Present sampling techniques are not capable of assessing both contamination with an organochlorine chemical and reproductive success without the need for removing individuals from that population. Experiments were performed to evaluate vitellogenin, vitamin A, and vitamin E as biomarkers of contamination with DDT, an organochlorine pesticide, and reproductive success in Japanese quail (Coturnix coturnix japonica) or northern bobwhites (Colinus virginianus). The utility of vitellogenin as a biomarker in northern bobwhites was investigated. Female northern bobwhites were dosed with 0, 1, 10, or 100 μg DDT/g body weight via corn oil intubation. The females were induced to lay eggs by providing the proper daylength, and number of eggs laid was monitored. No differences were found for number of eggs laid, egg morphology, or plasma concentration of vitellogenin among dose groups. Dietary supplementation and corn oil intubation were evaluated as alternative dosing techniques because handling involved with intubation may have caused decreased egg production in all treatment groups in the first experiment. Nonlaying female bobwhites were dosed with 0 μg DDT, 25 μg DDT/g food, or 25 μg/g body weight (intubation). Bobwhites receiving DDT via intubation accumulated more DDT in brains and livers than did bobwhites receiving DDT via their diet. The two techniques were found not to be equivalent. Vitamins A and E were investigated as biomarkers of DDT contamination and reproductive success in Japanese quail. Female Japanese quail were given 0, 1, 10, or 25 μg dietary DDT. Significant differences were found among dose groups for reproductive success (number of eggs showing embryonic development), and for plasma concentrations of vitamin A. Concentrations of vitamin E were not measurable. Correlation and regression analyses failed to show a relationship between reproduction and vitamin A concentrations. Vitamins A and E also were investigated as biomarkers in northern bobwhites. Female bobwhites were given 0, 1, 10, or 25 ppm dietary DDT. No significant differences were found among dose groups for reproductive success or for concentrations of either vitamin A or vitamin E. Correlation and regression analyses again failed to show any relationship between reproduction and plasma concentrations of vitamins A or E. During each experiment, concentrations of DDT and its metabolites were measured. DDE/DDT ratios in livers, a means of estimating liver enzyme induction, were calculated. The DDE/DDT ratios in livers of Japanese quail and northern bobwhites were found to be markedly different. More work needs to be done to better evaluate the relationship between differences in enzyme induction and reproductive success among different species. / Ph. D.

1. Reciprocal crosses were made between a DDT-resistant and a nonresistant strain of the German cockroach. 2. The techniques employed in rearing the roaches and obtaining toxicological data on the different strains, as well as the genetical and statistical procedures, are described. 3. The toxicological data are presented in the form of regression lines with appropriate statistical analyses. 4. A table showing the relative resistance to DDT of the various strains involved in this study is included. 5. Several conclusions are drawn relative to the inheritance of resistance to DDF in the German cockroach. / Master of Science

German cockroach, Blattella germanica L., females of resistant and non-resistant strains carrying oothecae were exposed to filter paper impregnated with propoxur, malathion, and diazinon. Premature oothecal drop was monitored during the exposure period and for 24 hours thereafter. Determinations of female mortality were also made 72 h post-exposure. Oothecae from exposed females were observed for percentage egg hatch, time from exposure to hatch, percentage nymphal emergence, nymphal survival, and the percentage of nymphs able to move about freely 24 hours post-emergence. The comparisons of these factors were made not only on prematurely dropped oothecae but also on oothecae retained by females, and oothecae that were manually detached from females. Premature oothecae dropped and those manually detached were hatched on an insecticide treated surface. Premature oothecal drop occurred in all experiments, but was delayed 24 h in experiments with organophosphates. The mortality of treated females which prematurely dropped their oothecae was higher than females retaining them (73% vs. 53%). Percentage nymphal emergence and survival were reduced when oothecae were placed on an insecticide treated surface. The significantly higher survival of resistant strain nymphs (Carver, 60%, and Lynn Haven, 99%) in a comparison to nymphs of a susceptible strain (VPI, 45%) on insecticide treated surfaces provides evidence for resistance in first instar nymphs. A procedure for the quantitative comparison of the effects of different insecticides on oothecal-bearing females has been developed. Insecticides which cause a higher percentage of premature oothecal drop may reduce regrowth of a cockroach population when compared to materials which do not, even if the insecticides cause similar mortality. The extent to which population regrowth occurs depends on whether oothecae hatch on a treated surface, oothecal age, and environmental conditions. / Master of Science

Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC₅₀ = 247 nM) than 20-HE (EC₅₀ = 13 μM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 μM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method. / Master of Science

The overall purpose of this study was to examine the effects of an insecticide vapor pulse on the dispersal response of the German cockroach, Blattella germanica (L). An apparatus was designed to expose test cockroaches to vapors from commercial pesticide formulations. Insecticide vapor-induced dispersal responses were recorded during a two hour period from different strains that had been allowed to acclimate to a harborage. The dispersal response of large nymphs from a pesticide susceptible laboratory strain (VPI) was compared to two propoxur resistant field strains (Carver, Kenly) after exposure to four propoxur formulations and their blanks. Vapors from the petroleum-based oil and aerosol formulations induced significantly more dispersal than vapors from water-based emulsifiable concentrate and wettable powder formulations. Vapors from formulations containing the toxicant generally induced a significantly faster dispersal response than did their blanks. Exposure of cockroaches to the vapors of diazinon, malathion, and cyfluthrin indicated that the class of pesticide can also influence the dispersal response. Strain differences were found in experiments with the propoxur formulations, their blanks, malathion, and cyfluthrin. Slow dispersal precluded demonstration of significant strain differences in experiments with diazinon. Exposure of mixed age groups of four strains to vapors from 1% propoxur-in-oil, an aerosol and their blanks indicated that dispersal patterns were similar to the single age class experiments. Inter- and intra-strain differences were found. Dispersal of the field strains was distinguished from that of the laboratory strain by more variable responses and differences among age classes. The strongest inter-and intra-strain differences were in response to the solvents rather than the complete formulation. The air concentration of propoxur from a 1% oil formulation was estimated at 146 pg/ml. Condensation of the toxicant onto the apparatus was also observed. Vapor pulse characteristics for the four propoxur formulations indicated that the equilibrium vaporization rate in the test apparatus was approximately 0.5 mg/min for the oil and aerosol formulations and 1.0 mg/min for the WP and EC formulations. Results suggest that effective control strategies must be tailored for each target population. / Ph. D.

Cette thèse a été initiée de part le contexte de résurgence et d'expansion de la dengue à travers le monde et notamment en France. Les stratégies actuelles de lutte contre le virus et contre son vecteur Ae. aegypti se heurtent à de nombreuses limitations. Elles sont d'ordres économiques, sociologiques, législatifs, environnementaux et biologiques. Cette étude a visé à répondre scientifiquement à un certain nombre de ces limitations en s'appuyant sur le contexte Martiniquais, qui à une échelle micro-géographique présente tous les paramètres requis pour étudier les causes et les conséquences de l'extension des résistances aux insecticides sur l'efficacité de la lutte antivectorielle. Dans un premier temps ce travail a visé à mieux comprendre les mécanismes métaboliques et moléculaires impliqués dans la résistance. Nous avons ensuite étudié la répartition et les mécanismes de résistance intervenant dans les différentes populations de moustiques à un niveau macro-géographique. Ceci a permis et d'étudier les facteurs environnementaux contribuant à leur sélection sur le terrain. Enfin une étude de génétique des populations nous a permis d'étudier les niveaux de variabilité génétique et de structuration de ces populations de vecteurs. La seconde partie de l'étude a visé à comprendre l'impact de cette résistance sur les actions de lutte antivectorielle. L'expression phénotypique de ces résistances a été étudiée au laboratoire et sur le terrain en mesurant notamment les taux de survie de moustiques exposés à différents types de traitement insecticides (larvicide/adulticide). Enfin, la dernière partie a été consacrée à la recherche de stratégies alternatives ou innovantes de lutte antivectorielle dans l'objectif de contribuer à une meilleure gestion des résistances au sein des populations de vecteurs. L'évolution et le futur de la recherche concernant la lutte contre Ae. aegypti et la dengue sont discutés dans cette étude. / This thesis was conducted in the context of the resurgence and expansion of dengue fever throughout the world, particularly concerning France. Current strategies of control of the virus and its vector Ae. aegypti encounter many limitations. These involve economic, sociological, legal, environmental and biological