The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.

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.

<p>The initiation of metamorphosis causes the cessation of the larval growth period which determines the final body size of adult insects. Because larval growth is roughly exponential, differences in timing the initiation of metamorphosis can cause large differences body size. Although many of the processes involved in metamorphosis have been well characterized, little is known about how the timing of the initiation of metamorphosis is determined. </p> <p>Using different strains from <italic>Tribolium castaneum<italic>, <italic>Tribolium freemani<italic>, and <italic>Manduca sexta<italic> and varied nutritional conditions, I was able to document the existence of a threshold size, which determines when the larva becomes competent to metamorphose. Threshold size, however, does not dictate the exact timing of initiation. The exact timing for the initiation of metamorphosis is determined by a pulse of the molting hormone, ecdysone, but only after threshold size has been reached. Ecdysone pulses before the larva attains threshold size only cause the larva to molt to another larval instar. These results indicate the timing of metamorphosis initiation is controlled by two factors: (1) attainment of threshold size, at which the larva becomes competent to initiate metamorphosis and (2) the timing of an ecdysone pulse after attaining threshold size. </p> <p>I hypothesize the attainment of threshold size, and therefore competence to metamorphose, is mediated by the effect of changing juvenile hormone concentrations caused by the increase in size of the larva. While the larval body grows nearly exponentially, the corpora allata, which secretes juvenile hormone, grows very little if at all. The difference in relative growth causes juvenile hormone concentrations to gradually become diluted. When juvenile hormone concentrations fall below a threshold, changes in protein-protein binding occur that can cause changes in signaling networks and ultimately gene expression. These changes make the larva competent for metamorphosis. </p> <p>I have demonstrated that only threshold size is consistently correlated with body size; other growth parameters such as growth rate, duration of instars, or number of instars do not consistently correlate with variation in body size. Using the black mutant strain of <italic>M. sexta<italic> I have shown that lower juvenile hormone titers correlate with lower threshold sizes. My hypothesis is consistent with the large body of literature indicating the involvement of juvenile hormone. I also hypothesize that the diversity of metamorphosis types in holometabolous insects can be explained by heterochronic shifts in the timing of threshold size and other developmental events related to metamorphosis. The heterochronic shifts affect not only the morphology of organs, but can also affect the overall phenotypic response of the larva to changes in the environment. The different phenotypic responses among species may make the more or less suited for certain types of niches.</p> / Dissertation

Behavior is a complex process resulting from the integration of genetic and environmental information. Thus, the genetically tractable Drosophila melanogaster was utilized to better understand the interplay between these factors since Drosophila males and females exhibit sex-specific courtship behaviors that are innate yet modifiable. These sex-specific behaviors, as well as sexually dimorphic development, are regulated, in part, by the somatic sex-determination hierarchy. Since reproductive behaviors rely on the rapid integration of multiple sensory cues, it is likely that the perception and integration of such cues and mating-induced physiological changes are mediated in part by changes in gene expression. Therefore, it was hypothesized that assaying gene expression changes in response to courtship or mating in Drosophila males would uncover new targets of the sex-determination hierarchy and other behaviorally important loci. We took a novel approach to find these behaviorally-responsive loci by utilizing microarray technology to assess courtship- or mating-induced gene expression changes in Drosophila male whole bodies or heads. Mutations in candidate loci were tested for effects on reproductive behaviors and present the first data showing that egghead (egh) and female-specific independent of transformer (fit) affect male reproductive behavior. egh is up regulated in male heads 20 min after courting and is required post-developmentally in a subset of neurons for robust male courtship behavior. fit, a fat body-expressed sex-determination hierarchy target gene, is up regulated in male whole bodies after 5 min of courtship. fit is also up regulated in male heads after 20 min of courtship or 2 hrs after mating. Mutations in fit result in male-male courtship; more specifically, fit mutants direct courtship towards males and also elicit courtship from wild-type males. By analyzing fit's role in courtship behavior, we also shed light on the role the fat body plays in modulating behavior. These studies provide the first pieces of evidence that gene expression changes occur in Drosophila males performing reproductive behaviors. This novel approach identified behaviorally important loci that are expressed in the nervous system and the fat body, indicating that both tissues modulate behavior. Also identified were sex-determination hierarchy target genes and it is likely that further analysis of the remaining candidates will reveal more members of this genetic cascade.

