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Sample records for ecdysone

  1. New players in the regulation of ecdysone biosynthesis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Insect ecdysone steroid hormone regulates major developmental transitions, such as molting and metamorphosis. The production of ecdysone correlates well with the timing of these transitions. Finding out how the ecdysone biosynthesis is regulated is crucial to fully understand these sophisticated developmental switches. Here we summarized recent findings in the regulation of ecdysone biosynthesis from the aspects of cell signaling, key biosynthetic enzymes and substrate cholesterol trafficking.

  2. Isolation of four ecdysones from Ajuga iva roots and a rapid semiquantitative method for ecdysone determination.

    Science.gov (United States)

    Sabri, N N; Asaad, A; Khafagy, S M

    1981-07-01

    The ethereal extract of AJUGA IVA (L.) S CHREB. roots, yielded 4 ecdysones. Three were proved to be cyasterone, makisterone A and ecdysterone, by using UV, IR, MS spectral methods and comparison with standard samples. Spectral data indicate that the fourth ecdysone is similar to cyasterone but having an additional hydroxy group in the side-chain. The chromatographic pattern of ecdysone content in roots, stems and leaves is presented as well as a rapid and simple Semiquantitative method for their estimation. The roots had the highest total content (0.381%).

  3. Isolation, characterization, and expression analyses of ecdysone receptor 1, ecdysone receptor 2 and ultraspiracle genes in varroa destructor mite

    Science.gov (United States)

    The varroa mite, Varroa destructor, is a honeybee ectoparasite considered the most important pest in apiaries throughout the US. Ecdysone receptor is a hormone secreted by the prothoracic gland of insects that controls ecdysis and stimulates metamorphosis. The ecdysone receptor is a nuclear receptor...

  4. Ecdysone Agonist: New Insecticides with Novel Mode of Action

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    Y. Andi Trisyono

    2002-12-01

    Full Text Available Development of insect resistance to insecticide has been the major driving force for the development of new insecticides. Awareness and demand from public for more environmentally friendly insecticides have contributed in shifting the trend from using broad spectrum to selective insecticides. As a result, scientists have looked for new target sites beyond the nervous system. Insect growth regulators (IGRs are more selective insecticides than conventional insecticides, and ecdysone agonists are the newest IGRs being commercialized, e.g. tebufenozide, methoxyfenozide, and halofenozide. Ecdysone agonists bind to the ecdysteroid receptors, and they act similarly to the molting hormone 20-hydroxyecdysone. The binding provides larvae or nymphs with a signal to enter a premature and lethal molting cycle. In addition, the ecdysone agonists cause a reduction in the number of eggs laid by female insects. The ecdysone agonists are being developed as selective biorational insecticides. Tebufenozide and methoxyfenozide are used to control lepidopteran insect pests, whereas halofenozide is being used to control coleopteran insect pests. Their selectivity is due to differences in the binding affinity between these compounds to the receptors in insects from different orders. The selectivity of these compounds makes them candidates to be used in combinations with other control strategies to develop integrated pest management programs in agricultural ecosystems. Key words: new insecticides, selectivity, ecdysone agonists

  5. Activation of Drosophila hemocyte motility by the ecdysone hormone

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    Christopher J. Sampson

    2013-11-01

    Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.

  6. The Mechanisms of the Ecdysone Pulses that Cause Metamorphosis

    DEFF Research Database (Denmark)

    Møller, Morten Erik

    Maturation in both mammals and insects is caused by pulses of steroid hormones released from glands in response to a brain-derived signal. The timing of this developmental transition is secured by the integration of many developmental cues, such as size, external environment and nutritional...... condition, with hardwired genetic programs. In holometabolous insects, pulses of the steroid hormone ecdysone cause the transition between the larval stages and the initiation of the metamorphosis, which transforms the juvenile larva to a sexually mature adult. Ecdysone is produced in the prothoracic gland...

  7. The Steroid Molting Hormone Ecdysone Regulates Sleep in Adult Drosophila melanogaster

    OpenAIRE

    Ishimoto, Hiroshi; Kitamoto, Toshihiro

    2010-01-01

    Ecdysone is the major steroid hormone in insects and plays essential roles in coordinating developmental transitions such as larval molting and metamorphosis through its active metabolite 20-hydroxyecdysone (20E). Although ecdysone is present throughout life in both males and females, its functions in adult physiology remain largely unknown. In this study we demonstrate that ecdysone-mediated signaling in the adult is intimately involved in transitions between the physiological states of slee...

  8. The Mechanisms of the Ecdysone Pulses that Cause Metamorphosis

    DEFF Research Database (Denmark)

    Møller, Morten Erik

    Maturation in both mammals and insects is caused by pulses of steroid hormones released from glands in response to a brain-derived signal. The timing of this developmental transition is secured by the integration of many developmental cues, such as size, external environment and nutritional...... condition, with hardwired genetic programs. In holometabolous insects, pulses of the steroid hormone ecdysone cause the transition between the larval stages and the initiation of the metamorphosis, which transforms the juvenile larva to a sexually mature adult. Ecdysone is produced in the prothoracic gland...... elucidated, many questions of how the system is regulated remain unknown. The first part of the thesis focuses on a genome-wide in-vivo RNAi screen, where 90 % of the protein coding genes in Drosophila were knocked down tissue-specifically in the cells of the PG. 15.2% of the genes led to developmental...

  9. The steroid molting hormone Ecdysone regulates sleep in adult Drosophila melanogaster.

    Science.gov (United States)

    Ishimoto, Hiroshi; Kitamoto, Toshihiro

    2010-05-01

    Ecdysone is the major steroid hormone in insects and plays essential roles in coordinating developmental transitions such as larval molting and metamorphosis through its active metabolite 20-hydroxyecdysone (20E). Although ecdysone is present throughout life in both males and females, its functions in adult physiology remain largely unknown. In this study we demonstrate that ecdysone-mediated signaling in the adult is intimately involved in transitions between the physiological states of sleep and wakefulness. First, administering 20E to adult Drosophila melanogaster promoted sleep in a dose-dependent manner, and it did so primarily by altering the length of sleep and wake bouts without affecting waking activity. Second, mutants for ecdysone synthesis displayed the "short-sleep phenotype," and this was alleviated by administering 20E at the adult stage. Third, mutants for nuclear ecdysone receptors showed reduced sleep, and conditional overexpression of wild-type ecdysone receptors in the adult mushroom bodies resulted in an isoform-specific increase in sleep. Finally, endogenous ecdysone levels increased after sleep deprivation, and mutants defective for ecdysone signaling displayed little sleep rebound, suggesting that ecdysone is involved in homeostatic sleep regulation. In light of the recent finding that lethargus--a period at larval-stage transitions in the nematode worm Caenorhabditis elegans--is a sleep-like state, our results suggest that sleep is functionally and mechanistically linked to a genetically programmed, quiescent behavioral state during development.

  10. Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles.

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    Zirin, Jonathan; Cheng, Daojun; Dhanyasi, Nagaraju; Cho, Julio; Dura, Jean-Maurice; Vijayraghavan, Krishnaswamy; Perrimon, Norbert

    2013-11-15

    One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.

  11. Warts signaling controls organ and body growth through regulation of ecdysone

    DEFF Research Database (Denmark)

    Møller, Morten Erik; Nagy, Stanislav; Gerlach, Stephan Uwe

    2017-01-01

    under different environmental conditions is poorly understood. In Drosophila, Hippo/Warts signaling functions intrinsically to regulate tissue growth and organ size [3, 4], whereas systemic growth is controlled via antagonistic interactions of the steroid hormone ecdysone and nutrient-dependent insulin....../insulin-like growth factor (IGF) (insulin) signaling [2, 5]. The interplay between insulin and ecdysone signaling regulates systemic growth and controls organismal size. Here, we show that Warts (Wts; LATS1/2) signaling regulates systemic growth in Drosophila by activating basal ecdysone production, which negatively...

  12. Pri peptides are mediators of ecdysone for the temporal control of development.

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    Chanut-Delalande, Hélène; Hashimoto, Yoshiko; Pelissier-Monier, Anne; Spokony, Rebecca; Dib, Azza; Kondo, Takefumi; Bohère, Jérôme; Niimi, Kaori; Latapie, Yvan; Inagaki, Sachi; Dubois, Laurence; Valenti, Philippe; Polesello, Cédric; Kobayashi, Satoru; Moussian, Bernard; White, Kevin P; Plaza, Serge; Kageyama, Yuji; Payre, François

    2014-11-01

    Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas we have a wealth of information about spatial patterning, the mechanisms underlying temporal control remain poorly understood. Here we show that Pri peptides, encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. We identify a previously uncharacterized enzyme of ecdysone biosynthesis, GstE14, and find that ecdysone triggers pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. We show that manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, we find that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing.

  13. Ash2 acts as an ecdysone receptor coactivator by stabilizing the histone methyltransferase Trr.

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    Carbonell, Albert; Mazo, Alexander; Serras, Florenci; Corominas, Montserrat

    2013-02-01

    The molting hormone ecdysone triggers chromatin changes via histone modifications that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with transcriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdysone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is required for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

  14. Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

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    Robert J Ihry

    Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

  15. Translational control by the DEAD Box RNA helicase belle regulates ecdysone-triggered transcriptional cascades.

    Directory of Open Access Journals (Sweden)

    Robert J Ihry

    Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

  16. Ecdysone signalling and ovarian development in insects: from stem cells to ovarian follicle formation.

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    Belles, Xavier; Piulachs, Maria-Dolors

    2015-02-01

    Although a great deal of information is available concerning the role of ecdysone in insect oogenesis, research has tended to focus on vitellogenesis and choriogenesis. As such, the study of oogenesis in a strict sense has received much less attention. This situation changed recently when a number of observations carried out in the meroistic polytrophic ovarioles of Drosophila melanogaster started to unravel the key roles played by ecdysone in different steps of oogenesis. Thus, in larval stages, a non-autonomous role of ecdysone, first in repression and later in activation, of stem cell niche and primordial germ cell differentiation has been reported. In the adult, ecdysone stimulates the proliferation of germline stem cells, plays a role in stem cell niche maintenance and is needed non-cell-autonomously for correct differentiation of germline stem cells. Moreover, in somatic cells ecdysone is required for 16-cell cyst formation and for ovarian follicle development. In the transition from stages 8 to 9 of oogenesis, ecdysone signalling is fundamental when deciding whether or not to go ahead with vitellogenesis depending on the nutritional status, as well as to start border cell migration. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

  17. Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori

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    Wei Sun, Yi-Hong Shen, Deng-Wei Qi, Zhong-Huai Xiang, Ze Zhang

    2012-01-01

    Full Text Available Molting hormone (ecdysteroid is one of the most important hormones in insects. The synthesis and inactivation of the ecdysteroid regulate the developmental process of insects. A major pathway of ecdysone inactivation is that ecdysone is converted to 3-dehydroecdysone, and then further to 3-epiecdysone in insects. Two enzymes (ecdysone oxidase: EO and 3DE-3α-reductase participate in this pathway. In this study, based on the previously characterized cDNAs in Spodoptera littoralis, we cloned and characterized EO and 3DE-3α-reductase genes in the silkworm, Bombyx mori. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and 3DE-3α-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3α-reductase gene, RT-PCR and western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized 3DE-3α-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the 5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer. The functional characterization of the enzymes participating in ecdysone inactivation in the silkworm provides hints for the artificial regulation of the silkworm development and biological control of pests.

  18. Structural requirement and stereospecificity of tetrahydroquinolines as potent ecdysone agonists.

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    Kitamura, Seiya; Harada, Toshiyuki; Hiramatsu, Hajime; Shimizu, Ryo; Miyagawa, Hisashi; Nakagawa, Yoshiaki

    2014-04-01

    Tetrahydroquinoline (THQ)-type compounds are a class of potential larvicides against mosquitoes. The structure-activity relationships (SAR) of these compounds were previously investigated (Smith et al., Bioorg. Med. Chem. Lett. 2003, 13, 1943-1946), and one of cis-forms (with respect to the configurations of 2-methyl and 4-anilino substitutions on the THQ basic structure) was stereoselectively synthesized. However, the absolute configurations of C2 and C4 were not determined. In this study, four THQ-type compounds with cis configurations were synthesized, and two were submitted for X-ray crystal structure analysis. This analysis demonstrated that two enantiomers are packed into the crystal form. We synthesized the cis-form of the fluorinated THQ compound, according to the published method, and the enantiomers were separated via chiral HPLC. The absolute configurations of the enantiomers were determined by X-ray crystallography. Each of the enantiomers was tested for activity against mosquito larvae in vivo and competitive binding to the ecdysone receptor in vitro. Compared to the (2S,4R) enantiomer, the (2R,4S) enantiomer showed 55 times higher activity in the mosquito larvicidal assay, and 36 times higher activity in the competitive receptor binding assay.

  19. Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

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    George Tzertzinis

    Full Text Available BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor and USP (Ultraspiracle. METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR. Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE. In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor homolog (Bma-RXR and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together

  20. Ecdysone Receptor (EcR Is Involved in the Transcription of Cell Cycle Genes in the Silkworm

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    Wenliang Qian

    2015-02-01

    Full Text Available EcR (ecdysone receptor-mediated ecdysone signaling pathway contributes to regulate the transcription of genes involved in various processes during insect development. In this work, we detected the expression of EcR gene in silkworm ovary-derived BmN4 cells and found that EcR RNAi result in an alteration of cell shape, indicating that EcR may orchestrate cell cycle progression. EcR RNAi and EcR overexpression analysis revealed that in the cultured BmN4 cells, EcR respectively promoted and suppressed the transcription of E2F-1 and CycE, two genes controlling cell cycle progression. Further examination demonstrated that ecdysone application in BmN4 cells not only changed the transcription of these two cell cycle genes like that under EcR overexpression, but also induced cell cycle arrest at G2/M phase. In vivo analysis confirmed that E2F-1 expression was elevated in silk gland of silkworm larvae after ecdysone application, which is same as its response to ecdysone in BmN4 cells. However, ecdysone also promotes CycE transcription in silk gland, and this is converse with the observation in BmN4 cells. These results provide new insights into understanding the roles of EcR-mediated ecdysone signaling in the regulation of cell cycle.

  1. Furostanol saponins and ecdysones with cytotoxic activity from Helleborus bocconei ssp. intermedius.

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    Rosselli, Sergio; Maggio, Antonella; Bruno, Maurizio; Spadaro, Vivienne; Formisano, Carmen; Irace, Carlo; Maffettone, Carmen; Mascolo, Nicola

    2009-09-01

    Two furostanol saponins helleboroside A (1) and helleboroside B (2) were isolated from the methanol extract of Helleborus bocconei Ten. subsp. intermedius (Guss.) Greuter and Burdet, along with the furospirostanol saponin 4 and two ecdysones: ecdysterone (5) and polypodyne B (6). Compound 2 was enzymatically hydrolysed to give product 3. The biological activity of all compounds was tested against rat C6 glioma cells showing a significant cytotoxicity for compounds 3, 4 and 6.

  2. The transcriptional corepressor SMRTER influences both Notch and ecdysone signaling during Drosophila development

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    Bryan W. Heck

    2011-12-01

    SMRTER (SMRT-related and ecdysone receptor interacting factor is the Drosophila homologue of the vertebrate proteins SMRT and N-CoR, and forms with them a well-conserved family of transcriptional corepressors. Molecular characterization of SMRT-family proteins in cultured cells has implicated them in a wide range of transcriptional regulatory pathways. However, little is currently known about how this conserved class of transcriptional corepressors regulates the development of particular tissues via specific pathways. In this study, through our characterization of multiple Smrter (Smr mutant lines, mosaic analysis of a loss-of-function Smr allele, and studies of two independent Smr RNAi fly lines, we report that SMRTER is required for the development of both ovarian follicle cells and the wing. In these two tissues, SMRTER inhibits not only the ecdysone pathway, but also the Notch pathway. We differentiate SMRTER's influence on these two signaling pathways by showing that SMRTER inhibits the Notch pathway, but not the ecdysone pathway, in a spatiotemporally restricted manner. We further confirm the likely involvement of SMRTER in the Notch pathway by demonstrating a direct interaction between SMRTER and Suppressor of Hairless [Su(H], a DNA-binding transcription factor pivotal in the Notch pathway, and the colocalization of both proteins at many chromosomal regions in salivary glands. Based on our results, we propose that SMRTER regulates the Notch pathway through its association with Su(H, and that overcoming a SMRTER-mediated transcriptional repression barrier may represent a key mechanism used by the Notch pathway to control the precise timing of events and the formation of sharp boundaries between cells in multiple tissues during development.

  3. Beta-ecdysone has bone protective but no estrogenic effects in ovariectomized rats.

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    Seidlova-Wuttke, Dana; Christel, David; Kapur, Priya; Nguyen, Ba Tiep; Jarry, Hubertus; Wuttke, Wolfgang

    2010-09-01

    Estrogens exert beneficial effects in the bone. Their chronic use however bares several risks. Therefore intensive search for non-estrogenic, bone protective compounds is going on. We observed that an extract of Tinospora cordifolia has antiosteoporotic effects and identified 20-OH-Ecdysone (beta-Ecdysone=Ecd) as a possible candidate for this action. Ovariectomized (ovx) rats were treated orally over 3 months with no Ecd (control) or 18, 57 or 121 mg Ecd/day/animal. Estradiol-17beta benzoate (E2) 159 microg/day/animal) fed animals served as positive controls. Bone mineral density (BMD) of tibia was measured by quantitative computer tomography, serum Osteocalcin and CrossLaps were measured in a ligand binding assay. Utilizing an estrogen receptor (ER) containing cytosolic extract of porcine uteri the capability of Ecd to bind to ER was tested. Ecd did not bind to ER. BMD was reduced by more than 50% in the control. In the Ecd animals BMD was dose dependently higher. Serum CrossLaps was lower in the Ecd and E2 group while serum Osteocalcin levels were decreased in the E2 but increased in the Ecd fed animals. Ecd has an antiosteoporotic effect which does not involve activation of ER.

  4. Development of an in vitro binding assay for ecdysone receptor of mysid shrimp (Americamysis bahia)

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    Yokota, Hirofumi, E-mail: h-yokota@mail.kobe-c.ac.jp [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Eguchi, Sayaka [Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505 (Japan); Nakai, Makoto [Hita Laboratory, Chemicals Evaluation and Research Institute (CERI), 3-822, Ishii-machi, Hita-shi, Oita 877-0061 (Japan)

    2011-10-15

    Highlights: We successfully performed cDNA cloning of EcR and USP of mysid shrimp. We then expressed the ligand-binding domains of the corresponding receptor peptides. The translated peptides could bind to ecdysone agonists as heterodimers. These results indicate that they are functional hormone receptors of mysid shrimp. - Abstract: A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [{sup 3}H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K{sub d}) = 2.14 nM. Competitive binding assays showed that the IC{sub 50} values for ponasterone A, muristerone A, 20-hydroxyecdysone, and {alpha}-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC{sub 50} values for two dibenzoylhydrazine ligands

  5. Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster.

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    Verma, Puja; Tapadia, Madhu G

    2015-08-01

    In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Structure-activity relationship of imidazothiadiazole analogs for the binding to the ecdysone receptor of insect cells.

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    Yokoi, Taiyo; Minami, Saki; Nakagawa, Yoshiaki; Miyagawa, Hisashi

    2015-05-01

    Diacylhydrazines are the first non-steroidal ecdysone agonists, and five compounds are used as insecticides in agriculture. After the discovery of diacylhydrazine-type compounds, numerous non-steroidal structures were reported as ecdysone agonists. Among various ecdysone agonists, imidazothiadiazoles are reported to be very potent in vitro; however, the experimental detail for the structure identification and bioassays are not stated in the paper (Holmwood and Schindler, Bioorg. Med. Chem. 17, 4064-4070, 2009). In our present study, we synthesized 18 imidazothiadiazole-type compounds and confirmed the chemical structures by spectrometric analyses. The binding activity of the synthesized compounds to the ecdysone receptor was evaluated in terms of the concentration required for 50% inhibition of [(3)H]ponasterone A incorporation [IC50 (M)] into lepidopteran (Sf-9), coleopteran (BCRL-Lepd-SL1), and dipteran (NIAS-AeAl2) cells. 6-(2-Chlorophenyl)-2-(trifluoromethyl)imidazo[2,1-b] [1,3,4]-thiadiazol-5-yl)acrylamide analogs with CONHR (secondary amide) were very potent against Sf-9 cells, but further alkylation (tertiary amide: CONR2) decreased the activity dramatically. Additionally, a primary amide analog (CONH2) was inactive. The activity also decreased 150-fold by the saturation of olefin region of the acrylamide moiety. In addition, various substituents were introduced at the 2-position of the imidazothiadiazole ring to disclose the physicochemical properties of the substituents which are important for receptor binding. The activity increased by 7500-fold with the introduction of the CF2CF2CF3 group compared to the unsubstituted compound against Sf-9 cells. Quantitative structure-activity relationship analysis for these substituents indicated that hydrophobic and electron-withdrawing groups were favorable for binding. Some of the compounds with strong receptor binding activity showed good larvicidal activity against Spodoptera litura. In contrast, the binding

  7. Ecdysone regulates morphogenesis and function of Malpighian tubules in Drosophila melanogaster through EcR-B2 isoform.

    Science.gov (United States)

    Gautam, Naveen Kumar; Verma, Puja; Tapadia, Madhu G

    2015-02-15

    Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and β-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump. B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.

  8. Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects.

    Science.gov (United States)

    Zotti, Moises João; Christiaens, Olivier; Rougé, Pierre; Grutzmacher, Anderson Dionei; Zimmer, Paulo Dejalma; Smagghe, Guy

    2012-04-01

    In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

  9. Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna.

    Science.gov (United States)

    Kato, Yasuhiko; Kobayashi, Kaoru; Oda, Shigeto; Tatarazako, Norihisa; Watanabe, Hajime; Iguchi, Taisen

    2007-04-01

    cDNAs encoding the ecdysone receptor (EcR) and ultra spiracle (USP) protein were cloned from the water flea Daphnia magna (Crustacea: Cladocera). The deduced EcR and USP amino acid sequences showed a high degree of homology to those of other crustaceans as well as insects. We isolated three isoforms of EcR that differ in the A/B domain. Quantitative PCR analysis indicated differing temporal expression patterns of the EcR isoforms during the molting period and demonstrated that the expression of one subtype correlated well with the timing of molt. Using cDNAs encoding EcR and USP, we constructed a Daphnia EcR/USP reporter based on a two-hybrid system. The gene fusions encoded the EcR ligand-binding domain (LBD) fused to the Gal4 DNA-binding domain, and the USP-LBD fused to the Vp16 activation domain. These chimeric genes were transfected with a luciferase reporter gene. Dose-dependent activation of the reporter gene could be observed when transfectants were exposed to Ec and other chemicals known to have Ec-like activities. This two-hybrid system may represent a useful reporter system for further examination of hormonal and chemical effects on Daphnia at the molecular level.

  10. Ecdysone receptor expression in developing and adult mushroom bodies of the ant Camponotus japonicus.

    Science.gov (United States)

    Nemoto, Michie; Hara, Kenji

    2007-09-01

    Mushroom bodies (MBs) are insect brain centers involved in sensory integration and memory formation. In social Hymenoptera, MBs are large and comprise larger number of Kenyon cells and have repeatedly been implied to underlie the social behaviors. In the present study, to facilitate our understanding of the neural basis of social behaviors, two complementary DNAs (cDNAs) encoding presumed ecdysone receptor isoforms (CjEcR-A and CjEcR-alpha) were identified in the developing brains of the carpenter ant Camponotus japonicus. Sequence comparison indicated that these CjEcR proteins had common DNA- and hormone-binding domains linked to different N-terminal regions. The alignment of the distinct regions with other insects EcRs indicated that CjEcR-A is the ant homologue of EcR-A, and CjEcR-alpha has a novel type of A/B region. Immunohistochemical analyses of the MBs of C. japonicus with the common region antibody demonstrated that these CjEcRs appear in all neuroblasts, neurons, and glia cells during neurogenesis, whereas expression is confined to the neurons, disappearing in the glia cells in newly emerged workers. Less expression was observed in the forager MBs. These findings suggest that CjEcRs are involved in maturation and development of ant MBs.

  11. Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

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    JM Albuquerque-Cunha

    2004-12-01

    Full Text Available The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM, and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.