dimensions. This study aimed to take into account some of these limitations in the setting of Martinique, which on a micro-geographical level presents all the necessary parameters to study the causes and consequences of the expansion of insecticide resistance on the effectiveness of vector control. Initial work aimed to increase our knowledge of the metabolic and molecular mechanisms implied in resistance. We then evaluated the distribution and mechanisms of resistance occurring in various populations of mosquitoes on a macro-geographical level. This allowed us to study the environmental factors contributing to selection of resistance mechanisms in the field. Finally a population genetic study enabled us to measure genetic variability and the structure of these vector populations. The second part of the study aimed at understanding the impact of resistance on vector control operations. The phenotypic expression of resistance was investigated in the laboratory and field by measuring the survival rates of mosquitoes exposed to various types of insecticide treatment (larvicide/adulticide). In the last part of this thesis we investigated alternative or innovative strategies of vector control with the objective of contributing towards improved management of resistance within vector populations. The evolution and future of research concerning the fight against Ae. aegypti and dengue is discussed in this study.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-10-17T19:20:47Z No. of bitstreams: 2 Dissertação - Diogo Nery Maciel - 2016.pdf: 6569625 bytes, checksum: 88b08e397d1fe85ad26417e8b0f2a08c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2016-10-18T17:16:36Z (GMT) No. of bitstreams: 2 Dissertação - Diogo Nery Maciel - 2016.pdf: 6569625 bytes, checksum: 88b08e397d1fe85ad26417e8b0f2a08c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-18T17:16:36Z (GMT). No. of bitstreams: 2 Dissertação - Diogo Nery Maciel - 2016.pdf: 6569625 bytes, checksum: 88b08e397d1fe85ad26417e8b0f2a08c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-13 / The increase in insecticide application for control the rice stalk stink bug Tibraca limbativentris Stal (Hemiptera: Pentatomidae) in irrigated rice in Brazil, has been reported by producers as responsible for loss of effectiveness of some insecticides commonly used to manage this insect and this is the base of this investigation. The insecticides used in this study were: bifenthrin, carbosulfan, etophenprox, λ-cyhalothrin, and thiametoxam. Six populations of insects were collected in producing areas of the states of Tocantins, Goiás and Santa Catarina, Brazil. Insect adults of each population were submitted to discriminant concentrations (LC95s) previously determined for the population collected in natural habitat in Santo Antônio de Goiás, and used as standard susceptible population. The frequency of individuals resistant in the population of Formoso do Araguaia was 76% for carbosulfan, 86% for bifenthrin, 72% for etofenprox, 80% for L-cyalothrin, and 80% for thyamethoxam. Although the frequencies of resistant individuals can be considered low to moderate, it is first report of resistance of T. limbativentris to insecticides. These results indicate the resistance development to other insecticides with different mode of actions, with emphasis to the stink bug populations of Formoso do Araguaia-TO. These results also indicates a possible occurrence of crossed a resistance or multiple in that population. / O aumento no uso de inseticidas ​​para o controle do percevejo-do- colmo Tibraca limbativentris Stal 1860 (Hemiptera: Pentatomidae) em lavouras de arroz irrigado no Brasil, associado a relatos de produtores sobre perda de eficácia dos produtos comumente usados, embasaram a condução desta investigação sobre a possível ocorrência de resistência aos inseticidas: bifentrina, carbosulfan, etofenproxi, lambda-cialotrina e tiametoxan. As populações de insetos foram coletadas em seis áreas de produção de arroz irrigado nos estados de Tocantins, Goiás e Santa Catarina, Brasil. Insetos adultos destas populações foram estabelecidos em gaiolas teladas de criação em casa de vegetação e em laboratório foram submetidos a concentrações discriminantes (LC 95 s) estimadas para a população de Santo Antônio de Goiás, coletada em ambiente natural e usada como população-padrão de susceptibilidade. As frequências de mortalidade de indivíduos para a população de Formoso do Araguaia foi de 76% para o inseticida carbosulfan, 86% para bifentrina, 72% para etofenproxi, 80% para L-cialotrina e de 80% para o inseticida tiametoxan. Embora as frequências de indivíduos resistentes encontradas sejam consideradas de baixas a moderadas, trata-se do primeiro registro de T. limbativentris resistentes a inseticidas e, já se percebe a extensão do problema com relação ao desenvolvimento de resistência a produtos de diferentes grupos e modos de ação, principalmente na região produtora de Formoso do Araguaia- TO. Os resultados desta pesquisa indicam uma possível ocorrência de resistência cruzada ou múltipla nessa população.