Matrix-assisted laser desorption/ionization (MALDI) has been celebrated as a soft ionization method for analyzing very diverse biological species including large proteins, peptides, carbohydrates, lipids and metabolites. The fact that MALDI is tolerant to salts and buffers and that it mostly produces singly charged ions from intact biomolecules is considered highly advantageous over electrospray ionization (ESI). Almost two decades after the introduction of vacuum MALDI, the technique was successfully implemented under atmospheric pressure (AP) conditions by Laiko and co-workers. Some of the most salient advantages of AP-MALDI over vacuum MALDI are its ability to generate intact ions from labile species with minimal fragmentation due to collisional cooling under AP, the ability of performing MSn experiments, and its exchangeability with other ion sources. However, AP-MALDI suffers from limited sensitivity due to low ion transmission efficiency under AP conditions. Because sensitivity is a function of the sample pretreatment method of choice, both preconcentration and selective sample fractionation can be used during the initial stages of the analytical pipeline to improve detectability. To that end, the first part of the work presented in this thesis is aimed at investigating various approaches to improve the sensitivity of AP-MALDI for mass spectrometric analysis of biomolecules. Chapter 1 reviews the history of laser desorption ionization (LDI), presenting salient features of vacuum MALDI and AP-MALDI, and concludes with a brief overview of leading ambient ionization techniques, such as Direct Analysis in Real Time (DART) ionization. Chapter 2 presents an investigation of an on chip sample preconcentration approach coupled to AP-MALDI for high-sensitivity analysis of neuropeptides extracted from Aedes aegypti mosquito heads. The theme of exploring efficient and reproducible purification methods for complex biosamples is continued in Chapter 3, where an evaluation of new on-tip solid-phase extraction (SPE) micro columns with various functional groups is presented. A second approach for enhancing AP-MALDI sensitivity by constructing a new pneumatically-assisted (PA) AP-MALDI ion source is presented in Chapter 4, where various factors affecting the performance of this device are investigated. Chapter 5 describes work involving the evaluation of DART ionization as a high-throughput method for the detection and identification of small terpene molecules central to the Aedes aegypti mosquito lifecycle.

abstract: A notable feature of advanced eusocial insect groups is a division of labor within the sterile worker caste. However, the physiological aspects underlying the differentiation of behavioral phenotypes are poorly understood in one of the most successful social taxa, the ants. By starting to understand the foundations on which social behaviors are built, it also becomes possible to better evaluate hypothetical explanations regarding the mechanisms behind the evolution of insect eusociality, such as the argument that the reproductive regulatory infrastructure of solitary ancestors was co-opted and modified to produce distinct castes. This dissertation provides new information regarding the internal factors that could underlie the division of labor observed in both founding queens and workers of Pogonomyrmex californicus ants, and shows that changes in task performance are correlated with differences in reproductive physiology in both castes. In queens and workers, foraging behavior is linked to elevated levels of the reproductively-associated juvenile hormone (JH), and, in workers, this behavioral change is accompanied by depressed levels of ecdysteroid hormones. In both castes, the transition to foraging is also associated with reduced ovarian activity. Further investigation shows that queens remain behaviorally plastic, even after worker emergence, but the association between JH and behavioral bias remains the same, suggesting that this hormone is an important component of behavioral development in these ants. In addition to these reproductive factors, treatment with an inhibitor of the nutrient-sensing pathway Target of Rapamycin (TOR) also causes queens to become biased towards foraging, suggesting an additional sensory component that could play an important role in division of labor. Overall, this work provides novel identification of the possible regulators behind ant division of labor, and suggests how reproductive physiology could play an important role in the evolution and regulation of non-reproductive social behaviors. / Dissertation/Thesis / Ph.D. Biology 2012

The present study considers the versatile role of adipokinetic hormones (AKHs). Using the firebug Pyrrhocoris apterus as a model insect, the hypothesis that AKHs mediate stress response mechanisms was explored. The outcome indicated that there is a positive feedback regulation between an oxidative stressor action and the level of AKH in insect body, and that AKHs might be involved in the activation of antioxidant protection mechanism. Further results revealed a functional homology between AKH and the mammalian hormone glucagon. The possible effects of glucagon on mobilisation of energy reserves and on elicitation of an antioxidant response to oxidative stress were investigated. As a result, glucagon-immunoreactive material was detected for the first time in the firebug central nervous system and gut. Antioxidant mechanisms are elicited after glucagon treatment but it did not involve mobilization of energy reserves or AKH level changes. As a complement, the existence of a feedback between juvenile hormone and AKH was investigated by topical application of the juvenile hormone analogue methoprene, which influenced the release of AKH from the central nervous system into the haemolymph and induced a partial reduction of lipid content in haemolymph.

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.

