Dissertations / Theses on the topic 'Insects - Metamorphosis'

The project consists in studying different aspects of the regulation of insect metamorphosis, using the cockroach Blattella germanica and the mayfly Cloeon dipterum, as laboratory models. In B. germanica the idea has been to study the possible role of Myoglianin in regulation of the decrease of juvenile hormone production that occurs at the beginning of the last nymphal instar. Also, to study the possible role of the adult specifier factor E93 in the destruction of the PG after the imaginal molt. In C. dipterum the plan has been to study the mechanisms that regulate metamorphosis, particularly during the formation of the subimago, and to compare these mechanisms with those operating in neopteran insects, which are condensed in the so-called MEKRE93 pathway.
El projecte consisteix en estudiar diferents aspectes de la regulació de la metamorfosi dels insectes, utilitzant la panerola Blattella germanica i l’efímera Cloeon dipterum com a models de laboratori. A B. germanica, la idea ha estat estudiar el possible paper de la mioglianina en la regulació de la disminució de la producció d’hormona juvenil que es produeix al començament de l’últim instar nimfal. També ha estat previst estudiar el possible paper del factor especificador de l’adult E93 en la destrucció de la glàndula protorácica després de la muda imaginal. A C. dipterum, el pla ha estat estudiar els mecanismes que regulen la metamorfosi, particularment durant la formació del subimago, i comparar aquests mecanismes amb els que operen en insectes neòpters, condensats en l'anomenada via MEKRE93

Buildings have identities. Like people, they have an essence that people can appreciate whether or not they can consciously evaluate that impression. Buildings can have personality and character. They can be amiable, reserved, even abrasive; we can enjoy or detest being in them. How does design imbue this character? Sometimes a building's identity is so sympathetic towards a program that the original use is apparent even after a change of use. Is this solely the result of conscious alteration, or can the original architect assist this constancy by so thoroughly infusing character into the design that, short of demolition, a semblance of identity will always be retained? Buildings have identities which can be enhanced or repressed through renovation. But can a building inform? Can a building be a devise for spreading information? What essential characteristics are necessary for identification? Would a building, possessing the characteristics of another object, be identifiable as that thing? Can a building be an insect?
Master of Architecture

Thesis (Ph. D.)--University of Oregon, 2004.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 99-113). Also available for download via the World Wide Web; free to University of Oregon users.

Heavy metals are of great concern when released into the environment, especially at high concentrations. Because of their persistence and toxicity, they have the ability to impact organisms both directly and indirectly via bioaccumulation in the food chain. In this report the effects on aquatic insect composition and abundance as well as possible effects on metamorphosis from larvae to adults were examined in six lakes – three with elevated Zn and Pb concentrations and three reference lakes - situated in Arjeplog municipality. Aquatic larvae and adult aquatic insects were sampled one year apart, and the number of individuals and community composition of both life stages were compared. Contrary to my hypothesis, the results showed no significant differences in abundance, taxa richness or number of individuals in pollution sensitive taxa (EPT) due to contamination. However, the result showed that the effect of contamination on the number of insects is different at different life stages (larval or adult), with fewer adults than expected emerging from contaminated lakes. This is likely explained by detrimental effects, caused by high metal concentrations, obstructing metamorphosis and decreasing emergence success. In this study, the negative effects on emergence could foremost be observed in chironomids (Chironomidae), which was the most abundant insect taxon in both reference and contaminated sites. This leads to the conclusion that including effects on metamorphosis can provide useful insights when assessing effects of a contaminant on the health of freshwater ecosystems.

