Investigations of host-associated differentiation of parasitoids have largely focused on the degree of molecular genetic differentiation, but a true test of species status must examine the mating patterns of differentiated populations to determine if they can interbreed in the wild and produce viable offspring. We examined possible mechanisms of isolation between two genetically distinct host-plant complex races of the braconid, Cotesia congregata, originating from hosts on tobacco and catalpa. We compared male responses to female pheromones, elements of male acoustic courtship signals, and breeding success between the two races. Males responded to pheromones from both sources and male courtship signals showed only subtle differences, suggesting that factors other than courtship behavior may be involved in isolation of the two races. However, nearly 90% of females from one hybrid cross failed to produce offspring, leading to post-zygotic isolation. Development time, emerged brood size, and sex ratios between the races also differed.

Cotesia congregata est une guêpe parasitoïde qui se développe à l’intérieur de la larve du Lépidoptère, Manduca sexta. Ce parasitoïde a développé une stratégie de virulence qui utilise un symbionte viral de la famille des Polydnavirus nommé Cotesia congregata bracovirus. Mon travail de thèse a permis de caractériser le dialogue moléculaire au cours de l’interaction par des approches de transcriptomique à haut débit et de physiologie. Ces travaux ont permis d’établir la première carte fonctionnelle du génome viral et de visualiser l’ensemble des gènes de M. sexta régulés au cours du parasitisme. La régulation des gènes de l’immunité a révélé que le parasitisme n’empêche pas l’induction de peptides antimicrobiens, mais entraine la sous-expression de gènes impliqués dans la réponse cellulaire. De plus j’ai pu mettre en évidence une réduction du nombre de cellules adhérentes au cours de l’interaction et décrire l’induction d’un phénotype de type apoptose d’une catégorie de cellules immunitaires. Ces résultats permettent d’identifier des gènes acteurs de l’interaction et apportent de nouvelles connaissances relatives aux interactions hôtes-parasitoïdes. / Cotesia congregata is a parasitoid wasp that develops inside the lepidopteran larvae, Manduca sexta. This parasitoid wasp has evolved virulence strategies using an obligate viral symbiont from the Polydnavirus family named Cotesia congregata bracovirus. My thesis work has allowed us to characterize the molecular dialogue during the interaction by physiological and high-throughput transcriptomic approaches. This work allowed to obtain the first functional map of the viral genome and to identify all M. sexta genes regulated during parasitism. Regulation of immune genes revealed that parasitism does not prevent induction of antimicrobial peptides, but leads to the down-regulation of genes involved in the cellular response. Moreover, I was able to demonstrate a reduction in the number of adherent cells during the interaction and to describe this induction as an apoptosis-like phenotype targeting a specific population of immune cells. These findings open the way to the identification of candidate genes involved in this particular interaction and provide new insights into host-parasitoid interactions in general.

Les Hyménoptères parasitoïdes ont un développement larvaire s'effectuant au détriment d'un organisme hôte. Pour exploiter au mieux la ressource que représente un hôte arthropode dont la biologie peut présenter certains obstacles tels que la mobilité et le système immunitaire, les parasitoïdes ont développé une diversité modes de vie et de stratégies de virulence. Ce manuscrit replace les parasitoïdes dans leur contexte évolutif afin de mieux comprendre la diversité surprenante de leurs modes de vie. Ces modes de vie conditionnent la nature des interactions dans les systèmes hôte/parasitoïde. Nous verrons comment, par l'utilisation de nombreux facteurs de virulence tel que le venin, les polydnavirus et bien d'autres encore, les parasitoïdes manipulent la physiologie de leur hôte afin de le rendre adéquat à leur propre développement. Ce travail s'est intéressé au modèle endoparasitoïde Hyposoter didymator (Hym., Ichneumonidae). Nous avons ainsi caractérisé les protéines produites dans la glande à venin des femelles et identifié l'ensemble des gènes du polydnavirus associé (HdIV; H. didymator Ichnovirus), grâce à des techniques de protéomique, génomique et transcriptomique. Nous avons également suivi et quantifié les altérations de la physiologie de l'hôte Spodoptera frugiperda au cours du parasitisme et évalué le rôle relatif de différents facteurs dans ces