Programas brasileiros de controle populacional do mosquito Aedes aegypti são caracterizados por incluírem ações integradas entre população e poder público, compreendendo suplementarmente a aplicação de inseticidas nos potenciais criadouros do mosquito. Um dos compostos utilizados para o controle populacional de mosquitos é o Pyriproxyfen, um regulador de crescimento de insetos que age durante os estágios imaturos impedindo a emergência das larvas para a fase adulta. Muitas pesquisas, em condições laboratoriais controladas, encontraram resultados eficientes na aplicação desse larvicida, inferindo a confirmação de eficácia para uso nos programas de controle. Contudo, esses trabalhos disponíveis desconsideram a interferência por fatores ambientais (e.g. temperatura, luminosidade, pH) o que, como consequência, pode apresentar diferenças nos resultados em ambiente natural. Dentre esses fatores, é importante o destaque a temperatura, que apresenta o potencial de produzir diferenças na eficiência do composto, uma vez que há evidências de interferência tanto na velocidade e no sucesso de desenvolvimento das larvas quanto da composição química do larvicida. Dessa forma, o presente trabalho avaliou o efeito da variação da temperatura na eficiência do larvicida Pyriproxyfen para controle populacional de larvas de Aedes aegypti. Para isso, foram realizados experimentos avaliando o efeito combinado de três diferentes temperaturas (20&#176;C, 25&#176;C, 30&#176;C) e cinco diferentes concentrações de Pyriproxyfen (0,0001; 0,001; 0,01; 0,1 e 1 mg.L-1), sendo as dosagens escolhidas em função da recomendação da Organização Mundial da Saúde (0,01 mg/L). Os testes foram realizados em incubadora BOD, com temperatura controlada desde a eclosão para evitar estresse térmico nas larvas. Também foi analisado o efeito sub letal do composto, nas respostas em longevidade dos mosquitos e do tamanho alar como indicador do tamanho das fêmeas provenientes dos tratamentos a 20&#176;C e a 30&#176;C. Quando aumentou-se a temperatura em 5&#176;C (de 25&#176;C para 30&#176;C), houve redução em 30% da eficiência do larvicida na menor dosagem ,redução de 10% da eficácia na concentração de 0,001 mg/L e redução em 20% na concentração de 0,01 mg/L. Para todas as concentrações testadas, os mosquitos sobreviventes tiveram sua longevidade reduzida significativamente se comparados aos mosquitos desenvolvidos em um ambiente sem o composto. Ainda, as fêmeas emergentes do ambiente a 30&#176;C foram significativamente maiores que as fêmeas que se desenvolveram a 20&#176;C. No que se refere ao tamanho de fêmeas, entre a mesma temperatura não houve diferença significativa com relação ao tamanho das asas. Estes resultados indicam que, apesar da alta eficiência do Pyriproxyfen, deve-se considerar com cautela a influência que os fatores ambientais têm sobre o efeito de compostos inseticidas no controle de vetores, uma vez que estes são capazes de interferir no cenário epidemiológico. Ademais, considerar dosagens diferentes de acordo com os fatores ambientais em campo pode trazer benefícios no uso mais racional de compostos inseticidas bem como serem mais eficientes no controle efetivo do vetor alvo. / Surveillance brazilian programs for populational control of the Aedes aegypti mosquito are characterized for applying integrated actions between the government and the municipalities population, comprising as a supplementary measure the application of pesticides. One of these compounds is Pyriproxyfen, a insect growth regulator that acts during the imature stages of the organismo, therefore preventing its emmergence to the adult stage. Many researches, at laboratory conditions, found efficient outcomes by testing this larvicide, inferring its confirmation of efficacy of application during the surveillance programs. However, these studies disconsider the possibility of interference by evironmental factors, such as temperature, luminosity and pH variations. Among these factors, the environmental temperature is highlighted, considering the existing evidence of interference in the larval development and at the chemical composition of the larvicide. Thus, this study aims to assess the temperature effect over the efficiency outcomes of the Pyriproxyfen as a larvicide for Aedes aegypti control. Were executed experiments analyzing the combined effect of three different temperatures (20&#176;C, 25&#176;C, 30&#176;C) and five concentrations of Pyriproxyfen (0,0001 mg/L, 0,001 mg/L, 0,01 mg/L, 0,1 mg/L and 1 mg/L). These tests were performed in a BOD incubator, with controlled and constant temperature, since the eclosion of the I instar larvae until the end of the test. Dead larvae were removed and registered daily. Were analyzed the sub lethal outcomes, by the longevity of the survivors and were calculated the wing size of the females from 20&#176;C and 30&#176;C as na indicator of body size of these female mosquitoes. When increased 5ºC in the temperature, from 25&#176;C to 30&#176;C, were observed an decrease in the efficiency of the larvicide by 30% at the lowest concentration, by 10% at a tem times higher concentration (0,001 mg/L) and a decrease by 20% at the concentration of 0,01 mg/L. For all concentrations assessed, the survivors lived less time than the mosquitoes which developed at na ambient without the Pyriproxyfen contamination. Yet, the females from the warmest environment, at 30ºC, were bigger than the females that developed in a cooler environment (20&#176;C). The results indicate that, despite of the high efficiency of this larvicide, we must consider with caution the influence by environmental factors in field conditions, since that they present the possibility of interfering directly or indirectly at the epidemiologic scenario. Furthermore, considering different dosages according to the environmental factors in the field can bring benefits in the more rational use of insecticidal compounds as well as being more efficient in the effective control of the target vector.