclogermacreno (15,41¡Ó0,23%-40 ¢XC/220 bar/isopropanol 3%), 14-oxi-£-muuroleno (21,23¡Ó0,10%-80 ¢XC/180 bar/etanol 3%), pipercalosidina (22,06¡Ó0,01%-40 ¢XC/180 bar/metanol 1%) e (E)-cariofileno (11,66¡Ó0,05%-40¢XC/220 bar/isopropanol 3%) foram os constituintes majoritarios presentes nos extratos obtidos com SC-CO2 ou com adicao de modificadores. Dentre as amostras testadas na IC50 frente as celulas HepG2 e HL60 as amostras P. klotzschianum oleo dos caules (94,77¡Ó1,93%-HepG2), P. klotzschianum oleo dos frutos (90,53¡Ó1,10%-HepG2, 76,64¡Ó7,44%-HL60) e P. klotzschianum oleo das folhas frescas (93,24¡Ó0,70%-HepG2) apresentaram atividade citotoxica. Os oleos obtidos das folhas secas da P. klotzschianum (122,372¡Ó1,247 £gg mL-1), folhas secas da P. hispidum (141,876¡Ó2,333 £gg mL-1), folhas frescas da P. arboreum (187,901¡Ó2,106 £gg mL-1) e folhas frescas da P. klotzschianum (223,051¡Ó1,253 £gg mL-1) apresentaram atividade larvicida contra o mosquito Aedes aegypti.

Malgré les efforts national et international pour contrôler le paludisme, de nombreux pays d’Afrique Sub-saharienne restent à risque d’infection, de morbidité et de décès liés au paludisme. L’objectif de cette thèse est d’évaluer l’efficacité « théorique » et l’efficacité « réelle » des outils de la lutte anti-vectorielle (LAV) en utilisant des indicateurs parasitologiques et cliniques. Pour évaluer l’efficacité « théorique » d’une mesure de LAV (étude expérimentale), il faut effectuer un essai contrôlé randomisé à unité de randomisation collective. L’évaluation de l’efficacité « réelle » des outils de LAV est possible grâce à l’utilisation d’un essai contrôlé randomisé à condition que le groupe témoin soit couvert au minimum par les moustiquaires imprégnées d’insecticide à longue durée d’action (MILD) de référence ou les outils de LAV déjà en utilisation dans la population d’étude. Mais, elle nécessite d’énormes moyens techniques, logistiques et financiers. Les résultats de cet essai ont permis de noter que ni la couverture universelle en MILD, ni les combinaisons de MILD+Pulvérisation intra-domiciliaire (PID) ou de MILD+bâches imprégnées d’insecticides n’ont pas apporté une protection supplémentaire contre l’infection et l’accès palustre non compliqué (APNC) par rapport à la MILD en couverture sélective. L’évaluation de l’efficacité réelle des outils de LAV en post-distribution requiert un type d’étude épidémiologique plus souple dans la faisabilité. Face aux contraintes éthiques et financières de l’étude longitudinale, nous avons validé l’utilisation d’une étude cas-témoin à cet effet. Elle a été réalisée en premier chez les enfants de 0-5 ans puis appliquée à toute la population. Les résultats obtenus indiquent que l’efficacité des MILD est variable d’une région à l’autre. En zone rurale, les MILD ont conféré sur l’APNC une efficacité de 40-50 % en ouverture sélective en MILD chez les enfants de zéro à cinq ans. Une réduction de 49 % des APNC a été notée dans toute la population si les MILD (en couverture universelle) sont associées à la PID. En zone urbaine, les MILD en couverture universelle n’ont pas permis de réduire le nombre d’APNC dans toute la population. Elles ont cependant permis une réduction de 50 % des infections palustres dans un seul quartier de la zone d’étude. Les limites de cette étude cas-témoin peuvent être intrinsèques aux mesures de lutte (défaut de couverture, résistance des vecteurs aux insecticides etc.). La mesure de l’exposition peut également être soumise à des biais. Plusieurs facteurs interférant avec la réussite de la LAV ont été évoqués. Le premier facteur défavorable à l’efficacité de la MILD est son défaut d’utilisation. De même, la description de la pièce où se joue la LAV montre que l’espace disponible pour que tous les acteurs puissent jouer convenablement leur rôle (MILD, PID, Homme et vecteur) est assez restreint. Cet espace est souvent mal éclairé. De même la présence des flammes libres participe à la dégradation de l’intégrité physique des MILD. La présence de trous sur les moustiquaires indique une perte de leur efficacité car à partir d’un certain indice > 100, les individus sont fortement exposés aux piqûres de vecteurs. En plus, An. funestus, un des principaux vecteurs assurant la transmission dans les zones d’études concernées peut piquer au-delà de 6 h du matin et assure une bonne partie de la transmission à l’extérieur des habitations. Enfin, les