Dissertations / Theses on the topic 'Hormonas juveniles'

La panerola Blattella germanica és un insecte de metamorfosi incompleta (hemimetàbol) i anautògen. Aquesta és una estratègia reproductiva per la qual les femelles no es poden reproduir fins que no han menjat. D'alguna manera els senyals nutricionals han d'arribar als diferents teixits i cèl•lules per informar-los que s'ha assolit un estat nutricional adequat per iniciar els processos reproductius. Tant la via de TOR com la via del receptor d'insulina (InR) semblen formar part conjunta d'aquesta senyalització nutricional. A B. germanica la vitel•logènesi s'activa mitjançant l'hormona juvenil (HJ), que es fabrica i secreta per unes glàndules retrocerebrals anomenades corpora allata (CA). Quan falta l'aliment, els CA no sintetitzen HJ i aquesta no pot activar la síntesi de vitel•logenina (Vg) en el cos gras (CG), que no pot ser incorporada als ovaris, impedint que aquests es desenvolupin correctament. Prèviament havia estat clonada la seqüència de TOR de B. germanica (BgTOR). En aquest treball s'han clonat les de BgInR, BgS6K i de BgPepck. Gràcies al dsRNAi s'ha estudiat la funció del InR com a regulador del creixement, veient que no només afecta a la maduresa sexual de les femelles sinó també la seva mida quan s'injecta en estadis nimfals. La interferència del InR també produeix problemes en el correcte desenvolupament i desplegament de les ales i les tegmines. Així mateix, s'ha comprovat que la interferència del InR fa baixar els nivells dels trànscrits de HMG-S1, HMG-S2, HMG-R i epoxidasa en els CA, implicats en la síntesi d'HJ, així com de la Vg en CG , independentment de l'efecte de la HJ sobre aquesta. També s'ha vist que la interferència del InR produeix una major longevitat en les femelles, a causa que aquestes no es reprodueixen, evitant el desgast que això produeix. Alhora, l'aplicació de dsInR confereix certa protecció davant situacions de dejuni. Altres resultats suggereixen que l'activació d'S6K, tal com succeeix en altres organismes, es realitza per modificacions post-transcripcionals, probablement per fosforilació de la proteïna. S'ha començat a posar a punt un sistema de western-blot per usar-lo per a la detecció de l'estat de fosforilació de S6K i per tant del seu estat d'activació. Mitjançant la tècnica del RNAi s'ha comprovat que la interferència per S6K fa baixar els transcrits de HMG-S1, HMGS2, HMG-R i epoxidasa en els CA, implicats en la síntesi d'HJ, així com la Vg en CG, indicant que està implicat en la transmissió del senyal nutricional als teixits implicats en la vitel•logènesi. Finalment s'ha establert Pepck com a marcador de l'estat nutricional, ja que s'incrementa la seva expressió en situacions de dejuni. No s'ha observat, però, que augmenti quan es redueixen els nivells de S6K o InR mitjançant RNAi, la qual cosa ens indica que la influència de la situació de dejuni sobre la transcripció de PEPCK es pot realitzar per vies alternatives a S6K i InR, si bé sembla que necessita de la presència de InR funcional.
The cockroach Blatella germanica is a hemimetabolous insect with anautogenous reproduction. TOR and Insulin Receptor (InR) pathways are responsible of the nutritional signaling and to control reproductive processes. In B. germanica the yolk protein synthesis is activated by juvenile hormone (JH), which is secreted by retrocerebrals glands known as corpora allata (CA). In starvation, CA are not able to synthesize JH and it can’t activate the synthesis of vitelogenin (Vg) in the fat body (FB), and it can’t be incorporated by the ovaries, preventing their proper development. In this work we have cloned the sequences of BgInR, BgS6K and BgPepck. Using RNAi, InR has been studied as a regulator of growth, affecting not only sexual maturity of females, but also their size when RNAi is injected at nymphs. InR interference also produces problems for the proper development and deployment of wings and elytra. It has also been observed that InR interference lowers the levels of HMG-S1, HMG-S2, HMG-R and epoxidase transcription in CA, involved on the synthesis of JH by CA, and consequently, reducing Vg levels. Independently of JH, InR has a direct effect on Vg expression. Besides, InR interference confers greater longevity in females and protection in front of fasting. We have also begun to implement western-blot technique to detect phosphorylation of S6K. The S6K interference reduce transcription levels of HMG-S1, HMG-S2, HMG-R and epoxidase in CA, and Vg in the CG , indicating that it is involved in nutritional signal transmission to the tissues involved in vitelogenesis. Finally, we established Pepck as a marker of the nutritional status, as its expression is increased in starvation. However, it has not been observed any increase when reducing the levels of S6K or InR using RNAi, indicating that the influence of starvation on Pepck transcription can be performed in alternative ways to S6K and InR, but needs the presence of functional InR.

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 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

Orientador: Hermione Elly Melara de Campos Bicudo
Banca: Maria Tercília Vilela de Azeredo-Oliveira
Banca: Francisco Chiaravalloti Neto
Resumo: O mosquito Aedes aegypti é vetor dos vírus causadores de doenças humanas entre as quais incluem-se a dengue, a dengue hemorrágica e a febre amarela. A transmissão dessas doenças é feita pelas fêmeas adultas, as quais necessitam de repasto sanguíneo para amadurecimento de seus óvulos. Ao picar indivíduos doentes e depois indivíduos não infectados, as fêmeas transmitem os vírus absorvidos dos primeiros, juntamente com o sangue. Vários estudos mostraram que o tratamento com cafeína (CAF) bloqueia o desenvolvimento e mata A. aegypti na fase larval, impedindo a produção de adultos e, consequentemente, a transmissão das doenças mencionadas. Paralelamente, trabalhos preliminares indicaram que o tratamento do mosquito com CAF altera o padrão de síntese das enzimas esterases. São enzimas que desempenham papel importante em vários processos fisiológicos dos organismos, estando inclusive envolvidas no controle da metamorfose dos insetos. No presente trabalho, as esterases foram analisadas em géis de poliacrilamida corados com α-naftil e β-naftil acetatos e Fast Blue RR. As amostras foram preparadas com larvas L2/L3, L4, pupas e adultos, porém, tendo em vista que a fase L4 foi a que permitiu melhor observação das bandas, o estudo praticamente restringiu-se à mesma. A comparação das amostras dos mosquitos tratados e controle envolveu a observação da presença e da frequência das bandas esterásicas nos géis. Foi também analisado o grau de expressão das bandas com base na intensidade de coloração avaliada pelo programa computacional Global Lab Image. A presente análise mostrou que a CAF altera cinco bandas β-esterásicas (EST-17A a EST-21) e um grupo de bandas α-esterásicas (EST-12 a EST-14). As bandas EST-17A a EST-20 apresentaram redução em ambas as características, enquanto EST-21 e as α-esterases apresentaram aumento. Tendo em vista as variações do grau de expressão das esterases, paralelamente ao ...
Abstract: The mosquito Aedes aegypti is a vector of human disease-causing virus, including dengue, dengue hemorrhagic fever and yellow fever. The transmission of these diseases is made by the adult females, which require blood repast for completing their eggs maturation. When females bite sick individuals and later bite healthy individuals, they transmit the virus absorbed with the blood from the first. Several studies have shown that treatment with caffeine (CAF) blocks development and kills A. aegypti in the larval phase, preventing the production of adults and, consequently, the transmission of the mentioned diseases. In addition, preliminary work indicated that CAF treatment of A. aegypti changes the pattern of synthesis of esterase enzymes. The esterase enzymes play important roles in various physiological processes of the organisms, including among them the control of the metamorphosis of insects. In the present study, the esterases were analyzed in polyacrylamide gels stained with α and ß naphthyl acetates and Fast Blue RR. The samples were prepared with L2, L3 and L4 larval stages, pupae and adults. However, considering that the L4 stage was the one that allowed better observation of the bands, the study was almost restricted to it. The comparison of the treated and control mosquitoes involved observations of the presence and frequency of the esterase bands in the gels. The degree of expression of the bands was also analyzed on the basis of the staining intensity evaluated by the Global Lab Image computer program. The analysis showed that CAF changed five β-esterase bands (EST-17A to EST-21) and a group of α-esterase bands (EST-12 to EST-14). The bands EST-17A to EST-20 showed reduction in both characteristics, while EST-21 and the α-esterases showed increase of them. In view of the changes occurred in the degree of expression of the esterases, parallel to the larval development block we thought in the hypothesis that these esterases ...
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O mosquito Aedes aegypti é vetor dos vírus causadores de doenças humanas entre as quais incluem-se a dengue, a dengue hemorrágica e a febre amarela. A transmissão dessas doenças é feita pelas fêmeas adultas, as quais necessitam de repasto sanguíneo para amadurecimento de seus óvulos. Ao picar indivíduos doentes e depois indivíduos não infectados, as fêmeas transmitem os vírus absorvidos dos primeiros, juntamente com o sangue. Vários estudos mostraram que o tratamento com cafeína (CAF) bloqueia o desenvolvimento e mata A. aegypti na fase larval, impedindo a produção de adultos e, consequentemente, a transmissão das doenças mencionadas. Paralelamente, trabalhos preliminares indicaram que o tratamento do mosquito com CAF altera o padrão de síntese das enzimas esterases. São enzimas que desempenham papel importante em vários processos fisiológicos dos organismos, estando inclusive envolvidas no controle da metamorfose dos insetos. No presente trabalho, as esterases foram analisadas em géis de poliacrilamida corados com α-naftil e β-naftil acetatos e Fast Blue RR. As amostras foram preparadas com larvas L2/L3, L4, pupas e adultos, porém, tendo em vista que a fase L4 foi a que permitiu melhor observação das bandas, o estudo praticamente restringiu-se à mesma. A comparação das amostras dos mosquitos tratados e controle envolveu a observação da presença e da frequência das bandas esterásicas nos géis. Foi também analisado o grau de expressão das bandas com base na intensidade de coloração avaliada pelo programa computacional Global Lab Image. A presente análise mostrou que a CAF altera cinco bandas β-esterásicas (EST-17A a EST-21) e um grupo de bandas α-esterásicas (EST-12 a EST-14). As bandas EST-17A a EST-20 apresentaram redução em ambas as características, enquanto EST-21 e as α-esterases apresentaram aumento. Tendo em vista as variações do grau de expressão das esterases, paralelamente ao ...
The mosquito Aedes aegypti is a vector of human disease-causing virus, including dengue, dengue hemorrhagic fever and yellow fever. The transmission of these diseases is made by the adult females, which require blood repast for completing their eggs maturation. When females bite sick individuals and later bite healthy individuals, they transmit the virus absorbed with the blood from the first. Several studies have shown that treatment with caffeine (CAF) blocks development and kills A. aegypti in the larval phase, preventing the production of adults and, consequently, the transmission of the mentioned diseases. In addition, preliminary work indicated that CAF treatment of A. aegypti changes the pattern of synthesis of esterase enzymes. The esterase enzymes play important roles in various physiological processes of the organisms, including among them the control of the metamorphosis of insects. In the present study, the esterases were analyzed in polyacrylamide gels stained with α and ß naphthyl acetates and Fast Blue RR. The samples were prepared with L2, L3 and L4 larval stages, pupae and adults. However, considering that the L4 stage was the one that allowed better observation of the bands, the study was almost restricted to it. The comparison of the treated and control mosquitoes involved observations of the presence and frequency of the esterase bands in the gels. The degree of expression of the bands was also analyzed on the basis of the staining intensity evaluated by the Global Lab Image computer program. The analysis showed that CAF changed five β-esterase bands (EST-17A to EST-21) and a group of α-esterase bands (EST-12 to EST-14). The bands EST-17A to EST-20 showed reduction in both characteristics, while EST-21 and the α-esterases showed increase of them. In view of the changes occurred in the degree of expression of the esterases, parallel to the larval development block we thought in the hypothesis that these esterases ...

