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.

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.

Sinefungin inhibited the S-adenosylmethionine-dependent farnesoic acid methyltransferase in a cell-free system containing a homogenate of corpora allata from female locusts, Locusta migratoria. The enzyme catalyzed the penultimate step of juvenile hormone biosynthesis in the insects. Culturing corpora allata in the presence of sinefungin greatly suppressed juvenile hormone production. The following in vivo effects were visible after injection of the inhibitor: increase in mortality and reduction of total haemolymph protein liter and ovary fresh weight, as well as length of terminal oocytes. Attempts to reverse these effects by topical application of the juvenile hormone analog ZR-515 (methoprene) were only partly successful. Therefore, the in vivo effects may be due to a general inhibition of methyltransferase enzymes in the insect. Sinefungin appeared to be of potential interest as the first representative of a new class of insect growth regulators.

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.

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.

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