  12. The insect ecdysone receptor is a good potential target for RNAi-based pest control.

    Science.gov (United States)

    Yu, Rong; Xu, Xinping; Liang, Yongkang; Tian, Honggang; Pan, Zhanqing; Jin, Shouheng; Wang, Na; Zhang, Wenqing

    2014-01-01

    RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.

  13. A regulatory pathway, ecdysone-transcription factor relish-cathepsin L, is involved in insect fat body dissociation.

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    Yao Zhang

    Full Text Available Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone-transcription factor (Relish-target gene (cathepsin L regulatory pathway.

  14. Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues

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    Seoghyun Lee

    2016-01-01

    Full Text Available Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet-regulated system. Exploiting a Drosophila ecdysone receptor (EcR-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+ and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site. Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

  15. Effects of both ecdysone and the acclimation to low temperature, on growth and metabolic rate of juvenile freshwater crayfish Cherax quadricarinatus (Decapoda, Parastacidae

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    Anouk Chaulet

    2013-06-01

    Full Text Available Growth, metabolic rate, and energy reserves of Cherax quadricarinatus (von Martens, 1868 juveniles were evaluated in crayfish acclimated for 16 weeks to either 25ºC (temperature near optimum or 20ºC (marginal for the species. Additionally, the modulating effect of ecdsyone on acclimation was studied. After 12 weeks of exposure, weight gain of both experimental groups acclimated to 25ºC (control: C25, and ecdysone treated: E25 was significantly higher than that of those groups acclimated to 20ºC (C20 and E20. A total compensation in metabolic rate was seen after acclimation from 25ºC to 20ºC; for both the control group and the group treated with ecdysone. A Q10value significantly higher was only observed in the group acclimated to 20ºC and treated with ecdysone. A reduction of glycogen reserves in both hepatopancreas and muscle, as well as a lower protein content in muscle, was seen in both groups acclimated to 20ºC. Correspondingly, glycemia was always higher in these groups. Increased lipid levels were seen in the hepatopancreas of animals acclimated to 20ºC, while a higher lipid level was also observed in muscle at 20ºC, but only in ecdysone-treated crayfish.

  16. Cloning and Expression of Ecdysone Receptor and Retinoid X Receptor from Procambarus clarkii: Induction by Eyestalk Ablation

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    Tian-Hao Dai

    2016-10-01

    Full Text Available Ecdysone receptor and retinoid X receptor are key regulators in molting. Here, full length ecdysone receptor (PcEcR and retinoid X receptor (PcRXR cDNAs from Procambarus clarkii were cloned. Full length cDNA of PcEcR has 2500 bp, encoding 576 amino acid proteins, and full length cDNA of PcRXR has 2593 bp, in which a 15 bp and a 204 bp insert/deletion splice variant regions in DNA binding domain and hinge domain were identified. The two splice variant regions in PcRXR result four isoforms: PcRXR1-4, encoding 525, 520, 457 and 452 amino acids respectively. PcEcR was highly expressed in the hepatopancreas and eyestalk and PcRXR was highly expressed in the eyestalk among eight examined tissues. Both PcEcR and PcRXR had induced expression after eyestalk ablation (ESA in the three examined tissues. In muscle, PcEcR and PcRXR were upregulated after ESA, PcEcR reached the highest level on day 3 after ESA and increased 33.5-fold relative to day 0, and PcRXR reached highest the level on day 1 after ESA and increased 2.7-fold relative to day 0. In the hepatopancreas, PcEcR and PcRXR dEcReased continuously after ESA, and the expression levels of PcEcR and PcRXR were only 0.7% and 1.7% on day 7 after ESA relative to day 0, respectively. In the ovaries, PcEcR was upregulated after ESA, reached the highest level on day 3 after ESA, increased 3.0-fold relative to day 0, and the expression level of PcRXR changed insignificantly after ESA (p > 0.05. The different responses of PcEcR and PcRXR after ESA indicates that different tissues play different roles (and coordinates their functions in molting.

  17. Effects of Quinizarin and Five Synthesized Derivatives on Fifth Larval Instar Midgut Ecdysone 20-Monooxygenase Activity of the Tobacco Hornworm Manduca sexta

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    Christopher A. Drummond

    2014-01-01

    Full Text Available The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone, and five anthraquinones that were synthesized from quinizarin, namely, 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect model Manduca sexta (tobacco hornworm. This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form. M. sexta fifth larval instar midgut homogenates were incubated with increasing concentrations (10−8 to 10−3 M of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibited I50’s of about 4×10-6 to 6×10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4-anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.

  18. Structural and evolutionary innovation of the heterodimerization interface between USP and the ecdysone receptor ECR in insects.

    Science.gov (United States)

    Iwema, Thomas; Chaumot, Arnaud; Studer, Romain A; Robinson-Rechavi, Marc; Billas, Isabelle M L; Moras, Dino; Laudet, Vincent; Bonneton, François

    2009-04-01

    Understanding how the variability of protein structure arises during evolution and leads to new structure-function relationships ultimately promoting evolutionary novelties is a major goal of molecular evolution and is critical for interpreting genome sequences. We addressed this issue using the ecdysone receptor (ECR), a major developmental factor that controls development and reproduction of arthropods. The functional ECR is a heterodimer of two nuclear receptors: ECR, which binds ecdysteroids, and its obligatory partner ultraspirade (USP), which is orthologous to the retinoid X receptor of vertebrates. Both genes underwent a dramatic increase of evolutionary rate in Mecopterida, the major insect terminal group containing Dipteras and Lepidopteras. We therefore questioned the implication of this event in terms of coevolution of their dimerization interface. A structural comparison revealed a 30% larger ligand-binding domain (LBD) heterodimerization surface in the Lepidoptera Heliothis when compared with basal insects, associated with a symmetrization of the interface, which is exceptional for nuclear receptors. Reconstruction of ancestral sequences and homology modeling of the ancestral Mecopterida ECR-USP reveal that this enlarged dimerization surface is a synapomorphy for Mecopterida. Furthermore, we show that the residues implicated in the new dimerization surface underwent specific evolutionary constraints in Mecopterida indicative of their new and conserved role in the dimerization interface. Most of all, the novel surface originates from a 15 degrees torsion of a subdomain of USP LBD toward its partner ECR, which is a long-range consequence of the peculiar position of a Mecopterida-specific insertion in loop L1-3, located outside of the interaction surface, in a less crucial domain of the partner protein. These results indicate that the coevolution between ECR and USP occurred through a novel mechanism of intramolecular epistasis that will undoubtedly be

  19. Development of a bipartite ecdysone-responsive gene switch for the oomycete Phytophthora infestans and its use to manipulate transcription during axenic culture and plant infection.

    Science.gov (United States)

    Gamboa-Meléndez, Heber; Judelson, Howard S

    2015-01-01

    Conditional expression systems have been proven to be useful tools for the elucidation of gene function in many taxa. Here, we report the development of the first useful inducible promoter system for an oomycete, based on an ecdysone receptor (EcR) and the ecdysone analogue methoxyfenozide. In Phytophthora infestans, the potato late blight pathogen, a monopartite transactivator containing the VP16 activation domain from herpes simplex virus, the GAL4 DNA-binding domain from yeast and the EcR receptor domain from the spruce budworm enabled high levels of expression of a β-glucuronidase (GUS) reporter gene, but unacceptable basal activity in the absence of the methoxyfenozide inducer. Greatly improved performance was obtained using a bipartite system in which transcription is activated by a heterodimer between a chimera of VP16 and the migratory locust retinoid X receptor, and a separate EcR-DNA-binding domain chimera. Transformants were obtained that exhibited >100-fold activation of the reporter by methoxyfenozide, with low basal levels of expression and induced activity approaching that of the strong ham34 promoter. Performance varied between transformants, probably as a result of position effects. The addition of methoxyfenozide enabled strong induction during hyphal growth, zoosporogenesis and colonization of tomato. No significant effects of the inducer or transactivators on growth, development or pathogenicity were observed. The technology should therefore be a useful addition to the arsenal of methods for the study of oomycete plant pathogens.

  20. Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila.

    Science.gov (United States)

    Yan, Yan; Wang, Hao; Chen, Hanqing; Lindström-Battle, Anya; Jiao, Renjie

    2015-09-20

    Body size is one of the features that distinguish one species from another in the biological world. Animals have developed mechanisms to control their body size during normal development. However, how animals cope with genetic alterations and/or environmental stresses to develop into normal-sized adults remain poorly understood. The ability of the animals to develop into a normal-sized adult after the challenges of genetic alterations and/or environmental stresses reveals a robustness of body size control. Here we show that the mutation of dGPAT4, a de novo synthase of lysophosphatidic acid, is a genetic alteration that triggers such a robust response of the animals to body size challenges in Drosophila. Loss of dGPAT4 leads to a severe delay of development, slow growth and resultant small-sized animals during the larval stages, but results in normal-sized adult flies. The robust body size adjustment of the dGPAT4 mutant is likely achieved by corresponding changes in ecdysone and insulin signaling, which is also manifested by compromised food intake. Thus, we propose that a strategy has been evolved by the animals to reach final body size when challenged by genetic alterations, which requires the coordinated ecdysone and insulin signaling.

  1. Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket.

    Science.gov (United States)

    Soin, Thomas; Iga, Masatoshi; Swevers, Luc; Rougé, Pierre; Janssen, Colin R; Smagghe, Guy

    2009-08-01

    Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

  2. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.;

    2006-01-01

    The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E......) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended......-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role...

  3. Ecdysone receptor (EcR) and ultraspiracle (USP) genes from the cyclopoid copepod Paracyclopina nana: Identification and expression in response to water accommodated fractions (WAFs).

    Science.gov (United States)

    Puthumana, Jayesh; Lee, Min-Chul; Han, Jeonghoon; Kim, Hui-Su; Hwang, Dae-Sik; Lee, Jae-Seong

    2017-02-01

    Ecdysteroid hormones are pivotal in the development, growth, and molting of arthropods, and the hormone pathway is triggered by binding ecdysteroid to a heterodimer of the two nuclear receptors; ecdysone receptors (EcR) and ultraspiracle (USP). We have characterized EcR and USP genes, and their 5'-untranslated region (5'-UTR) from the copepod Paracyclopina nana, and studied mRNA transcription levels in post-embryonic stages and in response to water accommodated fractions (WAFs) of crude oil. The open reading frames (ORF) of EcR and USP were 1470 and 1287bp that encoded 490 and 429 amino acids with molecular weight of 121.18 and 105.03kDa, respectively. Also, a well conserved DNA-binding domain (DBD) and ligand-binding domain (LBD) were identified which confirmed by phylogenetic analysis. Messenger RNA transcriptional levels of EcR and USP were developmental stage-specific in early post-embryonic stages (N3-4). However, an evoked expression of USP was observed throughout copepodid stage and in adult females. WAFs (40 and 80%) were acted as an ecdysone agonist in P. nana, and elicited the mRNA transcription levels in adults. Developmental stage-specific transcriptional activation of EcR and USP in response to WAFs was observed. USP gene was down-regulated in the nauplius in response to WAF, whereas up-regulation of USP was observed in the adults. This study represents the first data of molecular elucidation of EcR and USP genes and their regulatory elements from P. nana and the developmental stage specific expression in response to WAFs, which can be used as potential biomarkers for environmental stressors with ecotoxicological evaluations in copepods.

  4. Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: cloning and their expression patterns in eyestalks and Y-organs during the molt cycle.

    Science.gov (United States)

    Techa, Sirinart; Chung, J Sook

    2013-09-15

    Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3-5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5-4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5' UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera

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    Angel Roberto Barchuk

    2002-01-01

    Full Text Available The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids.

  6. Silencing of ecdysone receptor, insect intestinal mucin and sericotropin genes by bacterially produced double-stranded RNA affects larval growth and development in Plutella xylostella and Helicoverpa armigera.

    Science.gov (United States)

    Israni, B; Rajam, M V

    2017-04-01

    RNA interference mediated gene silencing, which is triggered by double-stranded RNA (dsRNA), has become a important tool for functional genomics studies in various systems, including insects. Bacterially produced dsRNA employs the use of a bacterial strain lacking in RNaseIII activity and harbouring a vector with dual T7 promoter sites, which allow the production of intact dsRNA molecules. Here, we report an assessment of the functional relevance of the ecdysone receptor, insect intestinal mucin and sericotropin genes through silencing by dsRNA in two lepidopteran insect pests, Helicoverpa armigera and Plutella xylostella, both of which cause serious crop losses. Oral feeding of dsRNA led to significant reduction in transcripts of the target insect genes, which caused significant larval mortality with various moulting anomalies and an overall developmental delay. We also found a significant decrease in reproductive potential in female moths, with a drop in egg laying and compromised egg hatching from treated larvae as compared to controls. dsRNA was stable in the insect gut and was efficiently processed into small interfering RNAs (siRNAs), thus accounting for the phenotypes observed in the present work. The study revealed the importance of these genes in core insect processes, which are essential for insect development and survival. © 2016 The Royal Entomological Society.

  7. Structural changes under low evolutionary constraint may decrease the affinity of dibenzoylhydrazine insecticides for the ecdysone receptor in non-lepidopteran insects.

    Science.gov (United States)

    Zotti, M J; Christiaens, O; Rougé, P; Grutzmacher, A D; Zimmer, P D; Smagghe, G

    2012-10-01

    Understanding how variations in genetic sequences are conveyed into structural and biochemical properties is of increasing interest in the field of molecular evolution. In order to gain insight into this process, we studied the ecdysone receptor (EcR), a transcription factor that controls moulting and metamorphosis in arthropods. Using an in silico homology model, we identified a region in the lepidopteran EcR that has no direct interaction with the natural hormone but is under strong evolutionary constraint. This region causes a small indentation in the three-dimensional structure of the protein which facilitates the binding of tebufenozide. Non-Mecopterida are considered much older, evolutionarily, than Lepidoptera and they do not have this extended cavity. This location shows differences in evolutionary constraint between Lepidoptera and other insects, where a much lower constraint is observed compared with the Lepidoptera. It is possible that the higher flexibility seen in the EcR of Lepidoptera is an entirely new trait and the higher constraint could then be an indication that this region does have another important function. Finally, we suggest that Try123, which is evolutionarily constrained and is up to now exclusively present in Lepidoptera EcRs, could play a critical role in discriminating between steroidal and non-steroidal ligands.

  8. RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants

    Science.gov (United States)

    Malik, Hassan Jamil; Raza, Amir; Amin, Imran; Scheffler, Jodi A.; Scheffler, Brian E.; Brown, Judith K.; Mansoor, Shahid

    2016-01-01

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread. PMID:27929123

  9. RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants.

    Science.gov (United States)

    Malik, Hassan Jamil; Raza, Amir; Amin, Imran; Scheffler, Jodi A; Scheffler, Brian E; Brown, Judith K; Mansoor, Shahid

    2016-12-08

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread.

  10. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

    Directory of Open Access Journals (Sweden)

    Kubo Takeo

    2010-02-01

    Full Text Available Abstract Background The ecdysone receptor (EcR regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. Results The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Conclusions Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional

  11. Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras.

    Science.gov (United States)

    Tavva, Venkata S; Dinkins, Randy D; Palli, Subba R; Collins, Glenn B

    2007-10-01

    Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.

  12. Effects of Insect Ecdysone and Juvenile Hormone on Reproduction of Bursaphelenchus xylophilus%昆虫蜕皮激素和保幼激素对松材线虫繁殖的影响

    Institute of Scientific and Technical Information of China (English)

    林峰; 袁冬菊; 赵博光; 王华光

    2014-01-01

    研究了外源昆虫蜕皮激素和保幼激素对松材线虫( Bursaphelenchus xylophilus)繁殖的影响。用10-6 g/mL到10-9 g/mL的7个连续几何级数浓度的保幼激素或蜕皮激素处理线虫。保幼激素处理6 d及以后的松材线虫繁殖数量显著高于空白对照和仅提供灰葡萄孢为营养的对照( P<0.05)。在观察时间内,保幼激素浓度以5×10-7 g/mL为线虫繁殖量的最高峰值,其线虫繁殖数量显著高于其他浓度( P<0.05)。蜕皮激素对松材线虫的繁殖同样具有明显的促进作用,激素处理6 d及以后的松材线虫繁殖数量显著高于两组对照( P<0.05)。在观察时间内,蜕皮激素浓度以5×10-7 g/mL为线虫繁殖量的最高峰值,且明显高于其他浓度的处理(P<0.05)。此外还发现保幼激素和蜕皮激素在浓度为5×10-9 g/mL处存在线虫繁殖的次高峰。%The effects of the insect 20-hydroxyecdysone and juvenile hormoneⅢ on reproduction of pine wood nematode,Bursaphelenchus xylophilus,were investigated.The nematodes were treated with 7 continuous geometric concentration series of 20-hydroxyecdysone or juvenile hormone Ⅲ.The numbers of the nematodes treated with 20-hydroxyecdysone or juvenile hormone Ⅲfrom the 6th day to the 9th were significantly higher than those of the blank control and the control treated with only the nematode food ,Botrytis cinerea ( P<0.05) . During the observing period the peak of the reproduction was at 5 ×10-7 g/mL of the ecdysone and the peak value was significantly higher than those in the other concentrations .During the observing period the peak of the number of the nematodes was at 5×10-7 g/mL of the two hormones and it was significantly higher than those in the other concentrations of the hormones .The second peak of the nematode number was also found at 5 ×10-9 g/mL in both the ecdysone and the juvenile hormone .

  13. Produção de β-ecdisona em Pfaffia glomerata (Spreng. Pedersen em função da adubação orgânica em 6 épocas de crescimento β-ecdysone production by Pfaffia glomerata (Spreng. Pedersen under organic fertilization in 6 growth periods

    Directory of Open Access Journals (Sweden)

    C.P.V. Guerreiro

    2009-01-01

    Full Text Available O trabalho teve como objetivo verificar a influência da adubação orgânica em 6 épocas de crescimento na produção de β-ecdisona por plantas de Pfaffia glomerata. O experimento foi conduzido na Fazenda Santo Antonio do Araquá, distrito de Catâneo Ângelo, município de São Manuel, São Paulo, Brasil. Utilizou-se o delineamento de blocos ao acaso, num esquema fatorial 5x6, com quatro repetições, considerando-se 8 plantas úteis por parcela. Os blocos foram constituídos de 6 épocas de crescimento (60, 120, 180, 240, 300 e 360 dias após a germinação e de 5 doses de esterco de galinha curtido [testemunha (sem adubação, 15, 30, 45 e 60 t ha-1]. Após cada colheita, as raízes das plantas foram secas em estufa com circulação de ar forçada a 40ºC e pesadas para posterior extração do β-ecdisona, seguindo metodologia desenvolvida por Magalhães (2000. Os resultados foram submetidos à análise de variância e ao teste de separação de médias de Scott Knott, todos a 5% de probabilidade. Quando ocorreu interação os resultados foram avaliados usando-se análise de regressão polinominal. O teor de β-ecdisona não foi influenciado pelas doses de adubo e nem pela época do crescimento das plantas. Porém a quantidade total de β-ecdisona por raiz foi influenciada pela época de crescimento, sendo que aos 360 dias após a emergência ocorreu uma maior quantidade do princípio ativo em todos os tratamentos. Apesar de não diferir estatisticamente dos demais tratamentos, aos 360 dias após a emergência das plantas, o tratamento 30 t ha-1 foi o que proporcionou maior quantidade de β-ecdisona.The aim of this study was to assess the influence of organic fertilization in 6 growth periods on β-ecdysone production by Pfaffia glomerata plants. The field work was conducted in "Santo Antonio do Araquá" Farm, Catâneo Ângelo District, São Manuel, São Paulo State, Brazil. The adopted design was in randomized blocks, in a 5x6 factorial

  14. Molecular cloning and expression analysis of an ecdysone receptor (EcR) gene in the wheat midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)%麦红吸浆虫蜕皮激素受体(EcR)基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    马康生; 李伯辽; 陈浩; 仵均祥

    2013-01-01

    In order to study the function of ecdysone receptor in the diapause of Sitodiplosis mosellana,the full-length cDNA sequence of an ecdysone receptor gene was amplified by using reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE),and the expression of the EcR gene was analyzed using real-time quantitative PCR (qPCR).The full-length cDNA sequence was named as SmEcR (GenBank accession number:KC491135).Its open reading frame (ORF) is 1 386 bp in length,encoding a 461-amino-acid protein,with a calculated molecular weight of 52.90 kD and the theoretical isoelectric point of 6.24.Multiple sequence alignment revealed that the deduced amino acid sequence of SmEcR has high identity with EcRs from other insect species,especially with that of Bradysia coprophila (92%).Real-time quantitative PCR showed that SmEcR transcripts were detected in all diapause and developmental stages.The expression of SmEcR was significantly different among different diapause stages,reaching the highest level in November and the lowest level in December.The larvae collected from wheat heads had a lower expression level of SmEcR while the adults had the highest SmEcR transcripts.This study lays the foundation for the further functional study of SmEcR in diapause regulation in S.mosellana.%为了研究蜕皮激素受体(EcR)在麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育活动中的作用,利用RT-PCR和RACE技术克隆得到了麦红吸浆虫蜕皮激素受体基因cDNA全长,并通过Real-time quantitative PCR研究了其表达情况.该cDNA全长序列被命名为SmEcR(GenBank登录号:KC491135),其开放阅读框长1 386 bp,编码461个氨基酸残基.其蛋白预测分子量52.90 kD,理论等电点6.24.该蛋白与其他已报道的昆虫EcR蛋白具有很高的同源性,其中与迟眼蕈蚊Bradysia coprophila中相应蛋白的氨基酸序列一致性高达92%.SmEcR在麦红吸浆虫不同滞育时期和不同虫态中均有表达,且

  15. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

    2006-01-01

    that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each...... larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression...

  16. Ecdysone receptor agonism leading to lethal molting disruption in arthropods: Review and adverse outcome pathway development

    Science.gov (United States)

    Molting is a key biological process in growth, development, reproduction and survival in arthropods. Complex neuroendocrine pathways are involved in the regulation of molting and may potentially become targets of environmental endocrine disrupting compounds (EDCs). For example, s...

  17. Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs.

    Science.gov (United States)

    Monteiro, Antónia; Tong, Xiaoling; Bear, Ashley; Liew, Seng Fatt; Bhardwaj, Shivam; Wasik, Bethany R; Dinwiddie, April; Bastianelli, Carole; Cheong, Wei Fun; Wenk, Markus R; Cao, Hui; Prudic, Kathleen L

    2015-01-01

    Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.

  18. Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects

    DEFF Research Database (Denmark)

    Hentze, Julie Lilith; Møller, Morten Erik; Jørgensen, Anne

    2013-01-01

    Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site...... of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis...... melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises...

  19. Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects

    DEFF Research Database (Denmark)

    Hentze, Julie Lilith; Møller, Morten Erik; Jørgensen, Anne;

    2013-01-01

    of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis...

  20. A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

    Directory of Open Access Journals (Sweden)

    Benjamin F B Costantino

    2008-06-01

    Full Text Available The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

  1. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    Science.gov (United States)

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  2. The Function and Evolution of the Halloween Genes; the Pathway to the Arthropod Molting Hormone. In Ecdysone, Structures and Functions

    DEFF Research Database (Denmark)

    Gilbert, Lawrence; Rewitz, Kim

    2009-01-01

    as mediating the final steps in the biosynthesis of the arthropod molting hormone, 20-hydroxyecdysone (20E). A fifth has now been studied in detail and shown to be required for ecdysteroidogenesis but its exact function has yet to be elucidated. Since both insects and crustaceans utilize 20E as their principal......, Coleoptera, Hymenoptera and other Diptera allowed the development of a phylogenetic scheme for this gene family and suggests that the Halloween genes and vertebrate steroidogenic P450s originated from common ancestors that were perhaps destined for steroidogenesis, and arose before the deuterostome-arthropod...

  3. Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

    OpenAIRE

    2012-01-01

    Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of p...