La maduració sexual dels animals està controlada per hormones. En els insectes alats aquesta maduració es coneix amb el nom de metamorfosi, i pot anar acompanyada d’una completa reorganització de la majoria d’òrgans i estructures juvenils (insectes holometàbols), o bé limitar-se a uns pocs teixits, principalment les ales i la genitàlia (insectes hemimetàbols). La metamorfosi dels insectes holometàbols (metamorfosi completa), que va aparèixer fa uns 300 milions d’anys a partir d’insectes hemimetàbols (metamorfosi incompleta), ha fet que aquest grup animal assolís un gran èxit evolutiu, ja que la diferent morfologia entre les formes juvenils i adultes els permet explotar hàbitats diferents i no competir entre elles. En tots els insectes els canvis metamòrfics estan controlats per dues hormones, la 20-hidroxiecdisona (20E) i l’hormona juvenil (HJ). La 20E s’encarrega d’induir les transicions entre les diverses mudes i estadis del desenvolupament, mentre que l’HJ modula la naturalesa d’aquestes transicions. La present tesi doctoral pretén aprofundir en el coneixement de la regulació molecular de la metamorfosi mitjançant l’estudi del procés de sumoilació, per una banda, i de la funció del gen E93 de l’altra. Per fer-ho s’han utilitzat tres models d’insectes: la panerola Blattella germanica (hemimetàbol), l’escarabat Tribolium castaneum (holometàbol basal) i la mosca Drosophila melanogaster (holometàbol modificat). La sumoilació és una modificació posttraduccional que consisteix en la unió de manera reversible d’una proteïna petita, anomenada Sumo (de l’anglès Small Ubiquitin-like MOdifier), a una proteïna diana. Aquesta unió comporta la modificació de les propietats de la proteïna diana canviant-ne la conformació, la localització subcel•lular, la capacitat d’interacció amb altres proteïnes o la capacitat d’unió al DNA (en el cas dels factors de transcripció), entre d’altres. En els insectes, l’estudi del procés de sumoilació s’havia centrat fins ara en l’holometàbol D. melanogaster, on s’ha vist que és imprescindible per a l’inici de la metamorfosi. En aquesta tesi doctoral s’ha descrit la funció d’aquest procés en el desenvolupament postembrionari d’un insecte molt més basal, B. germanica. Aquesta panerola té dos paràlegs Sumo, BgSumo1 i BgSumo3, mentre que D. melanogaster en té tan sols un, Smt3. Aquest fet permetia, doncs, estudiar l’evolució funcional de la sumoilació en els insectes, i com aquesta funció està distribuïda en animals amb dos proteïnes Sumo. Així, mitjançant la tècnica de l’RNA d’interferència in vivo, s’ha comprovat que la sumoilació és essencial per a la supervivència durant el desenvolupament postembrionari de B. germanica. A més, s’ha vist que el paràleg BgSumo1 és necessari per a que es doni correctament la muda imaginal, per a la transducció del senyal de la 20E durant aquesta muda i per a la proliferació de l’epiteli fol•licular de l’oòcit basal. D’altra banda, s’ha comprovat que diversos receptors nuclears que conformen la via de senyalització de la 20E, imprescindible per a l’inici de la metamorfosi, són capaços de sumoilar-se en condicions in vitro. La segona part del treball se centra en l’estudi funcional del factor de transcripció E93. Aquest, descrit prèviament en D. melanogaster com el responsable de la transmissió del senyal de la 20E en el procés de mort cel•lular programada durant la metamorfosi, s’ha comprovat en aquesta tesi que actua de regulador molt més general dels processos metamòrfics. Així, s’expressa fortament durant el període metamòrfic en els tres models estudiats (B. germanica, T. castaneum i D. melanogaster), i la seva absència bloqueja aquest procés. En l’hemimetàbol B. germanica, la manca de BgE93 durant el darrer estadi nimfal provoca la formació de nimfes supernumeràries, que mai assoleixen l’estadi adult. En l’holometàbol basal T. castaneum, per altra banda, l’absència de TcE93 impedeix la diferenciació adulta que es dóna durant la fase pupal i provoca la muda a un segon estadi pupal supernumerari. Finalment, en l’holometàbol modificat D. melanogaster també provoca un bloqueig general de la metamorfosi. A més, en aquests tres insectes E93 s’encarrega de reprimir l’expressió dels factors de transcripció Broad i Krüppel homolog-1, la presència dels quals impedeix la diferenciació adulta durant el darrer estadi juvenil dels insectes. Per tot això, aquest treball ha pogut descriure el factor de transcripció E93 com l’especificador adult dels insectes alats, pas essencial per avançar en el coneixement de la regulació de la metamorfosi i en l’estudi de l’aparició de la metamorfosi completa a partir d’insectes hemimetàbols.
All immature animals undergo remarkable morphological and physiological changes to become mature adults. In winged insects, metamorphic changes are either limited to a few tissues (hemimetaboly) or involve a complete reorganization of most tissues and organs (holometaboly). In both cases, adult differentiation requires a temporally regulated balance between cell death, tissue growth and morphogenesis. Two hormones control this balance, the steroid 20-hydroxyecdysone (20E) and juvenile hormone (JH). The main goal of this thesis is to characterize the molecular mechanisms underlying the metamorphic process in insects through (i) the study of sumoylation and (ii) the functional characterization of the E93 transcription factor. To this aim, the hemimetabolous cockroach Blattella germanica, as well as the basal holometabolous beetle Tribolium castaneum and the highly modified holometabolous fly Drosophila melanogaster were used. Sumoylation is a post-translational modification that consists on the covalent binding of a small protein, called Sumo (Small Ubiquitin-like MOdifier), to a target protein. This modification is involved in the regulation of various cellular processes such as nuclear-cytosolic transport, transcriptional regulation and progression of cell cycle, among others. Notably, whereas D. melanogaster has only one Sumo protein (Smt3), B. germanica has two, BgSumo1 and BgSumo3. In this thesis, by using RNAi in vivo experiments we have shown that, whereas BgSumo3 is dispensable for the correct development of B. germanica, reduction of BgSumo1 levels resulted in severe defects during the metamorphic transition, including a marked developmental delay due to impaired activation of the ecdysone-triggered signaling cascade. Furthermore, we have shown that all the proteins belonging to the ecdysone-dependent transcriptional cascade of nuclear hormone receptors (BgEcR, BgRXR, BgE75, BgHR3 and BgFTZ-F1) are SUMOylated in vitro. The second part of the thesis is focused on the functional characterization of the E93 gene. First described as a dedicated regulator of cell death, we have demonstrated that this factor controls all the metamorphic transformations in insects. Thus, in the hemimetabolous B. germanica the absence of E93 during the last nymphal instar causes the formation of supernumerary nymphal instars. Moreover, in the holometabolous T. castaneum and D. melanogaster the depletion of E93 impairs adult differentiation during the pupal period and, in the beetle, also causes the formation of a supernumerary pupal stage. Furthermore, E93 controls the essential downregulation of the anti-metamorphic factors Broad and Krüppel homolog-1, two proteins whose presence blocks adult metamorphosis during the pupal stage. In conclusion, our data demonstrate that, despite the evolutionary distance and the differences in the developmental strategies to reach adulthood, E93 is the universal adult specifier in winged insects.