perturbations et dans la réussite parasitaire. Nos résultats ont permis de montrer que seul le fluide du calice contenant HdIV est nécessaire au développement du parasitoïde. En parallèle, nous avons mis à jour une propriété immuno-évasive des œufs d'H. didymator liée à des protéines associées à l'exochorion. L'ensemble de ce travail a permis de dessiner un élégant schéma expliquant la complémentarité spatio-temporelle des facteurs de virulence durant le parasitisme. Finalement, nous avons cherché à mieux comprendre le déterminisme du spectre d'hôte d'H. didymator, ce qui nous a conduit à montrer que les deux stratégies de contournement de la réponse immunitaire (immuno-évasion et infection virale) se révèlent inefficaces chez les hôtes non-permissifs. / Parasitic wasps must deal with physiological features of their host such as mobility, an efficient immune system and a variable metabolism. To ensure successful parasitism in a large range of arthropod hosts, parasitoids display a huge diversity of lifestyle and rely in a variety of virulence factors. In this document, we introduce parasitoid lifestyle in an evolutionary context in order to better understand the parasitoid complexity. As the parasitoid lifestyle drives the host/parasitoid interaction outcome, we discuss for all how the virulence factors such as venom, polydnaviruses and many others are used to ensure successful development of the parasitoid. In this study, we focused on the endoparasitoid Hyposoter didymator (Hym., Ichneumonidae) virulence factors. We thus identified venom proteins and the genes from the associated polydnavirus, HdIV using proteomics, genomics and transcriptomics approaches. Studies on the effect of the venom and the calyx fluid containing the polydnavirus HdIV, reveal that only the calyx fluid is necessary for Spodoptera frugiperda host physiological alteration and parasitism success. Futhermore, this work presents the discovery of a local immune-evasive property of the H. didymator egg exochorion. All these data permitted us to design an effective spatio-temporal model of the virulence factor complementarity used by H. didymator during the parasitism time course. Finally, studies on the H. didymator host range reveals the inefficiency of the different virulence factors in non-permissive hosts, opening insights on the host permissiveness molecular mechanisms.

Cotesia flavipes (Cameron) (Hym.: Braconidae), como outros cenobiontes, é capaz de regular seu hospedeiro, criando um ambiente que sustenta e promove o desenvolvimento de suas larvas, comumente em detrimento do hospedeiro. Substâncias derivadas do trato reprodutivo das fêmeas (proteínas ovarianas, veneno e polidnavírus) são injetadas no hospedeiro, afetando a resposta imune e outros processos fisiológicos com o propósito de regular os níveis hormonais, nutrição e comportamento. O presente trabalho teve por objetivo avaliar o papel dessas substâncias no crescimento e desenvolvimento de Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), e avaliar como o parasitismo afeta a resposta imune do hospedeiro. Todas as substâncias derivadas da fêmea foram obtidas após a dissecação do parasitóide, através da coleta do reservatório de veneno ou dos ovários (proteínas ovarianas e polidnavírus) em tampão resfriado. As secreções foram processadas adequadamente e injetadas logo após a coleta. O veneno e as proteínas ovarianas + polidnavírus (PDV) foram injetados juntos ou separadamente em lagartas entre 0-12h do 6º instar. O efeito de cada um dos componentes isolados do parasitóide no desenvolvimento e crescimento do hospedeiro foi avaliado através de observações no ganho de peso, duração e viabilidade da fase larval e pupal. Os efeitos do parasitismo na resposta imune do hospedeiro foram avaliados tanto ao nível celular, através da contagem do número total de hemócitos e capacidade de encapsulação, como ao nível bioquímico, medindo-se a ativação da profenoloxidase e produção de óxido nítrico na hemolinfa das lagartas de D. saccharalis em diferentes estágios de desenvolvimento do parasitóide (0, 1, 3, 5, 7 e 9 dias após o parasitismo). As proteínas ovarianas do parasitóide e o PDV sozinho, ou co-injetado com o veneno, suspenderam o desenvolvimento larval do hospedeiro, enquanto que o veneno, sozinho, afetou o processo de metamorfose. A resposta imune do hospedeiro também foi afetada por C. flavipes, de maneira dependente do tempo. Lagartas parasitadas apresentaram declínio no número total de hemócitos a partir do 3º dia e a capacidade de encapsulação foi afetada ao longo do desenvolvimento do parasitóide. A atividade da fenoloxidase do hospedeiro foi alterada apenas no final do desenvolvimento imaturo do