  4. Developmental regulation of ecdysone receptor (EcR and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Tathyana Rachel Palo Mello

    2014-12-01

    Full Text Available Major developmental transitions in multicellular organisms are driven by steroid hormones. In insects, these, together with juvenile hormone (JH, control development, metamorphosis, reproduction and aging, and are also suggested to play an important role in caste differentiation of social insects. Here, we aimed to determine how EcR transcription and ecdysteroid titers are related during honeybee postembryonic development and what may actually be the role of EcR in caste development of this social insect. In addition, we expected that knocking-down EcR gene expression would give us information on the participation of the respective protein in regulating downstream targets of EcR. We found that in Apis mellifera females, EcR-A is the predominantly expressed variant in postembryonic development, while EcR-B transcript levels are higher in embryos, indicating an early developmental switch in EcR function. During larval and pupal stages, EcR-B expression levels are very low, while EcR-A transcripts are more variable and abundant in workers compared to queens. Strikingly, these transcript levels are opposite to the ecdysteroid titer profile. 20-hydroxyecdysone (20E application experiments revealed that low 20E levels induce EcR expression during development, whereas high ecdysteroid titers seem to be repressive. By means of RNAi-mediated knockdown (KD of both EcR transcript variants we detected the differential expression of 234 poly-A+ transcripts encoding genes such as CYPs, MRJPs and certain hormone response genes (Kr-h1 and ftz-f1. EcR-KD also promoted the differential expression of 70 miRNAs, including highly conserved ones (e.g. miR-133 and miR-375, as well honeybee-specific ones (e.g. miR-3745 and miR-3761. Our results put in evidence a broad spectrum of EcR-controlled gene expression during postembryonic development of honeybees, revealing new facets of EcR biology in this social insect.

  5. The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect

    Directory of Open Access Journals (Sweden)

    Antoine eAbrieux

    2014-09-01

    Full Text Available Olfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex of the primary olfactory center, the antennal lobe (AL is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. We recently showed that a G-protein-coupled receptor (GPCR, AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E, and dopamine (DA, present in the ALs, is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. Here we tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing combining receptor inhibition with intracellular recordings of AL neurons. We show that the sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover we tested the involvement of 20E and DA in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. We show that both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors.

  6. RNAi-mediated Mortality of the Whitefly through Transgenic Expression of Double-stranded RNA Homologous to Acetylcholinesterase and Ecdysone Receptor in Tobacco Plants

    Science.gov (United States)

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses affecting plants worldwide. Using RNA interference (RNAi) to downregulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus dise...

  7. Construction of a Yeast Model for Screening Aedes albopictus Ecdysone Agonist Pesticides%白纹伊蚊蜕皮激素拮抗剂筛选酵母模型的建立

    Institute of Scientific and Technical Information of China (English)

    顾金保; 孙彦涛; 彭鸿娟

    2007-01-01

    目的 在毕赤酵母体内构建白纹伊蚊蜕皮激素转录活化系统,建立高通量杀虫剂筛选模型,用于筛选蜕皮激素代谢途径拮抗药物.方法 人工合成果蝇蜕皮激素响应元件(EcRE)5次重复的序列,与果蝇热激蛋白基因启动子(pHSP27)序列连接,以绿色荧光蛋白(GFP)报告基因,将EcRE-pHSP27-GFP片段亚克隆入pPIC3.5k,整合入毕赤酵母染色体构建阴性酵母A.人工合成白纹伊蚊蜕皮激素受体(EcR)及超螺旋蛋白(USP)编码序列,两个基因以双表达盒形式亚克隆入组成型表达质粒pGAPZ,整合入酵母染色体的另一位点,使EcR与USP在酵母中组成型表达,构建模型酵母B.制备蜕皮激素拮抗剂虫酰肼悬液(浓度为0.83mg/ml),分别施加于模型酵母B与阴性酵母A,荧光显微镜下目测荧光强度,与未施加药物的对照组比较.同时提取各组RNA,半定量RT-PCR检验GFP基因的转录效率.结果 模型酵母B发出绿色荧光,而阴性酵母A与空白酵母GS115未见荧光,表明在模型酵母体内表达的EcR与USP形成复合二聚体,作用于EcRE启动GFP报告基因表达荧光蛋白.施用虫酰肼,模型酵母B荧光强度明显减弱,表明GFP的表达量减少.施用虫酰肼的模型酵母B,GFP与内参灰度比值(为0.614)低于对照组(1.134),表明虫酰肼可降低模型酵母B体内GFP基因的转录水平.结论 在酵母体内建立了白纹伊蚊蜕皮激素转录活化系统,该酵母模型可用于筛选作用于蜕皮激素代谢途径的药物.

  8. Contact and Systemic Activities of Three Diacylhydrazine Ecdysone Agonists to Chilo suppressalis (Lepidoptera: Pyralidae)%双酰肼类蜕皮促进剂对水稻二化螟的触杀与内吸作用比较

    Institute of Scientific and Technical Information of China (English)

    曹明章; 沈晋良

    2003-01-01

    采用点滴法和盆栽土壤处理法分别测定了双酰肼类杀虫剂虫酰肼、JS118和抑食肼(RH5849)对二化螟的触杀与内吸作用,并与氟虫腈和杀虫单进行了比较.结果表明,虫酰肼和JS118对二化螟4龄幼虫的触杀毒力相当,LD50分别为0.0252、0.0310μg/头,约是氟虫腈的十分之一、杀虫单的11~13倍;抑食肼的触杀毒力(LD50为0.2720μg/头)约是虫酰肼和JS118的十分之一.然而,抑食肼20、40g a.i./667m2处理盆栽土壤后3、7d,对二化螟、蚁螟的内吸杀虫效果分别达66.9%~89.0%和84.4%~96.7%,与氟虫腈(2 g a.i./667m2)、杀虫单(40g a.i./667m2)接近或相当,而虫酰肼和JS118(5、10 g a.i./667m2)无明显内吸杀虫作用.

  9. EST Table: NM_001043866 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available )|GO:0043565(sequence-specific DNA binding) 10/09/29 89 %/588 aa ref|NP_001037331.2| ecdysone receptor isofo...rm B2 [Bombyx mori] gb|AAA87340.1| 20-hydroxy-ecdysone receptor [Bombyx mori] 10/09/13 61 %/442 aa FBpp01170...0014267|gb|EFA10715.1| ecdysone receptor isoform B [Tribolium castaneum] NM_001043866 ...

  10. EST Table: FS894763 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS894763 E_FL_ftes_16A22_R_0 10/09/28 91 %/139 aa pdb|3C7T|A Chain A, Crystal Structure Of The Ecdyson...ain B, Crystal Structure Of The Ecdysone Phosphate Phosphatase, Eppase, From Bombix Mori In Complex With Tun...gstate pdb|3C7T|C Chain C, Crystal Structure Of The Ecdysone Phosphate Phosphatas...e, Eppase, From Bombix Mori In Complex With Tungstate pdb|3C7T|D Chain D, Crystal Structure Of The Ecdysone

  11. EST Table: BP124755 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BP124755 fbS20263 10/09/28 93 %/160 aa pdb|3C7T|A Chain A, Crystal Structure Of The Ecdyson...3C7T|C Chain C, Crystal Structure Of The Ecdysone Phosphate Phosphatase, Eppase, ...From Bombix Mori In Complex With Tungstate pdb|3C7T|D Chain D, Crystal Structure Of The Ecdysone Phosphate P...e Phosphate Phosphatase, Eppase, From Bombix Mori In Complex With Tungstate pdb|3C7T|B Chain B, Crys...tal Structure Of The Ecdysone Phosphate Phosphatase, Eppase, From Bombix Mori In Complex With Tungstate pdb|

  12. EST Table: BP179443 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BP179443 ovS302G11f 10/09/28 99 %/173 aa ref|NP_001106219.1| ecdysone 20-hydroxylas...e [Bombyx mori] dbj|BAE45332.1| ecdysone 20-hydroxylase [Bombyx mori] 10/08/29 47 %/182 aa FBpp0206947|DsecG

  13. EST Table: D49476 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available D49476 10/09/29 100 %/220 aa dbj|BAF76331.1| ecdysone receptor [Bombyx mori] 10/09/...3:38907568:1|gene:AGAP012211 10/09/10 61 %/217 aa gnl|Amel|GB30298-PB 10/09/10 61 %/216 aa gi|121308146|emb|CAL25730.1| ecdyson

  14. EST Table: CK559639 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK559639 rswpb0_001718.y1 10/09/29 41 %/206 aa gb|AAK56551.1| ecdysone oxidase [Spo...doptera littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 10/09/01 34 %/145 aa FBpp0145867

  15. EST Table: D49478 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available D49478 10/09/29 100 %/223 aa dbj|BAF76333.1| ecdysone receptor [Bombyx mori] 10/09/...98-PB 10/09/10 61 %/218 aa gi|121308146|emb|CAL25730.1| ecdysone receptor (isoform A) [Tribolium castaneum] BP118768 ...

  16. EST Table: DC556432 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available a ref|NP_001106219.1| ecdysone 20-hydroxylase [Bombyx mori] dbj|BAE45332.1| ecdysone 20-hydroxylase [Bombyx ...0005506(iron ion binding)|GO:0009055(electron carrier activity)|GO:0020037(heme binding) 10/09/28 87 %/170 a

  17. EST Table: DC544102 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available DC544102 E_FL_dpe-_24H08_F_0 10/09/28 64 %/120 aa gb|AAK56551.1| ecdysone oxidase [...Spodoptera littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 10/09/02 47 %/122 aa FBpp0261

  18. EST Table: CK564658 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK564658 rswpb0_010036.y1 10/09/29 51 %/118 aa gb|AAK56551.1| ecdysone oxidase [Spo...doptera littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 10/09/01 42 %/126 aa FBpp0073796

  19. Sequence Classification: 767795 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH TMB Non-TMB TMB Non-TMB >gi|17552276|ref|NP_497242.1| drosophila Ecdyson...e-induced 74EF Like transcription Factor with ets domain related, ELF1 Ecdysone-induced 74EF Like Facto

  20. EST Table: FY003163 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FY003163 bmov9o10 11/11/04 58 %/146 aa gb|AAK56551.1| ecdysone oxidase [Spodoptera ...littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 11/11/04 46 %/125 aa FBpp0261096|DyakGE1

  1. EST Table: D49477 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available D49477 10/09/29 80 %/310 aa ref|NP_001166848.1| ecdysone receptor isoform A [Bombyx...mel|GB30298-PA 10/09/10 53 %/220 aa gi|121308146|emb|CAL25730.1| ecdysone receptor (isoform A) [Tribolium castaneum] NM_001043866 ...

  2. EST Table: NM_001112748 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available NM_001112748 Cyp314a1 10/09/29 95 %/516 aa ref|NP_001106219.1| ecdysone 20-hydroxyl...ase [Bombyx mori] dbj|BAE45332.1| ecdysone 20-hydroxylase [Bombyx mori] 10/09/13 48 %/475 aa FBpp0206947|Dse

  3. EST Table: BB983310 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BB983310 ovS3018H05r 10/09/28 100 %/155 aa dbj|BAF76333.1| ecdysone receptor [Bomby...121308146|emb|CAL25730.1| ecdysone receptor (isoform A) [Tribolium castaneum] NM_001043866 ovS3 ...

  4. EST Table: BP179750 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BP179750 ovS306E05f 10/09/28 98 %/137 aa ref|NP_001106219.1| ecdysone 20-hydroxylas...e [Bombyx mori] dbj|BAE45332.1| ecdysone 20-hydroxylase [Bombyx mori] 10/08/29 46 %/138 aa FBpp0206947|DsecG

  5. EST Table: BB984320 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BB984320 ovS3032H12r 10/09/28 100 %/118 aa dbj|BAF76333.1| ecdysone receptor [Bomby...121308146|emb|CAL25730.1| ecdysone receptor (isoform A) [Tribolium castaneum] NM_001043866 ovS3 ...

  6. EST Table: CK563237 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK563237 rswpb0_006412.y1 10/09/29 53 %/103 aa gb|AAK56551.1| ecdysone oxidase [Spo...doptera littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 10/09/01 36 %/162 aa FBpp0145866

  7. EST Table: CK559801 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CK559801 rswpb0_001969.y1 10/09/29 52 %/144 aa gb|AAK56551.1| ecdysone oxidase [Spo...doptera littoralis] gb|AAK56552.1| ecdysone oxidase [Spodoptera littoralis] 10/09/01 40 %/149 aa FBpp0231217

  8. EST Table: BY931923 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 06 aa ref|NP_001166846.1| ecdysone receptor isoform B1 [Bombyx mori] sp|P49881.1|ECR_BOMMO RecName: Full=Ecdyson...e receptor; AltName: Full=Ecdysteroid receptor; AltName: Full=20-hydroxy-ecdysone receptor; Short=20E re...mber 1 gb|AAA87341.1| 20-hydroxy-ecdysone receptor [Bombyx mori] 10/08/29 65 %/253 aa FBpp0170502|DmojGI2128... 59 %/233 aa gnl|Amel|GB30298-PB 10/09/10 58 %/230 aa gi|121308146|emb|CAL25730.1| ecdysone receptor (isoform A) [Tribolium castaneum] BY931923 ovS0 ...

  9. EST Table: NM_001173375 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available NM_001173375 EcR 10/09/29 89 %/591 aa ref|NP_001166846.1| ecdysone receptor isoform... B1 [Bombyx mori] sp|P49881.1|ECR_BOMMO RecName: Full=Ecdysone receptor; AltName: Full=Ecdysteroid receptor;... AltName: Full=20-hydroxy-ecdysone receptor; Short=20E receptor; AltName: Full=EcRH; AltName: Full=Nuclear r...eceptor subfamily 1 group H member 1 gb|AAA87341.1| 20-hydroxy-ecdysone receptor ...57 %/420 aa gi|270014267|gb|EFA10715.1| ecdysone receptor isoform B [Tribolium castaneum] NM_001043866 ...

  10. Steroid hormone control of cell death and cell survival: molecular insights using RNAi.

    Directory of Open Access Journals (Sweden)

    Suganthi Chittaranjan

    2009-02-01

    Full Text Available The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087, was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-alpha3 and Smr with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2mbn cells (p<0.05 following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.

  11. Construction and availability test in eukaryocyte of ecdysone-inducible controlled transgene expressing truncated hIGF-Ⅰ%受控型蜕皮激素诱导表达截短型hIGF-Ⅰ转基因载体的构建及其在细胞水平上的有效性检测

    Institute of Scientific and Technical Information of China (English)

    绳纪坡; 藤艳; 杨晓; 邓继先

    2006-01-01

    目的:构建一种可以在哺乳动物细胞中蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体,为制备受控型蜕皮激素诱导表达截短型hIGF-Ⅰ的转基因小鼠奠定基础.方法:利用分子克隆技术构建受控型蜕皮激素诱导表达截短型hIGF-Ⅰ的转基因载体;将其电转至AM1菌中,利用其中的Cre重组酶将载体上两个同向LoxP序列锚定的新霉素(neomycin)基因删除,解除其对蜕皮激素诱导表达系统的阻断作用,利用PCR、酶切和测序鉴定删除情况;将重组后的载体转染至COS7细胞中进行瞬间表达,蜕皮激素诱导后回收培养上清和细胞裂解物进行Western印迹分析,检测hIGF-Ⅰ的表达情况.结果和结论:成功构建大小为13.6 kb的转基因载体pCE-IGF-Ⅰ;转化至AM1中后,PCR、酶切和测序的结果都证明其中的Cre酶能够将载体上neomycin基因删除;重组后的载体转染至COS7细胞中进行诱导表达,Western印迹实验证明截短型hIGF-Ⅰ能够在COS7细胞中顺利表达.上述结果证明该蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体能够用于转基因小鼠的制备.

  12. Tebufenozide effects on the reproductive potentials of the ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Khebbeb, M. E. H.*, Gaouaoui, R. and Bendjeddou, F. Laboratoire de ..... species (Stanley-Samuelson, 1994), the prostaglandins were detected in ... toxicity of three ecdysone agonist insecticides against the. Mediterranean ...

  13. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    Directory of Open Access Journals (Sweden)

    Tang Baozhen

    2012-10-01

    Full Text Available Abstract Background The diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM. In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists to their targets (ecdysone receptors leads to an adaptive response (resistance, is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

  14. Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

    Science.gov (United States)

    Moeller, Morten E.; Danielsen, E. Thomas; Herder, Rachel; O’Connor, Michael B.; Rewitz, Kim F.

    2013-01-01

    Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. Here we show that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). We show that ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, our study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation. PMID:24173800

  15. Larval diapause termination in the bamboo borer, Omphisa fuscidentalis

    Science.gov (United States)

    Suang, Suphawan; Manaboon, Manaporn; Singtripop, Tippawan; Hiruma, Kiyoshi; Kaneko, Yu; Tiansawat, Pimonrat; Neumann, Peter; Chantawannakul, Panuwan

    2017-01-01

    In insects, juvenile hormone (JH) and 20-hydroxyecdysone (20E) regulate larval growth and molting. However, little is known about how this cooperative control is terminating larval diapause especially in the bamboo borer, Omphisa fuscidentalis. In both in vivo and in vitro experiments, we here measured the expression levels of genes which were affected by juvenile hormone analogue (JHA: S-methoprene) and 20-hydroxyecdysone (20E) in diapausing O. fuscidentalis larvae. Corresponding mRNA expression changes in the subesophageal ganglion (SG) and prothoracic gland (PG) were evaluated using qRT-PCR. The data showed similar response patterns of JH receptor gene (OfMet), diapause hormone gene (OfDH-PBAN), ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes (OfBr-C, OfE75A, OfE75B, OfE75C and OfHR3). JHA induced the expressions of OfMet and OfDH-PBAN in both SG and PG, whereas ecdysone receptor genes and ecdysone inducible genes were induced by JHA only in PG. For 20E treatment group, expressions of ecdysone receptor genes and ecdysone inducible genes in both SG and PG were increased by 20E injection. In addition, the in vitro experiments showed that OfMet and OfDH-PBAN were up-regulated by JHA alone, but ecdysone receptor genes and ecdysone inducible genes were up-regulated by JHA and 20E. However, OfMet and OfDH-PBAN in the SG was expressed faster than OfMet and OfDH-PBAN in the PG and the expression of ecdysone receptor genes and ecdysone inducible genes induced by JHA was much later than observed for 20E. These results indicate that JHA might stimulate the PG indirectly via factors (OfMet and OfDH-PBAN) in the SG, which might be a regulatory mechanism for larval diapause termination in O. fuscidentalis. PMID:28369111

  16. Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster.

    Science.gov (United States)

    Parvy, Jean-Philippe; Wang, Peng; Garrido, Damien; Maria, Annick; Blais, Catherine; Poidevin, Mickael; Montagne, Jacques

    2014-10-01

    In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and βFtz-f1, representing a model for steroid hormone signalling. Here, we have combined genetic and imaging approaches to investigate the precise role of this signalling cascade within theprothoracic gland (PG), where ecdysone synthesis takes place. We show that these receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and βFtz-f1 act early in development through a forward process to moderate DHR3 levels. Our findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics.

  17. Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis

    Science.gov (United States)

    Nakaoka, Takayoshi; Iga, Masatoshi; Yamada, Tetsuya; Koujima, Ikumi; Takeshima, Mika; Zhou, Xiangying; Suzuki, Yutaka; Ogihara, Mari H.; Kataoka, Hiroshi

    2017-01-01

    Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. We analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. We found that the genes encoding δ-aminolevulinic acid synthase (CG3017/alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis. PMID:28257485

  18. Phytochemical analysis of Pfaffia glomerata inflorescences by LC-ESI-MS/MS.

    Science.gov (United States)

    Felipe, Daniele F; Brambilla, Lara Z S; Porto, Carla; Pilau, Eduardo J; Cortez, Diógenes A G

    2014-09-29

    Pfaffia glomerata contains high levels of β-ecdysone, which has shown a range of beneficial pharmacological effects. The present study demonstrated that inflorescences of P. glomerata contain other important bioactive compounds in addition to β-ecdysone. The identification of compounds from inflorescences using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was performed for the first time. The eight compounds identified were β-ecdysone, flavonoid glycosides such as quercetin-3-O-glucoside, kaempferol-3-O-glucoside and kaempferol-3-O-(6-p-coumaroyl)-glucoside, oleanane-type triterpenoid saponins such as ginsenoside Ro and chikusetsusaponin IV, in addition to oleanonic acid and gluconic acid. This study provided information on the phytochemicals contained in P. glomerata inflorescences revealing the potential application of this plant part as raw material for the phytotherapeutic and cosmetic industries.

  19. Phytochemical Analysis of Pfaffia glomerata Inflorescences by LC-ESI-MS/MS

    Directory of Open Access Journals (Sweden)

    Daniele F. Felipe

    2014-09-01

    Full Text Available Pfaffia glomerata contains high levels of β-ecdysone, which has shown a range of beneficial pharmacological effects. The present study demonstrated that inflorescences of P. glomerata contain other important bioactive compounds in addition to β-ecdysone. The identification of compounds from inflorescences using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS was performed for the first time. The eight compounds identified were β-ecdysone, flavonoid glycosides such as quercetin-3-O-glucoside, kaempferol-3-O-glucoside and kaempferol-3-O-(6-p-coumaroyl-glucoside, oleanane-type triterpenoid saponins such as ginsenoside Ro and chikusetsusaponin IV, in addition to oleanonic acid and gluconic acid. This study provided information on the phytochemicals contained in P. glomerata inflorescences revealing the potential application of this plant part as raw material for the phytotherapeutic and cosmetic industries.

  20. Transcriptional Control of Steroid Biosynthesis Genes in the Drosophila Prothoracic Gland by Ventral Veins Lacking and Knirps

    Science.gov (United States)

    Dorry, Elad; Komura-Kawa, Tatsuya; Fujimoto, Yoshinori; Troelsen, Jesper T.; Herder, Rachel; O'Connor, Michael B.; Niwa, Ryusuke; Rewitz, Kim F.

    2014-01-01

    Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development. PMID:24945799

  1. Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

    Directory of Open Access Journals (Sweden)

    E Thomas Danielsen

    2014-06-01

    Full Text Available Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl and the nuclear receptor Knirps (Kni, have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld, controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

  2. EST Table: BP181797 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available BP181797 ovS332H12f 10/09/28 90 %/122 aa ref|NP_001037331.2| ecdysone receptor isof...orm B2 [Bombyx mori] gb|AAA87340.1| 20-hydroxy-ecdysone receptor [Bombyx mori] 10/08/29 n.h 10/08/28 n.h 10/09/10 n.h 10/09/10 n.h 10/09/10 n.h NM_001043866 ovS3 ...

  3. EST Table: FS725349 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS725349 E_FL_bmmt_04G04_F_0 10/09/28 90 %/132 aa ref|NP_001037331.2| ecdysone rece...ptor isoform B2 [Bombyx mori] gb|AAA87340.1| 20-hydroxy-ecdysone receptor [Bombyx mori] 10/09/03 n.h 10/08/28 n.h 10/09/10 n.h 10/09/10 n.h 10/09/10 n.h NM_001043866 bmmt ...

  4. Developmental checkpoints and feedback circuits time insect maturation

    DEFF Research Database (Denmark)

    Rewitz, Kim Furbo; Yamanaka, Naoki; O'Connor, Michael B.

    2013-01-01

    Abstract The transition from juvenile to adult is a fundamental process that allows animals to allocate resource toward reproduction after completing a certain amount of growth. In insects, growth to a species-specific target size induces pulses of the steroid hormone ecdysone that triggers...... metamorphosis and reproductive maturation. The past few years have seen significant progress in understanding the interplay of mechanisms that coordinate timing of ecdysone production and release. These studies show that the neuroendocrine system monitors complex size-related and nutritional signals, as well...

  5. A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis

    DEFF Research Database (Denmark)

    Rewitz, Kim F; Larsen, Martin R; Lobner-Olesen, Anders

    2009-01-01

    In insects, the neuropeptide prothoracicotropic hormone (PTTH) stimulates production of ecdysone (E) in the prothoracic glands (PGs). E is the precursor of the principal steroid hormone, 20-hydroxyecdysone (20E), that is responsible for eliciting molting and metamorphosis. In this study, we used ...