La metamorfosi és un procés de canvi morfològic radical que succeeix en un període específic durant el desenvolupament postembrionari de diverses espècies animals, tals com amfibis o insectes. En el cas dels insectes, hi ha dos tipus principals de metamorfosi: de tipus hemimetàbol, on els individus fan una metamorfosi progressiva i les nimfes s’assemblen als adults, com succeeix en les xinxes, paneroles i saltamartins. En la metamorfosi holometàbola, en canvi, es dóna una transformació morfològica radical, de larva a pupa i de pupa a adult, com s’observa en papallones, escarabats i mosques. La metamorfosi holometàbola s’originà a partir d’ancestres hemimetàbols, i aquesta innovació va tenir molt d’èxit si considerem que més del 80% dels insectes actuals són espècies holometàboles. En tots dos tipus de metamorfosi la regulació és propiciada per l’acció de dues hormones, l’esteroide 20-hidroxiecdisona (20E), o hormona de la mud,a i el sesquiterpenoide hormona juvenil (HJ). Mentre que la 20E indueix les mudes, l’HJ reprimeix la metamorfosi. L’objectiu general d’aquesta tesi doctoral és ajudar a comprendre els mecanismes moleculars pels quals l’HJ reprimeix la metamorfosi, utilitzant com a model la panerola Blattella germanica, un insecte amb metamorfosi hemimetàbola poc modificada, i emmarcar aquests resultats en un context evolutiu. Així, hem estudiat el paper del receptor de l’HJ, el factor de transcripció Methoprene-tolerant (Met). Els estudis suprimint l’expressió d’aquest gen mitjançant RNA d’interferència (RNAi) en fases nimfals mostren que Met és necessari en la transducció del senyal hormonal, ja que la seva supressió provoca una metamorfosi precoç. El factor de transcripció Taiman (Tai) es postula com a millor candidat a actuar com a heterodímer de Met en la recepció de l’HJ, encara que cap experiment in vivo ha pogut demostrar aquesta funció degut a que la seva supressió en diversos models d’insectes resultà letal. A B. germanica Taiman s’expressa en quatre isoformes resultants de la combinació de dues insercions/delecions (indels) a la regió carboxi-terminal de la seqüència. La reducció de l'expressió de les isoformes que contenen la inserció-1 de Tai provoca una metamorfosi precoç. La presència d’aquesta inserció en isoformes de Tai d’altres espècies suggereix que el mecanisme de transducció de l’activitat antimetamòrfica de l’HJ mitjançant aquestes isoformes és un fenomen conservat en altres insectes. Un altre factor de transcripció que participa en la senyalització de l'HJ és Broad-Complex (BR-C). A les espècies holometàboles BR-C s’expressa al darrer estadi larvari i la seva expressió transitòria és essencial per una formació de la pupa. Els estudis a B. germanica revelen funcions ancestrals de BR-C relacionades amb divisió cel·lular i creixement de l’ala, alhora que aporten noves pistes que ajuden a entendre l’evolució de la metamorfosi dels insectes. Un altre element important que participa en la transducció del senyal de l’HJ en relació amb la metamorfosi és el factor de transcripció Krüppel-homolog 1 (Kr-h1). Hem mostrat que la reducció de nivells d'expressió de Kr-h1 a fases nimfals a B. germanica indueix una metamorfosi precoç. Aquests resultats, conjuntament amb els resultats previs obtinguts en espècies holometàboles, suggereixen que el paper repressor de Kr-h1 en la metamorfosi és una condició ancestral que s’ha conservat en espècies hemimetàboles i holometàboles. Els microRNAs (miRNAs) són una classe d’RNAs petits no codificants que regulen l’expressió de gens a nivell transcripcional mitjançant la regulació de l’mRNA. Per tal de desvetllar la funció dels miRNAs durant el desenvolupament de B. germanica, es van dur a terme experiments suprimint l’expressió de Dicer-1, enzim que participa en la biosíntesi dels miRNAs, i el resultat va ser una inhibició de la metamorfosi. Els resultats presentats en aquesta tesi suggereixen aquest fenotip és la conseqüència principal d’una davallada de l’expressió d’una sola família de miRNAs, miR-2. El conjunt d’experiments realitzats indiquen que miR-2 regula la davallada de l’expressió del transcrit de Kr- h1 a la darrera fase nimfal de B. germanica, la qual cosa propicia que la metamorfosi es desenvolupi correctament.
Metamorphosis is a process were a sudden and conspicuous morphological change occurs at a specific time point during the postembryonic development of several animal groups, like amphibians and insects. Insect metamorphosis proceeds in two modes: hemimetaboly, defined by a gradual change along the life cycle, as occurs in bugs, cockroaches and locusts, and holometaboly, characterized by an abrupt change from larvae to adult mediated by a pupal stage, has observed in butterflies, beetles and flies. Metamorphosis evolved from hemimetaboly to holometaboly and the latter innovation was most successful because more than 80% of present insects are holometabolan species. From an endocrine point of view, both hemimetabolan and holometabolan metamorphosis is regulated by two kinds of hormones: 20-hydroxyecdysone (20E), which induce molts, and juvenile hormone (JH), which inhibits metamorphic changes. Using the cockroach Blattella germanica as a basal hemimetabolous model, the general objective of this thesis is to study the molecular action of JH in repressing insect metamorphosis. One of the main players in hormonal signalling is Methoprene-tolerant (Met), which plays the role of JH receptor. Depletion of Met in young nymphal instars triggers precocious metamorphosis, suggesting that Met transduces the antimetamorphic signal of JH. Recent studies report that Met heterodimerizes with Taiman (Tai) forming the receptor complex of JH in metamorphosis repression. However, there is no data in vivo demonstrating a role of Tai in metamorphosis, because its depletion in a number of insect models resulted in 100% mortality. B. germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. RNAi depletion of insertion-1 isoforms results in a precocious adult development, demonstrating its involvement in metamorphosis. The insertion-1 of Tai is conserved in other insect species, which suggests that the mechanism of signal transduction of the antimetamorphic action of JH I conserved in other species. An important JH-dependent factor is BR-C, whose expression in holometabolan species is inhibited by JH in young larvae and enhanced in mature larvae to specify to pupal stage. The functional study of BR-C in cockroach reveal ancestral functions related to cell division and wing pad growth. Krüppel-homolog 1 (Kr-h1) is a transcription factor whose function as transductor of the antimetamorphic action of JH has been demonstrated in holometabolan species. RNAi experiments depleting Kr-h1 in young nymphal instars of B. germanica results in precocious metamorphosis, suggesting that their role as a JH transductor in metamorphosis is evolutionary conserved in hemimetabolan and holometabolan species. Finally, it has been reported that depletion of dicer-1, the enzyme that catalyzes the final step of miRNA biosynthesis, prevents metamorphosis in B. germanica. This thesis has addressed the question of how miRNAs act in metamorphosis and why their absence impairs it. The whole data of experiments reported here indicate that miR-2 scavenges Kr-h1 transcripts in the last nymphal instar of B. germanica, which contributes to the correct development of metamorphosis.