Juvenile hormone (JH) is an important insect hormone that controls diverse biological processes in postembryonic development and adult reproduction. JH exerts its effects through the nuclear receptor Methoprene-tolerant (MET). MET is a transcription factor of the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family. In the presence of JH, MET forms a heterodimer with its DNA-binding partner Taiman (TAI). The MET-TAI complex directly binds to the regulatory regions of some JH target genes and regulates their transcription. However many questions remain unanswered regarding the JH-regulated gene expression. The work in this report aims to determine the role of protein kinase C (PKC) in JH signaling in adult mosquitoes and to find the direct target genes of Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor encoded by a JH early response gene. We discovered that PKC is an essential component of a membrane-initiated JH signaling pathway. PKC was activated by JH in a phospholipase C (PLC)-dependent manner. Inhibition of PKC activity dramatically decreased the JH-induced gene expression. RNAi experiment indicated that several PKC isoforms were involved in the JH action in adult female mosquitoes. We showed that PKC modulated the transactivation activity of MET by enhancing the binding of MET and TAI to the promoters of JH target genes. Kr-h1 is rapidly upregulated by JH in newly emerged mosquitoes. RNAi-mediated depletion of AaKr-h1 caused a substantial decrease in oviposited eggs, indicating that this protein plays an essential role in mosquito reproduction. We combined chromatin immunoprecipitation (ChIP) with cloning of the generated DNA and have identified chromatin binding sites of AaKr-h1 in Aedes aegypti. After adult emergence, binding of AaKr-h1 to its in vivo targets increased with the JH-induced increase in AaKr-h1. Interestingly, depletion of AaKr-h1 in newly emerged mosquitoes led to considerable upregulation of some AaKr-h1 target genes but downregulation of other target genes. The results suggest that AaKr-h1 acts downstream of AaMET to regulate gene expression in response to JH and that AaKr-h1 can activate or repress the expression of individual target gene.
Ph. D.

Juvenile hormone (JH) is one of the principal hormones that regulate insect development and reproduction. Accumulating evidence suggests that Methoprene-tolerant (Met) protein is a nuclear receptor of JH. Work by others has shown that Met is capable of binding JH at physiological concentration. An RNAi knockdown of Met causes down-regulated expression of JH-responsive genes and a phenotype similar to that observed in JH-deficient insects, suggesting that Met is required for mediating physiological and molecular responses to JH. The work in this report aims to understand the mechanisms underlying gene regulation by JH via Met. Met is a bHLH-PAS (basic-helix-loop-helix Per-ARNT-Sim) family protein. Many proteins in this family function as heterodimers formed with other proteins of this family. In a yeast two-hybrid screening, we discovered that another bHLH-PAS family protein, FISC, interacts with Met in the presence of JH. FISC is also required for JH functions as an RNAi knockdown of FISC down-regulated JH-responsive genes. To elucidate how Met and FISC mediate JH functions in gene regulation, we employed molecular biology techniques and characterized the formation of a JH-Met-FISC complex and its actions in activating gene expression using mosquito Aedes aegypti as a model. My results demonstrated that Met and FISC forms a complex when JH is present via their conserved N-terminal domains. The complex then binds to E box-like sequences presented in the promoter of JH-responsive genes to activate gene expression. This mechanism also applies to the fruit fly Drosophila melanogaster, suggesting that it is a conserved action of JH in insects. Further studies showed that DNA-binding by Met and FISC requires the basic regions of the bHLH domains of both proteins. Lastly we identified a consensus binding-site of Met and FISC.
Ph. D.

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.

O Hormônio Juvenil (HJ) é um sesquiterpenóide que participa de diversas funções do ciclo de vida de insetos. Em Apis mellifera o HJ está envolvido também com o processo de diferenciação de castas e polietismo etário. Neste trabalho, genes participantes da degradação e das vias de síntese do HJ nos corpora allata (CA) foram identificados a partir das seqüências disponibilizadas pelo sequenciamento do genoma de A. mellifera. A identificação destes genes baseou-se em análises funcionais, como interferência por RNA fita dupla, similaridade entre seqüências, expressão tecido-específica e busca por motivos conservados. Análises de quantificação dos transcritos destes genes revelaram padrões condizentes com os títulos de HJ e mostraram que o balanço entre as vias de síntese e degradação deste hormônio age em conjunto para regular os títulos de HJ. Uma importante associação entre a degradação do HJ pelas enzimas esterase do HJ e epóxido hidrolase do HJ com o processo de diferenciação dos ovários, que ocorre durante o estágio larval, foi estabelecida. Estas enzimas parecem atuar ativamente na manutenção dos níveis de HJ durante o processo de diferenciação de castas. A alimentação mostrou ser um processo de suma importância sobre o metabolismo do HJ durante a vida adulta de operárias, em adição ao controle exercido pela alimentação já descrito durante o período larval, que leva à diferenciação de castas distintas. A execução deste trabalho contribuiu de maneira significativa para o conhecimento deste sistema instigante que controla toda a homeostasia em uma colônia do inseto social, Apis mellifera.
The sequisterpenoid, Juvenile Hormone (JH), is a key regulator in many aspects of insect life. In the Honey bee, Apis mellifera¸ JH is additionally involved in caste differentiation and also in age task performance during adult worker life. Herein, we identified genes coding to JH synthesis enzymes pathway in corpora allata and degradation in hemolymph and tissues based on sequences from Genome Sequencing Consortium. The identification of those genes involved functional assays as RNA interference, expression levels in specific tissues, search for functional motifs and also similarity among sequences. The results showed that a balance between synthesis and degradation occurs to the maintenance of hemolymph JH titers. An association between JH degradation by the enzymes, JH esterase and JH epoxide hydrolase, and ovary differentiation during larval stage was established. JH degradation showed to act together with the JH synthesis process to maintain the cast-specific titers of JH, which is essential to females development into castes. The nutrition status in Honey bee adult workers is an important mechanism controlling JH metabolism, in the same way it was observed previously for larvae development. The progress of this work contributed significantly to the knowledge of this amazing social insect life, A. mellifera.

Os hormônios juvenis (HJ) são uma classe de sesquiterpenóides que executam um papel crucial no desenvolvimento dos insetos. O HJ modula a ação de ecdisona, prevenindo a metamorfose nos estágios larvais. Os títulos deste honnônio são determinados pela sua síntese nos Corpora Allata e pela atividade hidrolítica de uma esterase específica (EHJ -Esterase do Hormônio Juvenil), um membro da família das carboxiesterases (3.1.1.1), que transforma o HJ em um metabólito considerado inativo (HJ-ácido). O HJ está intimamente envolvido no desenvolvimento e diferenciação de castas em A. mellifera; os títulos de hormônio diferem consideravelmente durante o desenvolvimento das castas. A metodologia ORESTES (Open-Reading-Frame-Expressed-Sequence- Tags) foi usada para a obtenção da seqüência do gene da EHJ. Vinte e seis clones que mostraram homologia com a seqüência da EHJ de outros insetos foram usados para a construção de primers para a análise da expressão do gene em experimentos de RT-PCR. O fragmento obtido pela amplificação utilizando estes primers mostrou alta identidade com as EHJ de Drosophila melanogaster e Tenebrio molitor em nível de aminoácidos. A primeira fita de cDNA foi sintetizada usando RNA total e usada como molde para PCR. A normalização foi feita utilizando-se a expressão do gene da actina de A. mellifera. O gene da EHJ é mais expresso em corpo gorduroso e epitélío do intestino. O pico de expressão do gene em operárias foi observado nos estágios que antecedem a metamorfose (L5F e L5S), após este período ocorre diminuição de expressão do gene em pré-pupas e pupas jovens e um aumento de expressão no final do período pupal e adultos de até 15 dias. A atividade do gene da EHJ está relacionada aos títulos de HJ durante o desenvolvimento, o que sugere a importância da EHJ para que a metamorfose ocorra normalmente. Os níveis de mRNA da EHJ foram quantificados nas castas e sexos. Operárias mostraram a maior expressão do gene durante os estágios de L3, L4, L5F1 e L5S1. Em rainhas, a expressão aumenta em pré-pupa, ao contrário do que ocorre em operárias. Os menores níveis de expressão ocorrem em zangões. A expressão do gene da EHJ é menor quando o HJ é essencial para o desenvolvimento das características de rainhas, o que ocorre nos estágios larvais mais jovens, podendo ser estabelecida relação direta entre o HJ e os níveis de mRNA da EHJ durante o desenvolvimento e manutenção de características de cada casta. O gene mostrou menor expressão em ovários de rainhas nos estágios larvais, isto pode ter importância na manutenção dos níveis de HJ para que este órgão seja protegido de degeneração, garantindo seu desenvolvimento normal. Já que os níveis de HJ são diferentes nas castas e sexos, a atividade diferencial do gene da EHJ aparentemente é um elemento chave na manutenção dos tipos morfológicos nesta complexa sociedade. O gene foi iníbido pela aplicação de 20E em pupas, assim, sugerimos que o gene é induzido pela presença de HJ, como ocorre nas fases larvais jovens e após a emergência, e inibido na presença de ecdisteróides, já que os resultados obtidos neste trabalho mostram que o gene da EHJ está reprimido quando os títulos de ecdisteróides estão elevados nas fases pupais.
The juvenile hormones (JH) are a class of sesquiterpenoids that play a crucial role in insect development. JH modulate the activity of ecdysone, preparing for metamorphosis at the end of the larval phase. The titers of this hormone are mainly determined by synthesis in the corpora allata and by the hydrolytic activity of a specific esterase (JHE - Juvenile Hormone Esterase), a carboxylesterase family member (3.1.1.1), which transforms JH into a metabolite considered inactive (JHacid). JH is intimately involved in Apis mellifera development and caste differentiation; the hormone titers differ considerably in developing queens and workers. The ORESTES (Open-Reading-Frame-Expressed-Sequence-Tags) methodology was used to obtain the JHE gene sequence. Twenty six clone sequences that showed homology with JHEs of other insects were used to construct specific primers to perform RT-PCR, in order to analyze JHE gene expression. The fragment amplified using these primers showed high identity with the JHE of Drosophila melanogaster and Tenebrio molitor at amino acid level. First strand cDNA was synthesized using total RNA and used as template for PCR. A. mellifera actin gene expression levels were used for normalization. The JHE gene is highly expressed in fat body and gut epithelium. The highest peak of JHE gene expression in workers was observed in the stages before metamorphosis, i.e. L5F and L5S, after which there is a decrease in the gene expression of pre-pupae and young pupae, with a increase at the end of pupal stages, and in the adult stages (until 15 days). The JHE gene activity is extremely related with the JH titers during the development, what suggests the importance of JHE enzyme activity to the normal metamorphosis. We quantified JHE mRNA levels in the castes and sexes of A. mellifera. Workers have the highest JHE gene expression levels during L3, L4, L5F1 and L5S1. In queens, there is an increase of JHE gene expression in pre-pupae, otherwise in works this stage shows a decrease in JHE expression. The lowest expression levels occur in drones. JHE expression is lower when JH is essential for the development of queen characteristics, what occurs during the early phases. Therefore it is possible to establish a direct relationship between JH and JHE mRNA levels during development and maintenance of the characteristics in each caste. The gene shows low expression levels in queens ovaries during larval stages where it may be important to the maintenance of JH levels, in order to protect this organ from degeneration, and to warrant a normal development. Since the levels of JH are different in the castes and sexes, the differential activity of the JHE gene apparently plays a key role in the maintenance of the morphotypes of this complex insect society. The gene was inhibited by 20E application in pupae, so we can suggest that the gene is induced by JH presence like we detected during larval stages and after emergence, and inhibited by ecdysteroids, since the data obtained in this work suggest that the JHE gene is repressed when the ecdysteroids titers are elevated.