  6. The expression profile and promoter analysis of ultraspiracle gene in the silkworm Bombyx mori.

    Science.gov (United States)

    Huang, Ming-xia; Du, Jie; Su, Bao-jin; Zhao, Guo-dong; Shen, Wei-de; Wei, Zheng-guo

    2014-12-01

    The nuclear receptor, ultraspiracle protein (USP), is a transcription factor and an essential component of a heterodimeric receptor complex with ecdysone receptor. However, the mechanisms underlying the transcriptional regulation of USP in silkworm are unknown. In this study, using dual-spike-in qPCR method, we examined the expression of Bombyx ultraspiracle gene (BmUSP) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. The results showed that the expression levels of BmUSP gene varied in different tissues and were increased 2 h after exposure to ecdysone. To identify the molecular mechanism underlying the regulation of USP gene expression in silkworm Bombyx mori, promoter truncation analyses were performed using the luciferase reporter assay and Bac-to-Bac expression system in several tissues of B. mori. BmUSP gene promoter with 5' end serial deletions showed different levels of activity in various tissues, higher in fat body and Malpighian tubule. Deletion of the region from -485 to -445 and -307 to -281 upstream of BmUSP gene abolished and increased its promoter activity, respectively. This region contains AP-1, Dfd transcription factor binding sites. These results indicate that BmUSP are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. This study would provide an important foundation for investigating the mechanism underlying the transcriptional regulation of BmUSP in the silkworm.

  7. RNAi Screen in Drosophila melanogastor Identifies Regulators of Steroidogenesis and Developmental Maturation

    DEFF Research Database (Denmark)

    Danielsen, Erik Thomas

    In contrast to humans, Drosophila melanogaster, commonly known as the fruit fly, only produces one major class of cholesterol-derived steroid hormones, the ecdysteroids. This makes Drosophila a simple but elegant model organism to study steroidogenesis. During development, pulses of ecdysone...

  8. Hormonal Interference with Pheromone Systems in Parasitic Acarines, Especially Ixodid Ticks.

    Science.gov (United States)

    1984-05-01

    hydroxyecdysone by treatment of frations in Locusta migratoria extracts with the enzymie glucuronidase. A second fraction, which did not respond to this enzyne...products of ecdysone in Locusta migratoria . Hoppe-Ze.l er’Is ;.. Z. Physiol. Chem. Bd. 354: 1043-1048.. Obenchain, F.D. and J.H. Oliver. 1975

  9. Dicty_cDB: VFL348 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 129 |pid:none) Drosophila melanogaster ecdysone d... 66 5e-10 AF108656_1( AF108656 |pid:none) Gallus gallus prosa...0 AK223290_1( AK223290 |pid:none) Homo sapiens mRNA for prosaposin (... 61 2e-08

  10. Dicty_cDB: VFN623 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available sophila melanogaster ecdysone d... 67 3e-10 AF108656_1( AF108656 |pid:none) Gallus gallus prosaposin mRNA, c...90 |pid:none) Homo sapiens mRNA for prosaposin (... 61 1e-08 (Q6IBQ6) RecName: Full=Proactivator polypeptide

  11. Regulation of Pattern Formation and Gene Amplification During Drosophila Oogenesis by the miR-318 microRNA

    DEFF Research Database (Denmark)

    Ge, Wanzhong; Deng, Qiannan; Guo, Ting

    2015-01-01

    and laid eggs with abnormal morphology. Removal of miR-318 disrupted the dorsal-anterior follicle cell patterning, resulting in abnormal dorsal appendages. miR-318 mutant females also produced thin and fragile eggshells, due to impaired chorion gene amplification. We provide evidence that the ecdysone...

  12. Unexpected role of the steroid-deficiency protein ecdysoneless in pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Ann-Katrin Claudius

    2014-04-01

    Full Text Available The steroid hormone ecdysone coordinates insect growth and development, directing the major postembryonic transition of forms, metamorphosis. The steroid-deficient ecdysoneless1 (ecd1 strain of Drosophila melanogaster has long served to assess the impact of ecdysone on gene regulation, morphogenesis, or reproduction. However, ecd also exerts cell-autonomous effects independently of the hormone, and mammalian Ecd homologs have been implicated in cell cycle regulation and cancer. Why the Drosophila ecd1 mutants lack ecdysone has not been resolved. Here, we show that in Drosophila cells, Ecd directly interacts with core components of the U5 snRNP spliceosomal complex, including the conserved Prp8 protein. In accord with a function in pre-mRNA splicing, Ecd and Prp8 are cell-autonomously required for survival of proliferating cells within the larval imaginal discs. In the steroidogenic prothoracic gland, loss of Ecd or Prp8 prevents splicing of a large intron from CYP307A2/spookier (spok pre-mRNA, thus eliminating this essential ecdysone-biosynthetic enzyme and blocking the entry to metamorphosis. Human Ecd (hEcd can substitute for its missing fly ortholog. When expressed in the Ecd-deficient prothoracic gland, hEcd re-establishes spok pre-mRNA splicing and protein expression, restoring ecdysone synthesis and normal development. Our work identifies Ecd as a novel pre-mRNA splicing factor whose function has been conserved in its human counterpart. Whether the role of mammalian Ecd in cancer involves pre-mRNA splicing remains to be discovered.

  13. Ascarosides coordinate the dispersal of a plant-parasitic nematode with the metamorphosis of its vector beetle

    Science.gov (United States)

    Zhao, Lilin; Zhang, Xinxing; Wei, Yanan; Zhou, Jiao; Zhang, Wei; Qin, Peijun; Chinta, Satya; Kong, Xiangbo; Liu, Yunpeng; Yu, Haiying; Hu, Songnian; Zou, Zhen; Butcher, Rebecca A.; Sun, Jianghua

    2016-01-01

    Insect vectors are required for the transmission of many species of parasitic nematodes, but the mechanisms by which the vectors and nematodes coordinate their life cycles are poorly understood. Here, we report that ascarosides, an evolutionarily conserved family of nematode pheromones, are produced not only by a plant-parasitic nematode, but also by its vector beetle. The pinewood nematode and its vector beetle cause pine wilt disease, which threatens forest ecosystems world-wide. Ascarosides secreted by the dispersal third-stage nematode LIII larvae promote beetle pupation by inducing ecdysone production in the beetle and up-regulating ecdysone-dependent gene expression. Once the beetle develops into the adult stage, it secretes ascarosides that attract the dispersal fourth-stage nematode LIV larvae, potentially facilitating their movement into the beetle trachea for transport to the next pine tree. These results demonstrate that ascarosides play a key role in the survival and spread of pine wilt disease. PMID:27477780

  14. Effect of acute exposure to cadmium on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic midge Chironomus riparius

    Energy Technology Data Exchange (ETDEWEB)

    Planello, R.; Martinez-Guitarte, J.L. [Grupo de Biologia y Toxicologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, UNED, Senda del Rey 9, 28040 Madrid (Spain); Morcillo, G., E-mail: gmorcillo@ccia.uned.es [Grupo de Biologia y Toxicologia Ambiental, Facultad de Ciencias, Universidad Nacional de Educacion a Distancia, UNED, Senda del Rey 9, 28040 Madrid (Spain)

    2010-03-01

    Cadmium is a widespread and highly toxic pollutant of particular ecotoxicological relevance for aquatic ecosystems where it accumulates. To identify biomarkers for ecotoxicity monitoring, the effect of cadmium on the expression of different genes related to the stress response as well as to the ecdysone hormone-signalling pathway was studied in the aquatic larvae of Chironomus riparius (Diptera, Chironomidae), a standard test organism in aquatic toxicology testing. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to evaluate the effects of acute and short-term cadmium exposures (10 mM CdCl{sub 2}, 12 h and 24 h) on the expression of hsp70, hsc70, hsp90 and hsp40 genes, as well as on that of the ecdysone hormonal-receptor genes (EcR and usp). A significant 3-fold increase in the level of hsp70 gene transcripts was induced by the treatment, whereas neither the other stress genes tested (hsp90 and hsp40) nor the constitutive form of hsp70, hsc70, was affected in the larvae exposed to cadmium. These results show that hsp70 is differentially activated to other environmentally regulated heat-shock genes, and constitutes a biomarker of exposure to this toxic metal. In addition, we also found that cadmium is able to alter the expression of the ecdysone receptor gene (EcR), whose mRNA level is significantly increased whereas usp levels remained unaltered. This finding, evidenced for the first time in invertebrates, supports the view that cadmium has the ability to mimic the effect of the hormone by the activation of the ecdysone nuclear receptor, which may partly explain the endocrine disruption capability that has been previously suggested for this toxic metal. Our research adds to the growing evidence implicating heavy metals, and cadmium in particular, as potential endocrine disruptive agents and may have significant implications for ecological risk assessment of endocrine-disrupting compounds in invertebrates.

  15. The POU factor ventral veins lacking/Drifter directs the timing of metamorphosis through ecdysteroid and juvenile hormone signaling.

    Directory of Open Access Journals (Sweden)

    CeCe Cheng

    2014-06-01

    Full Text Available Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae. RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3. In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3, were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm and spook (spo were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.

  16. Insulin signaling pathways in lepidopteran steroidogenesis

    Directory of Open Access Journals (Sweden)

    Wendy eSmith

    2014-02-01

    Full Text Available Molting and metamorphosis are stimulated by the secretion of ecdysteroid hormones from the prothoracic glands. Insulin-like hormones have been found to enhance prothoracic gland activity, providing a mechanism to link molting to nutritional state. In silk moths (Bombyx mori, the prothoracic glands are directly stimulated by insulin and the insulin-like hormone bombyxin. Further, in Bombyx , the neuropeptide prothoracicotropic hormone (PTTH appears to act at least in part through the insulin-signaling pathway. In the prothoracic glands of Manduca sexta, while insulin stimulates the phosphorylation of the insulin receptor and Akt, neither insulin nor bombyxin II stimulate ecdysone secretion. Involvement of the insulin-signaling pathway in Manduca prothoracic glands was explored using two inhibitors of phosphatidylinositol-3-kinase (PI3K, LY294002 and wortmannin. PI3K inhibitors block the phosphorylation of Akt and 4EBP but have no effect on ecdysone secretion, or on the phosphorylation of the MAPkinase, ERK. Inhibitors that block phosphorylation of ERK, including the MEK inhibitor U0126, and high doses of the RSK inhibitor SL0101, effectively inhibit ecdysone secretion. The results highlight differences between the two lepidopteran insects most commonly used to directly study ecdysteroid secretion. In Bombyx, the PTTH and insulin-signaling pathways intersect; both insulin and PTTH enhance the phosphorylation of Akt and stimulate ecdysteroid secretion, and inhibition of PI3K reduces ecdysteroid secretion. By contrast, in Manduca, the action of PTTH is distinct from insulin. The results highlight species differences in the roles of translational regulators such as 4EBP, and members of the MAPkinase pathway such as ERK and RSK, in the effects of nutritionally-sensitive hormones such as insulin on ecdysone secretion and molting.

  17. Control of target gene specificity during metamorphosis by the steroid response gene E93

    OpenAIRE

    Mou, Xiaochun; Duncan, Dianne M.; Baehrecke, Eric H; Duncan, Ian

    2012-01-01

    Hormonal control of sexual maturation is a common feature in animal development. A particularly dramatic example is the metamorphosis of insects, in which pulses of the steroid hormone ecdysone drive the wholesale transformation of the larva into an adult. The mechanisms responsible for this transformation are not well understood. Work in Drosophila indicates that the larval and adult forms are patterned by the same underlying sets of developmental regulators, but it is not understood how the...

  18. Arbutin in Serratula quinquefolia M.B. (Asteraceae)

    OpenAIRE

    Gerard Nowak; Joanna Nawrot; Karol Latowski

    2011-01-01

    Genus Serratula is known for the presence of steroid compounds which belong to the group of ecdysones. Former phytochemical works from the late sixties and early seventies indicate, in some Serratula species, the occurrence of a phenolic glycoside-arbutin. This has been confirmed in the present work through finding an α,β-arbutin anomer in Serratula quinquefolia M.B. New botanical data concerning the classification of genus Serratula suggest that the species in question should be regarded as ...

  19. Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jennifer C Regan

    2013-10-01

    Full Text Available Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of

  20. Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors.

    Directory of Open Access Journals (Sweden)

    Takayuki Ueno

    Full Text Available The hypopharyngeal glands (HPGs of worker honeybees undergo physiological changes along with the age-dependent role change from nursing to foraging: nurse bee HPGs secrete mainly major royal jelly proteins, whereas forager HPGs secrete mainly α-glucosidase III, which converts the sucrose in the nectar into glucose and fructose. We previously identified two other genes, Apis mellifera buffy (Ambuffy and Apis mellifera matrix metalloproteinase 1 (AmMMP1, with enriched expression in nurse bee and forager HPGs, respectively. In the present study, to clarify the molecular mechanisms that coordinate HPG physiology with worker behavior, we first analyzed whether Ambuffy, AmMMP1, mrjp2 (a gene encoding one of major royal jelly protein isoforms, and Hbg3 (a gene encoding α-glucosidase III expression, is associated with worker behavior in 'single-cohort colonies' where workers of almost the same age perform different tasks. Expression of these genes correlated with the worker's role, while controlling for age, indicating their regulation associated with the worker's behavior. Associated gene expression suggested the possible involvement of some hormonal factors in its regulation. We therefore examined the relationship between ecdysone- and juvenile hormone (JH-signaling, and the expression profiles of these 'indicator' genes (nurse bee HPG-selective genes: mrjp2 and Ambuffy, and forager HPG-selective genes: Hbg3 and AmMMP1. Expression of both ecdysone-regulated genes (ecdysone receptor, mushroom body large type Kenyon cell specific protein-1, and E74 and JH-regulated genes (Methoprene tolerant and Krüppel homolog 1 was higher in the forager HPGs than in the nurse bee HPGs, suggesting the possible roles of ecdysone- and JH-regulated genes in worker HPGs. Furthermore, 20-hydroxyecdysone-treatment repressed both nurse bee- and forager-selective gene expression, whereas methoprene-treatment enhanced the expression of forager-selective genes and repressed

  1. Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

    Science.gov (United States)

    Ozáez, Irene; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2013-07-01

    There is increasing evidence indicating that several UV filters might have endocrine disruptive effects. Numerous studies have evaluated hormonal effects in vertebrates, mainly reporting estrogenic and androgenic activities in mammals and fishes. There is only limited knowledge about potential endocrine activity in invertebrate hormonal systems. In this work, the effects on endocrine signaling genes of six frequently used UV filters were investigated in Chironomus riparius, a reference organism in aquatic toxicology. The UV filters studied were: octyl-p-methoxycinnamate (OMC) also called 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4-MBC); benzophenone-3 (BP-3); 4-hidroxybenzophenone (4-HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). After in vivo exposure at different dosages, expression levels of the genes coding for the ecdysone receptor (EcR), the ultraspiracle (usp, ortholog of the RXR) and the estrogen-related receptor (ERR) were quantified by Real Time PCR. The EcR gene was significantly upregulated by 4-MBC, OMC/EHMC and OD-PABA, with a dose-related response following 24h exposure. In contrast, the benzophenones, BP-3 and 4-HB, as well as OC did not alter this gene at the same exposure conditions. The transcription profiles of the usp and ERR genes were not significantly affected, except for BP-3 that inhibited the usp gene at the highest concentration. To our knowledge, this is the first experimental evidence in invertebrates of a direct effect of UV filters on endocrine-related genes, and is consistent with the known effects on vertebrate hormonal receptor genes. The capability of 4-MBC, OMC/EHMC and OD-PABA to stimulate the expression of the ecdysone receptor, a key transcription factor for the ecdysone-genomic response in arthropods, suggests the possibility of a broad and long-term effect on this hormonal pathway. These findings strengthen the need for further research about the ecotoxicological implications

  2. A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

    Science.gov (United States)

    Fagegaltier, Delphine; König, Annekatrin; Gordon, Assaf; Lai, Eric C; Gingeras, Thomas R; Hannon, Gregory J; Shcherbata, Halyna R

    2014-10-01

    MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.

  3. Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera).

    Science.gov (United States)

    Vafopoulou, Xanthe

    2009-12-01

    We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

  4. Eine neue Alternative für die Prävention und Therapie postmenopausaler Erkrankungen, insbesondere des metabolischen Syndroms

    Directory of Open Access Journals (Sweden)

    Wuttke W

    2015-01-01

    Full Text Available Viele postmenopausale, aber in zunehmendem Maße auch junge Frauen und Männer werden übergewichtig. Heute werden 2 Fetttypen unterschieden: der gynoide Birnentyp mit großen glutealen Fettdepots und der androide Apfeltyp mit viel viszeralem Fett. Das metabolische Syndrom entwickelt sich bei übergewichtigen bzw. fettleibigen Menschen in erster Linie durch Bildung großer viszeraler Fettdepots. Diese Patienten gehören also zu den Apfeltypen mit großen androiden Fettdepots, oder platt ausgedrückt: Sie haben einen „Bierbauch“. Bei übergewichtigen Frauen liegt in der Regel ein Mischtyp vor. Die viszeralen Fettzellen dieser Menschen sezernieren große Mengen an proinflammatorischen Zytokinen, welche den gesamten Körper in einen chronisch entzündlichen Zustand versetzen. Der daraus resultierende hohe oxidative Stress und die Zytokine führen zu einer Hyperlipidämie und es folgen Hypertonie und Arteriosklerose. Auch Insulinrezeptoren werden desensibilisiert und es entwickelt sich ein Typ-2-Diabetes. Das metabolische Syndrom hat aber nicht nur negative Auswirkungen auf das Herz-Kreislaufsystem, sondern auch auf die Muskulatur, da auch hier schädliche Fettzellen akkumulieren und den Aufbau von Muskelfasern hemmen. Fettzellen im Knochenmark und Fettzotten in Gelenken schädigen durch die dort sezernierten Zytokine die knochen- und knorpelaufbauenden Osteo- und Chondroblasten, sodass sich eine Osteoporose entwickelt und die sich durch Übergewicht anbahnende Arthrose gefördert wird. Ecdyson unterbindet den Aufbau von Fettzellen. Deshalb ist Ecdyson in der Lage, den Anteil an Körperfett zu reduzieren und dabei gleichzeitig die Muskelmenge und -kraft zu steigern. Ecdyson kommt in Spinat vor. In Kombination mit Substanzen, welche bekanntermaßen Knochen, Knorpel oder Arterien schützen, wie Kalzium, Vitamin D oder Antioxidanzien, verhindert oder reduziert ein Ecdyson-haltiges Spinatpräparat die Entwicklung des metabolischen Syndroms, einer

  5. Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori

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    Hong Kaili

    2007-07-01

    Full Text Available Abstract Background lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori. Results One member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities. Conclusion bmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The

  6. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris

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    Harindra E. Amarasinghe

    2015-07-01

    Full Text Available Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  7. Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

    DEFF Research Database (Denmark)

    Møller, Morten Erik; Danielsen, Erik Thomas; Herder, Rachel;

    2013-01-01

    Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating...... that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation....... these pulses are not known. Here we show that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression...

  8. Arbutin in Serratula quinquefolia M.B. (Asteraceae

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    Gerard Nowak

    2011-04-01

    Full Text Available Genus Serratula is known for the presence of steroid compounds which belong to the group of ecdysones. Former phytochemical works from the late sixties and early seventies indicate, in some Serratula species, the occurrence of a phenolic glycoside-arbutin. This has been confirmed in the present work through finding an α,β-arbutin anomer in Serratula quinquefolia M.B. New botanical data concerning the classification of genus Serratula suggest that the species in question should be regarded as belonging to genus Klasea (currently a section of genus Serratula. There has been an attempt to find a key to the chemical division within the taxons.

  9. Elimination of C-6-hydrogen during the formation of ecdysteroids from cholesterol in Locusta migratoria ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Y.; Hiramoto, M.; Kakinuma, K.; Ikekawa, N. (Tokyo Institute of Technology (Japan))

    1989-03-01

    Being administered to Locusta migratoria adult females, (6-{sup 3}H, 4-{sup 14}C)cholesterol was incorporated into ecdysone and 2-deoxyecdysone. The ratio of {sup 3}H/{sup 14}C of the two ecdysteroids isolated from newly laid eggs revealed that C-6-hydrogen of cholesterol was eliminated during the conversion to ecdysteroids in the ovaries of the insects. Thus, a hypothetical mechanism involving migration of the C-6-hydrogen to the C-5 position in the formation of A/B cis junction turned out to be less likely.

  10. INHIBITION OF FATTY ACID DESATURASES IN Drosophila melanogaster LARVAE BLOCKS FEEDING AND DEVELOPMENTAL PROGRESSION.

    Science.gov (United States)

    Wang, Yiwen; da Cruz, Tina Correia; Pulfemuller, Alicia; Grégoire, Stéphane; Ferveur, Jean-François; Moussian, Bernard

    2016-05-01

    Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program. © 2016 Wiley Periodicals, Inc.

  11. The interaction between a sexually transferred steroid hormone and a female protein regulates oogenesis in the malaria mosquito Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    Francesco Baldini

    2013-10-01

    Full Text Available Molecular interactions between male and female factors during mating profoundly affect the reproductive behavior and physiology of female insects. In natural populations of the malaria mosquito Anopheles gambiae, blood-fed females direct nutritional resources towards oogenesis only when inseminated. Here we show that the mating-dependent pathway of egg development in these mosquitoes is regulated by the interaction between the steroid hormone 20-hydroxy-ecdysone (20E transferred by males during copulation and a female Mating-Induced Stimulator of Oogenesis (MISO protein. RNAi silencing of MISO abolishes the increase in oogenesis caused by mating in blood-fed females, causes a delay in oocyte development, and impairs the function of male-transferred 20E. Co-immunoprecipitation experiments show that MISO and 20E interact in the female reproductive tract. Moreover MISO expression after mating is induced by 20E via the Ecdysone Receptor, demonstrating a close cooperation between the two factors. Male-transferred 20E therefore acts as a mating signal that females translate into an increased investment in egg development via a MISO-dependent pathway. The identification of this male-female reproductive interaction offers novel opportunities for the control of mosquito populations that transmit malaria.

  12. POLYPODIUM VULGARE LINN. A VERSATILE HERBAL MEDICINE: A REVIEW

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    Pervaiz Ahmad Dar*, G. Sofi and M. A. Jafri

    2012-06-01

    Full Text Available Polypodium vulgare Linn. also called as Bisfaij in Unani system of medicine is a perineal fern growing to a height of 30cm. Polypodium vulgare Linn. rhizome is used in European, American, Chinese, and Unani and Ayurveda traditions. It is claimed to be efficacious in jaundice, dropsy, scurvy and combined with mallows it removes hardness of the spleen. The distilled water of the roots and leaves is considered good for ague (malarial fever, and the fresh or dried roots, mixed with honey and applied to the nose, were used in the treatment of polypus The fresh root is used in the form of decoction, or powder for melancholia and also for rheumatic swelling of the joints. The rhizome extract was found to possess anti-epileptic activity. The ecdysones present in the rhizome (0.07%-1% dry weightwas seen to act topically on a wide variety of arthropods and caused abnormal molting and death, so ecdysone analogues may be useful not only as insecticides but also miticides. The aqueous extract of Polypodium vulgare Linn. was found to possess analgesic activity, protective effect in various neurological and neurodegenerative disorders, stimulatory effect on the adrenoceptors, and antioxidant properties.

  13. Identification of the molting hormone of the sweet potato (Bemisia tabaci) and greenhouse (Trialeurodes vaporariorum) whitefly.

    Science.gov (United States)

    Gelman, Dale B; Blackburn, Michael B; Hu, Jing S

    2005-01-01

    In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.

  14. Plant derived alternatives for hormone replacement therapy (HRT).

    Science.gov (United States)

    Seidlova-Wuttke, Dana; Jarry, Hubertus; Wuttke, Wolfgang

    2013-12-01

    Abstract Hormone replacement therapy (HRT) has undisputable positive effects on climacteric complaints, in the bone and on body weight but also several undesired side effects. Therefore, plant-derived alternatives are currently promoted. Phytoestrogens - primarily the isoflavones genistein, daidzein and coumestrol, stemming from soy (Glycine max) or red clover (Trifolium pratense) - were suggested to have the desired but not the undesired effects of estrogens. Most recently published placebo-controlled studies question the beneficial effects. When taken at the time of puberty however, phytoestrogens appear to protect against mammary cancer later in life. Extracts from the rhizome of Cimicifuga racemosa (black cohosh) have no estrogenic effects. In a narrow dose range they have beneficial effects on climacteric complaints, which are due to several compounds with dopaminergic, noradrenergic, serotoninergic and GABAergic actions that act together in the hypothalamus. Ecdysone is produced by several plants, including spinach (Spinacia oleracea) and was very early on shown to increase muscle mass. Later it became apparent that spinach extracts containing ecdysone decreased body fat load, thereby reducing secretion of proinflammatory cytokines by visceral adipocytes and oxidative stress. This had beneficial effects on body weight and serum lipids not only in obese postmenopausal but also in premenopausal women and in men. For the above-described plant extracts, solid placebo-controlled clinical trials are available. For other plant extracts claiming beneficial effects on climacteric complaints or postmenopausal diseases, no solid data are available.