parasitóide, enquanto que o óxido nítrico foi afetado nas 24 h iniciais após parasitismo. / Cotesia flavipes (Cameron) (Hym., Braconidae), as other koinobionts, is capable of regulating the host development to produce an suitable host environment to sustain and promote its own larval development at the host expenses. Female-derived substances from the reproductive tract (ovarian proteins, venom, polydnavirus) are injected into the host, affecting the host immune response and other physiological processes aiming to regulate the host hormone levels, nutrition and behavior. Our goal was to evaluate the role of these substances on Diatraea saccharalis (F.) (Lepidoptera: Crambidae) growth and development, and how the parasitism affects the host immune response. All female-derived substances were collected after parasitoid dissection by collecting the venom reservoir or the ovaries (ovarian proteins and polydnavirus). Dissections were carried out in ice-cold buffer, collected tissues were processed accordingly and the desired substances injected immediately after collection. Venom and ovarian proteins+polydnavirus (PDV) were injected jointly and separated in 0-12 hold 6th instars of D. saccharalis. The effect of these substances on host development and growth was evaluated by measuring the host weight gain, larval and pupal survivorship and developmental time. The effects of the parasitism on the host immune response was evaluated either at the cellular level, by measuring the total hemocyte count and the encapsulation capacity, and at the biochemical level, by measuring the prophenoloxidase activity and nitric oxide levels at different stages of parasitoid development (0, 1, 3, 5, 7 and 9 days after parasitism). Parasitoid ovarian proteins and PDV alone or co-injected with the venom arrested the host larval development, while the venom by itself only affected the host metamorphosis process. The host immune response was also affected by C. flavipes at a time-dependent manner. The total hemocyte count dropped at day 3 of parasitism, while the host encapsulation capacity was reduced during parasitoid development. The host prophenoloxidase activity was also affected mainly towards the end of parasitoid larval development, while the nitric oxide at the first 24 h after parasitism.

Parasitic wasps are highly diverse and play a major role in suppression of herbivorous pest populations, but relatively little is known of the mechanisms driving their diversity. Molecular studies indicate that cryptic species complexes resulting from adaptations to specific hosts or host-foodplants may be common. The gregarious endoparasitoid, Cotesia congregata (Braconidae), is a model system for understanding parasitic wasp biology. It is reported to attack at least 15 species of sphingid caterpillars, most of which are plant family specialists. Molecular studies have demonstrated genetic differentiation of two host-foodplant complex sources originating from Manduca sexta on tobacco (MsT) and Ceratomia catalpae on catalpa (CcC). Response to female pheromone and elements of their courtship songs differ. Wasps from both sources mated and produced F1 hybrid offspring in the laboratory; however, 90% of hybrid females resulting from one of the reciprocal crosses failed to produce offspring. I built on this previous work by evaluating an ecological barrier, the evolution of courtship songs within the genus, and patterns of hybrid sterility among four additional host-foodplant complexes, as well as differentiation of their symbiotic bracovirus. Tests of developmental tolerance to nicotine demonstrate that MsT wasps are highly adapted to hosts feeding on tobacco, whereas CcC wasps experience high mortality. Acoustic analysis of courtship songs among host-foodplant sources of C. congregata and eleven additional species of Cotesia demonstrates that songs are species specific and appear to be correlated with genetic relatedness. Cotesia congregata from all sources mated and produced F1 hybrid offspring in the laboratory; however, hybrid females resulting from specific reciprocal crosses failed to produce progeny. Dissections of hybrid females revealed that sterile wasps lacked mature ovaries and functional bracovirus, a symbiotic virus integrated into the wasp genome and necessary to suppress the host immune system. Relative in vivo expression of wasp bracovirus genes differs between MsT and CcC host-foodplant complexes. Cumulatively, these behavioral, ecological, and genetic barriers to reproduction indicate that C. congregata is diverged into two incipient species with limited gene flow, and provides insight into the role of varied reproductive barriers in speciation of parasitic wasps.