The evolutionary success of insects was particularly shaped by the innovation of the metamorphosis, especially by the transition from hemimetaboly to holometaboly. The mechanisms underlying this evolutionary transition represent an unsolved question, although different approaches have been used to study them. In the present thesis we followed a transcriptomic approach, comparing data on mRNA and miRNA expression in key developmental moments, comprising embryonic and postembryonic stages, in species representing the hemimetabolan and holometabolan modes. Most of the work has been carried out in the hemimetabolan species Blattella germanica, but we have used also other reference species for comparison, especially the holometabolan Drosophila melanogaster and Tribolium castaneum. The results show that there are not qualitative gene differences between holometabolan and hemimetabolan species, but differences in patterns of expression and potential networking of orthologous genes. Transcription factors, epigenetic modifiers, and miRNAs appear as important players in both developmental modes.
L'èxit evolutiu dels insectes ha estat mercat per la innovació de la metamorfosi i, en especial, per la transició de la metamorfosi hemimetàbola a holometàbola. Els mecanismes subjacents en aquesta transició evolutiva representen una qüestió no resolta. Per tal d'estudiar aquesta transició, en aquesta tesi hem utilitzat un enfocament transcriptomic comparant dades de mRNA i miRNA en estadis clau del desenvolupament, incloent-hi estadis embrionaris i post embrionaris en espècies representatives de metamorfosis hemimetàbola i holometàbola. La major part dels anàlisis s'han centrat en l'hemimetàbola Blattella germanica, tot i que s'han utilitzat dades d'altres espècies com a contrast, especialment dels holometàbols Drosophila melanogaster i Tribolium castaneum. Els resultats mostren que no hi ha diferències qualitatives en relació a gens dels hemimetàbols i holometàbols, en canvi les principals diferències consisteixen en els diferents perfils d'expressió de gens comuns i la seva xarxa de d'interacció. Els factors de transcripció, els modificadors epigenètics i els miRNAs emergeixen com a principals protagonistes dels mecanismes reguladors en ambdós models de desenvolupament.

Insect metamorphosis is controlled by several genes that are regulated by two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH). These genes, E93, Krüppel-homolog 1 (Kr-h1) and Broad-complex (Br-C), form a network of transcription factors defined as the “Gene Metamorphic Network” (MGN). Changes in the regulation of the MGN underlie evolution of complete metamorphosis. However, knowledge about the MGN in different insect types is scarce. This thesis works towards the characterization of the regulation and function of the MGN. First, we have characterized two larval size-assessment checkpoints that regulate the onset of metamorphosis in the coleopteran Tribolium castaneum by controlling the stage-specific expression of the MGN. Second, we have characterized the MGN in the neotenic Strepsipteran Xenos vesparum, and found that neoteny could result from modifications in E93, Br-C and Kr-h1 expression. Finally, we have characterized the role of the EGFR pathway in the regulation of 20E biosynthesis in T. castaneum.
La metamorfosi dels insectes està controlada per per dues hormones, l’hidroxiecdisona (20E) i l’hormona juvenil (HJ), que a la vegada regulen l’expressió d’una sèrie de gens. Aquests gens, E93, Krüppel-homolog 1 (Kr-h1) i Broad-complex (Br-C), formen una xarxa de factors de transcripció anomenada “Gene Metamorphic Network” (MGN). Canvis en la regulació de la MGN són la base de l’evolució de la metamorfosi completa, no obstant, el coneixement sobre la MGN en diferents tipus d’insectes és escàs. Aquesta tesi te com a objectiu la caracterització i la regulació de la MGN en diferents tipus d’insectes. En primer lloc, hem co-relacionat l’expressió dels gens de la MGN amb dos moments del desenvolupament associats a la mida de l’organisme que controlen l’inici de la metamorfosi en el coleòpter Tribolium castaneum. En segon lloc, hem caracteritzat la MGN en el desenvolupament neotènic de l’espècie Strepsiptera Xenos vesparum i hem trobat que la neotènia podria ser el resultat de modificacions en l’expressió de E93, Br-C and Kr-h1. Finalment, hem analitzat la funció de la via de senyalització EGFR en la regulació de la síntesis de l’20E en Tribolium castaneum.