Les maladies impliquant des micro-organismes représentent un problème majeur de santé publique en raison du fort taux de mortalité provoqué par l'accroissement et l'émergence de souches résistantes. De même, les régions tropicales du Monde et La Réunion doivent faire face depuis plusieurs années à la pression accrue d'arthropodes ravageurs de cultures et vecteurs de maladies. Pour lutter contre ces arthropodes, les pesticides synthétiques aux conséquences désastreuses sur l'Homme et l'environnement sont principalement utilisés. Depuis quelques années, on observe un phénomène grandissant de résistance aux molécules conventionnelles à la fois chez les micro-organismes et les arthropodes. Face à ce constat, il est primordial de proposer de nouvelles molécules bioactives capables de lutter efficacement contre les ravageurs et les micro-organismes. Dans ce cadre, un criblage préliminaire réalisé sur vingt plantes endémiques et indigènes de La Réunion a permis de mettre en évidence l'activité antibactérienne et acaricide des extraits acétate d'éthyle de plusieurs espèces végétales. En raison de son large spectre d'activités biologiques, Peperomia borbonensis Miq. (Piperaceae), a été sélectionnée pour une investigation chimique et biologique approfondie. La suite de nos travaux a été consacrée à l'isolement et la caractérisation des métabolites secondaires de l'extrait apolaire de cette espèce. Les fractionnements bio-guidés réalisés ont conduit à l'isolement de quatorze molécules dont un amide à la structure nouvelle. Par ailleurs, l'étude de la fraction volatile de cette espèce a montré le potentiel insecticide de l'huile essentielle des feuilles pour lutter contre la mouche du melon B. cucurbitae. / The emergence of resistant strains in infectious diseases is a major public health issue. Similarly, arthropods which caused important economic damages in tropical and subtropical areas are continuously developing resistance to chemicals. To counteract this situation, it is essential to find new bioactive molecules able to fight pests and micro-organisms. In this context a preliminary screening was performed on twenty endemic plants and native of Reunion. Twenty-six ethyl acetate extracts were tested for their antibacterial and acaricidal activities. Considering its broad spectrum of activity, Peperomia borbonensis Miq. (Piperaceae), was selected for further chemical and biological investigations. Two bio-guided fractionations were performed on its apolar extract and let to the isolation of fourteen molecules. Moreover, the study of the volatile fraction has shown the insecticidal potential of the leaf essential oil of P. borbonensis against the melon fly B. cucurbitae.

Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides. With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors. In vitro assay studies demonstrate that tri- and difluoromethyl ketones can potentially inhibit An. gambiae AChE (AgAChE). These compounds inhibit the enzyme by making a covalent adduct with the catalytic serine of AChE. Trifluoromethyl ketones however are poor inhibitors of the G119S resistant mutant of AgAChE. However difluoromethyl ketones can inhibit G119S AgAChE and compound 3-10g showed an IC₅₀ value of 25.1 nM after 23h incubation time. Despite this potent inhibition of AgAChE, the tri-, di-, and (mono)fluoroketones showed very low toxicity to An. gambiae, perhaps due to hydration and rapid clearance. In an attempt to improve An. gambiae toxicity, oximes and oxime ethers of these compounds were prepared as potential prodrugs. These structures identified trifluoromethyl ketone oxime 3-2d as a potent toxin against both wild-type (G3-strain) and a multiply resistant (Akron) strain of An. gambiae. This compound is within 3-fold of the toxicity of propoxur to wild type An. gambiae (LC₅₀ values of 106 and 39 µg/mL, respectively). Most significantly, 3-2d was much more toxic than propoxur to multiply-resistant (Akron) strain An. gambiae (LC₅₀ = 112 and >5,000 µg/mL, respectively). However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism. Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur over 22 h at pH 7.7 or 5.5. The mechanism of action of 3-2d remains unknown. Our enzyme inhibition studies have demonstrated that 3-2d does not hydrolyze to the trifluoromethyl ketone 2-9d at pH 7.7. The high Akron toxicity of 3-2d and poor inhibition of G119S AgAChE by 2-9d argue against enzyme mediated conversion of 3-2d to 2-9d within the mosquito. Thus, we can rule out an AChE inhibition mechanism for toxicity. Additional experiments by our collaborator (Dr. Jeffrey Bloomquist, University of Florida) also rule out inhibition of mitochondrial respiration or agonism of the muscarinic acetylcholine receptor. Future work will address other potential insecticidal modes of action. / Ph. D.