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. They are synthesized and secreted by a pair of small endocrine glands, the corpora allata (CA), which are intimately connected to the brain. The enzymes involved in the biosynthesis of JH are attractive targets for the control of mosquito populations. This dissertation is a comprehensive functional study of five Aedes aegypti CA enzymes, HMG-CoA synthase (AaHMGS), mevalonate kinase (AaMK), phosphomevalonate kinase (AaPMK), farnesyl diphosphate synthase (AaFPPS) and farnesyl pyrophosphate phosphatase (AaFPPase). The enzyme AaHMGS catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce HMG-CoA. The enzyme does not require any co-factor, although its activity is enhanced by addition of Mg2+. The enzyme AaMK is a class I mevalonate kinase that catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Activity of AaMK is inhibited by isoprenoids. The enzyme AaPMK catalyzes the cation-dependent reversible reaction of phosphomevalonate and ATP to form diphosphate mevalonate and ADP. The enzyme AaFPPS catalyzes the condensation of isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP). The enzyme AaFPPS shows an unusual product regulation mechanism, with chain length final product of 10 or 15 C depending on the metal cofactor present. The enzymes AaFPPase-1 and AaFPPase-2 efficiently hydrolyze FPP into farnesol, although RNAi experiments demonstrate that only AaFPPase-1 is involved in the catalysis of FPP into FOL in the CA of A. aegypti. This dissertation also explored the inhibition of the activity of some of the JH biosynthesis enzymes as tools for insect control. We described the effect of N-acetyl-S-geranylgeranyl-L-cysteine as a potent inhibitor of AaFPPase 1 and AaFPPase-2. In addition, inhibitors of AaMK and AaHMGS were also investigated using purified recombinant proteins. The present study provides an important contribution to the characterization of recombinant proteins, the analysis of enzyme kinetics and inhibition constants, as well as the understanding of the importance of these five enzymes in the control of JH biosynthesis rates.

Although feeding behavior is a matter of life and death for animals, the genetic factors that control it remain poorly understood. We have identified a novel regulator of hunger-induced behavior through comparison of transcriptomic changes in the fruit fly Drosophila melanogaster and the yellow fever mosquito Aedes aegypti. Head mRNA from each insect was sequenced at a roughly equivalent level of starvation. Using data gleaned from the protein orthology database OrthoDB, we looked for gene pairs in which both A. aegypti and D. melanogaster orthologs were significantly regulated by starvation. This identified Juvenile Hormone esterase (Jhe) as a possible modulator of hunger-induced behavior. Pan-neuronal knockdown of Jhe resulted in increased food consumption and caused enhancement of starvation-induced sleep suppression in Drosophila. These behavioral phenotypes were not caused by a developmental or metabolic defects, and were reproduced by feeding adult Drosophila methoprene, a synthetic Juvenile Hormone analog. Application of precocene I, an inhibitor of Juvenile Hormone biosynthesis, reversed the phenotype. Our analysis suggests that Jhe (and Juvenile Hormone by extension) is a novel and biologically relevant regulator of hunger-induced behavior.
Science, Faculty of
Zoology, Department of
Graduate

Ecdysis triggering hormone (ETH) is a neuropeptide known for its role in the orchestration of ecdysis. However, its role in the regulation of Juvenile Hormone (JH) synthesis is unknown. In Aedes aegypti, JH is synthesized by the corpora allata (CA) and titers are tightly regulated by allatoregulatory factors. In this study I describe the effect of ETH on JH synthesis during the late pupal stage and in the adult female after blood feeding. Analysis of ETH receptor (ETHRs) expression showed that ETHRs are present in both the CA and the corpora cardiaca (CC), a neurohemal organ. The data suggest that ETH regulates JH synthesis directly through its receptors in CA. Our results show that in pupa, ETH has a stimulatory effect on JH synthesis while in adult blood fed females, ETH is inhibitory. These findings constitute the first evidence of ETH as a regulatory peptide in mosquito JH synthesis.

In the present thesis, we have studied the metamorphosis of the cockroach Blattella germanica, focusing on two main periods of development, the embryonic and the postembryonic. In the embryonic development, we have studied the role of the juvenile hormone (JH), analysing the genes Methoprene-Tolerant (Met), Taiman (Tai), Krüppel homolog 1 (Kr-h1), and JH acid methyltransferase (JHAMT). We have also studied the transcription factor E93, which has a key role in adult morphogenesis, but that has never been studied in the embryo. Results have shown that both, JH and E93 play important roles during embryo development, especially in early stages. Regarding the postembryonic period, we studied the CREB binding protein (CBP) Nejire and the Transforming Growth Factor β (TGF-β) signalling pathway. Results have shown that both play relevant roles in the transition from the last nymphal stage to the adult.
En la presente tesis hemos estudiado la metamorfosis de la cucaracha Blattella germanica, centrándonos en dos períodos principales de desarrollo, el embrionario y el postembrionario. En el desarrollo embrionario, hemos estudiado el papel de la hormona juvenil (JH), analizando los genes Methoprene-Tolerant (Met), Taiman (Tai), el Krüppel homolog 1 (Kr-h1) y JH acid methyltransferase (JHAMT). También hemos estudiado el factor de transcripción E93, que tiene un papel clave en la morfogénesis adulta, pero que nunca se ha estudiado en el embrión. Los resultados han demostrado que tanto la JH como el factor E93 desempeñan papeles importantes durante el desarrollo del embrión, especialmente en etapas tempranas. En cuanto al período postembrionario, estudiamos la proteína de unión a CREB (CBP) Nejire y la vía de señalización Transforming Growth Factor β (TGF-β). Los resultados han demostrado que ambos desempeñan papeles relevantes en la transición entre la última fase ninfal y el adulto.

Isolated populations that inhabit various geographic and climatic ranges tend to diverge in their life history tactics. When development time is constrained by unfavorable seasons, often an organism must trade-off the investment of resource allocation between somatic and reproductive growth. The variation in reproductive tactics and juvenile hormone titers were studied among three populations of Romalea microptera from Athens, GA, Jacksonville, FL, and Miami, FL, all of which exist on a latitudinal cline. The Athens population was significantly younger at oviposition and gained significantly less body mass than both the Jacksonville and Miami populations, which did not differ from each other. Clutch mass did not differ across populations. With respect to both body size and oviposition age, Athens invested significantly more (measured by clutch size) to their first clutch than either Jacksonville or Miami, which did not differ from each other. Juvenile hormone and lipid profiles did not differ among populations. In response to the markedly reduced season length, results suggest that Athens grasshoppers respond with reproductive tactics that support terminal investment by investing more energy in less time to reproduction, at the expense of future reproduction.

Juvenile hormone (JH) plays pivotal roles in the development and reproduction of insects. Efforts to characterize the mechanisms of JH regulation are complicated due to JH pathways often being intertwined with those of 20-hydroxyecdysone (20E). Upon adult emergence, female Aedes aegypti enter a period of development during which they gain competence for mating, bloodfeeding, and egg production. JH levels rise dramatically and peak during the first 2-3 days post-emergence and remain relatively high until a bloodmeal is consumed, while 20E titers remain very low throughout the entire stage. Thus, post-emergence development offers a unique opportunity to study the effects of JH in the absence of 20E. In this study, four potential JH response genes were identified in newly emerged females. One such gene, AaKr-h1, is a homologue of Kr-h1, a zinc-finger transcription factor which has been characterized in Manduca sexta, Drosophila melanogaster, Tribolium castaneum, and Apis mellifera, and is involved in a diverse range of JH-regulated pathways. AaKr-h1 demonstrated a dose-dependent transcriptional response to JHIII as well as two JH mimics in abdominal ligation assays. The findings of this study indicate that Kr-h1 may be regulated by JH independently of any 20E regulation and suggests a fundamental, conserved role for Kr-h1 in JH-regulated pathways.
Master of Science in Life Sciences

Aedes aegypti is the primary vector for dengue, zika, chikungunya, and yellow fever viruses. Disease transmission through this mosquito places over 40% of the world's population at risk of contracting one or more of these pathogens. Current control strategies such as insecticide application have failed or carry additional burdens, such as off-target toxicity to mammals and birds. Our lab proposes utilizing a conserved arthropod hormone pathway, juvenile hormone (JH), related to growth and reproduction to curb these vector populations and reduce disease transmission. Additionally, JH is nontoxic to birds and mammals; it requires incredibly high doses to have lethal effects. We hypothesize that JH-responsive genes expressed early in the adult are responsible for her reproductive capacity and by manipulating the signaling downstream of the receptor, we will be able to decrease the female's fecundity and limit vector populations. Via bioinformatics screening of RNA-sequencing data using the New Tuxedo pipeline, we identified 47 potential transcription factor candidates. With the use of in vitro culturing of the mosquito's reproductive tissues in the presence of a translation inhibitor, we identified two early JH responsive gene candidates, FoxA and zinc finger 519, p-value <0.05. The functional characterization of these two remains to be seen, however, in Drosophila melanogaster, they both have roles in chromatin remodeling and require protein partners to carry out long range interactions.
Master of Science in Life Sciences
The mosquito, Aedes aegypti, is responsible for the spread of a myriad of viruses such as dengue, zika, and chikungunya. Currently, these infections have no vaccine or treatment available and transmission rates continue to steeply rise in response to the spread of breeding grounds. Popular insecticides carry detriments such as off-species toxicity and continuous application to treatment areas. Our lab proposes an alternative to these chemical insecticides by manipulating a developmental pathway in the mosquito. The Juvenile Hormone pathway is conserved in arthropods, responsible for growth and reproduction, and the hormone is nontoxic to mammals. Through the combination of bioinformatics and genomics studies, we have identified two JH-responsive gene candidates that are potential regulators of this pathway.