Les bracovirus forment une symbiose avec les guêpes parasitoïdes, demeurant dans leur génome et produits uniquement dans leurs ovaires. Nous avons caractérisé comment les cercles d’ADNdb contenu dans les particules étaient amplifiés depuis leur forme provirale avant leur encapsidation.Nous avons montré que le site d’intégration du génome viral est conservé chez les bracovirus et organisé dans le génome de la guêpe en un macrolocus regroupant la majorité des segments proviraux et 7 loci isolés. Nous avons mis en évidence 12 unités de réplication (UR) et que les 9 gènes viraux du cluster nudiviral étaient amplifiés sur une UR sans être encapsidés. Nous avons identifié des concatémères tête-tête et queue-queue comme étant les intermédiaires de réplication des UR, caractéristiques d’une réplication linéaire du génome viral. Enfin, nous avons montré que l’ADN polymérase B2 appartenait à un élément Maverick. L’absence de gènes viraux de la réplication du génome viral semble indiquer que la machinerie réplicative cellulaire serait impliquée. Il reste maintenant à mettre en évidence les différents facteurs cellulaires participant à l’amplification du génome viral. / Bracovirus form a symbiosis with parasitoid wasp, remaining in their genome and products only in their ovaries. We characterized how packaged dsDNA circles were amplified from their proviral genome before packaging in viral particles.We showed that viral genome integration site is conserved in bracovirus and organized in the wasp genome in a macrolocus where the majority of proviral segments was found and 7 isolated loci. We showed 12 replication units (UR) and the 9 nudiviral genes from cluster were amplified on a UR without being packaged. We identified concatemers head-head and tail-tail as the replication intermediates of UR, indicating a linear replication of the viral genome. Finally, we showed that DNA polymerase B2 belonged to a Maverick element.The absence of viral gene involved in the genome replication suggests that the cellular replication machinery is involved. It remains to highlight the different cellular factors involved in the amplification of the viral genome.

Cotesia flavipes (Cameron) (Hym.: Braconidae), como outros cenobiontes, é capaz de regular seu hospedeiro, criando um ambiente que sustenta e promove o desenvolvimento de suas larvas, comumente em detrimento do hospedeiro. Substâncias derivadas do trato reprodutivo das fêmeas (proteínas ovarianas, veneno e polidnavírus) são injetadas no hospedeiro, afetando a resposta imune e outros processos fisiológicos com o propósito de regular os níveis hormonais, nutrição e comportamento. O presente trabalho teve por objetivo avaliar o papel dessas substâncias no crescimento e desenvolvimento de Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), e avaliar como o parasitismo afeta a resposta imune do hospedeiro. Todas as substâncias derivadas da fêmea foram obtidas após a dissecação do parasitóide, através da coleta do reservatório de veneno ou dos ovários (proteínas ovarianas e polidnavírus) em tampão resfriado. As secreções foram processadas adequadamente e injetadas logo após a coleta. O veneno e as proteínas ovarianas + polidnavírus (PDV) foram injetados juntos ou separadamente em lagartas entre 0-12h do 6º instar. O efeito de cada um dos componentes isolados do parasitóide no desenvolvimento e crescimento do hospedeiro foi avaliado através de observações no ganho de peso, duração e viabilidade da fase larval e pupal. Os efeitos do parasitismo na resposta imune do hospedeiro foram avaliados tanto ao nível celular, através da contagem do número total de hemócitos e capacidade de encapsulação, como ao nível bioquímico, medindo-se a ativação da profenoloxidase e produção de óxido nítrico na hemolinfa das lagartas de D. saccharalis em diferentes estágios de desenvolvimento do parasitóide (0, 1, 3, 5, 7 e 9 dias após o parasitismo). As proteínas ovarianas do parasitóide e o PDV sozinho, ou co-injetado com o veneno, suspenderam o desenvolvimento larval do hospedeiro, enquanto que o veneno, sozinho, afetou o processo de metamorfose. A resposta imune do hospedeiro também foi afetada por C. flavipes, de maneira dependente do tempo. Lagartas parasitadas apresentaram declínio no número total de hemócitos a partir do 3º dia e a capacidade de encapsulação foi afetada ao longo do desenvolvimento do parasitóide. A atividade da fenoloxidase do hospedeiro foi alterada apenas no final do desenvolvimento imaturo do parasitóide, enquanto que