  15. Endocrine disruption in crustaceans due to pollutants: a review.

    Science.gov (United States)

    Rodríguez, Enrique M; Medesani, Daniel A; Fingerman, Milton

    2007-04-01

    The main endocrine-regulated processes of crustaceans have been reviewed in relation to the effects of endocrine-disrupting compounds (EDCs). Molting has been shown to be inhibited by several organic pollutants, such as xenoestrogens and related compounds, as well as by some pesticides. Most of these disrupters are thought to interfere with ecdysone at target tissues, although only for a few has this action been demonstrated in vitro. The heavy metal cadmium appears to inhibit some ecdysone secretion. Juvenoid compounds have also been shown to inhibit molting, likely by interfering with the stimulatory effect of methyl farnesoate. A molt-promoting effect of emamectin benzoate, a pesticide, has also been reported. As for reproduction, a variety of organic compounds, including xenoestrogens, juvenoids and ecdysteroids, has produced abnormal development of male and female secondary sexual characters, as well as alteration of the sex ratio. Cadmium and copper have been shown to interfere with hormones that stimulate reproduction, such as methyl farnesoate, as well as with secretion of the gonad inhibiting hormone, therefore affecting, for example, ovarian growth. Several heavy metals were able to produce hyperglycemia in crustaceans during short times of exposure; while a hypoglycemic response was noted after longer exposures, due to inhibition of secretion of the crustacean hyperglycemic hormone. The ecological relevance of EDCs on crustaceans is discussed, mainly in relation to the identification of useful biomarkers and sentinel species. New experimental approaches are also proposed.

  16. Hexokinase is a key regulator of energy metabolism and ROS activity in insect lifespan extension

    Science.gov (United States)

    Lin, Xian-Wu; Xu, Wei-Hua

    2016-01-01

    Developmental arrest (diapause) is a ‘non-aging’ state that is similar to the Caenorhabditis elegans dauer stage and Drosophila lifespan extension. Diapause results in low metabolic activity and a profound extension of insect lifespan. Here, we cloned the Helicoverpa armigera Hexokinase (HK) gene, a gene that is critical for the developmental arrest of this species. HK expression and activity levels were significantly increased in nondiapause-destined pupae compared with those of diapause-destined pupae. Downregulation of HK activity reduced cell viability and delayed pupal development by reducing metabolic activity and increasing ROS activity, which suggests that HK is a key regulator of insect development. We then identified the transcription factors Har-CREB, -c-Myc, and -POU as specifically binding the Har-HK promoter and regulating its activity. Intriguingly, Har-POU and -c-Myc are specific transcription factors for HK expression, whereas Har-CREB is nonspecific. Furthermore, Har-POU and -c-Myc could respond to ecdysone, which is an upstream hormone. Therefore, low ecdysone levels in diapause-destined individuals lead to low Har-POU and -c-Myc expression levels, ultimately repressing Har-HK expression and inducing entry into diapause or lifespan extension. PMID:26852422

  17. Transcriptional changes induced by in vivo exposure to pentachlorophenol (PCP) in Chironomus riparius (Diptera) aquatic larvae.

    Science.gov (United States)

    Morales, Mónica; Martínez-Paz, Pedro; Martín, Raquel; Planelló, Rosario; Urien, Josune; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2014-12-01

    Pentachlorophenol (PCP) has been extensively used worldwide as a pesticide and biocide and is frequently detected in the aquatic environment. In the present work, the toxicity of PCP was investigated in Chironomus riparius aquatic larvae. The effects following short- and long-term exposures were evaluated at the molecular level by analyzing changes in the transcriptional profile of different endocrine genes, as well as in genes involved in the stress response and detoxification. Interestingly, although no differences were found after 12- and 24-h treatments, at 96-h exposures PCP was able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the early ecdysone-inducible E74 gene, the estrogen-related receptor gene (ERR), the Hsp70 gene and the CYP4G gene. In contrast, the Hsp27 gene appeared to be downregulated, while the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor) was not altered in any of the conditions assayed. Moreover, Glutathione-S-Transferase (GST) activity was not affected. The results obtained show the ability of PCP to modulate transcription of different biomarker genes from important cellular metabolic activities, which could be useful in genomic approaches to monitoring. In particular, the significant upregulation of hormonal genes represents the first evidence at the genomic level of the potential endocrine disruptive effects of PCP on aquatic invertebrates. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. CYP450s analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development.

    Science.gov (United States)

    Ventura, Tomer; Bose, Utpal; Fitzgibbon, Quinn P; Smith, Gregory G; Shaw, P Nicholas; Cummins, Scott F; Elizur, Abigail

    2017-07-01

    Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  19. A biological timer in the fat body comprising Blimp-1, βFtz-f1 and Shade regulates pupation timing in Drosophila melanogaster.

    Science.gov (United States)

    Akagi, Kazutaka; Sarhan, Moustafa; Sultan, Abdel-Rahman S; Nishida, Haruka; Koie, Azusa; Nakayama, Takumi; Ueda, Hitoshi

    2016-07-01

    During the development of multicellular organisms, many events occur with precise timing. In Drosophila melanogaster, pupation occurs about 12 h after puparium formation and its timing is believed to be determined by the release of a steroid hormone, ecdysone (E), from the prothoracic gland. Here, we demonstrate that the ecdysone-20-monooxygenase Shade determines pupation timing by converting E to 20-hydroxyecdysone (20E) in the fat body, which is the organ that senses nutritional status. The timing of shade expression is determined by its transcriptional activator βFtz-f1. The βftz-f1 gene is activated after a decline in the expression of its transcriptional repressor Blimp-1, which is temporally expressed around puparium formation in response to a high titer of 20E. The expression level and stability of Blimp-1 is critical for the precise timing of pupation. Thus, we propose that Blimp-1 molecules function like sand in an hourglass in this precise developmental timer system. Furthermore, our data suggest that a biological advantage results from both the use of a transcriptional repressor for time determination and the association of developmental timing with nutritional status of the organism. © 2016. Published by The Company of Biologists Ltd.

  20. The Interaction between a Sexually Transferred Steroid Hormone and a Female Protein Regulates Oogenesis in the Malaria Mosquito Anopheles gambiae

    Science.gov (United States)

    Baldini, Francesco; Gabrieli, Paolo; South, Adam; Valim, Clarissa; Mancini, Francesca; Catteruccia, Flaminia

    2013-01-01

    Molecular interactions between male and female factors during mating profoundly affect the reproductive behavior and physiology of female insects. In natural populations of the malaria mosquito Anopheles gambiae, blood-fed females direct nutritional resources towards oogenesis only when inseminated. Here we show that the mating-dependent pathway of egg development in these mosquitoes is regulated by the interaction between the steroid hormone 20-hydroxy-ecdysone (20E) transferred by males during copulation and a female Mating-Induced Stimulator of Oogenesis (MISO) protein. RNAi silencing of MISO abolishes the increase in oogenesis caused by mating in blood-fed females, causes a delay in oocyte development, and impairs the function of male-transferred 20E. Co-immunoprecipitation experiments show that MISO and 20E interact in the female reproductive tract. Moreover MISO expression after mating is induced by 20E via the Ecdysone Receptor, demonstrating a close cooperation between the two factors. Male-transferred 20E therefore acts as a mating signal that females translate into an increased investment in egg development via a MISO-dependent pathway. The identification of this male–female reproductive interaction offers novel opportunities for the control of mosquito populations that transmit malaria. PMID:24204210

  1. Insect growth regulator activity of Cestrum parqui saponins: an interaction with cholesterol metabolism.

    Science.gov (United States)

    Ikbal, C; Ben, Halima-Kamel M; Ben, Hamouda M H

    2006-01-01

    Cestrum parqui is an ornamental shrub known for its insecticidal activity against some insect pests; this activity comes from the crude saponic extract of the leaves of this plant, the saponins cause insect growth regulator symptoms (development and moulting perturbation). In this work we try to demonstrate the hypothesis that saponins interact with ecdysone (moulting hormone) synthesis mechanisms by reducing diet cholesterol absorption (cholesterol forms the skeleton of ecdysone and of other ecdysteroids). To show the cholesterol/saponin interaction we used a stored product pest insect (Tribolium confuisurn), the larva of this insect are affected by saponins added in their diet, but the addition of cholesterol permits to reduce significatively this insecticidal propriety. Using Spodoptera littoralis larva model the tentative to detect a cholesterol rate reduction on the level of hemolymph is also unsuccessful. All these experiments shows that this type of reaction can't occur in the diet or in the digestive system but probably in insect cells. It is clear that Cestrurn parqui saponins affect the cholesterol metabolism but the exactly mechanism is still unknown. More investigations are necessary to develop this hypothesis and to envisage the use of Cestrum saponins as insect growth regulator bioinsecticide.

  2. Evaluation of Mosquito Repellent Activity of Isolated Oleic Acid, Eicosyl Ester from Thalictrum javanicum.

    Science.gov (United States)

    Gurunathan, Abinaya; Senguttuvan, Jamuna; Paulsamy, S

    2016-01-01

    To evaluate the traditional use, the mosquito repellent property of Thalictrum javanicum and to confirm the predicted larvicidal activity of the isolated compound, oleic acid, eicosyl ester from its aerial parts by PASS software, the present study was carried out using 4th instar stage larvae of the mosquitoes, Aedes aegypti (dengue vector) and Culex quinquefasciatus (filarial vector). Insecticidal susceptibility tests were conducted and the mortality rate was observed after 24 h exposure. The chitinase activity of isolated compound was assessed by using purified β-N-acetyl glucosaminidase (chitinase). Ecdysone 20-monooxygenase assay (radioimmuno assay) was made using the same larval stage of A. aegyptiand C. quinquefasciatus. The results were compared with the crude methanol extract of the whole plant. The isolated compound, oleic acid, eicosyl ester was found to be the most effective larvicide against A. aegypti (LC50/24 h -8.51 ppm) and C. quinquefasciatus (LC50/24 h - 12.5 ppm) than the crude methanol extract (LC50/24 h - 257.03 ppm and LC50/24 h - 281.83 ppm, respectively). The impact of oleic acid, eicosyl ester on reducing the activity of chitinase and ecdysone 20-monooxygenase was most prominent in both the target species, A. aegyptiand C. quinquefasciatus than the control. The results therefore suggest that the compound, oleic acid, eicosyl ester from Thalictrum javanicum may be considered as a potent source of mosquito larvicidal property.

  3. Evaluation of mosquito repellent activity of isolated oleic acid, eicosyl ester from Thalictrum javanicum

    Directory of Open Access Journals (Sweden)

    Abinaya Gurunathan

    2016-01-01

    Full Text Available To evaluate the traditional use, the mosquito repellent property of Thalictrum javanicumand to confirm the predicted larvicidal activity of the isolated compound, oleic acid, eicosyl ester from its aerial parts by PASS software, the present study was carried out using 4th instar stage larvae of the mosquitoes, Aedes aegypti(dengue vector and Culex quinquefasciatus(filarial vector. Insecticidal susceptibility tests were conducted and the mortality rate was observed after 24 h exposure. The chitinase activity of isolated compound was assessed by using purified β-N-acetyl glucosaminidase (chitinase. Ecdysone 20-monooxygenase assay (radioimmuno assay was made using the same larval stage of A. aegyptiand C. quinquefasciatus. The results were compared with the crude methanol extract of the whole plant. The isolated compound, oleic acid, eicosyl ester was found to be the most effective larvicide against A. aegypti (LC50/24 h -8.51 ppm and C. quinquefasciatus (LC50/24 h - 12.5 ppm than the crude methanol extract (LC50/24 h - 257.03 ppm and LC50/24 h - 281.83 ppm, respectively. The impact of oleic acid, eicosyl ester on reducing the activity of chitinase and ecdysone 20-monooxygenase was most prominent in both the target species, A. aegyptiand C. quinquefasciatusthan the control. The results therefore suggest that the compound, oleic acid, eicosyl ester from Thalictrum javanicummay be considered as a potent source of mosquito larvicidal property.

  4. Evaluation of Mosquito Repellent Activity of Isolated Oleic Acid, Eicosyl Ester from Thalictrum javanicum

    Science.gov (United States)

    Gurunathan, Abinaya; Senguttuvan, Jamuna; Paulsamy, S.

    2016-01-01

    To evaluate the traditional use, the mosquito repellent property of Thalictrum javanicum and to confirm the predicted larvicidal activity of the isolated compound, oleic acid, eicosyl ester from its aerial parts by PASS software, the present study was carried out using 4th instar stage larvae of the mosquitoes, Aedes aegypti (dengue vector) and Culex quinquefasciatus (filarial vector). Insecticidal susceptibility tests were conducted and the mortality rate was observed after 24 h exposure. The chitinase activity of isolated compound was assessed by using purified β-N-acetyl glucosaminidase (chitinase). Ecdysone 20-monooxygenase assay (radioimmuno assay) was made using the same larval stage of A. aegyptiand C. quinquefasciatus. The results were compared with the crude methanol extract of the whole plant. The isolated compound, oleic acid, eicosyl ester was found to be the most effective larvicide against A. aegypti (LC50/24 h -8.51 ppm) and C. quinquefasciatus (LC50/24 h - 12.5 ppm) than the crude methanol extract (LC50/24 h - 257.03 ppm and LC50/24 h - 281.83 ppm, respectively). The impact of oleic acid, eicosyl ester on reducing the activity of chitinase and ecdysone 20-monooxygenase was most prominent in both the target species, A. aegyptiand C. quinquefasciatus than the control. The results therefore suggest that the compound, oleic acid, eicosyl ester from Thalictrum javanicum may be considered as a potent source of mosquito larvicidal property. PMID:27168688

  5. DNA damage and transcriptional changes induced by tributyltin (TBT) after short in vivo exposures of Chironomus riparius (Diptera) larvae.

    Science.gov (United States)

    Morales, Mónica; Martínez-Paz, Pedro; Ozáez, Irene; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2013-08-01

    Tributyltin (TBT) is a widespread environmental contaminant in aquatic systems whose adverse effects in development and reproduction are related to its well-known endocrine-disrupting activity. In this work, the early molecular effects of TBT in Chironomus riparius (Diptera) were evaluated by analyzing its DNA damaging potential and the transcriptional response of different endocrine-related genes. Twenty-four-hour in vivo exposures of the aquatic larvae, at environmentally relevant doses of TBT, revealed genotoxic activity as shown by significant increases in DNA strand breaks quantified with the comet assay. TBT was also able to induce significant increases in transcripts from the ecdysone receptor gene (EcR), the ultraspiracle gene (usp) (insect ortholog of the retinoid X receptor), the estrogen-related receptor (ERR) gene and the E74 early ecdysone-inducible gene, as measured by real-time RT-PCR. In contrast, the expression of the vitellogenin (vg) gene remained unaltered, while the hsp70 gene appeared to be down-regulated. The ability of TBT to up-regulate hormonal target genes provides the first evidence, at genomic level, of its endocrine disruptive effects and also suggests a mechanism of action that mimics ecdysteroid hormones in insects. These data reveal for the first time the early genomic effects of TBT on an insect genome.

  6. Comprehensive study of the phenolics and saponins from Helleborus niger L. Leaves and stems by liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Duckstein, Sarina M; Stintzing, Florian C

    2014-02-01

    The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MS(n) ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non-acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, β-ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and β-ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plant's defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.

  7. Transcriptional deregulation of genetic biomarkers in Chironomus riparius larvae exposed to ecologically relevant concentrations of di(2-ethylhexyl) phthalate (DEHP)

    Science.gov (United States)

    Morcillo, Gloria; Planelló, Rosario

    2017-01-01

    Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental pollutant used worldwide as a plasticizer and solvent in many formulations. Based on available toxicological data, it has been classified as toxic for reproduction and as an endocrine disruptor. Despite this, ecotoxicological studies in aquatic wildlife organisms are still scarce. In the present work, the toxic molecular alterations caused by DEHP in aquatic larvae of the midge Chironomus riparius have been studied, by analyzing the transcriptional activity of genes related to some vital cellular pathways, such as the ribosomal machinery (rpL4, rpL13), the cell stress response (hsc70, hsp70, hsp40, hsp27), the ecdysone hormone pathway (EcR), the energy metabolism (GAPDH), and detoxication processes (CYP4G). Environmentally relevant concentrations (10−3 to 105 μg/L) and exposure conditions (24 to 96 h) have been tested, as well as the toxic effects after DEHP withdrawal. Although the compound caused no mortality, significant changes were detected in almost all the studied biomarkers: e.g. strong repression of hsp70; general inhibition of EcR; GAPDH activity loss in long exposures; among others. Our data show a general transcriptional downregulation that could be associated with an adaptive response to cell damage. Besides, the activity of the compound as an ecdysone antagonist and its delayed effects over almost all the biomarkers analyzed are described as novel toxic targets in insects. PMID:28166271

  8. MODIFICATIONS ULTRASTRUCTURALES DES CELLULES SÉCRÉTRICES DE LA GLANDE PROTHORACIQUE DE VERS À SOIE AU COURS DES DEUX DERNIERS ÂGES LARVAIRES

    Science.gov (United States)

    Beaulaton, J. A.

    1968-01-01

    Ultrastructural study of the prothoracic glands of silkworms (Antheraea pernyi and Bombyx mori) at the last two larval stages has shown that the essential modifications which take place during each intermolt affect the chondriome of secretory cells. A description is given of the differentiation of macromitochondria from typical mitochondria by a general swelling, a clearing of the matrix, and the formation of a complex tubular network. The hypothesis of fixation or anaesthesia artifacts has been dismissed because of the persistence of these transformations after different fixations and because of the existence of numerous intermediary stages between these two types of chondriosomes which imply the progressiveness of differentiation. The cytochemical demonstration of mitochondrial DNA fibers suggests that the genetic information, probably present in this type of nucleic acid, controls the differentiation and the specific metabolic activity of these organelles. The frequent relationships observed in Antheraea between the tubules of agranular reticulum and the macromitochondria which are reminiscent of the vacuoles-mitochondria associations of the adrenal cortex, may be related to the transfer of cholesterol and other precursors of steroidogenesis. In the last stages, the macromitochondria become transformed into vacuoles by a disappearance of the tubular network. The correlation revealed between mitochondrial transformations and the cyclical release of ecdysone (65) leads to the conclusion that a prominent fraction of chondriosomes is involved, in relation to the agranular reticulum, in the elaboration of steroid hormones such as ecdysone. PMID:5699930

  9. Plum, an immunoglobulin superfamily protein, regulates axon pruning by facilitating TGF-β signaling.

    Science.gov (United States)

    Yu, Xiaomeng M; Gutman, Itai; Mosca, Timothy J; Iram, Tal; Ozkan, Engin; Garcia, K Christopher; Luo, Liqun; Schuldiner, Oren

    2013-05-08

    Axon pruning during development is essential for proper wiring of the mature nervous system, but its regulation remains poorly understood. We have identified an immunoglobulin superfamily (IgSF) transmembrane protein, Plum, that is cell autonomously required for axon pruning of mushroom body (MB) γ neurons and for ectopic synapse refinement at the developing neuromuscular junction in Drosophila. Plum promotes MB γ neuron axon pruning by regulating the expression of Ecdysone Receptor-B1, a key initiator of axon pruning. Genetic analyses indicate that Plum acts to facilitate signaling of Myoglianin, a glial-derived TGF-β, on MB γ neurons upstream of the type-I TGF-β receptor Baboon. Myoglianin, Baboon, and Ecdysone Receptor-B1 are also required for neuromuscular junction ectopic synapse refinement. Our study highlights both IgSF proteins and TGF-β facilitation as key promoters of developmental axon elimination and demonstrates a mechanistic conservation between MB axon pruning during metamorphosis and the refinement of ectopic larval neuromuscular connections.

  10. Steroid hormone regulation of the voltage-gated, calcium-activated potassium channel expression in developing muscular and neural systems.

    Science.gov (United States)

    Garrison, Sheldon L; Witten, Jane L

    2010-11-01

    A precise organization of gene expression is required for developing neural and muscular systems. Steroid hormones can control the expression of genes that are critical for development. In this study we test the hypothesis that the steroid hormone ecdysone regulates gene expression of the voltage-gated calcium-activated potassium ion channel, Slowpoke or KCNMA1. Late in adult development of the tobacco hawkmoth Manduca sexta, slowpoke (msslo) levels increased contributing to the maturation of the dorsal longitudinal flight muscles (DLMs) and CNS. We show that critical components of ecdysteroid gene regulation were present during upreglation of msslo in late adult DLM and CNS development. Ecdysteroid receptor complex heterodimeric partner proteins, the ecdysteroid receptor (EcR) and ultraspiracle (USP), and the ecdysone-induced early gene, msE75B, were expressed at key developmental time points, suggesting that ecdysteroids direct aspects of gene expression in the DLMs during these late developmental stages. We provide evidence that ecdysteroids suppress msslo transcription in the DLMs; when titers decline msslo transcript levels increase. These results are consistent with msslo being a downstream gene in an ecdysteroid-mediated gene cascade during DLM development. We also show that the ecdysteroids regulate msslo transcript levels in the developing CNS. These results will contribute to our understanding of how the spatiotemporal regulation of slowpoke transcription contributes to tailoring cell excitability to the differing physiological and behavioral demands during development.

  11. Comparative effects of butyl benzyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP) on the aquatic larvae of Chironomus riparius based on gene expression assays related to the endocrine system, the stress response and ribosomes.

    Science.gov (United States)

    Planelló, Rosario; Herrero, Oscar; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2011-09-01

    In this work, the effects of butyl benzyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP), two of the most extensively used phthalates, were studied in Chironomus riparius under acute short-term treatments, to compare their relative toxicities and identify genes sensitive to exposure. The ecotoxicity of these phthalates was assessed by analysis of the alterations in gene expression profiles of selected inducible and constitutive genes related to the endocrine system, the cellular stress response and the ribosomal machinery. Fourth instar larvae, a model system in aquatic toxicology, were experimentally exposed to five increasing concentrations (0.01, 0.1, 1, 10, and 100mg/L) of DEHP and BBP for 24h. Gene expression was analysed by the changes in levels of transcripts, using RT-PCR techniques with specific gene probes. The exposures to DEHP or BBP were able to rapidly induce the hsp70 gene in a concentration-dependent manner, whereas the cognate form hsc70 was not altered by either of these chemicals. Transcription of ribosomal RNA as a measure of cell viability, quantified by the levels of ITS2, was not affected by DEHP, but was slightly, yet significantly, downregulated by BBP at the highest concentrations tested. Finally, as these phthalates are classified as endocrine disruptor chemicals (EDCs), their potential effect on the ecdysone endocrine system was studied by analysing the two genes, EcR and usp, of the heterodimeric ecdysone receptor complex. It was found that BBP provoked the overexpression of the EcR gene, with significant increases from exposures of 0.1mg/L and above, while DEHP significantly decreased the activity of this gene at the highest concentration. These data are relevant as they show for the first time the ability of phthalates to interfere with endocrine marker genes in invertebrates, demonstrating their potential capacity to alter the ecdysone signalling pathway. Overall, the study clearly shows a differential gene-toxin interaction

  12. Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter.

    Science.gov (United States)

    Dunlop, J; Lou, Z; Zhang, Y; McIlvain, H B

    1999-12-01

    1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.

  13. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

    Science.gov (United States)

    Rewitz, Kim F; Yamanaka, Naoki; Gilbert, Lawrence I; O'Connor, Michael B

    2009-12-04

    Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal-regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation of the Torso/ERK pathway.

  14. A cytochrome p450 conserved in insects is involved in cuticle formation.

    Directory of Open Access Journals (Sweden)

    Tamar Sztal

    Full Text Available The sequencing of numerous insect genomes has revealed dynamic changes in the number and identity of cytochrome P450 genes in different insects. In the evolutionary sense, the rapid birth and death of many P450 genes is observed, with only a small number of P450 genes showing orthology between insects with sequenced genomes. It is likely that these conserved P450s function in conserved pathways. In this study, we demonstrate the P450 gene, Cyp301a1, present in all insect genomes sequenced to date, affects the formation of the adult cuticle in Drosophila melanogaster. A Cyp301a1 piggyBac insertion mutant and RNAi of Cyp301a1 both show a similar cuticle malformation phenotype, which can be reduced by 20-hydroxyecdysone, suggesting that Cyp301a1 is an important gene involved in the formation of the adult cuticle and may be involved in ecdysone regulation in this tissue.