Els resultats d'aquesta Tesi posen de manifest el paper del cervell en el control del desenvolupament larvari de Sesamia nonagrioides. Així, S. nonagrioides és el primer lepidòpter on s'ha trobat que es pot desenvolupar de larva a adult sense cervell. Les nostres dades mostren que les mudes depenen de l'alliberament d'ecdisteroides per les glàndules protoràciques (GPs) però poden tenir lloc sense la hormona protoracicotròpica (PTTH) del cervell.
Mentre que les larves decapitades (larves sense cervell ni corpora allata, (CA)) pupen, les larves de les que s'ha privat del cervell (però s'ha mantingut el CA) sofreixen sovint varies mudes successives; la primera muda pot ser de larva o pupa segons l'edat a la que la larva ha estat privada del cervell i el fotoperíode al qual la larva s'ha desenvolupat. Moltes de les larves L6 privades del cervell un dia després de la muda van mudar a larva independentment de les condicions de fotoperíode durant el desenvolupament, de dia llarg (DL) o curt (DC), mentre que 5 dies després totes les larves desenvolupades en DL van pupar i les de la mateixa edat però desenvolupades en DC van mudar a larva. Els implants de cervell acceleren les mudes a pupa però no alteren les mudes a larva. S. nonagrioides no sembla tenir cap font alternativa als CA de hormona juvenil (HJ). Les larves decapitades no van mostrar quantitats significades de HJ mentre que les larves privades de cervell van mostrar concentracions que es poden detectar fins i tot 10 dies després de la manipulació quirúrgica, però els implants de cervell no activen els CA essent aparentment neural l'activació d'aquests factors. El cervell podria ser el responsable del manteniment de l'estat larvari per inhibició neural de la pupació; quan les larves d'edat variada van ser privades del cervell, la majoria van pupar. De manera similar, el cervell també podria ser el responsable del manteniment de la diapausa per inhibició de la pupació, conseqüentment, quan les larves van ser privades del cervell (tot mantenint o no els CA) les diferencies entre larves dipausants o no diapausants van desaparèixer.
El nivell d'ecdisteroides en les larves decapitades augmentà 10 dies després de la manipulació, aproximadament el temps necessari per a pupar en aquelles larves en absència de HJ, encara que l'extracció de les GPs va impedir la muda, i això probà que la presencia de GPs és essencial per al procés de muda. Les GPs en les larves de S. nonagrioides poden funcionar sense estimulació del cervell i la PTTH pot ser alliberada per una font de fora del cap; a S. nonagrioides hem identificat la PTTH mRNA i una font alternativa en l'intestí. La qPCR confirmà que el gen de la PTTH a S. nonagrioides s'expressa molt al cervell de l'instar 6è amb un màxim al dia 5è i un mínim en la prepupa, però el nivell d'expressió de la PTTH es va detectar també en l'intestí de les larves intactes i encara més en el de las decapitades amb una expressió màxima en la prepupa.
La més gran part de les larves decapitades muden a pupa sense cap senyal de desenvolupament d'adult mentre que la majoria de pupes provades de cervell sofreixen metamorfosi a adult, lo que suggereix que la transformació de pupa a adult depèn d'un factor desconegut present en les larves privades de cervell però no en les decapitades. En S. nonagrioides la HJ aplicada tòpicament no solament no va inhibir la metamorfosi de pupa a adult però la pogué haver afavorit mentre que l'aplicació d'un agonista d'ecdisteroides a les pupes no va tenir cap efecte en el desenvolupament d'adult.
La ingestió per les larves de S. nonagrioides de quantitats subletals de la proteïna Cry1Ab continguda en les fulles de panís o en la dieta va produir un perllongament del seu desenvolupament acompanyat d'un augment en el nombre de mudes abans de pupar però només en les larves criades en condicions de DL, no en les criades en condicions DC. Aquests resultats són deguts a un augment del nivell de HJ en la hemolimfa de les larves no diapausants (DL) alimentades amb fulles de panís Bt o amb la proteïna Bt afegida a la dieta; pel contrari, no es va detectar el possible lleuger increment de HJ causat per la ingestió de la proteïna Bt en les larves diapausants (DC). A més, l'efecte de la proteïna Bt en la concentració d'ecdisteroides en les larves no diapausants va ser suprimir l'augment de la hormona que era necessari per la pupació i per tant va retardar la pupació en les larves tractades. Aquestes respostes poden ser considerades com un mecanisme de defensa que permet que algunes larves puguin mudar i sobreviure a la ingestió de la toxina.
Los resultados de esta Tesis ponen de manifiesto el papel del cerebro en el control del desarrollo larvario de Sesamia nonagrioides. Así, S. nonagrioides es el primer lepidóptero en el que se demuestra que el desarrollo de larva a adulto puede producirse sin la presencia del cerebro. Nuestros datos demuestran que aunque las mudas dependen de la liberación de ecdisteroides por las glándulas protorácicas (GPs), estas pueden ser activadas sin hormona protoracicotrópica (PTTH) del cerebro.
Mientras que las larvas decapitadas (larvas sin cerebro ni corpora allata, (CA)) pupan, las larvas descerebradas, de las que se ha extraído el cerebro pero mantienen el CA, sufren a menudo varias mudas sucesivas, la primera a otra larva o pupa según la edad a la que la larva ha sido desprovista del cerebro y según el fotoperiodo bajo el cual la larva se había desarrollado. Muchas larvas desprovistas del cerebro un día después de la muda a sexto estadio mudan a larva independientemente de las condiciones de fotoperiodo, día largo (DL) o corto (DC), recibidas durante su desarrollo, mientras que 5 días después todas las larvas desarrolladas en día largo puparán y las de la misma edad pero desarrolladas en día corto mudaran a larva. Los implantes de cerebro aceleran las mudas a pupa pero no alteran las mudas a larva. S. nonagrioides no parece tener ninguna fuente de hormona juvenil (HJ) alternativa a los CA. Las larvas decapitadas no mostraron cantidades significativas de HJ mientras que las desprovistas de cerebro mostraron cantidades de HJ detectables incluso 10 días después de la manipulación quirúrgica indicando así que los CA siguieron liberando HJ en ausencia del cerebro. Los implantes de cerebro no activaron los CA por lo que su activación es aparentemente neural. El cerebro debe ser el responsable del mantenimiento del estado larvario por inhibición neural de la pupación ya que cuando se extrajo el cerebro a larvas de diferentes edades la mayoría puparon. De manera similar, el cerebro también debe ser el responsable del mantenimiento de la diapausa ya que cuando las larvas fueron desprovistas del cerebro (manteniendo o no los CA) las diferencias entre larvas dipausantes o no diapausantes desaparecieron.
El nivel de ecdisteroides en las larvas decapitadas aumentó 10 días después de la extracción del cerebro, este tiempo es aproximadamente el tiempo que las larvas de último estadio larvario necesitan para pupar. La extracción de las GPs impidió la muda, lo que demuestra que su presencia es esencial para que tenga lugar el proceso de la muda. Ante la evidencia de que las GPs de las larvas de S. nonagrioides podían activarse sin la estimulación por hormona PTTH del cerebro se buscaron fuentes alternativas de la hormona y se identificó PTTH mRNA en el intestino de la larva. La qPCR confirmó que el gen de la PTTH en S. nonagrioides se expresa de forma elevada en el cerebro de la larva de 6º estadio con un máximo el 5º día del mismo y un mínimo en la prepupa, pero la expresión de la PTTH se detectó también en el intestino de las larvas intactas y mucho más en las decapitadas con máxima expresión en el periodo de prepupa.
De forma general se asume que la metamorfosis pupa-adulto en los insectos se produce en ausencia de HJ y en el caso de S. nonagrioides la mayoría de las larvas decapitadas mudan a pupa sin mostrar posteriormente ningún indicio de desarrollo a adulto mientras que la mayoría de pupas desprovistas de cerebro pero que mantiene su CA sufren metamorfosis a adulto. Este hecho sugirió que la transformación de pupa a adulto en esta especie depende de algún factor presente en las larvas descerebradas pero no en las decapitadas. En S. nonagrioides la HJ aplicada tópicamente no solamente no inhibió la metamorfosis de pupa a adulto sino que la favoreció mientras que la aplicación de un agonista de ecdisteroides a las pupas no tuvo efecto sobre el desarrollo a adulto.
La ingestión por las larvas de S. nonagrioides de cantidades subletales de la proteína Cry1Ab contenida en hoja de maíz o en dieta produjo un prolongamiento de su desarrollo acompañado de un aumento en el número de mudas larvarias antes de pupar, pero sólo en las larvas desarrolladas en condiciones de DL, no en las desarrolladas en condiciones de DC. Estos resultados son consecuencia del aumento de HJ en la hemolinfa de las larvas no diapausantes alimentadas con hoja de maíz Bt o con la proteína Bt añadida. Sin embargo, no se detectó el posible ligero aumento de HJ causado por la ingestión de la proteína Bt en las larvas diapausantes (desarrolladas en DC). Otro efecto de la ingestión de la proteína Bt en las larvas no-diapausantes fue suprimir el aumento en la concentración de ecdisteroides necesario para la pupación que por tanto se retrasó en las larvas tratadas. Estas respuestas pueden ser consideradas como un mecanismo de defensa que permite que algunas larvas puedan mudar y así sobrevivir a la ingestión de la toxina.
The results of this Thesis highlight the role of brain in the control of larval development in Sesamia nonagrioides. Thus, S. nonagrioides is the first lepidopteran found to develop from larvae to adult without brain. Our data show that molts depend on the release of ecdysteroids by prothoracic glands (PGs) but they can occur without prothoracicotropic hormone (PTTH) from the brain.
While all decapitated larvae (larvae without brain nor corpora allata, (CA)) pupate, the debrained larvae (larvae with no brain but with CA) often undergo several successive molts; first molt could be to larva or pupa, depending on the age at which the larva has been debrained and on the photoperiod under which the larvae have developed: many of the L6 larvae debrained 1 day after molting, molted to larvae independently of the photoperiod conditions of development, long (LD) or short (SD) day, but 5 days later all larvae developed under LD conditions pupated whereas larvae of the same age developd under SD conditions molted to larvae. Brain implants slightly accelerate pupal molts but do not alter the timing of larval molts. S. nonagrioides does not seem to have any alternative source of juvenile hormone (JH) to the CA. Decapitated larvae did not show noticeable amounts of JH while debrained larvae showed detectable concentrations of JH still 10 days after the surgical manipulation but the brain implants do not activate CA, apparently being neural the activation of these factors. The brain might be responsible for larval stage maintenance by neural inhibition of pupation; when the larvae of any age were deprived of their brain, the majority pupated. In the same way, the brain might be also responsible of diapause maintenance by neural inhibition of pupation; consequently, when the larvae were deprived of their brain (maintaining or not their CA) differences between diapausing and non-diapausing larvae disappeared.
The level of ecdysteroids in the decapitated larvae increased ten days after manipulation, approximately the time needed to pupate in these larvae in absence of JH, but the removal of PGs prevented molting, proving that the presence of PGs is essential for the molting process. The PGs of S. nonagrioides larvae can function without brain stimulation and PTTH could be released by a source outside the head; in S. nonagrioides we have identified the PTTH mRNA and an alternative PTTH source in the gut. The qPCR confirmed that the PTTH gene of S. nonagrioides is strongly expressed in the brain of the 6th instar with a maximum on day 5 and a minimum in prepupa, but the level of PTTH expression was also detected in the gut of intact and even more in decapitated larvae with a maximum expression in prepupa.
Most decapitated larvae molt to pupa with no sign of adult development while the majority of debrained pupae suffer metamorphosis to adult thus suggesting that pupal-adult transformation depends on an unknown factor present in the debrained but not in the decapitated larvae. In S. nonagrioides JH applied topically not only did not inhibit the pupal-adult metamorphosis but could have favored it while the application of an ecdysteroids agonist to the pupae had no effect on the adult development.