o óxido nítrico foi afetado nas 24 h iniciais após parasitismo. / Cotesia flavipes (Cameron) (Hym., Braconidae), as other koinobionts, is capable of regulating the host development to produce an suitable host environment to sustain and promote its own larval development at the host expenses. Female-derived substances from the reproductive tract (ovarian proteins, venom, polydnavirus) are injected into the host, affecting the host immune response and other physiological processes aiming to regulate the host hormone levels, nutrition and behavior. Our goal was to evaluate the role of these substances on Diatraea saccharalis (F.) (Lepidoptera: Crambidae) growth and development, and how the parasitism affects the host immune response. All female-derived substances were collected after parasitoid dissection by collecting the venom reservoir or the ovaries (ovarian proteins and polydnavirus). Dissections were carried out in ice-cold buffer, collected tissues were processed accordingly and the desired substances injected immediately after collection. Venom and ovarian proteins+polydnavirus (PDV) were injected jointly and separated in 0-12 hold 6th instars of D. saccharalis. The effect of these substances on host development and growth was evaluated by measuring the host weight gain, larval and pupal survivorship and developmental time. The effects of the parasitism on the host immune response was evaluated either at the cellular level, by measuring the total hemocyte count and the encapsulation capacity, and at the biochemical level, by measuring the prophenoloxidase activity and nitric oxide levels at different stages of parasitoid development (0, 1, 3, 5, 7 and 9 days after parasitism). Parasitoid ovarian proteins and PDV alone or co-injected with the venom arrested the host larval development, while the venom by itself only affected the host metamorphosis process. The host immune response was also affected by C. flavipes at a time-dependent manner. The total hemocyte count dropped at day 3 of parasitism, while the host encapsulation capacity was reduced during parasitoid development. The host prophenoloxidase activity was also affected mainly towards the end of parasitoid larval development, while the nitric oxide at the first 24 h after parasitism.

Parasitoides criam condições favoráveis para seu desenvolvimento regulando processos fisiológicos do hospedeiro via a utilização de moléculas produzidas por tecidos maternos ou por tecidos derivados do parasitoide imaturo ou de simbiontes associados (vírus de poliDNA - PDV). Assim, essas moléculas se destacam como uma fonte preciosa para o desenvolvimento de métodos alternativos de controle de pragas. O objetivo desse trabalho foi o de explorar a interação hospedeiro-parasitoide, visando à identificação de moléculas que participem do processo de regulação hospedeira para o desenvolvimento de métodos alternativos de controle de pragas. A busca pela identificação dessas moléculas foi concentrada na interação Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) pela avaliação i) do efeito do parasitismo na fisiologia digestiva do hospedeiro e ii) da contribuição da glândula de veneno e do ovário na produção de moléculas bioativas e iii) pela caracterização de transcritos produzidos por teratócitos desse parasitoide. O potencial de aplicação de moléculas derivadas da interação hospedeiro-parasitoide foi verificado pela caracterização da atividade de uma quitinase putativa isolada da associação Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae), via expressão heteróloga e transformação genética de plantas de fumo. Os resultados demonstraram que o parasitismo afeta a utilização do alimento por lagartas parasitadas, reduzindo as taxas de crescimento (RGR), consumo (RCR) e metabólica (RMR) relativas, enquanto aumenta a eficiência de conversão do digerido (ECD). O parasitismo por C. flavipes também aumentou a permanência do alimento no intestino do hospedeiro e a atividade de -amilase, sacarase e trealase em lagartas de D. saccharalis. Análises do transcriptoma da glândula de veneno e do ovário de C. flavipes indicaram a produção de muitas proteínas desconhecidas. Porém, foi possível identificar proteases, um inibidor de proteinase e uma proteína com ação antimicrobiana, produzidas pela glândula de veneno, e uma proteína de expressão precoce (ep1), como produto do ovário de C. flavipes. Dois transcritos isolados de teratócitos de C. flavipes foram putativamente identificados como serpina e fator de inibição