  15. Effects of hormone agonists on Sf9 cells, proliferation and cell cycle arrest.

    Science.gov (United States)

    Giraudo, Maeva; Califano, Jérôme; Hilliou, Frédérique; Tran, Trang; Taquet, Nathalie; Feyereisen, René; Le Goff, Gaëlle

    2011-01-01

    Methoxyfenozide and methoprene are two insecticides that mimic the action of the main hormones involved in the control of insect growth and development, 20-hydroxyecdysone and juvenile hormone. We investigated their effect on the Spodoptera frugiperda Sf9 cell line. Methoxyfenozide was more toxic than methoprene in cell viability tests and more potent in the inhibition of cellular proliferation. Cell growth arrest occurred in the G2/M phase after a methoprene treatment and more modestly in G1 after methoxyfenozide treatment. Microarray experiments and real-time quantitative PCR to follow the expression of nuclear receptors ultraspiracle and ecdysone receptor were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment with no gene in common between the two treatments. Our results suggest two different signalling pathways in Sf9 cells.

  16. Effects of hormone agonists on Sf9 cells, proliferation and cell cycle arrest.

    Directory of Open Access Journals (Sweden)

    Maeva Giraudo

    Full Text Available Methoxyfenozide and methoprene are two insecticides that mimic the action of the main hormones involved in the control of insect growth and development, 20-hydroxyecdysone and juvenile hormone. We investigated their effect on the Spodoptera frugiperda Sf9 cell line. Methoxyfenozide was more toxic than methoprene in cell viability tests and more potent in the inhibition of cellular proliferation. Cell growth arrest occurred in the G2/M phase after a methoprene treatment and more modestly in G1 after methoxyfenozide treatment. Microarray experiments and real-time quantitative PCR to follow the expression of nuclear receptors ultraspiracle and ecdysone receptor were performed to understand the molecular action of these hormone agonists. Twenty-six genes were differentially expressed after methoxyfenozide treatment and 55 genes after methoprene treatment with no gene in common between the two treatments. Our results suggest two different signalling pathways in Sf9 cells.

  17. Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans.

    Science.gov (United States)

    Ikekawa, Nobuo; Fujimoto, Yoshinori; Ishiguro, Masaji

    2013-01-01

    Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970-1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids.

  18. Effects of precocene and azadirachtin in Rhodnius prolixus: some data on development and reproduction

    Directory of Open Access Journals (Sweden)

    Eloi S. Garcia

    1987-01-01

    Full Text Available The results presented in this paper clearly indicate that precocene and azadirachtin are effective inhibitors of moulting and reproduction in the hemipteran Rhodnius prolixus. The time of application is important and only applications of these substances early in the intermoulting period cause their effects in nymphs. The inhibition of moulting is fully reversed by ecdysone therapy. Precocene and azadirachtin also affected drastically the oogenesis and egg deposition in this insect. Precocene-induced sterilization is reversed by application of juvenile hormone III. However, this hormone is unable to reverse the effect of azadirachtin on reproduction. Ecdysteroid titers in nymphs and adult females are decreased by these treatments. In vitro analysis suggest that precocene and azadirachtin may act directly on the prothoracic glands and ovaries producing ecdysteroids. Based on these and other findings the possible mode of action of these compounds on the development and reproduction of Rhodnius prolixus is discussed.

  19. [Effects of dimilin, a chitin inhibitor 1 (4 chlorophenyl) 3 (2-6 difluorobenzoyl) urea on the oenocytes and molting in the processionary caterpillar (Thaumetopoea pityocampa Schiff.) (Lepidoptera) (author's transl)].

    Science.gov (United States)

    Denneulin, J C; Lamy, M

    1977-01-01

    Te oenocytes of the processionary caterpillar show histophysiological variations during their developing cycle. Grafting experiments and culture in vitro, have not so far allowed us to reveal the least participation of the oenocytes in the determinism of molting and in the transformation of cholesterol into ecdysone. On the other hand, histochemical studies of the oenocytes during the last period of their larval state, reveal, just before nymphosis, the existence of polysaccharides which probably correspond to the synthesis of pre-cuticular substance. When the caterpillars are treated with a chitin inhibitor (pH - 60-40 = Dimilin), the polysaccharides are not longer to be seen in the oenocytes. This deficiency in cuticular material could well be the consequence of one of the most spectacular effects of this product that is a profound perturbation in the formation of the cuticle that leads to the death of animals when molting.

  20. Proximate mechanism of behavioral manipulation of an orb-weaver spider host by a parasitoid wasp

    Science.gov (United States)

    Gonzaga, Marcelo Oliveira; de Oliveira, Leandro Licursi; Sperber, Carlos Frankl

    2017-01-01

    Some ichneumonid wasps induce modifications in the web building behavior of their spider hosts to produce resistant “cocoon” webs. These structures hold and protect the wasp’s cocoon during pupa development. The mechanism responsible for host manipulation probably involves the inoculation of psychotropic chemicals by the parasitoid larva during a specific developmental period. Recent studies indicate that some spiders build cocoon webs similar to those normally built immediately before ecdysis, suggesting that this substance might be a molting hormone or a precursor chemical of this hormone. Here, we report that Cyclosa spider species exhibiting modified behavior presented higher 20-OH-ecdysone levels than parasitized spiders acting normally or unparasitized individuals. We suggest that the lack of control that spiders have when constructing modified webs can be triggered by anachronic activation of ecdysis. PMID:28158280

  1. Nitric Oxide Synthase Regulates Growth Coordination During Drosophila melanogaster Imaginal Disc Regeneration.

    Science.gov (United States)

    Jaszczak, Jacob S; Wolpe, Jacob B; Dao, Anh Q; Halme, Adrian

    2015-08-01

    Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. Here we describe a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This both produces a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. Here we demonstrate that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals.

  2. Role of elongator subunit Elp3 in Drosophila melanogaster larval development and immunity

    DEFF Research Database (Denmark)

    Walker, Jane; Kwon, So Yeon; Badenhorst, Paul

    2011-01-01

    The Elongator complex has been implicated in several cellular processes, including gene expression and tRNA modification. We investigated the biological importance of the Elp3 gene in Drosophila melanogaster. Deletion of Elp3 results in larval lethality at the pupal stage. During early development......, larval growth is dramatically impaired, with progression to the third instar delayed for ~24 hr, and pupariation occurring only at day 14 after egg laying. Melanotic nodules appear after 4 days. Microarray analysis shows that stress response genes are induced and ecdysone-induced transcription factors...... are severely repressed in the mutant. Interestingly, the phenotypes of Elp3 flies are similar to those of flies lacking the domino gene, encoding a SWI/SNF-like ATP-dependent chromatin-remodeling enzyme. Indeed, the gene expression profiles of these mutants are also remarkably similar. Together, these data...

  3. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  4. Molecular determinants of juvenile hormone action as revealed by 3D QSAR analysis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Denisa Liszeková

    Full Text Available BACKGROUND: Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH. While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 A or longer than 13.5 A, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. CONCLUSIONS/SIGNIFICANCE: The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.

  5. An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster.

    Science.gov (United States)

    Minami, Ryunosuke; Sato, Chiaki; Yamahama, Yumi; Kubo, Hideo; Hariyama, Takahiko; Kimura, Ken-Ichi

    2016-12-01

    The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

  6. Control of target gene specificity during metamorphosis by the steroid response gene E93.

    Science.gov (United States)

    Mou, Xiaochun; Duncan, Dianne M; Baehrecke, Eric H; Duncan, Ian

    2012-02-21

    Hormonal control of sexual maturation is a common feature in animal development. A particularly dramatic example is the metamorphosis of insects, in which pulses of the steroid hormone ecdysone drive the wholesale transformation of the larva into an adult. The mechanisms responsible for this transformation are not well understood. Work in Drosophila indicates that the larval and adult forms are patterned by the same underlying sets of developmental regulators, but it is not understood how the same regulators pattern two distinct forms. Recent studies indicate that this ability is facilitated by a global change in the responsiveness of target genes during metamorphosis. Here we show that this shift is controlled in part by the ecdysone-induced transcription factor E93. Although long considered a dedicated regulator of larval cell death, we find that E93 is expressed widely in adult cells at the pupal stage and is required for many patterning processes at this time. To understand the role of E93 in adult patterning, we focused on a simple E93-dependent process, the induction of the Dll gene within bract cells of the pupal leg by EGF receptor signaling. In this system, we show that E93 functions to cause Dll to become responsive to EGF receptor signaling. We demonstrate that E93 is both necessary and sufficient for directing this switch. E93 likely controls the responsiveness of many other target genes because it is required broadly for patterning during metamorphosis. The wide conservation of E93 orthologs suggests that similar mechanisms control life-cycle transitions in other organisms, including vertebrates.

  7. The homeodomain transcription factors antennapedia and POU-M2 regulate the transcription of the steroidogenic enzyme gene Phantom in the silkworm.

    Science.gov (United States)

    Meng, Meng; Cheng, Dao-Jun; Peng, Jian; Qian, Wen-Liang; Li, Jia-Rui; Dai, Dan-Dan; Zhang, Tian-Lei; Xia, Qing-You

    2015-10-01

    The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.

  8. EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

    Science.gov (United States)

    Shergill, J K; Cammack, R; Chen, J H; Fisher, M J; Madden, S; Rees, H H

    1995-01-01

    EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase. PMID:7741702

  9. Induced Expression of Cytochrome P450 CYP305 B1V1 Gene in Different Tissues of Wild Mulberry Silkworm (Bombyx mandarina)%野桑蚕不同组织细胞色素P450 CYP305B1V1基因的诱导表达特征研究

    Institute of Scientific and Technical Information of China (English)

    路爱成; 卫正国; 李兵; 沈卫德

    2009-01-01

    [Objective] The aim of this study was to investigate effects of various inducers on the expression of cytochrome P450 CYP305 B1V1 Gene in different tissues of wild mulberry silkworm. [Method] Referring to the mRNA sequence of CYP305 B1V1 Gene published in GenBank for wild mulberry silkworm, one pair of primers was designed, and the expression of cytochrome P450 CYP305 B1V1 Gene in different tissues of wild mulberry silkworm treated by NaF, rutin, cypermethrin and ecdysone was also analyzed by the semi-quantitative RT-PCR. Furthermore, homology comparison and phylogenetic analysis for amino acid sequences of this gene were studied. [Result] Rutin, cypermethrin and NaF had effects on the expression of P450 CYP305 B1V1 Gene in different tissues of wild mulberry silkworm, while ecdysone had no significant effect. Homology comparison for amino acids indicated that the amino acid sequence of this gene was the most similar to that of CYP305 B1 gene in Bombyx mori with 100% amino acid identity, and highly similar to those of Tribolium casmneum CYP305A1, Apis mellifera CYP305A1, Drosophila melanogaster CYP305A1, Anopheles gambiae CYP305A2 and Culex pipiens quinquefasciatus CYP2L1. [Conclusion] CYP305 B1V1 Gene of wild mulberry silkworm is likely to mainly take part in the metabolism of exogenous compounds, which is of great significance for revealing the function of cytochrome P450 and the metabolic mechanism of different drugs.

  10. Fungal ecdysteroid-22-oxidase, a new tool for manipulating ecdysteroid signaling and insect development.

    Science.gov (United States)

    Kamimura, Manabu; Saito, Hitoshi; Niwa, Ryusuke; Niimi, Teruyuki; Toyoda, Kinuko; Ueno, Chihiro; Kanamori, Yasushi; Shimura, Sachiko; Kiuchi, Makoto

    2012-05-11

    Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 μM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.

  11. The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

    Science.gov (United States)

    Gündner, Anna Lisa; Hahn, Ines; Sendscheid, Oliver; Aberle, Hermann; Hoch, Michael

    2014-01-01

    Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar

  12. Development of a methoxyfenozide-responsive gene switch for applications in plants.

    Science.gov (United States)

    Tavva, Venkata S; Dinkins, Randy D; Palli, Subba R; Collins, Glenn B

    2006-02-01

    The ecdysone receptor (EcR) has been used to develop gene switches for conditional regulation of transgene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require micromolar concentrations of ligand for activation of the transgene; this has limited the use of these gene switches. We have developed a Choristoneura fumiferana ecdysone receptor (CfEcR)-based two-hybrid gene switch that works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of the chemical ligand methoxyfenozide. Methoxyfenozide is already registered for field use with an excellent safety profile, and it has potential as a gene switch ligand for applications in the field. The receptor constructs were prepared by fusing DEF domains (hinge region plus ligand-binding domain) of CfEcR to the GAL4 DNA-binding domain and EF domains (ligand-binding domain) of ultraspiracle from Choristoneura fumiferana (CfUSP) or RXR from Locusta migratoria (LmRXR), Mus musculus (MmRXR) or Homo sapiens (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase gene placed under the control of 5x GAL4 response elements and -46 35S minimal promoter in tobacco, corn and soybean protoplasts and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the CfEcR + LmRXR two-hybrid switch was 25 to 625 times greater than the monopartate gene switch, depending on the plant species tested.

  13. The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

    Directory of Open Access Journals (Sweden)

    Anna Lisa Gündner

    Full Text Available Phosphoinositide-3-kinase enhancer (PIKE proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH domain, a GTPase-activating (GAP domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to

  14. Mutants for Drosophila Isocitrate Dehydrogenase 3b Are Defective in Mitochondrial Function and Larval Cell Death

    Science.gov (United States)

    Duncan, Dianne M.; Kiefel, Paula; Duncan, Ian

    2017-01-01

    The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three “type” alleles of E93 cause a near-complete block in salivary gland cell death. Here, we show that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the β subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germline mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells. PMID:28104670

  15. Molecular adaptation and resilience of the insect’s nuclear receptor USP

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    Chaumot Arnaud

    2012-10-01

    Full Text Available Abstract Background The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1 and USP (NR2B4, was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD of USP during evolution of Mecopterida. Results We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation. Conclusions In order to explain the episodic mode of

  16. Insect hormones regulate expression of diapause hormone receptor gene of the silkworm (Bombyx mori) in vitro%昆虫激素体外调节家蚕滞育激素受体基因的表达

    Institute of Scientific and Technical Information of China (English)

    王力刚; 朱娟; 王猛; 唐顺明; 沈兴家

    2013-01-01

    为了深入研究家蚕滞育的分子机理,从家蚕基因组中扩增了1395 bp和972 bp 2个不同长度的滞育激素受体基因Bmdhr启动子片段,以pGL3.0 Basic为载体,分别构建荧光素酶报告质粒,利用家蚕细胞瞬时表达系统分析其转录活性以及昆虫保幼激素类似物(JHA)、蜕皮激素(20-OH-ecdysone)和滞育激素(DH)对其活性的影响.结果表明:1395 bp的Bmdhr启动子活性显著低于972 bp的Bmdhr启动子,这说明Bmdhr启动子在-941~-1364 nt区间存在负调控元件.JHA浓度为2,4,6μg/mL时,极显著地增强启动子活性;浓度为1μg/mL时活性显著减弱,而为8μg/mL时则活性极显著减弱.20-OH-ecdysone对Bmdhr启动子活性的影响具有剂量效应,低浓度(1,2μg/mL)时显著增强启动子活性;浓度为4μg/mL时,启动子的活性显著减弱;但当浓度继续升高时,启动子活性又极显著增强.DH对Bmdhr启动子活性的影响同样具有剂量效应,其浓度为10~40 nM时,显著增强启动子活性;而当其浓度达到60 nM时,启动子活性变化不显著.%To study the molecular mechanism of silkworm (Bombyx mori) diapause, two heterogeneous promoter fragments,1 395 bp and 972 bp,of diapause hormone receptor gene (Bmdhr) were cloned into pGL3.0 basic vector to construct reporter plasmids , respectively .The effects of foreign insect hormones including juvenile hormone ana -logue ( JHA) , 20-OH-ecdysone and diapause hormone ( DH) on regulation of Bmdhr promoter activities were ana-lyzed in BmN cells .The results showed that the activity of 1 395 bp promoter fragment was significantly lower than that of 972 bp one, implying that there is a negative regulation element between -941 and-1364 nt of Bmdhr pro-moter region.JHA of 2, 4 and 6μg/mL increased the promoter activity by 1.5~2.1 folds, but JHA of 8μg/mL decreased the promoter activity significantly .The effects of ecdysone on the Bmdhr

  17. 三疣梭子蟹核受体基因HR38的克隆及其在蜕皮中的表达分析%Cloning and expression analysis in molting cycle of nuclear receptor HR38 gene inPortunus trituberculatus

    Institute of Scientific and Technical Information of China (English)

    张龙涛; 吕建建; 高保全; 刘萍; 付萍

    2016-01-01

    The nuclear receptor superfamily contains a large number of transcription factors that mediate many physio-logical process, such as cell differentiation, growth and development. Family members have a conserved functional domain that includes a DNA-binding domain and a ligand-binding domain. HR38, a member of the nuclear receptor superfamily, is an orphan receptor for which no ligand is known. HR38 is involved in an unusual ecdysteroid signaling pathway in Drosophila.Portunus trituberculatus is a major Chinese aquaculture species and is also an important Chi-nese export. The crustacean molting cycle is a physiologically important growth process. To investigate its function in the molting cycle ofPortunus trituberculatus, cDNA encoding nuclear receptor HR38 ofPortunus trituberculatus was cloned by rapid amplification of cDNA ends (RACE) and namedPHR38. The full-lengthPTHR38 cDNA is 2950 bp, including a 101-bp 5′ untranslated region (UTR) and a 551-bp 3′ UTR. The 2298-bp open reading frame (ORF) encodes a 765-amino acid polypeptide. Bioinformatic analysis revealed that PTHR38 is anunstable protein and has no trans-membrane domains. Homology analysis showed thatPTHR38 ofPortunus trituberculatus has the highest homology to the HR38 gene ofDaphnia magna. Six crabs ateach main molting stage (inter-, pre- and post-molting) were selected by morphological feature observation. Eyestalks were excised, the crabs were depigmented, their exoskeletons were re-moved, and different tissues were sampled before being stored in Trizol Reagent for RNA extraction. Quantitative real-time PCR showed different expression patterns ofPTHR38at each stage of the molting cycle, which suggested that PTHR38 functions in the molting cycle ofPortunus trituberculatus. It also showed a similar expression tendency to ecdysone in hemocytes and thus might be associated with ecdysone. In muscle and the hepatopancreas,PTHR38was upregulated in the post-molting stage. Previous studies showed that HR38

  18. Insect Development in Altered Gravitational Environment

    Science.gov (United States)

    Tischler, Marc E.

    1996-01-01

    When tobacco hornworm (Manduca sexta) larvae burrow underground (25-30 cm) to pupate, they reorient themselves to a relatively horizontal position indicating an ability to sense gravity. To evaluate their sensitivity to gravitational environment during metamorphosis, Manduca (pharate adults) were placed in a vertical (head-up) position. Distinct morphological changes, each one reflecting ensuing phases, were used to follow adult development. Five days after pupation, the vertical group showed accelerated (P less than 0.05) development and were nearly 4 phases ahead (P less than 0.0001) after 10 days. Differences in development in the vertical group were characterized further by increased (7-48%) hemolymph concentrations of 13 amino acids, but a decrease in cys and pro and no change in arg, his, met and val (trp, undetectable). Decreased (36%) turnover of injected H-3 - phenylalanine suggested slower utilization of amino acids contributed, at least partly, to the increased concentrations. Vertically-oriented Manduca also exhibited a greater (20 %, P less than 0.001) protein content in their flight muscles near the end of development. Analysis of hemolymph sugar levels showed a redistribution of sugars from the monosaccharide glucose to the disaccharide trehalose. Since injection of 20-hydroxyecdysone decreased (49%) turnover of H-3- phenylalanine in pharate adults and since ecdysteroids are known to increase flight muscle size and control adult development, these results are consistent with our measuring a greater (+80%, P less than 0.05) ecdysteroid titer in the vertically-oriented insects. These results suggest that gravity environment influences ecdysone output by the pharate adult. When we evaluated hemolymph flow in the head-up and control positions, we found that injected C-14-inulin was distributed somewhat more rapidly in the head-up group irrespective of the sight of injection (head or abdomen) likely because in the head-up position flow of the hemolymph is

  19. Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway.

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    Qiong Yao

    Full Text Available BACKGROUND: 20-hydroxyecdysone (20E and its receptor complex ecdysone receptor (EcR and ultraspiracle (USP play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis. PRINCIPAL FINDINGS: Here, we show that injection-based RNA interference (RNAi directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes. CONCLUSIONS: We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.

  20. PSM, a mediator of PDGF-BB-, IGF-I-, and insulin-stimulated mitogenesis.

    Science.gov (United States)

    Riedel, H; Yousaf, N; Zhao, Y; Dai, H; Deng, Y; Wang, J

    2000-01-06

    PSM/SH2-B has been described as a cellular partner of the FcepsilonRI receptor, insulin receptor (IR), insulin-like growth factor-I (IGF-I) receptor (IGF-IR), and nerve growth factor receptor (TrkA). A function has been proposed in neuronal differentiation and development but its role in other signaling pathways is still unclear. To further elucidate the physiologic role of PSM we have identified additional mitogenic receptor tyrosine kinases as putative PSM partners including platelet-derived growth factor (PDGF) receptor (PDGFR) beta, hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor. We have mapped Y740 as a site of PDGFR beta that is involved in the association with PSM. We have further investigated the putative role of PSM in mitogenesis with three independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adapter in normal NIH3T3 and baby hamster kidney fibroblasts. (1) PSM expression from cDNA using an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I-, and insulin- but not EGF-induced DNA synthesis in an ecdysone dose-responsive fashion; (2) Microinjection of the (dominant negative) PSM SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis; and (3) A peptide mimetic of the PSM Pro-rich putative SH3 domain-binding region interfered with PDGF-BB-, IGF-I-, and insulin- but not with EGF-induced DNA synthesis in NIH3T3 fibroblasts. This experiment was based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. These experimental strategies independently suggest that PSM functions as a positive, stimulatory, mitogenic signaling mediator in PDGF-BB, IGF-I, and insulin but not in EGF action. This function appears to involve the PSM SH2 domain as well as the Pro-rich putative SH3 domain binding region. Our findings support the model that PSM

  1. Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons

    Directory of Open Access Journals (Sweden)

    Chwalla Barbara

    2008-07-01

    candidate pathway controlled by the ecdysone receptor, and found that it promotes branching and growth of developing da neuron dendrites, but a role in RP2 dendrite development during embryonic and early larval stages was not apparent. Conclusion We identified commonalities (for example, growth and branching and distinctions (for example, targeting and ecdysone response in the molecular and organizational framework that underlies dendrite development of peripheral and central neurons.

  2. Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development.

    Science.gov (United States)

    Connahs, Heidi; Rhen, Turk; Simmons, Rebecca B

    2016-03-31

    Butterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui. We identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all

  3. Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    Full Text Available Many secondary metabolites have insecticidal efficacy against pests and may be affected by abiotic stress. However, little is known of how plants may respond to such stress as pertains the growth and development of pests. The objective of this study was to determine if and how salt stress on cotton plants affects the population dynamics of aphids. The NaCl treatment (50 mM, 100 mM, 150 mM and 200 mM increased contents of gossypol in cotton by 26.8-51.4%, flavonoids by 22.5-37.6% and tannic by 15.1-24.3% at 7-28 d after salt stress. Compared with non-stressed plants, the population of aphids on 150 and 200 mM NaCl stressed plants was reduced by 46.4 and 65.4% at 7d and by 97.3 and 100% at 14 days after infestation. Reductions in aphid population were possibly attributed to the elevated secondary metabolism under salt stress. A total of 796 clones for aphids transcriptome, 412 clones in the positive- library (TEST and 384 clones in the reverse-library (Ck, were obtained from subtracted cDNA libraries and sequenced. Gene ontology (GO functional classification and KEGG pathway analysis showed more genes related to fatty acid and lipid biosynthesis, and fewer genes related to carbohydrate metabolism, amino acid metabolism, energy metabolism and cell motility pathways in TEST than in Ck library, which might be the reason of aphids population reduction. A comparative analysis with qRT-PCR indicated high expression of transcripts CYP6A14, CYP6A13, CYP303A1, NADH dehydrogenase and fatty acid synthase in the TEST group. However, CYP307A1 and two ecdysone-induced protein genes were down regulated. The results indicate that genes of aphids related to growth and development can express at a higher level in reaction to the enhanced secondary metabolism in cotton under salinity stress. The expression of CYP307A1 was positively correlated with the population dynamics of aphids since it was involved in ecdysone synthesis.