Ingestion by S. nonagrioides larvae of sub-lethal amounts of Cry1Ab protein contained in maize leaves or the diet produced a prolonged development accompanied by an increase in the number of molts before pupating only in the larvae reared under LD conditions but not in the larvae reared under SD conditions. These results are due to an increase of the level of JH in the hemolymph in the non-diapausing larvae fed with Bt maize leaves or with Bt protein in the diet; on the contrary, in diapausing (SD) larvae the possible low increase of JH due to the Bt toxin ingested was not detected. In addition, the effect of Bt toxin on the ecdysteroids titer in non diapausing larvae was to suppress the increase of the hormone necessary for the pupation of and thus delaying pupation in the treated larvae. These responses may be considered as a defense mechanism allowing some larvae to molt and to survive to the toxin ingestion.

The antibiotic fungal metabolite sinefungin is a potent inhibitor of S-adenosylmethionine-acceptor methyltransferases. Its effect on insect metabolism and especially on corpora allata farnesoic acid methyltransferase, which catalyzes the penultimate step of juvenile hormone biosynthesis, was investigated in Locusta migratoria. Injection of sinefungin results in a delay of imaginal molt and in suppression of ovary development. Isolated corpora allata are unable to synthesize juvenile hormone III in the presence of more than 1.0 mM sinefungin. In a cell-free system containing the S-adenosylmethionine-dependent farnesoic acid methyltransferase from corpora allata sinefungin is a competitive inhibitor of the synthesis of methylfarnesoate with Ki of 1 μM.

In Aedes aegypti, development and reproduction are regulated by juvenile hormone III (JH). This master regulatory hormone is synthesized by the corpora allata (CA), a pair of endocrine glands with neural connections to the brain. JH titers are largely determined by the rate of biosynthetic activity of the CA and are regulated by inhibitory and stimulatory factors. Like JH, the ecdysteroid 20-hydroxyecdysone (20E) is a key hormonal regulator and has been proposed as an allatoregulator in other insects. However, its part in the regulation of JH biosynthesis of mosquitoes was unknown. The specific aims of this dissertation were to (1) evaluate if 20E plays a role in the activation of the late pupal CA and (2) evaluate if 20E plays a role in the reactivation of JH synthesis in blood-fed females. To this end, we evaluated if 20E could prematurely activate JH biosynthesis in the CA of an early pupa (24h prior to eclosion or -24h). Remarkably, in vitro stimulation with 20E at -24h initiated JH synthesis at a time when transcript levels for most JH biosynthetic enzymes are low. Moreover, the application of 20E correlated with an increase in the enzymatic activity of juvenile hormone acid methyltransferase (JHAMT), a critical enzyme of the biosynthetic pathway. Additionally, separation of the CA from the brain increased JH synthesis. Together, these results indicate that 20E acts as a developmental mediator of CA maturation which overrides an inhibitory effect of the brain. In our previous aim we demonstrated that 20E mediates activation of the pupal CA which ensures the development of ovarian follicles of the newly emerged female. For mosquitoes, a blood-meal is required to complete vitellogenesis and results in suppression of CA activity. However, the CA must be reactivated to initiate the second gonotrophic cycle. Our findings show that in vitro stimulation with 20E at 24h post blood feeding reactivates the gland. Again, stimulation with the ecdysteroid resulted in increased activity of another key enzyme, farnesal dehydrogenase (FALDH). These results suggest a stimulatory role of 20E on the biosynthetic activity of the CA in the blood fed female.

Orientador: Eduardo Antônio Sanches
Resumo: O Brasil apresenta um grande potencial para produção de peixes em ambientes marinhos/estuarinos, entretanto, isso ainda não é realidade por vários fatores, dentre eles a tecnologia de produção ainda limitada de algumas espécies nativas promissoras, como é o caso do robalo-flecha Centropomus undecimalis. Assim, o objetivo deste estudo foi avaliar o desempenho zootécnico e desenvolvimento gonadal de juvenis de C. undecimalis após alimentação com ração (45% PB) contendo 17β-estradiol (E2) e 17α-metiltestosterona (MT). Para tanto, 450 peixes (um ano de idade) foram submetidos a um delineamento experimental inteiramente casualizado contendo três tratamentos em triplicata. Os tratamentos foram: 1ª) ração sem adição de hormônio (grupo controle); 2ª) ração com 100 mg de E2 por kg; 3ª) ração com 60 mg de MT por kg,, fornecidas durante 55 dias. Após o término da alimentação com hormônio os peixes foram alimentados com a mesma ração, sem adição de hormônio, durante 313 dias, totalizando 368 dias. Não se verificou efeito (P>0,05) dos tratamentos sobre os parâmetros zootécnicos avaliados. Não foram encontradas estruturas morfológicas da completa diferenciação sexual ou não se observou células especificas de ovários ou testículos ao final dos 368 dias. Conclui-se que as alimentações contendo 17βestradiol e 17α-metiltestosterona não influenciaram o desempenho zootécnico de juvenis de C. undecimalis após um ano de alimentação.
Abstract: The Brazil has great potential for fish production in marine/estuarine environments, however, this is not yet reality due to several factors, including the still limited production technology of some promising native species, such as the common snook, Centropomus undecimalis. Thus, the objective of this study was to evaluate the zootechnical performance and gonadal development of juveniles of C. undecimalis after feeding with feed (45% CP) containing 17β-estradiol (E2) and 17α-methyltestosterone (MT). To that end, 450 fish (one year old) were submitted to a completely randomized experimental design containing three treatments in triplicate. The treatments were: 1st) ration without addition of hormone (control group); 2nd) ration with 100 mg of E2 per kg; 3rd) ration with 60 mg MT per kg, provided for 55 days. After the end of the feeding with hormone the fish were fed the same feed, without addition of hormone, during 313 days, totaling 368 days. There was no effect (P>0.05) of the treatments on the zootechnical parameters evaluated. No morphological structures were found of complete sexual differentiation or no specific ovary or testis cells were observed at the end of 368 days. It was concluded that feeds containing 17β-estradiol and 17αmethyltestosterone did not influence the performance of juveniles of C. undecimalis after one year of feeding. KEY-WORDS: feminization, masculinization, hormonal administration, marine fish farming.
Mestre

AbstractFETTEROLF, BRANDON JOHN. Synthesis and Analysis of Mechanism Based Inhibitors of Juvenile Hormone Epoxide Hydrolase from Insect Trichoplusia ni. (Under the direction of Russell J. Linderman) The research presented here entails a multi-disciplinary advance at understanding the role of juvenile hormone epoxide hydrolase in the metabolism of juvenile hormone in last stadium Trichoplusia ni. The work combines synthetic organic chemistry and biochemistry.A series of juvenile hormone analogs have been synthesized and their effectiveness to inhibit insect JHEH has been ascertained. Some of the key elements in the synthetic sequence for the fluorinated compounds include chemoselective oxidation of a primary alcohol in the presence of an alpha-fluorinated alcohol, introduction of a geminal difluoro group via a Reformatsky reaction or use of the DAST reagent, and selective nucleophilic and electrophilic epoxidation reactions. The synthesis of some of the non-fluorinated compounds employed regio-selective opening of an epoxide, Sonogashira palladium catalyzed cross coupling, nucleophilic epoxidation, and a variety of protecting group manipulations. Of all the inhibitors tested, the alkene 10 was the most effective alternative substrate with an I50 of 4.29 mM, slightly more potent than the benchmark MEMD. We also found that the introduction of fluorine increased the potency of the inhibitors compared to the non-fluorinated analogs. Lastly, inhibitor 5 provided insight to support a novel mechanism based inhibition of juvenile hormone epoxide hydrolase by formation of a tetrahydrofuran intermediate.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第11645号
農博第1501号
新制||農||910(附属図書館)
学位論文||H17||N4038(農学部図書室)
23288
UT51-2005-D394
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 田中 克, 教授 矢野 秀雄, 助教授 田川 正朋
学位規則第4条第1項該当

Juvenile hormone (JH) is the central hormonal regulator of life-history trade-offs in many insects. In Aedes aegypti, JH regulates reproductive development after emergence. Little is known about JH’s physiological functions after reproductive development is complete or JH’s role in mediating life-history trade-offs. By examining the effect of hormones, nutrition, and mating on ovarian physiology during the previtellogenic resting stage, critical roles were determined for these factors in mediating life-history trade-offs and reproductive output. The extent of follicular resorption during the previtellogenic resting stage is dependent on nutritional quality. Feeding females a low quality diet during the resting stage causes the rate of follicular resorption to increase and reproductive output to decrease. Conversely, feeding females a high quality diet causes resorption to remain low. The extent of resorption can be increased by separating the ovaries from a source of JH or decreased by exogenous application of methoprene. Active caspases were localized to resorbing follicles indicating that an apoptosis-like mechanism participates in follicular resorption. Accumulations of neutral lipids and the accumulation of mRNA’s integral to endocytosis and oocyte development such as the vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) were also examined under various nutritional and hormonal conditions. The abundance of mRNA's and neutral lipid content increased within the previtellogenic ovary as mosquitoes were offered increasing sucrose concentrations or were treated with methoprene. These same nutritional and hormonal manipulations altered the extent of resorption after a blood meal indicating that the fate of follicles and overall fecundity depends, in part, on nutritional and hormonal status during the previtellogenic resting stage. Mating female mosquitoes also altered follicle quality and resorption similarly to nutrition or hormonal application and demonstrates that male accessory gland substances such as JH III passed to the female during copulation have a strong effect on ovarian physiology during the previtellogenic resting stage and can influence reproductive output. Taken together these results demonstrate that the previtellogenic resting stage is not an inactive period but is instead a period marked by extensive life-history and fitness trade-offs in response to nutrition, hormones and mating stimuli.