da tradução (CfHTIF). Ambos os transcritos foram detectados em teratócitos após cinco dias do parasitismo, sendo que transcritos do CfHTIF também foram observados em hemócitos de hospedeiros parasitados. CfHTIF apresentou alta homologia a proteínas produzidas por PDVs associados a outras espécies de Cotesia, sugerindo a sua origem viral, sendo este o primeiro relato da utilização de teratócitos para a produção de proteínas virais voltadas à regulação hospedeira. A caracterização da atividade da quitinase de teratócitos de T. nigriceps e a avaliação do seu potencial biotecnológico via transgenia de plantas para o controle de lagartas de H. virescens indicou que, apesar dessa quitinase possuir capacidade de ligação à quitina coloidal e cristalina em ensaios in vitro, a mesma não demonstra qualquer atividade quitinolítica. Avaliações de plantas de fumo transformadas com o Tnchi não indicaram qualquer efeito deletério ao desenvolvimento de lagartas de H. virescens. Apesar da ausência de atividade quitinolítica, essa quitinase apresentou atividade antimicrobiana. A possibilidade de exploração dessa proteína na produção de plantas de interesse econômico é discutida. / Parasitoids regulate their hosts physiological processes in order to produce suitable conditions for their own development by employing molecules produced by maternal tissues, the immature parasitoid and derived tissues, and associated simbionts (polidnavirus PDV). These molecules represent, therefore, an untapped resource of new molecules for the development of alternative pest control methods. Our objective was to exploit the hostparasitoid interactions aiming at the identification of molecules involved in host regulation for their use in the development of alternative strategies for pest control. The search for new molecules was concentrated on the interaction Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) by assessing i) the effects of parasitization on the host digestive physiology and ii) the contribution of the venom gland and ovary in producing regulatory molecules, and by iii) the characterization of cDNAs isolated from teratocytes of C. flavipes. The potential application of molecules from hostparasitoid associations was verified by characterizing the activity of a putative chitinase isolated from the interaction Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) using recombinant proteins and geneticallymodified tobacco plants. Data obtained demonstrated parasitized larvae had reduced relative growth (RGR), consumption (RCR) and metabolic (RMR) rates, while an increased efficiency in the conversion of the digested food (ECD) was obtained as compared to unparasitized larvae. Larvae parasitized by C. flavipes retained the food in the gut for a longer period and had higher activities for -amylase, sacarase and trehalase if compared to control larvae. Analysis of the transcriptome of the venom gland and ovary of C. flavipes predicted a large number of unknown proteins. Nevertheless, several proteases, a protease inhibitor and an antimicrobial protein from the venom gland, and an early-expressed protein (ep1), with a putative role in the process of host regulation from the ovaries, were identified. Two cDNAs isolated from teratocytes of C. flavipes were putatively identified as a serpin and an eukaryote translation inhibitory factor (CfHTIF). Both were detected in teratocytes from day 5 of parasitism and onwards, with the CfHTIF being also detected in hemocytes of parasitized hosts. Comparative analysis of the CfHTIF indicated it is highly homologous to host translation inhibitory factors produced by PDVs associated with other species of Cotesia, indicating its possible viral origin and suggesting this to be the first record of teratocytes as a source of PDV-derived proteins for host regulation. The characterization of the activity from the chitinase isolated from teratocytes of T. nigreceps (Tnchi) and the evaluation of its biotechnological potential via plant transgenesis to control larvae of H. virescens indicated this chitinase can bind to colloidal and crystalline chitins in in vitro assays, but it does not display chitinolytic activity. Assays with transformed tobacco plants expressing Tnchi did not result in any effect to H. virescens larval development and survival. Although Tnchi did not have any chitinolytic activity, it did show to have an antimicrobial effect. The possible exploitation of this protein for production of economically-important plants is dicussed.