Trabajos previos llevados a cabo en el laboratorio de acogida, usando como modelo el insecto basal Blattella germanica, mostraron que los microRNAs (miRNAs) son cruciales para completar la metamorfosis. El objetivo general de esta tesis fue identificar miRNAs que estuviesen implicados en este proceso. Como primer paso, se estableció un catálogo general de miRNAs en B. germanica mediante secuenciación con Solexa. A partir de ahí, se prepararon dos librerías de miRNAs, una en la etapa metamórfica y otro en la etapa no metamórfica, para distinguir miRNAs diferencialmente expresados entre las dos etapas y evaluar la influencia de las hormonas principales de la metamorfosis sobre la expresión de estos miRNAs. Nuestros experimentos también mostraron que los factores de transcripción Broad-complex inducen la expresión de let-7 y miR-100, y que estos miRNAs desempeñan un papel en la regulación del tamaño y del patrón de venas e intervenas de las alas de B. germanica. Por último, se estudió el papel de miR-8-3p y miR-8-5p en la regulación de los niveles de transcrito de atrofina, un factor implicado en la coordinación neuromuscular, que es importante para asegurar una adecuada ecdisis en la muda metamórfica.
Previous work carried out in the host laboratory, using the basal insect Blattella germanica as model, showed that microRNAs (miRNAs) are crucial to complete metamorphosis. The general goal of this thesis was to identify particular miRNAs involved in this process. As a first step, we established a general catalogue of miRNAs in B. germanica using high throughput Solexa sequencing. Thereafter, we prepared two miRNA libraries; one in the metamorphic stage and other one in the non-metamorphic stage, to distinguish miRNAs differentially expressed between the two stages, and to assess the influence of the main metamorphosis hormones on the expression of these miRNAs. Our experiments also showed that Broad complex transcription factors induce the expression of let-7 and miR-100, and that these miRNAs play a role in regulating the size and the vein-intervein patterning of B. germanica wings. Finally, we studied the role of miR-8-3p and miR- 8-5p in regulating the transcript levels of atrophin, a factor involved in neuromuscular coordination, which is important to ensure a proper ecdysis in the metamorphic molt.

Thesis (Ph. D.)--University of Oregon, 2005.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 118-133). Also available for download via the World Wide Web; free to University of Oregon users.

This doctoral thesis in creative writing comprises a novella — Order of Our Lady Cicada — and an exegesis which maps representations of metamorphoses, tricksters and insects through seven stories. The creative writing component is a literary artefact and a piece of research in itself. The exegesis is a further construction of new knowledge, addressing the novella among six other literary texts. The two pieces — novella and exegesis — are imbricated and both draw on theories from post-humanism, including New Materialisms and critical animal studies. Overall, this thesis problematises both representation and liberal humanism, seeking a corporeal relation with insects, tricksters and metamorphoses in the texts without settling on easy meaning-making. I have formed the two components of the dissertation by engaging methodologies of rhizomatic research, phenomenological research, “agnostic” research, voice-hearing and ficto-criticism.

La symbiose est un phénomène ubiquitaire dans la nature et joue un rôle évolutif majeur. Alors que la communauté scientifique reconnaît désormais l’importance des associations symbiotiques dans de nombreux processus biologiques et pathologiques chez les animaux, la compréhension des mécanismes de contrôle, de tolérance et de modulation des populations symbiotiques est un enjeu majeur. Pour aborder ces questions, j’ai étudié l’association entre le charançon Sitophilus et la bactérie intracellulaire Sodalis pierantonius. Sitophilus héberge son endosymbiote dans des cellules spécialisées, les bactériocytes, regroupées en un organe, le bactériome. En retour, S. pierantonius fournit à son hôte des nutriments présents en faibles quantités dans son alimentation, les céréales. S. pierantonius étant immunogène pour son hôte, dans un premier chapitre, j’ai étudié les régulations immunitaires spécifiques au bactériome assurant le maintien de l’homéostasie immunitaire. J’ai dans un premier temps montré que la compartimentalisation des endosymbiotes, limitant les contacts immunitaires avec l’hôte, repose sur l’expression IMD-dépendante d’un peptide antimicrobien, une régulation similaire aux réponses immunitaires aux pathogènes. Ensuite, j’ai montré comment l’immunogénicité des endosymbiotes, via son peptidoglycane, est limitée par des Protéines de Reconnaissance du PeptidoGlycane (PGRP). Le peptidoglycane symbiotique ne semble pas être reconnu dans le bactériome, et sa reconnaissance systémique est contenue par l’action locale de PGRP-LB. Cette protéine clive le peptidoglycane symbiotique, empêchant ainsi l’activation chronique du système immunitaire systémique. Dans un deuxième chapitre, j’ai étudié comment, au cours de la métamorphose, le bactériome se réorganise complètement. Le bactériome larvaire se dissocie, les bactériocytes migrent le long de l’intestin et forment de multiples nouveaux bactériomes. Une approche de dual-RNAseq nous a permis de révéler l’implication à la fois de l’hôte et du symbiote dans ce remodelage morphologique. Les résultats obtenus durant cette thèse montrent l’impact incommensurable des bactéries sur des processus immunitaires et développementaux, et sur l’évolution des animaux en général
Symbiosis is ubiquitous in nature and plays a crucial role in evolution. As the scientific community is becoming increasingly aware of the importance of such associations in both biological and pathological processes in animals, understanding how symbiotic populations are controlled, tolerated, and modulated, is becoming a major stake. To address these questions, I studied the mutualistic association between the weevil Sitophilus and the intracellular bacterium Sodalis pierantonius. Sitophilus houses S. pierantonius in specialized host cells, the bacteriocytes, which group together in an organ, the bacteriome. In return, S. pierantonius provides its host with nutrients scarecely present in its cereal-based diet. S. pierantonius being immunogenic for its host, I studied in a first chapter how specific bacteriome immune regulations ensure the maintenance of host immune homeostasis. In a first part, I showed that endosymbiont compartmentalization, which limits host-endosymbiont immune contacts, relies on the IMD-dependent expression of one antimicrobial peptide, a regulation similar to that of immune responses in pathogenic conditions. Then, I showed how endosymbiont immunogenicity, via its peptidoglycan, is tamed by PeptidoGlycan Recognition Proteins (PGRPs). While symbiotic peptidoglycan would not be recognized within the bacteriome, its systemic recognition is circumscribed by PGRP-LB local action. PGRP-LB cleaves symbiotic peptidoglycan, thereby preventing a chronical and detrimental activation of the host systemic immunity. In a second chapter, I studied how, during metamorphosis, the bacteriome is completely remodeled. The larval bacteriome dissociates, bacteriocytes migrate along the midgut, and settle in multiple new bacteriomes. A dual-RNAseq approach allowed us to pinpoint both host and symbiont implication in this drastic morphological reorganization. The results obtained during this PhD show the immeasurable impact bacteria bear on host immune and developmental processes, and more generally on animal evolution