Introdução: Hexamerinas são proteínas de estocagem sintetizadas pelo corpo gorduroso de larvas de insetos e secretadas na hemolinfa, onde se acumulam. A função canônica das hexamerinas consiste em servir de reserva de aminoácidos e energia para a reconstrução de tecidos e órgãos durante a metamorfose. Este trabalho teve como objetivo a busca por evidências de funções alternativas das hexamerinas durante o ciclo de vida de abelhas A. mellifera. Resultados: Os perfis temporais de expressão das quatro hexamerinas (HEX 70a, HEX 70b, HEX 70c e HEX 110), verificados por meio de SDS-PAGE e western blot, corroboram sua função canônica na metamorfose. Consistente com esta função, as quatro hexamerinas foram localizadas no citoplasma das células do corpo gorduroso utilizando-se anticorpos específicos e microscopia confocal. No entanto, funções adicionais puderam ser inferidas com base nos seguintes resultados: (1) Foci das quatro hexamerinas foram localizados nos núcleos de algumas células do corpo gorduroso em metamorfose, levando à hipótese de que têm função anti-apoptótica durante este período crítico do desenvolvimento; (2) Além disso, HEX 70a e HEX 110 foram localizadas no citoplasma e núcleo de células ovarianas e testiculares, indicando função no desenvolvimento e maturação das gônadas; (3) A co-localização de um análogo de timidina (EdU) e HEX 70a nos núcleos das células dos ovaríolos, sugeriu fortemente uma função na proliferação celular. O knockdown de HEX 70a in vivo por meio de injeção de anticorpo específico prejudicou o crescimento dos ovaríolos de rainhas, reforçando a hipótese de função na proliferação celular, (4) interferiu na esclerotização da cutícula de operárias, indicando função na formação do exoesqueleto e (5) provocou a antecipação da ecdise adulta, provavelmente em resposta à ausência (ou diminuição) dos aminoácidos derivados das hexamerinas. Foram investigados também aspectos da regulação dos genes de hexamerinas. A manipulação experimental da dieta alimentar e dos títulos do hormônio juvenil (HJ) interferiram claramente na expressão dos genes de hexamerinas. A potencial ação reguladora do HJ foi reforçada pelos resultados de análises por bioinformática da região 5 UTR de cada gene de hexamerina (Martins et al., 2010) que revelaram potencial motivo de ligação à proteína Ultraspiracle (Usp), um membro do complexo receptor do HJ no DNA. Procedimentos para expressar as hexamerinas in vitro em sistema de bactérias e purificá-las estão em progresso visando a caracterização da estrutura e de interações entre as subunidades. Conclusão: Estes resultados ressaltam que as hexamerinas têm outras funções no ciclo de vida de A. mellifera, além da função já bem estabelecida de reserva de aminoácidos para a metamorfose.
Background: Insect hexamerins are storage proteins synthesized by the larval fat body and secreted into the hemolymph, where they accumulate. The canonical function of hexamerins is to provide amino acids and energy for the reconstruction of tissues and organs during pupal-to-adult development. The aim of the current study was to search for evidence of alternative roles for the hexamerins in the life cycle of the honey bee, A. mellifera. Results: The canonical role of insect hexamerins received support from our data on the temporal expression profiles of the four honey bee hexamerin subunits (HEX 70a, HEX 70b, HEX 70c and HEX 110), as verified by SDS-PAGE and western blot using hemolymph and fat body samples. Consistent with the canonical function, the four hexamerins were localized in the cytoplasm of fat body cells, during metamorphosis, by using specific antibodies and confocal laser-scanning microscopy. However, additional functions could be inferred by the following findings: (1) The four hexamerins were also localized in the nuclei of some fat body cells, thus tentatively suggesting an anti-apoptotic role during metamorphosis; (2) Furthermore, HEX 70a and HEX 110 were localized in the cytoplasm and nucleus of ovarian and testicular cells, pointing to a role in gonad development and maturation. Co-labeling of the thymidine analog EdU and HEX 70a in the ovariole cell nuclei, strongly suggested a role in cell proliferation; HEX 70a depletion via injection of the specific antibody in queen pupae impaired ovariole growth, thus strengthening our hypothesis on a role in cell proliferation, (3) HEX 70a depletion also impaired cuticle sclerotization, indicating a function in exoskeleton formation, and (4) led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. We also investigated aspects of the regulation of hexamerin genes. The experimental manipulation of diet consumption and juvenile hormone (JH) titer clearly interfered in the expression of hexamerin genes. Regulation by JH was also supported by a previous bioinformatics analysis of the 5 UTR region of each hexamerin gene (Martins et al., 2010), which revealed a potential binding site for Ultraspiracle (Usp), a member of the JH receptor complex in the DNA. Experiments are in progress for in vitro expression and purification of the four hexamerins aiming to further characterize their structures and interactions. Conclusion: Taken together, these results imply in novel roles for hexamerins in the life cycle of A. mellifera in addition to their well-established role as amino acids sources for metamorphosis.

Em sete adolescentes com dermatomiosite (DM) juvenil (DMJ) foi avaliada a função gonadal através do estadiamento puberal, aspectos da sexualidade, exame físico da genitália e exames complementares: análise seminal (duas amostras com intervalo de um mês), anticorpos anti-espermatozóides, ultra-sonografia escrotal e dosagens hormonais (testosterona, hormônio estimulante do folículo, hormônio luteinizante, prolactina, T3, T4, T4 livre e TSH). Todos os pacientes apresentaram terazospermia, dois tiveram varicocele e um anticorpo anti-espermatozóide localizado em peça intermediária. A futura fertilidade destes pacientes é incerta e estudos de prevalência de função gonadal em populações de jovens e adultos do sexo masculino com DM são necessários
In seven adolescents with dermatomyositis (MD) juvenile (JDM), gonadal function was evaluated through the puberal estadiamento, aspects of the sexuality, examination of the genitalia, semen analysis (two semen samples over a period of one month), anti-sperm, testicular ultrasound and hormones (testosterone, follicle stimulating hormone, luteinizing hormone, prolactin, T3, T4, free T4 and TSH).

Wax secretion in worker honeybees is significantly related to the age of the worker and, while the secretion pattern remains the same, the absolute amount of wax secreted varies seasonally. Comb building festoons, previously thought to be the site of wax secretion, contain only a fraction of the newly-secreted wax in the nest. Festooning behaviour was also found to be seasonal. The amount of wax secreted by workers was significantly affected by hive. Although age-related changes in behaviour and physiology of worker honeybees appears to be modulated by juvenile hormone (JH), wax secretion is not dependent on JH. Manipulating JH III titres by injecting the hormone and manipulating the only source of the hormone (the corpora allata: CA) did not affect wax secretion. Increasing haemolymph JH titre shortly after ec1osion did not affect the amount of wax produced by workers aged 3 to 21 days, nor could a critical period be found during which elevated hormone titres would affect the rate of wax secretion. Allatectomy of newly eclosed workers did not affect wax production. Removing the putative neural feedback inhibition on the CA did not result in a change in wax production. Implanting CA from older workers into younger workers had no significant effect. Methoprene, a widely-used JH analog, caused reduced wax secretion in workers. It is suggested that methoprene poisons worker honeybees. The results obtained are consistent with an alternative model for wax secretion proposed by Butler (1954). The methodological problems found in this work are present in many other studies. When viewed in this light, the role of JH in polyethism appears dubious and there are alternative models of polyethism that do not have these shortcomings.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-graduação em Aquicultura, Florianópolis, 2013
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A manipulação do sexo gonadal em peixes de cultivo é uma importante técnica utilizada na aquicultura, pois permite obter benefícios associados a um dos sexos. Os hormônios esteroides são utilizados para controlar o sexo gonadal. Apesar desta técnica já ser utilizada com várias espécies pouco se sabe a respeito do uso do 17ß-estradiol (E2) com o robalo-peva Centropomus parallelus e robalo-flecha Centropomus undecimalis. Estas duas espécies são nativas, eurialinas e com alto valor de mercado. Sendo assim, o objetivo desta Tese foi avaliar o efeito de rações com diferentes concentrações de E2 na proporção dos sexos, na sobrevivência e crescimento dos juvenis de robalo-peva e robalo-flecha. Os experimentos foram divididos em duas etapas: feminização e pós feminização. No final dos experimentos foi possível obter um alto percentual de fêmeas. Também foi possível observar que o crescimento dos peixes só foi reduzido durante o tratamento hormonal. Após o fim da oferta da ração com hormônio os peixes recuperaram o crescimento. Além disso, o hormônio não afetou a sobrevivência dos peixes. Com este trabalho concluiu-se que o uso de E2 na ração é eficiente na feminização de juvenis de robalo-peva e robalo-flecha.

The manipulation of gonadal sex in fish farming is an important technique used in aquaculture, since it gives benefits associated with one sex. The steroid hormones are used to control the gonadal sex. Although this technique is already used in several species, little is known about the use of 17ß-estradiol (E2) with fat snook Centropomus parallelus and common snook Centropomus undecimalis. These two species are native, and eurialinas with high market value. Therefore, the aim of these experiments was to evaluate the effect of diets with different concentrations of E2 in the sex ratio, survival and growth of juvenile fat snoook and common snook. The experiments were divided into two stages: feminization and post-feminization. At the end of the experiments was to obtain a high percentage of females. The growth of the treated fish was significantly retarded during the period of treatment, while there was no side effect detected post-tretament and the retarded fish caught up after the E2 treatment. Furthermore, the hormone did not affect survival. With this work it was concluded that the use of E2 in the diet is effective in feminization of juvenile fat snook and comoon snook.