Les associations mutualistes, en permettant l'acquisition de nouvelles fonctions, ont joué un rôle majeur dans l'évolution des espèces. Pour comprendre en quoi ces associations sont impliquées dans l'adaptation des espèces nous avons étudié un cas unique de mutualisme associant un virus de type polydnavirus et une guêpe parasitoïde. Dans cette association, le virus est injecté dans l'hôte lépidoptère lors de l'oviposition et joue un rôle majeur dans le succès parasitaire en induisant une altération des fonctions physiologiques de l'hôte. En regard du nombre d'espèces de guêpes caractérisées par cette association, le virus doit constituer une innovation adaptative majeure et jouer un rôle déterminant dans l'évolution et la diversification des espèces de guêpes. En quoi cette association joue t-elle un rôle déterminant dans l'évolution et l'adaptation des guêpes ? Quelles sont les fonctions physiologiques ciblées chez l'hôte ? Pour répondre à ces questions nous avons étudié l'évolution de deux familles de gènes codant pour des facteurs de virulence potentiels et nous avons exploré les fonctions physiologiques d'une protéine potentiellement ciblée lors du parasitisme. Nous avons mis en évidence le rôle important de la sélection naturelle dans l'évolution des familles de gènes viraux. Par modélisation de la structure tridimensionnelle d'un facteur de virulence codant pour des cystatines, nous avons montré que cette sélection agissait préférentiellement au niveau des sites d'interaction avec les protéines cibles. De plus, cette étude souligne le caractère dynamique de l'évolution des facteurs de virulence incluant de multiples évènements de duplication, caractérisés par des processus de perte et d'acquisition au cours de l'évolution de l'association. Le caractère adaptatif et dynamique de l'évolution des gènes viraux a aussi été étudié en regard de l'évolution des espèces de guêpes et de leur spectre d'hôte. Par une approche fonctionnelle, nous avons étudié le rôle physiologique de protéases à cystéine qui constituent des cibles potentielles des cystatines virales. Nous avons montré que ces protéases sont régulées spécifiquement au cours du parasitisme au niveau protéique et transcriptionnel. Nous avons également montré que l'activité de ces protéases est modifiée après parasitisme. L'évolution adaptative et dynamique des facteurs de virulence reflètent leur rôle important dans le parasitisme. Il reste maintenant à montrer comment ces facteurs interagissent sur la physiologie de l'hôte lépidoptère. Des protéases à cystéine sont spécifiquement ciblées par le parasitisme, en étudiant les mécanismes d'interaction de ces protéases avec les cystatines virales et les processus coévolutifs mis en jeu, nous

Parasitoides criam condições favoráveis para seu desenvolvimento regulando processos fisiológicos do hospedeiro via a utilização de moléculas produzidas por tecidos maternos ou por tecidos derivados do parasitoide imaturo ou de simbiontes associados (vírus de poliDNA - PDV). Assim, essas moléculas se destacam como uma fonte preciosa para o desenvolvimento de métodos alternativos de controle de pragas. O objetivo desse trabalho foi o de explorar a interação hospedeiro-parasitoide, visando à identificação de moléculas que participem do processo de regulação hospedeira para o desenvolvimento de métodos alternativos de controle de pragas. A busca pela identificação dessas moléculas foi concentrada na interação Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) pela avaliação i) do efeito do parasitismo na fisiologia digestiva do hospedeiro e ii) da contribuição da glândula de veneno e do ovário na produção de moléculas bioativas e iii) pela caracterização de transcritos produzidos por teratócitos desse parasitoide. O potencial de aplicação de moléculas derivadas da interação hospedeiro-parasitoide foi verificado pela caracterização da atividade de uma quitinase putativa isolada da associação Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae), via expressão heteróloga e transformação genética de plantas de fumo. Os resultados demonstraram que o parasitismo afeta a utilização do alimento por lagartas parasitadas, reduzindo as taxas de crescimento (RGR), consumo (RCR) e metabólica (RMR) relativas, enquanto aumenta a eficiência de conversão do digerido (ECD). O parasitismo por C. flavipes também aumentou a permanência do alimento no intestino do hospedeiro e a atividade de -amilase, sacarase e trealase em lagartas de D. saccharalis. Análises do transcriptoma da glândula de veneno e do ovário de C. flavipes indicaram a produção de muitas proteínas desconhecidas. Porém, foi possível identificar proteases, um inibidor de proteinase e uma proteína com ação antimicrobiana, produzidas pela glândula de veneno, e uma proteína de expressão