moustiquaires peuvent également créer des dommages corporels à partir d’incendie auxquelles elles participent de part leur caractère inflammable. A l’étape actuelle de la lutte contre le paludisme, il est nécessaire de trouver de meilleurs outils pour améliorer la qualité de la prévention par la LAV. La recherche opérationnelle nous semble en outre primordiale dans la mesure où les outils de LAV utilisés à large échelle ont déjà fourni de bons résultats d’efficacité au laboratoire. / Despite national and international efforts, malaria remains a major public health in many countries. Health systems are hindered by the lack of information on the actual burden of malaria and the effectiveness of vector control tools. Vector-control measures are a component of integrated malaria control strategies. The objective of our thesis was to evaluate the efficacy and the effectiveness of malaria vector control tools using parasitological and clinical criteria.With a block randomized control trial, we investigated whether the combination of long-lasting insecticidal mosquito nets (LNs) with indoor residual spraying (IRS) or Carbamate-treated Plastic Sheeting (CTPS) conferred better protection against malaria vectors than did LNs alone. The clinical incidence density of malaria was not reduced in the children from the "Universal LN" group (incidence density rate (0.95, 95% CI 0.67–1.36, p=0.79), nor in those from the "Target LN + IRS" group (1.32, 0.90–1.93, p=0.15) or from the "Universal LN + CTPS group (1.05, 0.75–1.48, p=0.77) compared with the reference group "Target LN". The same trend was observed with the prevalence and parasite density of asymptomatic infections. The evaluation of the effectiveness of vector control tools is possible but requires enormous technical, logistic and financial resources. The evaluation of the effectiveness of malaria vector control tools after distribution requires a more flexible epidemiological study. Considering the ethical and financial constraints of the longitudinal study, we validated the use of a case-control study to this purpose. It was conducted primarily among children aged 0-5 years old and then applied to the entire population. Results were spatial dependant when taking into account age and compliance to chemoprophylaxis as confusion factors, use of other vector control tools, sex, and economic status, school level of the mother or the head of the house. In the rural area, the use of LNs provided significant level of protection (40-50%) against clinical cases among children aged 0-5 years old. This significant protection was obtained among all population only if the LNs were associated to IRS. In the urban area, the use of LN was not provided protection against clinical cases but reduce 50% of the risk of Plasmodium falciparum infection in one neighbourhood. The limits of this case-control study may be intrinsic to control measures (lake of coverage, vector resistance to insecticides etc.). The exposure of vector control tools measures may also be subject to bias. Several factors interfering with the success of malaria control were discussed. The first negative factor to the effectiveness of vector control tools was the "no use of LNs". Then follows the immediate environment where the fight against vectors took place. The description of the room where played vector control fighting shows that the space available for all the actors (LNs, IRS, humans and vectors) to play their role properly is quite limited. This space is often poorly enlightened. Similarly the presence of open flames is involved in the degradation of the physical integrity of LNs. The presence of holes on the bed-nets indicates a loss of effectiveness because from a hole index above 100, individuals are highly exposed to the bites of vectors. In addition, An. funestus, one of the main vectors of malaria transmission in the study area, bitted after 6:00 am and provides much of the transmission in outdoor. Finally, the nets can also create personal injury as fire. At the end our work, we conclude that the innovative vector control tools are required to improve malaria vector control. But, operational research seems now essential as the vector control tools used on a large scale have provided good efficacy results in the laboratory. The challenge then is to obtain comparable results in real condition of use and look for effectiveness barriers.