Em abelhas eussociais o controle das condições internas do ninho é importante para a sobrevivência da colônia e um dos principais mecanismos para controle da temperatura colonial é a escolha do local de nidificação, como ocos de troncos de árvores, tal qual ocorre nas abelhas sem ferrão Melipona quadrifasciata, sistema biológico deste estudo. Além de mecanismos comportamentais, em Hymenoptera são também conhecidos mecanismos fisiológicos de termorregulação (termogênese) como a ocorrência de ciclos fúteis (descritos para mamangavas Bombus) e o tremor muscular. A termogênese em abelhas ocorre principalmente durante incubação da cria, atividades sociais e aquecimento pré-vôo. As aminas biogênicas desempenham um amplo espectro de funções em abelhas, e é sugerido que o sistema noradrenérgico/adrenérico de vertebrados é funcional e estruturalmente equivalente ao sistema octopaminérgico/tiraminérgico de insetos. O emprego de bloqueador beta-adrenérgico em abelhas pode levar a hipotermia, emergindo a hipótese de que haveria envolvimento de uma via octopaminérgica na energética e termogênese de abelhas. Os três principais aspectos da termorregulação focados neste trabalho são a termorregulação colonial, a energética individual e mecanismos de termogênese. Com relação à termorregulação colonial, o objetivo principal foi descrevê-la em duas regiões do ninho (discos de cria e potes de alimento), por meio de registro de séries temporais de temperatura e umidade relativa, e estudo da atividade externa de vôo. Foi detectada ritmicidade nas séries temporais de colônias abertas, com influência das periodicidades da temperatura ambiente, umidade relativa e luminosidade, sobre as séries temporais registradas dentro da colônia. As séries temporais de atividade de vôo apresentavam periodicidades similares às ambientais, mas também eram relacionadas às da cria. Em colônias fechadas, as séries temporais dos discos de cria apresentavam periodicidades próximas a 24h e 12h, sugerindo aspecto endógeno da regulação de temperatura e umidade próximo à cria. Também, objetivou-se caracterizar o efeito do tipo de iluminação e de diferentes temperaturas. Foram estudados, por meio de respirometria intermitente, os efeitos do ciclo claro/escuro a 28°C (fotoperíodo normal, fotoperíodo invertido, claro e escuro constante) sobre a taxa metabólica (TM), e os efeitos de diferentes temperaturas ambientais (10 a 40°C) sobre a TM e quociente respiratório (QR). Os diferentes ciclos de claro/escuro afetaram a TM, sendo observados, em fotoperíodo normal e claro constante valores de TM baixos entre 19h e 5h. Em fotoperíodo invertido, valores diminuídos foram registrados entre 6h e 18h, sugerindo um ritmo exógeno em resposta a mudanças fotoperiódicas. Entre 7h e 17h59, no claro, ressalta-se que não há diferenças estatisticamente significativas na TM. A temperatura tem um efeito significativo, com valores diminuídos a 35 e 40°C e mais ainda a 10°C. Entre 15 e 30°C não há diferenças significativas. Na terceira parte do trabalho objetivou-se verificar o efeito tempo-dependente de bloqueadores beta-adrenérgicos e octopaminérgicos (Alprenolol e Mianserina) sobre a TM e temperatura torácica de forrageadoras e verificar se a octopamina e methoprene (análogo de hormônio juvenil) compensam os efeitos dos bloqueadores. Também foi analisado se há alterações dos substratos energéticos catabolizados, considerando o QR e a atividade enzimática (Vmax de hexoquinase, trealase, HOAD e glicogênio fosforilase). Com o emprego dos bloqueadores, foram detectadas alterações no QR e na atividade das quatro enzimas. A diminuição da temperatura torácica obtida em forrageadoras tratadas com Alprenolol sugere o envolvimento de vias octopaminérgicas na termogênese em M. quadrifasciata; todavia, não se sabe se tais efeitos são decorrentes de interferências na produção de calor e/ou um subproduto da diminuição da TM. No nível colonial, ninhos tratados com os mesmos bloqueadores parecem apresentar periodicidades de séries temporais mais similares à da umidade relativa do ambiente e menos à da temperatura ambiente, sugerindo menor resposta às variações de temperatura. Os resultados não provam que há mecanismos de termogênese sem tremor muscular em abelhas, envolvendo octopamina e hormônio juvenil, mas indicam um possível envolvimento destas substâncias na taxa metabólica e oxidação de diferentes substratos energéticos. Sob uma visão comparativa, pode-se apontar semelhanças entre efeitos de modificações em vias octopaminérgicas/tiraminérgicas de insetos e noradrenérgicas/tiraminérgicas de vertebrados, quando à energética e termogênese. Os resultados não provam tal afirmação, mas apontam evidências do papel da via octopaminérgica na taxa metabólica e temperatura torácica de forrageadoras.
In eusocial insects, such as stingless bees, the control of nest conditions and maintenance of the colonial microclimate are important to brood incubation, development of eggs, larvae and pupae, and survival of the colony. One of the main thermoregulatory mechanisms to control nest temperature is the microhabitat selection to build the nest, such as tree holes, characteristic of the stingless bee Melipona quadrifasciata, the biological system chosen for this study. In addition to the behavioral mechanisms involved in thermoregulation, physiological mechanisms underlying thermogenesis are also found in Hymenoptera, such as futile cycles (in bumblebees) and shivering thermogenesis. Thermogenesis in bees is detected mainly during brood incubation, social interactions and pre-flight warming; additional possible physiological mechanisms have not yet been investigated with enough detail. Biogenic amines, especially octopamine, play important physiological roles in bees. Although octopamine and norepinephrine are chemically not identical, it appears that octopaminergic systems of invertebrates and noradrenergic systems of vertebrates are homologous. Oral treatment with beta-blockers can cause hypothermia, leading to the hypothesis that an octopaminergic pathway involved in bee energetics and thermogenesis might exist. The main aspects investigated in the present study are the colonial thermoregulation, the energetics of individual bees, and the mechanisms associated with thermogenesis in M. quadrifasciata. With regards to the colonial thermoregulation, temperature and humidity, time series were recorded inside and outside the nest, in two colonial compartments, i.e., brood and pots. In addition, flight activity was also recorded. Flight activity time series presented similar periodicities in both ambient and brood series. In closed colonies, maintained under constant conditions, the brood time series presented periodicities close to 24h and 12h, suggesting endogenous aspects and rhythm. Another goal of this study was to characterizing the effects of the light-dark cycles and ambient temperatures. This was achieved using intermittent respirometry, and the effects of the light-dark cycles at 28°C (normal photoperiod, inverted photoperiod, constant light and constant dark) on metabolic rate (MR), and the effects of different temperatures (10 40°C) on MR and respiratory quotient (RQ) were measured. The different light-dark cycles affected the MR under normal photoperiod and constant light. Two \"platforms,\" with low MR values were detected between 19h and 5h. When the photoperiod was inverted, lower values were recorded in the dark phase, between 6h and 18h, resulting in an inverted pattern of MR, thus suggesting an exogenous response to photoperiodic changes. Between 7h and 17h59, in the light phase, metabolic rate did not change significantly. The temperature has a significant effect on MR and RQ of the foragers, and reduced values at 10, 35 and 40°C were detected. Between 15 and 30°C no significant differences were detected. In the third part of this work the aim was to verify the time-dependent effects of beta-blockers (Alprenolol and Mianserina) on MR and thoracic temperature of foragers, and to verify whether octopamine and methoprene (analogous to juvenile hormone) compensate the effects of beta-blockers. Alterations of substrates oxidation, considering the RQ and the activity (Vmax) of specific enzymes, such as hexokinase, trehalase, HOAD and glycogen phosphorylase, were also investigated. Treatments with blockers caused alterations in the RQ and in the enzyme activities of hexokinase, HOAD and glycogen phosphorylase. The reduced values of thoracic temperature in foragers treated with Alprenolol suggests the involvement of octopaminergic pathways in thermogenesis; however, it is not yet known if such effects are due to interferences in the heat production and/or represent a by-product of a reduced metabolic rate. At the colonial level, nests treated with blockers presented colonial time series periodicities more similar to the ambient humidity series than to the ambient temperature series, suggesting therefore that the responses to ambient temperature are reduced. These results do not prove that mechanisms of nonshivering thermogenesis are present in bees, involving both octopamin and juvenile hormone, but they can indicate possible involvements of these substances in metabolic rate, energetics and fuel utilization. Under a comparative approach, one can suggest similarities between the effects caused by modifications in the noradrenergic and octopaminergic pathways on the energetics and thermogenesis of M. quadrifasciata. The results, however, do not prove such hypothesis, but they suggest an octopaminergic influence on both metabolic rate and thoracic temperature of M. quadrifasciata foragers.

Broad Complex (BRC) is an ecdysone-pathway gene essential for entry into and progression through metamorphosis in D. melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. Alternative splicing, of a protein-binding BTB-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC), produces four BRC isoforms. Highly conserved orthologs of BTBBRC and all four ZBRC were found in silico from Diptera, Lepidoptera, Hymenoptera and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometalolous orders. Five Tramtrack subfamily members were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, revealing that Z3BRC is the basal member. All four ZBRC domains, including Z4BRC which has no known essential function, are evolving in a manner consistent with selective constraint. Transgenic rescue and immunohistochemistry were used to explore how different BRC isoforms contribute to their shared tissue-morphogenesis functions at the onset of metamorphosis, when BRC is required for CNS reorganization. As predicted, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. Each isoform had a unique expression pattern in the CNS, with no substantial three-way overlap among them. Z4 is strongly expressed in a novel subset of CNS neurons. The most prominent localizations of BRC-Z1, -Z2, -Z3 corresponded with glia, neuroblasts and neurons, respectively. There appears to be a switch from BRC-Z2 in proliferating cells to BRC-Z1 and BRC-Z3 in differentiating cells. The temporal-requirement and spatial-distribution data suggest that BRC-dependent CNS morphogenesis is the result of multicellular interactions among different cell types at different times. BRC-Z1-expressing glia in prepupae may mediate the final steps of CNS morphogenesis. Lastly, BRC is required for migration and programmed cell death of the ring gland, the site of ecdysone and juvenile hormone production. Therefore, BRC may function in ecdysone auto-regulation.