  4. An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.

    Science.gov (United States)

    Pacchia, A L; Adelson, M E; Kaul, M; Ron, Y; Dougherty, J P

    2001-03-30

    Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

  5. Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Vassilis Stratoulias

    Full Text Available Understanding the control of stem cell (SC differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. Here, we report critical roles for lin-28 during oogenesis. We found that let-7 maturation was increased in lin-28 null mutant fly ovaries. We showed that lin-28 null mutant female flies displayed reduced fecundity, due to defects in egg chamber formation. More specifically, we demonstrated that in mutant ovaries, the egg chambers fuse during early oogenesis resulting in abnormal late egg chambers. We also showed that this phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. We suggest a model in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. Our results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation.

  6. X-ray cone beam microtomography for quantitative assessment of tracheal and pharyngeal volumes of Rhodnius prolixus; Utilizacao da microtomografia computadorizada com feixe de raios-X conico para a determinacao quantitativa do volume da traqueia e faringe do Rhodnius prolixus

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Izabella Soares de

    2017-07-01

    In the past decade microcomputerized tomography imaging using synchrotron radiation has become a powerful technique to generate high resolution images of Rhodinus prolixus. Images of soft tissues (protocerebrum and muscles) and dense structures (pharynx, trachea and esophagus) of R. prolixus head have been obtained using synchrotron radiation microtomography in mono and polychromatic configuration, respectively. Advancements in conventional microtomography have increased the achievable resolution and contrast, making this relatively inexpensive and widely available technology potentially useful for studies of insect internal morphology. The main goal of this work was to provide a new set of high quality microtomographic images of R. prolixus achieved by means of a desktop X-ray microtomograph. It allows the three-dimensional visualization of important chitinized structures: pharynx and tracheae. Pharyngeal and tracheal volumes were quantitatively evaluated at different days (1, 4, 10, 15 and 20) after feeding. The results suggest that variation of average volumes could be attributed to insect hormone 20-hydroxy-ecdysone (20-OH-Ec) pulse at 11 days after feeding. Pharyngeal volumes decrease 3.80 times. On the other hand, tracheal volumes increase 1.78 times. Head total volume showed similar trends than trachea. (author)

  7. Effects of insect growth regulators on the mosquito-parasitic nematode Romanomermis iyengari.

    Science.gov (United States)

    Suman, Devi Shankar; Brey, Christopher W; Wang, Yi; Sanad, Manar; Shamseldean, Muhammed S M; Gaugler, Randy

    2013-02-01

    Pyriproxyfen, a juvenile hormone analogue, diflubenzuron, a chitin synthesis inhibitor, and azadirachtin, an ecdysone agonist, are three insect growth regulators (IGRs) considered as selective and effective insecticides for mosquitoes. Romanomermis iyengari (Welch) is a mosquito-parasitic mermithid that can provide biological control against many medically important mosquito species. The compatibility of these two control tactics was tested by evaluating the sublethal effects of exposure to IGR on nematode developmental stages (preparasitic, parasitic, and preparasitic + parasitic) using Culex pipiens larvae as the host. Sublethal concentrations of IGRs were 90 % emergence inhibition of host mosquito. Preparasitic exposure to pyriproxyfen, azadirachtin, and diflurbenzuron had no effect on infectivity, parasite load, sex ratio, or male size but reduced nematode female length and increased male sex ratio at one parasite/larva. When IGRs treatments were made against the parasitic and preparasitic + parasitic stages, pyriproxyfen and azadirachtin reduced R. iyengari infectivity, parasite load, and male nematode length, whereas pyriproxyfen exposure increased male sex ratio and reduced the female R. iyengari length. Thus, IGRs have significant negative impacts on different stages of mosquito mermithid that can destabilize the balance of host-parasite population interaction. Therefore, IGRs should be used with caution in mosquito habitats where these parasites have established.

  8. The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

    Science.gov (United States)

    Montagne, Jacques; Lecerf, Caroline; Parvy, Jean-Philippe; Bennion, Janis M; Radimerski, Thomas; Ruhf, Marie-Laure; Zilbermann, Frederic; Vouilloz, Nicole; Stocker, Hugo; Hafen, Ernst; Kozma, Sara C; Thomas, George

    2010-05-06

    S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

  9. The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jacques Montagne

    2010-05-01

    Full Text Available S6 kinases (S6Ks act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR, Drosophila hormone receptor 3 (DHR3, a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

  10. Environmental endocrine disruptors: A proposed classification scheme

    Energy Technology Data Exchange (ETDEWEB)

    Fur, P.L. de; Roberts, J. [Environmental Defense Fund, Washington, DC (United States)

    1995-12-31

    A number of chemicals known to act on animal systems through the endocrine system have been termed environmental endocrine disruptors. This group includes some of the PCBs and TCDDs, as well as lead, mercury and a large number of pesticides. The common feature is that the chemicals interact with endogenous endocrine systems at the cellular and/or molecular level to alter normal processes that are controlled or regulated by hormones. Although the existence of artificial or environmental estrogens (e.g. chlordecone and DES) has been known for some time, recent data indicate that this phenomenon is widespread. Indeed, anti-androgens have been held responsible for reproductive dysfunction in alligator populations in Florida. But the significance of endocrine disruption was recognized by pesticide manufacturers when insect growth regulators were developed to interfere with hormonal control of growth. Controlling, regulating or managing these chemicals depends in no small part on the ability to identify, screen or otherwise know that a chemical is an endocrine disrupter. Two possible classifications schemes are: using the effects caused in an animal, or animals as an exposure indicator; and using a known screen for the point of contact with the animal. The former would require extensive knowledge of cause and effect relationships in dozens of animal groups; the latter would require a screening tool comparable to an estrogen binding assay. The authors present a possible classification based on chemicals known to disrupt estrogenic, androgenic and ecdysone regulated hormonal systems.

  11. Insecticidal Constituents and Activity of Alkaloids from Cynanchum mongolicum.

    Science.gov (United States)

    Ge, Yang; Liu, Pingping; Yang, Rui; Zhang, Liu; Chen, Hongxing; Camara, Ibrahima; Liu, Yiqing; Shi, Wangpeng

    2015-09-21

    Based on MS and NMR data and bioassay-guided tracing, three insecticidal alkaloids I, II and III from Cynanchum mongolicum were identified to be antofine N-oxide, antofine and tylophorine. Alkaloid I was more toxic than alkaloids II and III, but they were less active against Spodoptera litura than total alkaloids. The contact toxicity from these alkaloids against the aphid Lipaphis erysimi was significant, as the 24 h-LC50 values of alkaloids I, II, III and total alkaloids were 292.48, 367.21, 487.791 and 163.52 mg/L, respectively. The development disruption of S. litura larvae was tested, the pupation and emergence rates of S. litura decreased and the acute mortality of S. litura increased significantly by day 3 after being injected in their body cavity with 10-40 mg/L of total alkaloid. The ecdysone titer of treated S. litura larvae and prepupae declined with increasing alkaloid concentration. The alkaloids of Cynanchum mongolicum are potential insect growth inhibitors.

  12. The spliceosome-associated protein Mfap1 binds to VCP in Drosophila.

    Science.gov (United States)

    Rode, Sandra; Ohm, Henrike; Zipfel, Jaqueline; Rumpf, Sebastian

    2017-01-01

    Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP) regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da) neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.

  13. ROLE OF SERUM AND ION CHANNEL BLOCK ON GROWTH AND HORMONALLY-INDUCED DIFFERENTIATION OF Spodoptera frugiperda (Sf21) INSECT CELLS.

    Science.gov (United States)

    Jenson, Lacey J; Bloomquist, Jeffrey R

    2015-11-01

    A neuronal morphological phenotype can be induced in cultured Spodoptera frugiperda insect cells (Sf21) by supplementing serum-containing media with 20-hydroxyecdysone (20-HE) and/or insulin. In this study, the primary objectives were to determine any role of ion channels in mediating the morphological change in cells treated with 20-HE and insulin, and whether serum was required to observe this effect. Results showed serum-free media also induced growth of processes in Sf21 cells, but at a lower percentage than that found previously in cells bathed in serum-containing media. Veratridine, a sodium channel activator, increased cell survival when applied in combination with 20-HE to Sf21 cells, and the effect was blocked by tetrodotoxin (1 μM) a known sodium channel blocker. Cobalt, a calcium channel blocker, showed significant inhibition of cell process growth when applied in combination with both 20-HE and 20-HE plus veratridine. Cobalt also showed significant inhibition of cell process growth when applied in combination with insulin. Thus, some type of sodium channel, as well as a mechanism for transmembrane calcium ion movement, are apparently expressed in Sf21 cells and are involved in the differentiation process. These cell lines may be used in a wide variety of endeavors, including the screening of insecticides, as well as foster basic studies of neurodevelopment and ecdysone action.

  14. An adaptive transposable element insertion in the regulatory region of the EO gene in the domesticated silkworm, Bombyx mori.

    Science.gov (United States)

    Sun, Wei; Shen, Yi-Hong; Han, Min-Jin; Cao, Yun-Feng; Zhang, Ze

    2014-12-01

    Although there are many studies to show a key role of transposable elements (TEs) in adaptive evolution of higher organisms, little is known about the molecular mechanisms. In this study, we found that a partial TE (Taguchi) inserted in the cis-regulatory region of the silkworm ecdysone oxidase (EO) gene, which encodes a crucial enzyme to reduce the titer of molting hormone (20-hydroxyecdysone, 20E). The TE insertion occurred during domestication of silkworm and the frequency of the TE insertion in the domesticated silkworm (Bombyx mori) is high, 54.24%. The linkage disequilibrium in the TE inserted strains of the domesticated silkworm was elevated. Molecular population genetics analyses suggest that this TE insertion is adaptive for the domesticated silkworm. Luminescent reporter assay shows that the TE inserted in the cis-regulatory region of the EO gene functions as a 20E-induced enhancer of the gene expression. Further, phenotypic bioassay indicates that the silkworm with the TE insertion exhibited more stable developmental phenotype than the silkworm without the TE insertion when suffering from food shortage. Thus, the inserted TE in the cis-regulatory region of the EO gene increased developmental uniformity of silkworm individuals through regulating 20E metabolism, partially explaining transformation of a domestication developmental trait in the domesticated silkworm. Our results emphasize the exceptional role of gene expression regulation in developmental transition of domesticated animals.

  15. Functional analysis of the larval serum protein gene promoter from silkworm,Bombyx mori.

    Institute of Scientific and Technical Information of China (English)

    TANG Shunming; YI Yongzhu; SHEN Xingjia; ZHANG Zhifang; LI Yiren; HE Jialu

    2003-01-01

    The regulation region of larval serum protein gene, Bombyx mori. (BmLSP), consisting of the first intron, the first exon, the central promoter region and 5′-upstream region, is cloned from genomic DNA from the silkworm variety of Suju×Minghu. Using PCR and restriction endonuclease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are constructed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hormones on the BmLSP promoter activity are investigated. The results demonstrate that the promoter activity of BmLSP is 5.8- or 4.4-fold higher than that of BmLSPs whose first intron or the element in 5′-upstream region harboring the homologous sequence with the first intron of light-chain fibroin gene (EHIF) is deleted, respectively, suggesting that both the first intron and EHIF contain the main positive cis-acting elements. However, the inactive mariner transposable element (MTE) in 5′-upstream region presents a negative effect. Furthermore, the effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity show a typical dose-dependent manner, that is, low concentration treatments increase the BmLSP promoter activity and high concentration treatments decrease it. Meanwhile, insect ecdysone (MH) treatments present no significant effect.

  16. Essential role of grim-led programmed cell death for the establishment of corazonin-producing peptidergic nervous system during embryogenesis and metamorphosis in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Gyunghee Lee

    2013-01-01

    In Drosophila melanogaster, combinatorial activities of four death genes, head involution defective (hid, reaper (rpr, grim, and sickle (skl, have been known to play crucial roles in the developmentally regulated programmed cell death (PCD of various tissues. However, different expression patterns of the death genes also suggest distinct functions played by each. During early metamorphosis, a great number of larval neurons unfit for adult life style are removed by PCD. Among them are eight pairs of corazonin-expressing larval peptidergic neurons in the ventral nerve cord (vCrz. To reveal death genes responsible for the PCD of vCrz neurons, we examined extant and recently available mutations as well as RNA interference that disrupt functions of single or multiple death genes. We found grim as a chief proapoptotic gene and skl and rpr as minor ones. The function of grim is also required for PCD of the mitotic sibling cells of the vCrz neuronal precursors (EW3-sib during embryonic neurogenesis. An intergenic region between grim and rpr, which, it has been suggested, may enhance expression of three death genes in embryonic neuroblasts, appears to play a role for the vCrz PCD, but not for the EW3-sib cell death. The death of vCrz neurons and EW3-sib is triggered by ecdysone and the Notch signaling pathway, respectively, suggesting distinct regulatory mechanisms of grim expression in a cell- and developmental stage-specific manner.

  17. Ecdysteroid receptor from the American lobster Homarus americanus: EcR/RXR isoform cloning and ligand-binding properties.

    Science.gov (United States)

    Tarrant, Ann M; Behrendt, Lars; Stegeman, John J; Verslycke, Tim

    2011-09-01

    In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.

  18. Strongyloides stercoralis Infection in Alcoholic Patients

    Science.gov (United States)

    Pacheco, Flavia T. F.; Souza, Joelma N.; Silva, Mônica L. S.; Inês, Elizabete J.; Soares, Neci M.

    2016-01-01

    The course of Strongyloides stercoralis infection is usually asymptomatic with a low discharge of rhabditoid larva in feces. However, the deleterious effects of alcohol consumption seem to enhance the susceptibility to infection, as shown by a fivefold higher strongyloidiasis frequency in alcoholics than in nonalcoholics. Moreover, the association between S. stercoralis infection and alcoholism presents a risk for hyperinfection and severe strongyloidiasis. There are several possible mechanisms for the disruption of the host-parasite equilibrium in ethanol-addicted patients with chronic strongyloidiasis. One explanation is that chronic ethanol intake stimulates the hypothalamic-pituitary-adrenal (HPA) axis to produce excessive levels of endogenous cortisol, which in turn can lead to a deficiency in type 2 T helper cells (Th2) protective response, and also to mimic the parasite hormone ecdysone, which promotes the transformation of rhabditiform larvae to filariform larvae, leading to autoinfection. Therefore, when untreated, alcoholic patients are continuously infected by this autoinfection mechanism. Thus, the early diagnosis of strongyloidiasis and treatment can prevent serious forms of hyperinfection in ethanol abusers. PMID:28105424

  19. A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis.

    Science.gov (United States)

    Banerjee, Shamik; Chinthapalli, Balaji

    2014-09-01

    Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with 'mitochondria-shaping' proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.

  20. Transcriptome analysis of sexually dimorphic Chinese white wax scale insects reveals key differences in developmental programs and transcription factor expression.

    Science.gov (United States)

    Yang, Pu; Chen, Xiao-Ming; Liu, Wei-Wei; Feng, Ying; Sun, Tao

    2015-01-01

    The Chinese white wax scale insect, Ericerus pela, represents one of the most dramatic examples of sexual dimorphism in any insect species. In this study, we showed that although E. pela males display complete metamorphosis similar to holometabolous insects, the species forms the sister group to Acyrthosiphon pisum and cluster with hemimetabolous insects. The gene expression profile and Gene Ontology (GO) analyses revealed that the two sexes engaged in distinct developmental programs. In particular, female development appeared to prioritize the expression of genes related to cellular, metabolic, and developmental processes and to anatomical structure formation in nymphs. By contrast, male nymphal development is characterized by the significant down-regulation of genes involved in chitin, the respiratory system, and neurons. The wing and appendage morphogenesis, anatomical and tissue structure morphogenesis programs activated after male nymphal development. Transcription factors (that convey juvenile hormone or ecdysone signals, and Hox genes) and DNA methyltransferase were also differentially expressed between females and males. These results may indicate the roles that these differentially expressed genes play in regulating sexual dimorphism through orchestrating complex genetic programs. This differential expression was particularly prominent for processes linked to female development and wing development in males.

  1. Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation.

    Science.gov (United States)

    Chung, J Sook

    2010-01-01

    The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210-330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. (c) 2009 Wiley Periodicals, Inc.

  2. Changes in ecdysteroids during embryogenesis of the blue crab, callinectes sapidus rathbun

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, J.F.; Skinner, D.M.

    1979-01-01

    Total ecdysteroid titers (estimated by radioimmunoassay (RIA)) in embryos of the blue crab increased from approx. 6 ng 20-hydroxyecdysone equivalents/g in the immature embryo to a maximum of approx. 500 ng 20-hydroxyecdysone equivalents/g in maturing embryos; titers dropped to approx. 300 ng 20-hydroxyecydsone equivalents/g in prehatch embryos. High-pressure reverse-phase chromatography of the embryo extracts resolved five RIA-active components, ..cap alpha..-Ecdysone and the polar conjugate of 20-hydroxyecdysone were present in low quantities. The concentration of 20-hydroxyecdysone increased during embryogenesis to a maximum of approx. 160 ng/g in maturing embryos and decreased only slightly in the prehatch embryos. Two unidentified components were also detected and the changes in their concentrations were estimated. One, an apolar component (peak III), accounted for as much as 63% of the total RIA activity as the embryos matured. The estimated concentration of this component increased from 85 ng/g in early embryos to 475 ng/g in maturing embryos, then decreased by 50% in the prehatch embryos. The level of the other, more polar component (peak II) increased from 7.5 to 75 ng/g as the embryos developed. The increase in the concentration of ecdysteroids during embryogenesis indicates that crab embryos have the capacity to synthesize ecdysteroids and suggests that these hormones may have a physiological role in the embryonic development of crustaceans.

  3. Perspectives on the integration of a supercritical fluid extraction plant to a sugarcane biorefinery: thermo-economical evaluation of CO2 recycle systems

    Directory of Open Access Journals (Sweden)

    Juliana Q. ALBARELLI

    Full Text Available Abstract In the present study, the software Aspen Plus® was used to analyse two different systems for CO2 recycle in a SFE process for extraction of more polar compounds using ethanol as co-solvent, the most common co-solvent used due to its environment-friendly nature. The extraction process of β-ecdysone from Brazilian ginseng roots was considered as example in the computational simulations. The first CO2 recycle system, named Recycle A, considered the compression of the CO2 separated in the second flash to the recycle pressure assumed at the first flash tank, its cooling to 25 °C and recirculation, while the second recycle system, named Recycle B, considered the cooling and pumping of the CO2 separated in the second flash, its heating to 25 °C and recirculation. The best techno-economic condition to operate the recycling step would be using Recycle A at 40 bar and 30 °C considering a stand-alone SFE process; and using Recycle B at 40 bar and 40 °C, considering this process in close proximity of a hypothetical sugarcane biorefinery. Therefore, these results suggest that the selection where would be located the SFE plant should be taken into account during the first steps of the process design.

  4. Comparative Proteomic Profiling Reveals Molecular Characteristics Associated with Oogenesis and Oocyte Maturation during Ovarian Development of Bactrocera dorsalis (Hendel)

    Science.gov (United States)

    Li, Ran; Zhang, Meng-Yi; Liu, Yu-Wei; Zhang, Zheng; Smagghe, Guy; Wang, Jin-Jun

    2017-01-01

    Time-dependent expression of proteins in ovary is important to understand oogenesis in insects. Here, we profiled the proteomes of developing ovaries from Bactrocera dorsalis (Hendel) to obtain information about ovarian development with particular emphasis on differentially expressed proteins (DEPs) involved in oogenesis. A total of 4838 proteins were identified with an average peptide number of 8.15 and sequence coverage of 20.79%. Quantitative proteomic analysis showed that a total of 612 and 196 proteins were differentially expressed in developing and mature ovaries, respectively. Furthermore, 153, 196 and 59 potential target proteins were highly expressed in early, vitellogenic and mature ovaries and most tested DEPs had the similar trends consistent with the respective transcriptional profiles. These proteins were abundantly expressed in pre-vitellogenic and vitellogenic stages, including tropomyosin, vitellogenin, eukaryotic translation initiation factor, heat shock protein, importin protein, vitelline membrane protein, and chorion protein. Several hormone and signal pathway related proteins were also identified during ovarian development including piRNA, notch, insulin, juvenile, and ecdysone hormone signal pathways. This is the first report of a global ovary proteome of a tephritid fruit fly, and may contribute to understanding the complicate processes of ovarian development and exploring the potentially novel pest control targets. PMID:28665301

  5. Strongyloides stercoralis Infection in Alcoholic Patients

    Directory of Open Access Journals (Sweden)

    Marcia C. A. Teixeira

    2016-01-01

    Full Text Available The course of Strongyloides stercoralis infection is usually asymptomatic with a low discharge of rhabditoid larva in feces. However, the deleterious effects of alcohol consumption seem to enhance the susceptibility to infection, as shown by a fivefold higher strongyloidiasis frequency in alcoholics than in nonalcoholics. Moreover, the association between S. stercoralis infection and alcoholism presents a risk for hyperinfection and severe strongyloidiasis. There are several possible mechanisms for the disruption of the host-parasite equilibrium in ethanol-addicted patients with chronic strongyloidiasis. One explanation is that chronic ethanol intake stimulates the hypothalamic-pituitary-adrenal (HPA axis to produce excessive levels of endogenous cortisol, which in turn can lead to a deficiency in type 2 T helper cells (Th2 protective response, and also to mimic the parasite hormone ecdysone, which promotes the transformation of rhabditiform larvae to filariform larvae, leading to autoinfection. Therefore, when untreated, alcoholic patients are continuously infected by this autoinfection mechanism. Thus, the early diagnosis of strongyloidiasis and treatment can prevent serious forms of hyperinfection in ethanol abusers.

  6. DsRNA degradation in the pea aphid (Acyrthosiphon pisum) associated with lack of response in RNAi feeding and injection assay.

    Science.gov (United States)

    Christiaens, Olivier; Swevers, Luc; Smagghe, Guy

    2014-03-01

    Over the past decade, RNA interference (RNAi), the sequence-specific suppression of gene expression, has proven very promising for molecular research in many species, including model insects as Tribolium castaneum and Apis mellifera. It showed its usefulness to analyze gene function and its potential to manage pest populations and reduce disease pathogens. However, in several insects, the efficiency of RNAi is low or very variable at best. One of the factors that could influence RNAi efficiency in insects is degradation of dsRNA after administration to the insect. In this paper, we report on the importance of dsRNA breakdown in the pea aphid (Acyrthosiphon pisum) associated with the absence of an RNAi response upon oral feeding and injection with dsRNA targeting different genes such as the ecdysone hormone receptor and ultraspiracle. In essence, we discovered that both the salivary secretions of aphids and the hemolymph were able to degrade the dsRNA. In parallel, introduction of dsRNA in the aphid body was not able to provoke a response in the expression of the siRNA core machinery genes.

  7. Cross-talk between the fat body and brain regulates insect developmental arrest.

    Science.gov (United States)

    Xu, Wei-Hua; Lu, Yu-Xuan; Denlinger, David L

    2012-09-04

    Developmental arrest, a critical component of the life cycle in animals as diverse as nematodes (dauer state), insects (diapause), and vertebrates (hibernation), results in dramatic depression of the metabolic rate and a profound extension in longevity. Although many details of the hormonal systems controlling developmental arrest are well-known, we know little about the interactions between metabolic events and the hormones controlling the arrested state. Here, we show that diapause is regulated by an interplay between blood-borne metabolites and regulatory centers within the brain. Gene expression in the fat body, the insect equivalent of the liver, is strongly suppressed during diapause, resulting in low levels of tricarboxylic acid (TCA) intermediates circulating within the blood, and at diapause termination, the fat body becomes activated, releasing an abundance of TCA intermediates that act on the brain to stimulate synthesis of regulatory peptides that prompt production of the insect growth hormone ecdysone. This model is supported by our success in breaking diapause by injecting a mixture of TCA intermediates and upstream metabolites. The results underscore the importance of cross-talk between the brain and fat body as a regulator of diapause and suggest that the TCA cycle may be a checkpoint for regulating different forms of animal dormancy.