Fleas are one of the major external parasites affecting free range chickens, causing irritation, tissue damage, blood loss and toxicosis which in turn affect quality and quantity of meat and egg production. There are many commercial insecticides available that are effective against fleas. These commercial insecticides have, however become expensive to most resourcelimited farmers and therefore unaffordable, causing farmers to seek low cost alternatives. This study was conducted to document, determine the existence of external parasites in freerange chickens and validate the alternative remedies used in controlling free-range chicken fleas by resource-limited farmers. A questionnaire survey was used to collect data on external parasites of free-range chickens and ethno-veterinary control remedies used by resourcelimited farmers to control these parasites at Amatola basin, in Eastern Cape, South Africa. Resource-limited farmers perceived that mites (79.6%), fleas (64.5%), lice (10.8%) and ticks (6.5%) were problematic parasites of chickens. To control these parasites, resource-limited farmers commonly use ash (28%) madubula (13% carbolic acid) (26.7%), Jeyes fluid (13% carbolic acid) (10%), paraffin (8.4%), used engine oil (2.8%), plants Tagetes minuta, Clutia pulchella, Calpurnia aurea (5.2%) and a few (4.2%) used commercial drugs Karbadust (Carbarly 5%) and mercaptothion 5%. About 7.5% of the respondents used neither of the remedies. To confirm the farmers’ perception on problematic external parasites of free-range chickens a diagnostic survey was conducted. Fifty free-range chickens were randomly selected and examined for external parasites. About 96% of the free-range chickens examined harboured at least one species of external parasites. Fleas (Echidnophaga gallinacea) (50.7%) were the most prevalent followed by lice, Menopon gallinae (12.4%); Menacanthus stramineus (5.3%) and Knemidocoptes mutans (0.57%). Given that Jeyes fluid, used engine oil, paraffin, C. aurea, C. pulchella and T. minuta were mainly used in controlling these parasites, it was important to determine the potential dermal irritant effects of these plants. A dermal irritant effect test was, therefore, conducted using a rat model. Forty eight rats, with parts of the backs shaven, were used to screen the materials for irritation. All the materials tested did not cause any visible skin irritation on rats (p>0.05). Regarding that ethnoveterinary materials were non irritant on rat skin in vitro repellency and contact bio-assay models were used to assess the insecticidal properties of Tagetes minuta, Calpurnia aurea, Clutia pulchella, used engine oil, paraffin and Jeyes fluid on fleas. For the in vitro bioassay T. minuta and Jeyes fluid at 100% concentration demonstrated a repellency level of 76 and 83%, respectively. Tagetes minuta was the most effective among plant materials (p<0.05). Clutia pulchella, C. aurea, used engine oil and paraffin showed insignificant repellency (p>0.05). For the contact bio-assay, C. pulchella, C. aurea and T. minuta at a concentration of 100% resulted in flea mortality of 83.5, 73.3 and 42.5%, respectively. The efficacy of Clutia pulchella compared well with that of Karbadust which had a mean mortality of 97.5%. Paraffin, used engine oil, and Jeyes fluid (19.2%) caused higher flea mortality of more than 82%. In the in vivo study 60 free-range chickens were artificially infested with fleas and test materials were topically applied on infested sites. Test materials exhibited varying flea load reduction efficacy. Used engine oil and Jeyes fluid at 76.8% concentration had a reduction efficacy of 100 and 96% after 3 days post application of test materials. These were not significantly different to that of the positive control Kabadust (carbaryl 5%). The plants C. pulchella and C aurea at 100% concentration had an efficacy of 92 and 77%, respectively. The commonly used remedies by resource-limited farmers to control fleas vary in efficacy. Some of the materials are as effective as the commercial insecticides hence they have a potential to be exploited as insecticides. Further investigations on plant compounds with insecticidal properties and their toxicity, need to be conducted before the plants are recommended for use.

This research deals with impacts of pesticide substances on spider populations. In this case pesticides of the neonicotinoids classes were tested. Specific neonicotinoids were applied to different of spiders which were collected in various localities and at some stage of development - nymph, adult or subadult individuals. The diploma thesis analyzes and the documents the effects of neonicotinoids to spiders. The results show that some pesticides can have a significant negative effect to locomotion, predation, ballooning, drop or to spiders mortality. Keywords: spider, arthropod, pesticides, insecticides, neonicotinoids, sublethal effects, mortality, EthoVision XT, locomotion, drop, ballooning, predation, Philodromus, Oedothorax, Pardosa, Xerolycosa, Phylloneta

No description available.

Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe. / Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.

Algunas plagas causan daños importantes en productos y granos almacenados, lo cual conlleva consecuentemente a pérdidas de producción y calidad en estos productos.Las principales plagas del arroz almacenado en España son, Sitophilus oryzae L. (Coleoptera Curculionidae), Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae), y Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae). Las dos primeras son plagas primarias que atacan directamente el grano y resultan bastante destructivas debido a que sus larvas se alimentan y desarrollan dentro de él. C.pusillus es, sin embargo, una plaga secundaria que se beneficia de granos que ya están dañados y rotos.Actualmente, el uso de fumigantes e insecticidas de síntesis sigue siendo el principal método de lucha para controlar las plagas de almacén, si bien recientemente (dadas las continuas restricciones al uso de agroquímicos) existe un gran interés en la utilización de otras alternativas tales como el control biológico, el almacenamiento a bajas temperaturas, o los tratamientos con calor entre otros.Igualmente muchos productos obtenidos principalmente de plantas y que derivan del metabolismo secundario de las mismas ofrecen una fuente de bioinsecticidas que podrían representar una alternativa ecológica frente a los insecticidas de síntesis ya que su uso masivo e indiscriminado ha ocasionado problemas tales como la aparición de resistencias en determinadas especies de insectos frente a diferentes materias activas, desequilibrios ecológicos y problemas medioambientales sin olvidar el riesgo que entrañan para la salud humana.Con este trabajo se ha pretendido estudiar la actividad plaguicida de los aceites esenciales extraídos de tres plantas: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) y Ocimum basilicum L. (Labiatae) y su posterior fraccionamiento para identificar dentro de estos aceites los compuestos químicos responsables de esta actividad insecticida sobre tres plagas de almacén de arroz (S. oryzae, R.dominica y C.pusillus).Del estudio de estos monoterpenoides, linalol, S-carvona y estragol resultaron tener una alta actividad insecticida sobre estas plagas. Sin embargo el E-anetol fue más selectivo para R.dominica y C.pusillus, así como el limoneno, &#947;-terpineno, geraniol y eucaliptol sólo resultaron activos frente a C.pusillus.Algunos monoterpenoides podrían actuar de sinergistas potenciando la actividad de otros, como podría ser el caso del alcanfor, acetato de geranilo y E-anetol con linalol en R.dominica y C.pusillus, o el caso del metoxicinamaldehido, p-anisaldehido y linalol que pueden tener efecto sinergista sobre el estragol.Este trabajo también abarcó el estudio de un posible modo de acción de estos monoterpenoides, la inhibición de la acetilcolinesterasa, para alcanzar un mayor entendimiento del comportamiento de estas sustancias en el interior del insecto.Así se pudo observar como la mayoría de monoterpenoides estudiados inhibían en cierta medida esta enzima, siendo fenchona, S-carvona y linalol los monoterpenoides que mayor inhibición originaron.Del mismo modo se observó como fenchona, &#947;-terpineno, geraniol y linalol inhibían competitivamente la acetilcolinesterasa, mientras que S-carvona, estragol y alcanfor producían una inhibición mixta para esta enzima.Sin embargo no se observó inhibición de la acetilcolinesterasa por parte del E-anetol a las concentraciones de monoterpenoides ensayadas.Para completar este trabajo se examinó de igual forma la capacidad que tenían estos bioinsecticidas de generar resistencia en estas tres plagas así como el mecanismo de resistencia implicado en el desarrollo de este proceso. Para ello se fueron seleccionando las poblaciones de insectos mediante la aplicación de los diferentes monoterpenoides a dosis crecientes durante 7 generaciones. De este modo se pudo calcular el factor de resistencia en cada una de las plagas y para cada uno de los monoterpenoides comparando las concentraciones letales 50 de las poblaciones seleccionadas con las poblaciones iniciales (sensibles).A continuación se analizaron tres posibles sistemas de detoxificación enzimáticos gracias al uso de sinergistas para estudiar el mecanismo de resistencia que podría estar involucrado.De tal forma se observó como estos monoterpenoides inducían lentamente resistencias resultando ventajosos en un futuro para el control de estas plagas.Esta Tesis ha englobado un estudio íntegro y profundo de estos insecticidas ecológicos desde la extracción de los aceites esenciales, seguido de la identificación