Neste estudo, foi investigado o efeito da dieta larval no desenvolvimento de características morfológicas casta-específicas e na expressão de alguns genes durante o desenvolvimento da abelha sem ferrão Scaptotrigona aff. depilis . Nesta espécie, as castas femininas são determinadas pela quantidade de alimento consumida durante o desenvolvimento larval. Grupos experimentais larvas criadas in vitro foram feitos com duas quantidades diferentes de alimento larval, obtido de favos recém provisionados nesta espécie. As larvas do primeiro grupo receberam 32l de alimento larval, correspondente a quantidade média de alimento recebida por larvas de operárias naturalmente, já as larvas do segundo grupo receberam 130l, correspondente a quantidade média encontrada naturalmente em células reais. Todas as larvas criadas com 130l de alimento larval se desenvolveram em rainhas, como esperado; da mesma forma, a maioria das larvas criadas com 32l se desenvolveram em operárias. Interessantemente, porém, algumas larvas deste grupo se desenvolveram em rainhas miniaturas, sugerindo que outros fatores, além do trófico, estejam envolvidos na determinação de castas em S. aff. depilis. Subsequentemente, analisamos os títulos de hormônio juvenil (HJ) na hemolinfa por radioimunoensaio durante quatro estágios do último instar larval e encontramos que as larvas do grupo de 130l de dieta apresentaram maiores títulos no estágio defecante (LD) (p=0,034, t-test), se comparado com as larvas do grupo de 32l. Os níveis de expressão de determinados genes que foram previamente descritos em outras espécies como expressos preferencialmente em rainhas. Um destes genes, dnmt-3, codifica a DNA metiltransferase envolvida na metilação do DNA. Para este gene, encontramos maiores níveis de transcritos nas larvas alimentadas como rainhas (130l) nos estágios LPD e LD (p=0,029, Mann-Whitney), do que as larvas que receberam 32l. Analisamos também dois genes envolvidos no metabolismo do HJ, e para um deles, jheh, codificador de uma epóxido-hidrolase do hormônio juvenil, não encontramos diferença nos níveis de mRNA entre larvas de rainhas e operárias. Para jhe, o qual codifica a esterase do hormônio juvenil, encontramos maiores níveis de transcritos no estágio LD nas larvas criadas com 130l de alimento. Isso indica que a expressão de jhe pode ser induzida pelo aumento dos títulos de HJ neste estágio. Os dois genes, hmgr e mfe, envolvidos no passo inicial e final da síntese do HJ, respectivamente, o primeiro mostrou uma pequena variação nos níveis de expressão, sendo que a expressão de mfe foi menor nas larvas criadas com 32l de alimento larval e foi encontrado um pico de expressão no estágio LPD nas larvas alimentadas com 130l. O produto desse gene, metilfarnesoato epoxidase, está envolvido no passo limitante da síntese de HJ em Apis mellifera. Dois outros genes analisados foram o homólogo ao EcR, codificador do receptor de ecdisona e o usp, codificador do potencial receptor do HJ. Seus níveis de expressões foram maiores nos estágios LPD e LD, respectivamente, nas larvas alimentadas com 130l de alimento e os níveis de EcR se correlacionaram com o perfil dos títulos de ecdisona e os de usp com os títulos de HJ, publicados para esta espécie. Não houve diferença nos níveis de expressão dos genes selecionados que pudessem relacionar ou permitir distinguir as rainhas miniaturas das operárias durante os estágios do desenvolvimento estudados. Uma análise morfométrica de adultos faratos indica claramente que as rainhas miniaturas são rainhas autênticas.
In this work we investigated the role played by the larval diet in the development of caste-specific morphological traits and in the expression of candidate genes during the development of the stingless bees, Scaptotrigona aff. depilis. In this species, the female castes are determinate by the amount of food consume during larval development. Experimental groups of larvae were reared in vitro on two different quantities of larval food obtained from newly provisioned brood cells of this species. Larvae of the first group received 32l of larval food, corresponding to the quantity usually received by the workers larva, whereas larvae of the second group received 130l, corresponding to the quantity normally deposited in queen cells. All larvae reared on 130l of larval food developed into queens, as expected, and similarly, most of the larvae reared on 32l developed into workers. Interestingly, however, some larvae of this group developed into miniature queens, suggesting that factors additional to the trophic ones may be involved in caste determination in S. aff. depilis. We subsequently analyzed the hemolymph juvenile hormone (JH) titers by radioimmunoassay during four stages of the last larval instar and we found that larvae of the 130l diet group had higher JH titer in the defecating stange (LD) (p=0,034, t-test) than larvae of the 32l diet group. Next we analyzed the expression levels of set of candidate genes that had previously been described as preferentially expressed in queens of others bee species. One of these genes dnmt-3, encodes DNA methyltransferase involved in DNA methylation. For this gene we found higher transcripts levels in prospective queens during the final larval stage (both in pre-defecating larvae LPD and defecating larvae- LD) (p=0,029, Mann-Whitney). We also analyzed two genes involved in JH metabolism, and for one of them jheh, encoding a juvenile hormone epoxide hydrolase, we did not find any differences in mRNA levels for queens and workers larvae. For jhe, which codes juvenile hormone esterase we found higher transcript levels in the LD stage in larvae reared on 130l of larval food. This indicates that jhe expression may be induced by an elevated JH titer during this stage. For two genes, hmgr and mfe, involved in an initial and a final step of JH synthesis, respectively, the first one showed little variation in expression levels, whereas mfe expression was lower in larvae reared on 32l of larval food and we found an expression peak in the LPD stage (p=0,029, Mann-Whitney) of larvae reared on 130l larval food. The product of this gene, a methylfarnesoate epoxidase, has been shown to be involved in a rate-limiting step of JH sunthesis in the honey bee, Apis mellifera. The other two genes analyzed were the EcR homolog encoding an ecdysone receptor and usp coding for potencial JH receptor. Their expressions were higher during LPD (p=0,006, t-test) stages and LD (p=0,026, t-test), respectively, in larvae fed with 130l of food and the levels of transcripts of EcR correlated with changes in the ecdysone titer and usp with the JH titer published for this species. We did not find differences in expressions levels for any of these candidate genes that could related to and allow to distinguish between prospective miniature queens and workers in these stages. A morphometric analysis of pharate adults, however, clearly demonstrated that the miniature queens are authentic queens.

Insect juvenile hormone (JH) regulates the growth and development of insects. Synthetic analogues of JH (JHAs) have been used as agents of pest control, disrupting the metamorphosis of insects. The purpose of the present study was to determine physiological and biochemical effects of methoprene, a commercial JHA, on certain organs and tissues of the African migratory locust (Locusta mi- gratoria migratorioides, phase gregaria). Methoprene was topically applied to newly moulted, fifth (final) instar larvae and the subsequent development of the animals was followed. Cytological development of fat body and dorsal longitudinal flight muscle was studied by light and electron microscopy. Fat body cells of control insects were synthetically active early in the fifth instar, and stored lipid and glycogen in the latter half of the instar. Fat bodies of 8-day old adults were sexually dimorphic, female cells showing high levels of RNA and protein synthesis while male cells were filled with lipid and glycogen stores. Methoprene treatment stimulated the synthetic activity of the cells in fifth instar and adult stadia, especially in female tissue. Cell nuclei were abnormally enlarged, suggesting increased ploidy levels. Levels of lipid and carbohydrate were measured in fat body and haemolymph but methoprene had no obvious effect on them, nor on glycogen phosphorylase activity. However, the JHA affected rates of incorporation of [(^14)C]glucose into fat body lipids during the first four days of the fifth instar. Separation of haemolymph proteins by gel electrophoresis revealed an extra protein band in the blood of treated female locusts from the middle of the fifth instar onwards. The same band appeared in the blood of control females only when they reached sexual maturity. Methoprene treatment disrupted normal development of dorsal longitudinal flight muscles during the fifth instar and early adult life. The JHA reduced muscle fibre growth but seemed to accelerate myofibril and mitochondrial differentiation in the fifth instar. Treatment also inhibited formation of interfibrillar tracheoles and caused disruption of the myofilaments and sarcoplasmic reticulum in adult muscle. Mitochondria were isolated from flight muscles of mature adults and their respiratory metabolism was measured using an oxygen electrode. Mitochondria from control animals showed high rates of oxygen consumption and good respiratory control. Mitochondria from treated locusts had poor respiratory control and low respiratory rates. Similar results were obtained by in vitro applications of methoprene or juvenile hormone to mitochondria.

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The bees of Melipona genus have trofogenetic mechanism to determination of caste. The level of juvenile hormone (JH) in several stages of larval development determines the characteristics of females. Diploid males and females of Melipona quadrifasciata that, in last instar larvae, received topical JH develop external morphological characteristics of queen. This work was based on morphometric data to investigate the effect of JH III in the development of haploid and diploid males of Melipona quadrifasciata and compare morphological characteristics of them with morphological characteristics already known in females of this species. Ten individuals were used for each treatment; workers, natural queens, queens produced from the topical application of JH, natural haploid males, haploid males received application of JH but did not differ, haploid males that received topical application of JH and developed features of queens (QLM - Queen-Like Male), natural diploid males, diploid males that received topical application of JH but did not differ and diploid QLM. The anatomical averages analyzed were: width of compound eyes, width of tibia, length of mouthparts, length of mandible, intertegule distance and interorbit distance. All averages allowed the separation of individuals into groups. However, the lengths of the mouthpart averages were differences most significantly between treatments. Natural queens and queens produced from the effect of JH had similar averages. Diploid and haploid QLM showed average variables with similar size in the natural queens and queens produced from topical application of JH. Workers were more similar to males than the queens in the most averages variables and, in general, the workers presented average size more than queens. Natural diploid and haploid males had close averages, which does not allow visual differentiation of these individuals. The dates confirm that the JH is primarily responsible for the differentiation of specific characteristics of each caste, and, in the high levels of it causes the appearance of the characteristics of queen, independent on genotype.
Abelhas do gênero Melipona possuem mecanismo de determinação das castas trofogenético. O nível de hormônio juvenil (HJ) em certas fases do desenvolvimento larval determina as características das fêmeas. Fêmeas e machos diplóides de Melipona quadrifasciata que, na fase pré defecante, recebem aplicação tópica de HJ desenvolvem características morfológicas externas de rainha. Este trabalho baseou-se em dados morfométricos para investigar o efeito do HJ III no desenvolvimento de machos haplóides e diplóides de Melipona quadrifasciata e comparar estes efeitos com os efeitos já conhecidos em fêmeas desta espécie. Foram utilizados dez indivíduos de cada tratamento, sendo operárias, rainhas naturais, rainhas produzidas a partir da aplicação tópica de HJ, machos haplóides naturais, machos haplóides que receberam aplicação de HJ mas não se diferenciaram, machos haplóides que receberam aplicação tópica de HJ e desenvolveram características de rainha (QLM – Queen-Like Male), machos diplóides naturais, machos diplóides que receberam aplicação tópica de HJ mas não se diferenciaram e QLM diplóides. As medidas anatômicas analizadas foram largura do olho composto, largura da tíbia, comprimento do aparelho bucal, comprimento da mandíbula, distância intertégula e distância interorbital. Todas as medidas possibilitaram a separação dos indivíduos em grupos, no entanto o comprimento do aparelho bucal foi a medida com diferenças mais significativas entre os tratamentos. Rainhas naturais e rainhas produzidas a partir do efeito do HJ possuem medidas semelhantes. QLM haplóides e diplóides apresentaram as variáveis medidas com tamanhos semelhantes as das rainhas naturais e produzidas a partir da aplicação tópica de HJ. Operárias se mostraram mais semelhantes aos machos do que as rainhas quanto a maior parte das maioria das variáveis medidas e, em geral, as operárias apresentam médias de tamanho maiores e as rainhas menores. Macho diplóides e haplóides naturais possuem medidas próximas, o que não permite a diferenciação visual desses indivíduos. Os dados corroboram que o HJ é o principal responsável pela diferenciação das características específicas de cada castas, assim, em altos níveis ele acarreta o aparecimento das características de rainha, independente do genótipo.