precoce (ep1), como produto do ovário de C. flavipes. Dois transcritos isolados de teratócitos de C. flavipes foram putativamente identificados como serpina e fator de inibição da tradução (CfHTIF). Ambos os transcritos foram detectados em teratócitos após cinco dias do parasitismo, sendo que transcritos do CfHTIF também foram observados em hemócitos de hospedeiros parasitados. CfHTIF apresentou alta homologia a proteínas produzidas por PDVs associados a outras espécies de Cotesia, sugerindo a sua origem viral, sendo este o primeiro relato da utilização de teratócitos para a produção de proteínas virais voltadas à regulação hospedeira. A caracterização da atividade da quitinase de teratócitos de T. nigriceps e a avaliação do seu potencial biotecnológico via transgenia de plantas para o controle de lagartas de H. virescens indicou que, apesar dessa quitinase possuir capacidade de ligação à quitina coloidal e cristalina em ensaios in vitro, a mesma não demonstra qualquer atividade quitinolítica. Avaliações de plantas de fumo transformadas com o Tnchi não indicaram qualquer efeito deletério ao desenvolvimento de lagartas de H. virescens. Apesar da ausência de atividade quitinolítica, essa quitinase apresentou atividade antimicrobiana. A possibilidade de exploração dessa proteína na produção de plantas de interesse econômico é discutida. / Parasitoids regulate their hosts physiological processes in order to produce suitable conditions for their own development by employing molecules produced by maternal tissues, the immature parasitoid and derived tissues, and associated simbionts (polidnavirus PDV). These molecules represent, therefore, an untapped resource of new molecules for the development of alternative pest control methods. Our objective was to exploit the hostparasitoid interactions aiming at the identification of molecules involved in host regulation for their use in the development of alternative strategies for pest control. The search for new molecules was concentrated on the interaction Diatraea saccharalis (F.) (Lepidoptera: Crambidae) Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) by assessing i) the effects of parasitization on the host digestive physiology and ii) the contribution of the venom gland and ovary in producing regulatory molecules, and by iii) the characterization of cDNAs isolated from teratocytes of C. flavipes. The potential application of molecules from hostparasitoid associations was verified by characterizing the activity of a putative chitinase isolated from the interaction Heliothis virescens (F.) (Lepidoptera: Noctuidae) Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) using recombinant proteins and geneticallymodified tobacco plants. Data obtained demonstrated parasitized larvae had reduced relative growth (RGR), consumption (RCR) and metabolic (RMR) rates, while an increased efficiency in the conversion of the digested food (ECD) was obtained as compared to unparasitized larvae. Larvae parasitized by C. flavipes retained the food in the gut for a longer period and had higher activities for -amylase, sacarase and trehalase if compared to control larvae. Analysis of the transcriptome of the venom gland and ovary of C. flavipes predicted a large number of unknown proteins. Nevertheless, several proteases, a protease inhibitor and an antimicrobial protein from the venom gland, and an early-expressed protein (ep1), with a putative role in the process of host regulation from the ovaries, were identified. Two cDNAs isolated from teratocytes of C. flavipes were putatively identified as a serpin and an eukaryote translation inhibitory factor (CfHTIF). Both were detected in teratocytes from day 5 of parasitism and onwards, with the CfHTIF being also detected in hemocytes of parasitized hosts. Comparative analysis of the CfHTIF indicated it is highly homologous to host translation inhibitory factors produced by PDVs associated with other species of Cotesia, indicating its possible viral origin and suggesting this to be the first record of teratocytes as a source of PDV-derived proteins for host regulation. The characterization of the activity from the chitinase isolated from teratocytes of T. nigreceps (Tnchi) and the evaluation of its biotechnological potential via plant transgenesis to control larvae of H. virescens indicated this chitinase can bind to colloidal and crystalline chitins in in vitro assays, but it does not display chitinolytic activity. Assays with transformed tobacco plants expressing Tnchi did not result in any effect to H. virescens larval development and survival. Although Tnchi did not have any chitinolytic activity, it did show to have an antimicrobial effect. The possible exploitation of this protein for production of economically-important plants is dicussed.