de compuestos puros (CG-EM) con actividad insecticida junto con el estudio de un modo de acción de estos insecticidas, la selección de resistencia en las poblaciones de insectos y los posibles mecanismos de resistencia que pudieran estar implicados en este proceso. / Some pests cause serious damage to stored grains and stored products and consequently production and quality losses in these products.The rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae), the lesser grain borer, Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae) and Cryptolestes pusillus Schönherr (Coleoptera: Cucujidae) were the main damaging pests found in stored rice in Spain.S.oryzae and R.dominica are primary pests attacking directly the intact grain and are quite destructive because their larvi feed and develop inside the grain whereas C.pusillus is a secondary pest which benefits from grains previously damaged.At the present time, organic synthetic pesticides are still the main method to control stored grain pests, however, recently (due to restriction in agrochemicals use) there is a great interest in using other altenatives such as biological control, storage at low-temperatures, or heat treatment.Likewise, many products obtained mainly from plants and derived from secondary metabolism have insecticidal activity against insects, such as monoterpenoids, which present a broad variety of bioinsecticide products which could turn out to be an ecologic alternative to synthetic pesticides since the majority of alternative products are not harmful for the human healthy and they become less environmentally damaging, exhibiting a low impact on the environment.In addition, it cannot be ignored the different difficulties related to resistance due to several active compounds from organic pesticides found in some species of insects.With this work, we have considered remarkable to study the insecticide activity of essential oils extracted from three plants: Coriandrum sativum L. (Umbelliferae), Carum carvii L. (Umbelliferae) and Ocimum basilicum L. (Labiatae) and carry out a bioassay-guided fractionation of their essential oils to identify which compounds were responsible for the volatile toxicity shown on three stored rice pests (S. oryzae, R. dominica and C. pusillus).Linalool, S-carvone and estragole turned out to have a high insecticide activity on these pests. Nevertheless E-anetol was more selective to R. dominica and C. pusillus, being only active on C. pusillus limonene, &#947;-terpinene, geraniol and eucalyptol.Some monoterpenoids were found as synergists, increasing the activity of the other ones, like for instance, camphor, geranyl acetate and E-anethole with linalool in R.dominica and C.pusillus or metoxycinnamaldehyde, p-anysaldehyde and linalool which could activate to estragole.In this work, the inhibition of acetylcholinesterase as a posible mode of action was studied as well, to reach a clear understanding about the action of these products inside the insects.The majority of monoterpenoids inhibited the enzyme acetylcholinesterase being fenchone, S-carvone and linalool the monoterpenoids that produced a higher inhibition.Furthermore, it was observed how fenchone, &#947;-terpinene, geraniol and linalool showed a competitive inhibition whereas S-carvone, estragole and camphor produced a mixed inhibition for this enzyme. However the enzyme acetylcholinesterase was not inhibited by E-anethole.To finish up this work, the selection for monoterpenoid resistance on these pests as well as the metabolic mechanisms implicated was studied.The resistant strains were selected from susceptible insect populations and survivors were reared separately for each monoterpenoid and successive generations were treated with higher concentrations. These populations were selected until seven times.As a result we could calculate the resistance factor on each pest (comparing lethal concentration 50 values of susceptible and resistant strains).Next, three enzymatic systems detoxifying these monoterpenoids were analysed to study the metabolic mechanism implicated.In this way we could realize that all monoterpenoids induced resistance slowly, concluding that these pesticides will be appropriated to control these pests in the future.This Thesis has concerned a study in depth about ecological insecticides from extraction of essential oils, identification of compounds (GC-MS) with insecticide activity, mode of action and study of resistance and mechanism of insecticide resistance involved in this process.

Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe. / Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.