A cutícula dos insetos é composta principalmente por uma variedade de proteínas que interagem com filamentos de quitina, um polímero de N-acetilglicosamina, para formar um envoltório rígido que protege e dá forma ao organismo. O crescimento dos insetos depende da renovação periódica da cutícula, que se desprende durante a apólise e é digerida enquanto a epiderme sintetiza uma nova cutícula substituta. Tal renovação caracteriza a muda e metamorfose e é coordenada por hormônios, com destaque para os ecdisteróides. O atual trabalho objetivou caracterizar a expressão diferencial de genes do tegumento (cutícula e epiderme subjacente), além de elucidar aspectos de regulação e função no contexto da muda e metamorfose, com foco nos genes codificadores de proteínas estruturais e enzimas cuticulares. Para este fim, utilizamos o tegumento de fases específicas da muda pupal-adulta, isto é, de pupas (Pw), de pupas em apólise (Pp) e de adultas faratas (Pbl) para análises de microarrays de cDNA. As análises dos microarrays mostraram 761 e 1173 genes diferencialmente expressos nos tegumentos de adultas faratas (Pbl) em comparação com pupas (Pw) ou pupas em apólise (Pp), respectivamente. A categorização destes genes, segundo os critérios do Gene Ontology, distinguiu totalmente o tegumento de adultas faratas (Pbl) dos tegumentos de pupas (Pw) ou pupas em apólise (Pp) tanto em relação ao critério Processo Biológico quanto em relação à Função molecular, evidenciando grande mudança na expressão gênica durante a construção do exoesqueleto definitivo nas adultas faratas (Pbl). Os microarrays mostraram aumento estatisticamente significante da expressão de 24 genes cuticulares no tegumento de adultas faratas. Este resultado foi validado por RT-PCR em tempo real (qRT-PCR) para 23 destes genes (AmelCPR3, AmelCPR4, AmelCPR6, AmelCPR14, AmelCPR15, AmelCPR17, AmelCPR23, AmelCPR24, AmelCPR25, AmelCPR28, AmelCPR29, AmelCPR30, apd-1, apd-2, apd-3, CPLCP1, Am-C, Am-D, AmelTwdl1, AmelTwdl2, GB12449, GB12811 e GB11550), e por RT-PCR semiquantitativa para o gene Amlac2. Além disto, a maior expressão de outros 2 genes cuticulares (AmelCPR1 e AmelCPR2) em adultas faratas foi demonstrada por qRT-PCR. Estes genes cuticulares positivamente regulados no tegumento de adultas faratas (Pbl) devem estar envolvidos com a formação e diferenciação do exoesqueleto definitivo. O aumento da expressão gênica neste período da muda (Pbl) é regulado pela variação do título de ecdisteróides e ocorre enquanto o título deste hormônio decai, após ter atingido o pico indutor da apólise na fase de desenvolvimento precedente (Pp). Ao contrário, as análises por qRT-PCR mostraram que 2 outros genes cuticulares (AmelCPF1 e AmelCPR1) são negativamente regulados no tegumento de adultas faratas em comparação com pupas, sugerindo que são específicos de cutícula pupal. Estes genes foram inibidos pelo aumento dos níveis de ecdisteróides, que induz a apólise. Vinte e um entre os 24 genes cuticulares diferencialmente expressos nos microarrays codificam proteínas pertencentes às famílias CPF, CPR, Apidermina, CPLCP, Análoga a peritrofina e Tweedle. Os outros 3 genes diferencialmente expressos (GB12449, GB12811, GB11550) não tinham sido ainda caracterizados como genes cuticulares. Dois deles, GB12449 e GB12811, foram sequenciados para validação da predição e para a caracterização das respectivas estruturas genômicas. Experimentos de hibridação in situ com sonda fluorescente (FISH) nos permitiram localizar altos níveis de transcritos destes genes no citoplasma de células da epiderme de adultas faratas, sugerindo fortemente sua natureza cuticular e envolvimento na construção do exoesqueleto definitivo. O presente estudo consiste na primeira análise global de expressão de genes do tegumento de uma espécie de himenóptero social. Os resultados apresentados levaram à identificação de genes com expressão associada à muda pupal-adulta e formação do exoesqueleto definitivo. Este trabalho contribui com novos dados moleculares para o aprofundamento do conhecimento da metamorfose de A. mellifera.
The insect cuticle is mainly composed of proteins that interact with chitin filaments to form a rigid structure that protects and shapes the organism. Insects grow through the periodic renewal of the cuticle, which is shed at each apolysis episode, and subsequently digested while the epidermis synthesizes the cuticle of the next stage. These molting events are coordinated by hormones, mainly ecdysteroids. The current work aimed to characterize differential gene expression in the integument (cuticle and underlying epidermis) during the ecdysteroid-regulated pupal-to-adult molt. Special attention was given to the structure and expression of genes encoding proteins and enzymes involved in cuticle formation and differentiation. To achieve these goals, we used thoracic integument of newly-ecdysed pupae (Pw), pupae in apolysis (Pp) and pharate adults (Pbl) in cDNA microarray analyses. The microarray analysis showed 761 and 1173 differentially expressed genes in the pharate adult integument (Pbl) in comparison to pupae (Pw) or pupae in apolysis (Pp), respectively. Gene Ontology terms for Biological Process and Molecular Function completely distinguished the integument of pharate adults (Pbl) from the integument of pupae (Pw) or pupae in apolysis (Pp). The microarray analysis discriminated 24 cuticular genes with a significant expression increase in the pharate adult integument. This was validated by real time RT-PCR analysis (qRT-PCR) for 23 of these genes (AmelCPR3, AmelCPR4, AmelCPR6, AmelCPR14, AmelCPR15, AmelCPR17, AmelCPR23, AmelCPR24, AmelCPR25, AmelCPR28, AmelCPR29, AmelCPR30, apd-1, apd-2, apd-3, CPLCP1, Am-C, Am-D, AmelTwdl1, AmelTwdl2, GB12449, GB12811 and GB11550), and by semiquantitative RT-PCR for Amlac2. In addition, the increased expression of other two cuticular genes (AmelCPR1 and AmelCPR2) was confirmed by qRT-PCR. These up-regulated cuticular genes in pharate adult integument apparently are involved in adult cuticle formation and differentiation, which occurs while the ecdysteroids titers decay, after reaching the peak that induces apolysis in the preceding phase (Pp). In contrast, two cuticular genes (AmelCPF1 e AmelCPR1) were confirmed by qRT-PCR analysis as negatively regulated in the integument of pharate adults compared to pupae, suggesting that they are specific to pupal cuticle. Therefore, these genes were inhibited by the increasing ecdysteroid levels that induce apolysis. Twenty one of the 24 cuticular genes differentially expressed in the microarrays encode proteins belonging to the CPF, CPR, Apidermin, CPLCP, Analogous to peritrofins and Tweedle families. The other three differentially expressed genes (GB12449, GB12811, GB11550) had not yet been assigned as cuticular genes. Two of them (GB12449 and GB12811) were sequenced, thus allowing prediction validation and gene structure characterization. In situ hybridization experiments using fluorescent probe (FISH) localized high expression of these genes in the pharate adult epidermis, strongly suggesting their involvement in the construction of the adult exoskeleton. This study is the first global gene expression analysis of the integument from a social hymenopteran species. The expression of genes in the integument was associated to the molting process and to the adult exoskeleton formation. This work contributes with new molecular data for a deeper understanding of A. mellifera metamorphosis.