Malaria is one of the deadliest diseases known to mankind. In 2010, 219 million cases were reported, and 666,000 deaths were attributed to this disease. In the past, pyrethroid-treated mosquito nets have shown efficacy in reducing malaria transmission in many malaria endemic regions. However, an upsurge in the mosquito population that is resistant to pyrethroids threatens to compromise the efficacy of pyrethroid-treated bed nets. In an effort to develop another class of insecticide with a different mode of action, we have explored three classes of five membered heterocyclic carbamates (isoxazol-3-yl, pyrazol-5-yl, and pyrazol-4-yl), and 3-oxoisoxazole- 2(3H)-carboxamide as acetylcholinesterase inhibitors (AChE) targeting wild type (G3) and resistant (Akron) malaria mosquito Anopheles gambiae (Ag). Isoxazole carboxamide and carbamates were obtained regioselectively through judicious use of two different protocols. The final products were characterized and identified using ¹H and ¹³C NMR, and mass spectroscopy. In addition, the carboxamide structure was confirmed using X-ray diffraction. Several of the novel carbamates and carboxamides evaluated exhibited excellent toxicity towards susceptible G3 and resistant Akron strain An. gambiae (48f LC₅₀ G3 = 41 μg/mL, LC₅₀ Akron = 58 μg/mL, and 47i LC₅₀ G3 = 38 μg/mL, LC₅₀ Akron = 40 μg/mL). Hence, achieving the resistance- breaking goal. On the contrary, the commercial aryl methylcarbamates currently approved for indoor residual sprays (IRS) showed no potency towards the resistant strain An. gambiae (LC₅₀ G3 = 16-42 μg/mL, and LC₅₀ Akron >5,000 μg/mL). Further, we observed low toxicological cross-resistance ratios (RR) for the toxic isoxazol-3-yl and pyrazol-4-yl carbamates, and 3- oxoisoxazole-2(3H)-carboxamides (RR = 0.5-2.0). Amongst the commercial AChE inhibitors approved for IRS, only aldicarb exhibited such low RR (RR = 0.5), whereas the RR for commercial aryl methylcarbamates exceed 130-fold. The low RR observed for these novel heterocyclic inhibitors would certainly be favorable for a new anticholinesterase-based mosquitocide targeting both the susceptible and resistant strain mosquitoes. Although the overall selectivity (Ag vs human) did not exceed 24-fold, the heterocyclic carbamates and carboxamides synthesized by the author showed appreciable inhibition of resistant AChE (G119S) in comparison to commercial aryl carbamates, which showed no inhibition at all. During the course of this project, the isoxazol-3-yl and pyrazol-5-yl methylcarbamates proved to be unstable, and thus could not be isolated. The synthesis of pyrazol-4-yl methylcarbamates using N-methylcarbamoyl chloride proved particularly challenging due to the formation of by-products called allophanates. The similar Rf of the by-product and the desired final product made the isolation laborious and time-consuming. We have successfully overcome this problem by employing a new protocol, where triphosgene served as the carbonylating agent and N-methylamine in THF was used as the amine source. In addition, we have also developed another one-pot protocol for a safer synthesis of pyrazol-4-yl methylcarbamates utilizing 1,1- carbonyldiimidazole (CDI), and N-methylamine hydrogen chloride salt. With the pyrazol-4-yl core, apart from achieving excellent toxicity towards both strains of An. gambiae, we have also achieved excellent AgAChE vs hAChE selectivity (Ag vs h >100-fold). Due to our continued interest in developing this core, we have devised a convenient, scalable, no-column approach for the synthesis an intermediate 103 that can be utilized to synthesize these compounds more efficiently. / Ph. D.