Resumo: Recentemente as abelhas têm sido devidamente valorizadas como importantes polinizadoras de flores silvestres e cultivadas. A densidade populacional de muitos polinizadores tem diminuído devido, principalmente, à intensificação agrícola e ao uso de pesticidas, prejudicando os serviços de polinização. A metodologia clássica para estimar a toxicidade dos produtos químicos para insetos é determinar a dose letal média (DL50) ou a concentração letal média (CL50), podendo então estabelecer doses que sejam mais seguras aos organismos não-alvo ou benéficos. Além dos efeitos de toxicidade aguda, levando a morte das abelhas, doses subletais dos inseticidas podem provocar alterações comportamentais e fisiológicas nos indivíduos, que ao longo do tempo acarretarão em sérios prejuízos na manutenção da colônia. Um dos inseticidas amplamente utilizado é o fipronil, este atua ligando-se aos receptores do ácido gama-aminobutírico (GABA), interrompendo os canais de cloro, resultando na perda de sinalização inibitória neural. Na literatura pode-se encontrar diversos trabalhos que utilizam como modelo principal a abelha Apis mellifera, porém, é importante ressaltar a diversidade existente entre as abelhas nativas no Brasil, os meliponíneos, e sua participação na conservação da biodiversidade, assim como na polinização de áreas de cultivo, o que torna extremamente importante estudos com essa abelha. Com a finalidade de entender como o fipronil interfere morfo e fisiologicamente em abelhas sem ferrão, a região de interesse deste estudo foram os corpos pedunculados, já que estes são centros cerebrais complexos e tidos como local de convergência multisensorial. Para auxiliar no mapeamento metabólico, utilizou-se como marcador a enzima citocromo oxidase e a enzima caspase-3, técnicas utilizadas na observação de atividade neural... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A few decades the bees are considered an important indicator of high environmental sensitivity, and appreciated as important pollinators of wildflowers and cultivated. The population density of many pollinators have declined to harmful levels to pollination services manly due to agricultural intensification and the use of pesticides. The classic methodology of estimating the effects of chemicals for insects is to determine the median lethal dose (LD50) or median lethal concentration (LC50) that can then establish doses that do not harm non-target organisms or beneficial. Besides the effects of acute toxicity, leading to death bees, sublethal doses of insecticides can cause physiological and behavior changes of individuals over time, resulting in serious harm to maintain the colony. One of the widely used insecticides is fipronil, its acts by binding to gamma-aminobutyric acid (GABA) disrupting chloride channels, resulting in loss of inhibitory neural signaling. In the literature one can find several works using as main bee model Apis mellifera, however, it is important to highlight the diversity of Brazilian native bees, the stingless bees, and their participation in biodiversity conversation, as well as in the pollination of cultivated land. In order to understand how fipronil affect morpho and physiologically the stingless bee S. postica, the region of interest in this study were the mushroom bodies, since these are complex brain centers and used as a place of multisensory convergence. This work established the contact LD50 and Ingestion LC50 to the fipronil insecticide for foragers workers stingless bee Scaptotrigona postica in 0.54ng/bee and 0.24ng/μL of the food after 24 hours, respectively, confirming the high toxicity of this phenylpyrazole, in the groups submitted to contact contamination, were identify morphological... (Complete abstract click electronic access below) / Orientador: Osmar Malaspina / Coorientador: Roberta Cornélio Ferreira Nocelli / Banca: Elaine Cristina Mathias da Silva Zacarin / Banca: Thaisa Cristina Roat / Mestre

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-06-18T11:32:10Z No. of bitstreams: 1 ELVIRA BEZERRA PESSOA - TESE PPGEA 2012..pdf: 49929920 bytes, checksum: 3b211bebb087afcfef048700e63d31fb (MD5) / Made available in DSpace on 2018-06-18T11:32:10Z (GMT). No. of bitstreams: 1 ELVIRA BEZERRA PESSOA - TESE PPGEA 2012..pdf: 49929920 bytes, checksum: 3b211bebb087afcfef048700e63d31fb (MD5) Previous issue date: 2012-04 / Dentre as pragas que atacam as sementes de feijão durante o armazenamento, destaca-se o Zabrotes subfasciatus (gorgulho-do-feijão) por reduzir a qualidade e o valor nutricional das sementes. A redução da qualidade dos grãos/sementes durante o armazenamento está associada, principalmente, ao grau de infestação e às condições ambientais cm que se encontra a massa de