Au cours d’une infection, l’intensité et la durée de la réaction immunitaire doivent être étroitement contrôlées au risque de devenir délétère pour l’hôte. Chez la drosophile Drosophila Melanogaster, une infection bactérienne conduit à l’activation de voies de signalisation de type NFκ-B, conservées au cours de l'évolution, via la détection du peptidoglycane, un composant de la paroi bactérienne, par des récepteurs PGRPs (« Peptidoglycan Recognition Proteins »). Durant ma thèse, j’ai étudié les conséquences d’une activation non contrôlée d'une de ces voies, la voie IMD, sur la physiologie de la drosophile. Le premier projet auquel j’ai participé montre que l’inactivation de PGRP-LF, un régulateur négatif de la voie, conduit à l'activation de la voie IMD dans les tissus larvaires dérivés de l’ectoderme en absence d’infection. Cette activation entraine l’expression ectopique dans ces tissus de la protéines DIAP1 («Drosophila Inhibitor of Apoptosis 1»), suffisante pour induire la malformation de certains tissus de la drosophile adulte démontrant une fonction développementale jusqu'alors inconnue de la voie IMD au cours de la métamorphose de la drosophile. Dans la seconde partie de ma thèse, j’ai pu observer que durant une infection chronique, le maintien de l’activation de la voie IMD entraine l’apparition de nombreux phénotypes tels qu’une baisse de l’espérance de vie, des troubles locomoteurs, de la neurodégénérescence et l’atrophie du corps gras et des ovaires. Mes résultats montrent que l’activation de la voie IMD spécifiquement au niveau de la glie périneuriale, un composant de la barrière hématoencéphalique, participe activement à l’apparition des phénotypes observés
Upon infection, the intensity and duration of the immune response must be tightly controlled at the risk of becoming harmful for the host. In the fruit fly Drosophila Melanogaster, a bacterial infection leads to the activation of evolutionary conserved NFk-B signalling pathways, through detection of a bacterial cell wall component, the peptidoglycan (PG), by PGRPs ("Peptidoglycan Recognition Proteins") receptors. During my thesis, I studied the consequences of uncontrolled activation of one of these NFk-B pathways, the IMD pathway, on the physiology of Drosophila. The first project I took part in shows that inactivation of PGRP-LF, a negative regulator of the pathway, leads to the activation of the IMD pathway in ectodermal derived larval tissues without any infection. This pathway activation in those tissues leads to an ectopic expression of the DIAP1 ("Drosophila Inhibitor of Apoptosis 1") protein, wich is sufficient to induce some adult Drosophila structures malformations, demonstrating a previously unknown developmental function of the IMD pathway during Drosophila metamorphosis. In the second part of my thesis, I was able to demonstrate that during chronic infection, maintenance of IMD pathway activation leads to the appearance of many deleterious phenotypes such as a decreased lifespan, locomotor disorders, neurodegeneration and atrophy of the fat body and ovaries. My results show that IMD pathway activation specifically in the perineurial glia, a component of the blood-brain barrier, actively contributes to the development of the observed phenotypes. Demonstrating for the first time that fhe fly's brain is able to detect circulating PG in the hemolymph

This thesis consists of two published articles in both of which I am the first author, and unpublished results. My studies using RNA interference in the model beetle Tribolium castaneum (Coleoptera) and non-model insects contribute to defining the core of juvenile hormone signalling in insect metamorphosis. Results of studies in Tribolium presented in my first publication identify the JH-resistance gene Methoprene-tolerant (Met) as the first known transducer of the anti-metamorphic effect of juvenile hormone; my unpublished studies on a true bug Pyrrhocoris apterus (Hemiptera) demonstrate that role of Met in metamorphosis is shared by insects with hemimetabolous and holometabolous type of metamorphosis. The second publication demonstrates that Met exerts its function by regulating the Broad-Complex (BR-C) gene, and studies in Tribolium and the lacewing Chrysopa perla (Neuroptera) show that its central role of BR-C in holometabolan metamorphosis has changed during Holometabola evolution. My unpublished results show that the gene Krüppel-homolog 1 is another Met target whose function in preventing precocious metamorphosis has been conserved between the holometabolous beetle and the hemimetabolous true bug Rhodnius prolixus.

