京都大学 / 新制・課程博士 / 博士(農学) / 甲第24912号 / 農博第2575号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Mosquito reproduction is tightly regulated by the endocrine system. The sesquiterpenoid insect hormone, Juvenile hormone (JH), plays a crucial role in mosquito reproductive maturation. JH signaling pathways consist of a hierarchy of transcriptional regulators that modulate the transcriptional responses to this hormone. Genomic action of JH is mediated through the intracellular receptor Methoprene tolerant (Met) and Krüppel homolog 1 (Kr-h1), an intermediate effector that acts downstream of Met. Kr-h1 is an essential transcription factor for proper oogenesis and egg production in several insects. However, the regulatory mechanism of Kr-h1 in mosquito reproduction has not been well studied. In the current study, we performed global analyses of the Kr-h1 binding sites at multiple time points before and after a blood meal. In addition to known JH-regulated genes, we identified the binding of Kr-h1 to several genes that are controlled by the insect steroid hormone 20-hydroxyecdysone (20E). Kr-h1 seemed to have different roles in regulating the 20E-responsive genes before and after the blood-feeding. RNAi mediated Kr-h1 silencing demonstrated the activator role of Kr-h1 on several 20E-regulated genes in the previtellogenic stage, while Kr-h1 mostly repressed those genes after blood feeding. On the genes that were activated by Kr-h1 in the previtellogenic stage, the binding of Kr-h1 was associated with an increase of the histone marker H3K27ac. For the first time, we demonstrated that the regulatory action of Kr-h1 involves histone modification on the 20E-responsive genes. This study significantly extends our understanding of the regulatory mechanism of Kr-h1, and the cross-talk of JH and 20E in coordinating mosquito reproduction. JH analogs are commonly used as mosquito larvicides. Recent studies reported that the application of a JH analog, pyriproxyfen (PPF), on adult female mosquitoes substantially reduces their reproduction. A big knowledge gap was the poor understanding of the mechanism underlying this sterilizing effect of PPF. Here, with our customized laboratory setup that mimics the bed net intervention, we established a dose-dependent effect of PPF in compromising mosquito fecundity and fertility. We carefully assessed the effects of PPF exposure on mosquito physiology and follicular development. PPF induced excessive growth of primary follicles during the previtellogenic stage. However, the follicular development in the PPF-treated mosquitoes was severely impaired after blood feeding. The primary follicles were much smaller than their counterparts in the control groups and their development stopped at Christopher's stage III. Moreover, PPF triggered the atypical premature growth of secondary follicles at ~36 h PBM. In addition to the follicular developmental reprogramming, PPF also altered the levels of storage metabolites, enhancing the accumulation of glycogen and triglyceride (TAG) before a blood meal and speeding their depletion after blood-feeding. Consistent with the observed phenotypical changes and relevant metabolic genes, several 20E-responsive genes were significantly altered in their expression as a result of PPF exposure. Furthermore, RNAi experiments demonstrated that the JH receptor Met is required in the PPF-induced sterilization. In summary, we evaluated the sterilizing effects of PPF on mosquito reproduction, investigated the molecular action of PPF in regulating mosquito gene expression, and determined the signaling pathway involved in the PPF-induced sterilization of female mosquitoes. / Doctor of Philosophy / Among different insect-borne diseases, mosquito causes the highest disease burden with almost 700 million infections and over a million deaths every year. Aedes aegypti mosquitoes are the major vehicle to transmit several viral diseases including dengue, yellow fever, chikungunya, and Zika fever. They pose a global threat to public health and economic sectors. Different mosquito control strategies are used, and a very quick, powerful, and popular strategy is using chemical insecticides to decrease mosquito populations. However, insecticide resistance in mosquitoes and non-specific toxicity to other animals are great challenges associated with the commonly used insecticides. To resolve this problem, new insecticides are urgently needed. If we can broaden our understanding of mosquito reproductive biology, new targets will be identified and can be exploited to develop new insecticides. In our study, we investigated an insect-specific hormone, Juvenile hormone (JH), to understand its regulatory action in mosquito reproduction. Also, this study improved our knowledge of the molecular understanding of the insecticide (synthetic JH-like compound) in decreasing mosquito egg numbers and reducing the hatching rate. Overall, we gained a significant understanding of the hormonal regulation of mosquito reproduction. This knowledge can be used in the future to develop new insecticides with better efficiency to decrease the mosquito population and mosquito-borne disease burden.

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.

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.

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.

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.

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.

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.

The haemolymph of the adult Colorado potato beetle, Lepinotarsa decemlineata Say, contains a high molecular weight (MW > 200,000) JH-III specific binding protein. The Kd value of the protein for racemic JH-III is 1.3 ± 0.2 × 10−7 M. It has a lower affinity for racemic JH-I and it does not bind JH-III-diol or JH-III-acid. The binding protein does discriminate between the enantiomers of synthetic, racemic JH-III as was determined by stereochemical anaysis of the bound and the free JH-III. Incubation of racemic JH-III with crude haemolymph results in preferential formation of (10S)-JH-III-acid, the unnatural configuration. The JH-esterase present in L. decemlineata haemolymph is not enantioselective. It is concluded that the most important function of the binding protein is that of a specific carrier, protecting the natural hormone against degradation by esterases. The carrier does not protect JH-I as efficiently as the lower homologue.

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.