feijão. Assim, buscou-se com este trabalho produzir e avaliar o potencial de extratos vegetais de Aspidosperma pyrifolium (pereiro), Anadenanthera colubrina (angico) e Licania rígida (oiticica) no controle do Zabrotes subjàsciatus, bem como avaliar a qualidade física e fisiológica das sementes de feijão Phaseolus tratadas com extratos, obtidos da folha c da casca dessas espécies, durante 180 dias acondicionadas em embalagens do tipo pet em ambiente não controlado de temperatura e umidade relativa do ar. O trabalho foi realizado cm quatro experimentos, onde no primeiro foram produzidos os extratos e realizados a bbatividade inseticida dos extratos vegetais hidroalcoolico e seco, cm que o extrato pereiro casca a partir da dose de 3 ml, foi 100% eficiente em matar o Zabrotes subfasciatus e, nas doses de 4 e 5 ml a mortalidade de 100% deu-se para todos os extratos. No segundo experimento se estudou a repelência/atralividade e mortalidade do Zabrotes subfasciatus pelos extratos pós das mesmas plantas, tendo-sc observado que o número de insetos adultos repelidos pelos extratos pós tbi maior com o extrato oiticica casca, seguido dos demais (pereiro casca, angico casca, pereiro folha e angico folha) que estatisticamente se igualaram e, o menor número de insetos repelidos deu-se com o extrato oiticica folha, ocorrendo o contrário com a atratividade. Os bioensaios para prospecção lltoquímica dos extratos vegetais obtidos das folhas e das cascas dessas espécies, correspondeu ao terceiro experimento, em que os extratos de pereiro revelaram os mais elevados índices de compostos secundários (alcalóides, esteróides, taninos, flavonóides e saponinas) seguidos de angico e oiticica e, no quarto experimento os extratos botânicos foram utilizados no controle deste inseto praga durante o armazenamento, onde constatou-se atividade inseticida no controle do Zabrotes subfasciatus, matando-os e/ou inibindo o seu desenvolvimento. No tratamento das sementes de feijão Phaseolus, a dose de 3 ml tbi a de melhor controle com melhor resultados para os extratos oiticica folha c pereiro casca. Os procedimentos adotados no tratamento das sementes com os extratos das espécies vegetais demonstraram eficiência na manutenção da viabilidade não afetando a germinação durante os 180 dias do armazenamento. / Among the pests that attack bean seeds during storage, we highlight the 'Zabrotes subfasciatus (bean weevil) by reducing the quality and nutritional value of seeds. lhe reduction of the quality of grains / seeds during storage is mainly associated with the degree of infestai ion and environmental conditions in which it is the mass of beans. Thus. we attempted to produce this work and assess the potential of plant extracts of Aspidosperma pyrifolium (pereiro), Anadenanthera colubrína (angico) and Licania rígida (oiticica) of Zabrotes subfasciatus in control and evaluate the physical and physiological quality of seeds Phaseolus beans treated with extracts obtained from leaves and bark of these species, 180 days packed in like pet under uncontrolled temperature and relative humidity. The study was conducted in four experimenls. where the first extracts werc produccd and performed the bioactivky of insecticide hydroalcoholic plant extracts and dry in the extract from the bark pereiro dose of 3 ml. was 100% effective in killing the Zabrotes subfasciatus and, in doses of 4 and 5 ml 100% mortaliiy are given for ali the extracts. In the second experiment we studied the rcpcllcncy / attractiveness and mortality of Zabrotes subfasciatus post by extracts of these plants and has been observed that the number of adult inseets repelled by the extracts was higher with post myrtle bark extract, followcd by the others (pereiro shcll angico bark, leaf and angico pereiro sheet) that are staiisticaily similar, and the fewer inseets gave repelled with myrtle leaf extract. but the contrary to the attractiveness. The bioassays for phytochemical screening of plant extracts obtained from leaves and bark of these species corresponded to the third experiment, in which extracts pereiro revcaled the highest leveis of secondary compounds (alkaioids, steroids, tannins, flavonoids and saponins) followed by mimosa and myrtle. and lhe fourth experiment, the botanical extracts were used to control this insect pest during storage, where it was found in control insccticidal activity of Zabrotes subfasciatus, killing them and / or inhibiting its development. In the treatment of seeds o f Phaseolus beans, the dose of 3 ml was the best results for better control extracts oiticica, pereiro sheet and the shell. The procedures used in seed treatment with the extracts of plant species were efficient in maintaining the viability did not affect germination during 180 days of storage.