This thesis comprises three published papers and one manuscript, all focused on the role of juvenile hormone (JH), the JH receptor Methoprene-tolerant (Met) and its target gene Krüppel-homolog 1 (Kr-h1) in insect development and reproduction. The JH-Met-Kr-h1 pathway is critical for metamorphic transition in hemimetabolan Pyrrhocoris apterus (Hemiptera) and holometabolan Bombyx mori (Lepidoptera) but seems to be dispensable during early larval postembryonic development. The results also show that Met and its heterodimeric partner Taiman (Tai) but not Kr-h1 are critical for ovarian development and vitellogenesis in Pyrrhocoris females. In vitro, in vivo and cell-based techniques in Drosophila melanogaster have demonstrated that Met and its paralog Gce are a bona fide receptor for JH. Only Gce capable of binding JH rescued Drosophila deficient for Met and Gce proteins, and the capacity of Gce to bind JH was necessary for JH-dependent transcriptional activation by Gce and Tai.

The initiation of metamorphosis causes the cessation of the larval growth period which determines the final body size of adult insects. Because larval growth is roughly exponential, differences in timing the initiation of metamorphosis can cause large differences body size. Although many of the processes involved in metamorphosis have been well characterized, little is known about how the timing of the initiation of metamorphosis is determined.

Using different strains from Tribolium castaneum, Tribolium freemani, and Manduca sexta and varied nutritional conditions, I was able to document the existence of a threshold size, which determines when the larva becomes competent to metamorphose. Threshold size, however, does not dictate the exact timing of initiation. The exact timing for the initiation of metamorphosis is determined by a pulse of the molting hormone, ecdysone, but only after threshold size has been reached. Ecdysone pulses before the larva attains threshold size only cause the larva to molt to another larval instar. These results indicate the timing of metamorphosis initiation is controlled by two factors: (1) attainment of threshold size, at which the larva becomes competent to initiate metamorphosis and (2) the timing of an ecdysone pulse after attaining threshold size.

I hypothesize the attainment of threshold size, and therefore competence to metamorphose, is mediated by the effect of changing juvenile hormone concentrations caused by the increase in size of the larva. While the larval body grows nearly exponentially, the corpora allata, which secretes juvenile hormone, grows very little if at all. The difference in relative growth causes juvenile hormone concentrations to gradually become diluted. When juvenile hormone concentrations fall below a threshold, changes in protein-protein binding occur that can cause changes in signaling networks and ultimately gene expression. These changes make the larva competent for metamorphosis.

I have demonstrated that only threshold size is consistently correlated with body size; other growth parameters such as growth rate, duration of instars, or number of instars do not consistently correlate with variation in body size. Using the black mutant strain of M. sexta I have shown that lower juvenile hormone titers correlate with lower threshold sizes. My hypothesis is consistent with the large body of literature indicating the involvement of juvenile hormone. I also hypothesize that the diversity of metamorphosis types in holometabolous insects can be explained by heterochronic shifts in the timing of threshold size and other developmental events related to metamorphosis. The heterochronic shifts affect not only the morphology of organs, but can also affect the overall phenotypic response of the larva to changes in the environment. The different phenotypic responses among species may make the more or less suited for certain types of niches.

Dissertation

The series of two fossil species belonging to the order Palaeodictyoptera from the Late Carboniferous of Kuznetsk Basin in Russia were re-examined. The two species as Tchirkovaea guttata and Paimbia fenestrata were investigated with emphasis on the wing growth and development in comparison with the structure of developing wings in recent mayflies. This fossil material of T. guttata and P. fenestrata was long considered by previous authors as undisputed evidence for a unique type of wing development in the Palaeozoic insects. The idea was that the larvae of these insects possessed the wings, which became articulated and fully movable already earlier during the postembryonic development and that these gradually growing wings changed their position from longitudinal to perpendicular to the body axis. Moreover, the development was supposed to include two or more subimaginal instars, implying that the fully winged instars moulted several times during postembryonic development. After detailed study of the available fossils and subsequent comparison of the fossil evidence with the development of wings in the recent mayfly Cloeon dipterum it was discovered, that the alleged series of immature, subimaginal and imaginal wings of T. guttata and P. fenestrata do not provide clear evidence that would support...

The purpose of this thesis has been to explore in what ways Franz Kafka’s ”The Metamorphosis” can be read as a story of gender. By bringing together Judith Butler’s theory of materialization and Lennard J. Davis’s crip theory I have spoken of Dismodernity as the domain of abject bodies that have been repudiated by (post)modern societies as untintelligible and dysfunctional. From this vantage point ”The Metamorphosis” can be seen as an allegory of Dismodernity and the protagonist, Gregor Samsa, can be seen as a political figure of Dismodernity. Therefore, I have tried to draw a feminist insect politics out of his metamorphosis from (hu)man into insect. By doing a close reading, through the theoretical lenses of Judith Butler, Lennard J. Davis and Donna Haraway, Gregor Samsa can be read as an abject non-masculinity which is both produced and made impossible by a heterosexual matrix’s need of intelligible genders and a capitalist system’s need of functional workers. As an abject non-masculinity Gregor Samsa works as a queer (unintelligible) and dismodern (dysfunctional) trickster that both disturbs and makes visible the established gendered norms of (un)intelligibility and (dis)ability through a blurring of the boundaries between human/animal, public/private and masculinity/femininity. As an involuntary trickster he also challenges gender studies and its seeking for ultimate representations for oppositional consciousness pure in their radical potential.