  8. Whole transcriptome responses among females of the filariasis and arbovirus vector mosquito Culex pipiens implicate TGF-β signaling and chromatin modification as key drivers of diapause induction.

    Science.gov (United States)

    Hickner, Paul V; Mori, Akio; Zeng, Erliang; Tan, John C; Severson, David W

    2015-07-01

    Culex pipiens mosquitoes are important disease vectors inhabiting temperate zones, worldwide. The seasonal reduction in temperature and photoperiod accompanying late summer and early fall prompts female mosquitoes to enter diapause, a stage of developmental arrest and physiological conditioning that enhances survival during the winter months. To investigate the molecular mechanisms underlying diapause induction, we used custom whole transcriptome microarrays to identify differences in gene expression following exposure to nondiapause (long days, 25 °C) and diapause-inducing (short days, 18 °C) environmental conditions. Using a two-way ANOVA, we identified 1130 genes that were differentially expressed. We used the expression of these genes across three time points to construct a gene co-expression network comprising five modules. Genes in modules 1, 2, and 3 were largely up-regulated, while genes in modules 4 and 5 were down-regulated when compared to nondiapause conditions. Pathway enrichment analysis of the network modules revealed some potential regulatory mechanisms driving diapause induction. Module 1 was enriched for genes in the TGF-ß and Wnt signaling pathways; module 2 was enriched for genes involved in insect hormone biosynthesis, specifically, ecdysone synthesis; module 3 was enriched for genes involved in chromatin modification; and module 5 was enriched for genes in the circadian rhythm pathway. Our results suggest that TGF-β signaling and chromatin modification are key drivers for the integration of environmental signals into the diapause induction phase in C. pipiens mosquitoes.

  9. The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause.

    Science.gov (United States)

    Zhang, Tian-Yi; Sun, Jiu-Song; Zhang, Qi-Rui; Xu, Jun; Jiang, Rong-Jing; Xu, Wei-Hua

    2004-06-01

    FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.

  10. Hormonal mechanisms underlying termination of larval diapause by juvenile hormone in the bamboo borer, Omphisa fuscidentalis.

    Science.gov (United States)

    Singtripop, Tippawan; Manaboon, Manaporn; Tatun, Nujira; Kaneko, Yu; Sakurai, Sho

    2008-01-01

    Topical application of methoprene, a juvenile hormone analogue (JHA), induces pupation by activating the prothoracic glands (PGs) in diapausing larvae of the bamboo borer, Omphisa fuscidentalis. To determine the minimum stimulation period for PG activation, we transplanted PGs of JHA-treated larvae (donors) into non-treated larvae (recipients) on successive days after JHA treatment and observed the recipients for pupation. JHA stimulation for 1 day was sufficient to induce pupation. In recipient larvae, the hemolymph ecdysteroid titer increased transiently on day 18 after transplantation and significantly on days 24-28, prior to pupation. Secretory activity of recipient PGs increased transiently on day 16 and days 22-28. Because the recipient PG activity was too low to account for an increased ecdysteroid titer, the JHA-stimulated donor PGs must produce the major part of hemolymph ecdysteroids. In addition, the ecdysteroid produced by the donor PGs might have stimulated the recipient PGs. We examined the possible involvement of two ecdysone receptor (EcR) isoforms, OfEcR-A and OfEcR-B1, in PG activation by JHA, and found that although both isoforms were up-regulated, accompanied by an increased ecdysteroid titer in the hemolymph, the isoform mRNA levels were not altered at all before the increase in PG secretory activity. Thus, EcR expression might not be involved in feedback activation of PGs.

  11. Minocycline treatment suppresses juvenile development and growth by attenuating insulin/TOR signaling in Drosophila animal model

    Science.gov (United States)

    Yun, Hyun Myoung; Noh, Sujin; Hyun, Seogang

    2017-01-01

    Minocycline is a broad spectrum, semi-synthetic tetracycline analog that is used to treat bacterial infection. Recently, this drug has been receiving increasing attention for its non-antibiotic properties, including anti-inflammatory, tumor suppressive, and neuroprotective effects. Drosophila is a useful model organism for studying human metabolism and disease. In this study, we investigated the effects of minocycline on juvenile development and growth in Drosophila. Feeding minocycline to Drosophila larvae suppresses larval body growth and delays the timing of pupation in a dose-dependent manner. We found that the drug treatment decreased the activated form of Akt and S6K in peripheral tissues, which suggested that the insulin/target of rapamycin (TOR) signaling had been attenuated. Specifically enhancing TOR activity in the prothoracic gland (PG), the ecdysone-generating organ, attenuated the drug-induced developmental delay, which is consistent with the critical role of PG’s TOR signaling in determining pupation time. Our results reveal previously unrecognized effects of minocycline and offer a new potential therapeutic opportunity for various pathological conditions associated with insulin/TOR signaling. PMID:28317899

  12. Isolation of arginine kinase from Apis cerana cerana and its possible involvement in response to adverse stress.

    Science.gov (United States)

    Chen, Xiaobo; Yao, Pengbo; Chu, Xiaoqian; Hao, Lili; Guo, Xingqi; Xu, Baohua

    2015-01-01

    Arginine kinases (AK) in invertebrates play the same role as creatine kinases in vertebrates. Both proteins are important for energy metabolism, and previous studies on AK focused on this attribute. In this study, the arginine kinase gene was isolated from Apis cerana cerana and was named AccAK. A 5'-flanking region was also cloned and shown to contain abundant putative binding sites for transcription factors related to development and response to adverse stress. We imitated several abiotic and biotic stresses suffered by A. cerana cerana during their life, including heavy metals, pesticides, herbicides, heat, cold, oxidants, antioxidants, ecdysone, and Ascosphaera apis and then studied the expression patterns of AccAK after these treatments. AccAK was upregulated under all conditions, and, in some conditions, this response was very pronounced. Western blot and AccAK enzyme activity assays confirmed the results. In addition, a disc diffusion assay showed that overexpression of AccAK reduced the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, our results indicated that AccAK may be involved of great significance in response to adverse abiotic and biotic stresses.

  13. Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae

    Science.gov (United States)

    Shaw, W. Robert; Teodori, Eleonora; Mitchell, Sara N.; Baldini, Francesco; Gabrieli, Paolo; Rogers, David W.; Catteruccia, Flaminia

    2014-01-01

    Anopheles gambiae mosquitoes are major African vectors of malaria, a disease that kills more than 600,000 people every year. Given the spread of insecticide resistance in natural mosquito populations, alternative vector control strategies aimed at reducing the reproductive success of mosquitoes are being promoted. Unlike many other insects, An. gambiae females mate a single time in their lives and must use sperm stored in the sperm storage organ, the spermatheca, to fertilize a lifetime's supply of eggs. Maintenance of sperm viability during storage is therefore crucial to the reproductive capacity of these mosquitoes. However, to date, no information is available on the factors and mechanisms ensuring sperm functionality in the spermatheca. Here we identify cellular components and molecular mechanisms used by An. gambiae females to maximize their fertility. Pathways of energy metabolism, cellular transport, and oxidative stress are strongly regulated by mating in the spermatheca. We identify the mating-induced heme peroxidase (HPX) 15 as an important factor in long-term fertility, and demonstrate that its function is required during multiple gonotrophic cycles. We find that HPX15 induction is regulated by sexually transferred 20-hydroxy-ecdysone (20E), a steroid hormone that is produced by the male accessory glands and transferred during copulation, and that expression of this peroxidase is mediated via the 20E nuclear receptor. To our knowledge, our findings provide the first evidence of the mechanisms regulating fertility in Anopheles, and identify HPX15 as a target for vector control. PMID:24711401

  14. Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods.

    Science.gov (United States)

    Ho, Raimana; Girault, Jean-Pierre; Cousteau, Pierre-Yves; Bianchini, Jean-Pierre; Raharivelomanana, Phila; Lafont, René

    2008-02-01

    The Polynesian medicinal fern Microsorum membranifolium contains very large amounts of ecdysteroids, including ecdysone, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. It also contains large amounts of unusual ecdysteroids which have been unambiguously identified by mass spectrometry and nuclear magnetic resonance. A new class of ecdysteroid conjugates (3-glucosyl-ferulates of 2-deoxyecdysone and 2-deoxy-20-hydroxyecdysone) is isolated, together with a new glycoside (2-deoxyecdysone 25-rhamnoside). The simultaneous presence of a sugar and an aromatic moiety results in a very particular chromatographic behavior of these conjugates. They behave like flavonoids and polyphenols when using the classical purification on polyamide, aimed at removing the latter from crude plant extracts, and would therefore be lost. They elute as non-polar ecdysteroids on reversed-phase high-performance liquid chromatography (RP-HPLC), whereas their behavior on normal-phase (NP) HPLC is strongly dependent on the mobile phase composition. Our data highlight the importance of selectivity in the choice of HPLC methods used for ecdysteroid separations.

  15. A Study on Chemical Constituents of Tinospora sagittata%青牛胆化学成分的研究

    Institute of Scientific and Technical Information of China (English)

    王世平; 吴艳俊; 李玲; 张金娟; 吴君

    2011-01-01

    Objective: To study the chemical constituents of Tinospora sagittata. Methods: Various chromatographic techniques including silica gel, Sephadex LH-20, and RP-18 trabeculum were used to isolate the constituents of T. sagittata, and their structures were identified by spectral and chemical methods. Result: Five compounds were isolated from T. sagittata, and their structures were identified as 20β-hydroxy-ecdysone ( Ⅰ ), tinoside ( Ⅱ ), columbamine ( m ), acutumine ( Ⅳ ), and jatrorrhizine( Ⅴ ). Conclusion: This is the first report that compound Ⅴ being isolated from T. sagittata.%目的:对青牛胆的化学成分进行研究.方法:应用硅胶、Sephadex LH-20、RP-18柱色谱进行分离、纯化,根据理化常数和光谱分析鉴定结构.结果:从青牛胆中分离出5个化合物,经鉴定为:20β-羟基蜕皮素(Ⅰ)、金果榄苷(Ⅱ)、非洲防己碱(Ⅲ)、尖防己碱(Ⅳ)、药根碱(Ⅴ).结论:化合物Ⅴ为首次从该植物中分离得到.

  16. Mechanisms regulating nutrition-dependent developmental plasticity through organ-specific effects in insects

    Directory of Open Access Journals (Sweden)

    Takashi eKoyama

    2013-09-01

    Full Text Available Nutrition, via the insulin/insulin-like growth factor (IIS/Target of Rapamycin (TOR signaling pathway, can provide a strong molding force for determining animal size and shape. For instance, nutrition induces a disproportionate increase in the size of male horns in dung and rhinoceros beetles, or mandibles in staghorn or horned flour beetles, relative to body size. In these species, well-fed male larvae produce adults with greatly enlarged horns or mandibles, whereas males that are starved or poorly fed as larvae bear much more modest appendages. Changes in IIS/TOR signaling plays a key role in appendage development by regulating growth in the horn and mandible primordia. In contrast, changes the IIS/TOR pathway produces minimal effects on the size of other adult structures, such as the male genitalia in fruit flies and dung beetles. The horn, mandible and genitalia illustrate that although all tissues are exposed to the same hormonal environment within the larval body, the extent to which insulin can induce growth is organ specific. In addition, the IIS/TOR pathway affects body size and shape by controlling production of metamorphic hormones important for regulating developmental timing, like the steroid molting hormone ecdysone and sesquiterpenoid hormone juvenile hormone. In this review, we discuss recent results from Drosophila and other insects that highlight mechanisms allowing tissues to differ in their sensitivity to IIS/TOR and the potential consequences of these differences on body size and shape.

  17. Krüppel homolog 1 and E93 mediate Juvenile hormone regulation of metamorphosis in the common bed bug, Cimex lectularius

    Science.gov (United States)

    Gujar, Hemant; Palli, Subba Reddy

    2016-01-01

    The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs. PMID:27185064

  18. Juvenile hormone regulation of female reproduction in the common bed bug, Cimex lectularius

    Science.gov (United States)

    Gujar, Hemant; Palli, Subba Reddy

    2016-01-01

    To begin studies on reproduction in common bed bug, Cimex lectularius, we identified three genes coding for vitellogenin (Vg, a protein required for the reproductive success of insects) and studied their hormonal regulation. RNA interference studied showed that expression of Vg3 gene in the adult females is a prerequisite for successful completion of embryogenesis in the eggs laid by them. Juvenile hormone (JH) receptor, Methoprene-tolerant (Met), steroid receptor coactivator (SRC) and GATAa but not ecdysone receptor (EcR) or its partner, ultraspiracle (USP) are required for expression of Vg genes. Feeding and mating working through Vg, Met, SRC, EcR, and GATAa regulate oocyte development. Knockdown of the expression of Met, SRC, EcR, USP, BR-C (Broad-Complex), TOR (target of rapamycin), and GATAa in female adults resulted in a reduction in the number eggs laid by them. Interestingly, Kruppel homolog 1 (Kr-h1) knockdown in the adult females did not reduce their fecundity but affected the development of embryos in the eggs laid by females injected with Kr-h1 double-stranded RNA. These data suggest that JH functioning through Met and SRC regulate both vitellogenesis and oogenesis in C. lectularius. However, JH does not work through Kr-h1 but may work through transcription factors not yet identified. PMID:27762340

  19. Transcriptome Analysis of Drosophila melanogaster Third Instar Larval Ring Glands Points to Novel Functions and Uncovers a Cytochrome p450 Required for Development

    Science.gov (United States)

    Christesen, Danielle; Yang, Ying Ting; Somers, Jason; Robin, Charles; Sztal, Tamar; Batterham, Philip; Perry, Trent

    2016-01-01

    In Drosophila melanogaster larvae, the ring gland (RG) is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum, and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the RG’s broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval RGs has provided a fascinating insight into the diversity of developmental signaling in this organ. We have found strong enrichment of expression of two gene pathways not previously associated with the RG: immune response and fatty acid metabolism. We have also uncovered strong expression for many uncharacterized genes. Additionally, RNA interference against RG-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency, respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the RG in governing insect development. PMID:27974438

  20. Ecdysteroids: the overlooked sex steroids of insects? Males: the black box

    Institute of Scientific and Technical Information of China (English)

    ARNOLD DE LOOF

    2006-01-01

    The paradigm, still around in textbooks, that 'in insects sex is strictly genetic,thus that they do not have sex hormones', is mainly based on a wrong interpretation of the 'gynandromorph argument'. It is no longer tenable. Given the fact that vertebrates and invertebrates probably had a common, sexually reproducing ancestor, there is no reason to assume that only vertebrates need sex hormones. The major function of sex hormones is to inform the somatoplasm about developmental changes that take place in the gonads. In contrast to juvenile hormone and neuropeptides, ecdysteroids meet all criteria to act as sex hormones, which was probably their ancient role. Their much better documented role in moulting and metamorphosis was a secondary acquisition that enabled arthropods to cope with growth problems, imposed by a rigid cuticle. Female insects use 20-hydroxyecdysone (20E), secreted by the follicle cells of the ovary, in a similar way as females of egg-laying vertebrates use estrogens. For a variety of reasons, the possibility that ecdysteroids, in particular ecdysone (E), might also act as sex hormones in male insects, thus as the counterpart of testosterone of vertebrates, has been very much overlooked. Thanks to the recent discovery of the molecular basis of the haploid-diploid system of sex determination in the honeybee, the characterization of Halloween genes, proteomics, RNAi and so on, it now becomes possible to verify whether in insects, as with vertebrates, males are the endocrinologically default gender form.

  1. Effects of methoxyfenozide on Lobesia botrana Den & Schiff (Lepidoptera: Tortricidae) egg, larval and adult stages.

    Science.gov (United States)

    Sáenz-de-Cabezón Irigaray, Francisco-Javier; Marco, Vicente; Zalom, Frank G; Pérez-Moreno, Ignacio

    2005-11-01

    The effect of the non-steroidal ecdysone agonist methoxyfenozide was evaluated against different developmental stages of the grape berry moth, Lobesia botrana Dennis & Schiffermuller (Lep, Tortricidae). Methoxyfenozide administered orally reduced the fecundity and fertility of adults treated with 1, 5 and 10 mg litre(-1); longevity was not affected. An LC(50) value of 4.5 mg litre(-1) was obtained when applied to eggs of less than 1 day old. Surface treatment was more effective than when applied by spraying. Administered into the diet, methoxyfenozide had a larvicidal effect; older larvae were more susceptible than younger larvae, with LC(50) values of 0.1 mg litre(-1) for L(1), 0.04 for L(3) and 0.02 for L(5). Larvae treated with sub-lethal doses throughout their lives did not emerge as adults at the highest doses (0.08, 0.04, 0.02 and 0.01 mg litre(-1)), with 65% and 40% emergence occurring for the lowest (0.005 and 0.0025 mg litre(-1)). Mortality occurred only in the larval stage.

  2. Lethal and sublethal effects of methoxyphenozide on the development, survival and reproduction of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

    Energy Technology Data Exchange (ETDEWEB)

    Zarate, N.; Diaz, O. [Universidad Autonoma de San Luis Potosi (Mexico). Facultad de Agronomia; Martinez, A.M.; Figueroa, J.I.; Pineda, S., E-mail: spineda_us@yahoo.co [Universidad Michoacana de San Nicolas de Hidalgo, Tarimbaro, Michoacan (Mexico). Inst. de Investigaciones Agropecuarias y Forestales; Schneider, M.I. [National Council of Scientific and Technical Research (CEPAVE/CCT/CONICET), La Plata (Argentina). Centro de Estudios Parasitologicos y de Vectores. Centro Cientifico Tecnologico; Smagghe, G. [Ghent University, Ghent (Belgium). Faculty of Bioscience Engineering. Lab of Agrozoology; Vinuela, E.; Budia, F. [Escuela Tecnica Superior de Ingenieros Agronomos, Madrid (Spain). Escuela Tecnica Superior de Ingenieros Agronomos. Proteccion de Cultivos

    2011-01-15

    The lethal and sublethal effects of the ecdysone agonist methoxyphenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith), were investigated by feeding a methoxyphenozide-treated diet to fifth instars until pupation in doses corresponding to the LC{sub 10} and LC{sub 25} for the compound. Larval mortality reached 8% and 26% in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12% for the LC{sub 10} and 60% for the LC{sub 25} was observed before pupation. Treated larvae exhibited lower pupal weights, higher pupal mortality, presence of deformed pupae, and more deformed adults than untreated larvae. The incorporation of methoxyfenozide into the diet had a significant effect on the timing of larval development. The development period for males and females was about seven days longer than the controls for both concentrations tested. In contrast, the compound affected neither pupae nor adult longevity. Finally, S. frugiperda adults that resulted from fifth instars treated with methoxyfenozide were not affected in their mean cumulative number of eggs laid per female (fecundity), nor percentages of eggs hatched (fertility), or the sex ratio. Our results suggest that the combination of lethal and sublethal effects of methoxyfenozide may have important implications for the population dynamics of the fall armyworm. (author)

  3. Dopamine is a key regulator in the signalling pathway underlying predator-induced defences in Daphnia.

    Science.gov (United States)

    Weiss, Linda C; Leese, Florian; Laforsch, Christian; Tollrian, Ralph

    2015-10-07

    The waterflea Daphnia is a model to investigate the genetic basis of phenotypic plasticity resulting from one differentially expressed genome. Daphnia develops adaptive phenotypes (e.g. morphological defences) thwarting predators, based on chemical predator cue perception. To understand the genomic basis of phenotypic plasticity, the description of the precedent cellular and neuronal mechanisms is fundamental. However, key regulators remain unknown. All neuronal and endocrine stimulants were able to modulate but not induce defences, indicating a pathway of interlinked steps. A candidate able to link neuronal with endocrine responses is the multi-functional amine dopamine. We here tested its involvement in trait formation in Daphnia pulex and Daphnia longicephala using an induction assay composed of predator cues combined with dopaminergic and cholinergic stimulants. The mere application of both stimulants was sufficient to induce morphological defences. We determined dopamine localization in cells found in close association with the defensive trait. These cells serve as centres controlling divergent morphologies. As a mitogen and sclerotization agent, we anticipate that dopamine is involved in proliferation and structural formation of morphological defences. Furthermore, dopamine pathways appear to be interconnected with endocrine pathways, and control juvenile hormone and ecdysone levels. In conclusion, dopamine is suggested as a key regulator of phenotypic plasticity. © 2015 The Author(s).

  4. Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy.

    Science.gov (United States)

    Martínez-Andújar, Cristina; Ordiz, M Isabel; Huang, Zhonglian; Nonogaki, Mariko; Beachy, Roger N; Nonogaki, Hiroyuki

    2011-10-11

    Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

  5. Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

    Science.gov (United States)

    Ameku, Tomotsune; Niwa, Ryusuke

    2016-06-01

    Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

  6. Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila.

    Science.gov (United States)

    Venkatesh, K; Siddhartha, G; Joshi, R; Patel, S; Hasan, G

    2001-05-01

    Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.

  7. Cross-talk between the fat body and brain regulates insect developmental arrest

    Science.gov (United States)

    Xu, Wei-Hua; Lu, Yu-Xuan; Denlinger, David L.

    2012-01-01

    Developmental arrest, a critical component of the life cycle in animals as diverse as nematodes (dauer state), insects (diapause), and vertebrates (hibernation), results in dramatic depression of the metabolic rate and a profound extension in longevity. Although many details of the hormonal systems controlling developmental arrest are well-known, we know little about the interactions between metabolic events and the hormones controlling the arrested state. Here, we show that diapause is regulated by an interplay between blood-borne metabolites and regulatory centers within the brain. Gene expression in the fat body, the insect equivalent of the liver, is strongly suppressed during diapause, resulting in low levels of tricarboxylic acid (TCA) intermediates circulating within the blood, and at diapause termination, the fat body becomes activated, releasing an abundance of TCA intermediates that act on the brain to stimulate synthesis of regulatory peptides that prompt production of the insect growth hormone ecdysone. This model is supported by our success in breaking diapause by injecting a mixture of TCA intermediates and upstream metabolites. The results underscore the importance of cross-talk between the brain and fat body as a regulator of diapause and suggest that the TCA cycle may be a checkpoint for regulating different forms of animal dormancy. PMID:22912402

  8. Biorational agents--mechanism and importance in IPM and IRM programs for controlling agricultural pests.

    Science.gov (United States)

    Ishaaya, I; Kontsedalov, S; Mazirov, D; Horowitz, A R

    2001-01-01

    Among the new approaches for controlling agricultural pests is the development of novel compounds affecting specific processes in insects such as chitin synthesis inhibitors, juvenile hormone mimics and ecdysone agonists. In addition, efforts have been made to develop compounds acting selectively on groups of insects by inhibiting or enhancing biochemical sites such as respiration (diafenthiuron), the nicotinyl acetylcholine receptors (imidacloprid and acetamiprid), the GABA receptors (avermectins), the salivary glands of sucking pests (pymetrozine) and others. Among the most recent novel insecticides with selective properties are novaluron, thiamethoxam, emamectin and spinosad. Novaluron (Rimon) is a novel chitin synthesis inhibitor that acts by both ingestion and contact. It is a powerful suppressor of lepidopteran larvae such as Spodoptera littoralis and Helicoverpa armigera (by ingestion) and of whiteflies such as Bemisia tabaci and Trialeurodes vaporariorum (by contact). Thiamethoxam (Actarn), a novel neonicotinoid acts specifically on aphids and whiteflies. Emamectin (Proclaim), an avermectin derivative acts on GABA receptor affecting diversity of insects such as mites, lepidopterans and thrips. Spinosad (Tracer) seems to act on both acetylcholine and GABA receptors affecting diversity of insect species and is considered an important agent for controlling the western flower thrips.

  9. Desiccation stress induces developmental heterochrony in Drosophila melanogaster

    Indian Academy of Sciences (India)

    LEENA THORAT; DASHARATH P OULKAR; KAUSHIK BANERJEE; BIMALENDU B NATH

    2016-09-01

    Stressful environments are known to perturb developmental patterns in insects. In the purview of desiccation as astressor, relatively little is known about the developmental consequences linked with desiccation tolerance. In thisstudy, we have particularly focused on the exploration of the temporal profile of postembryonic development inresponse to desiccation exposure in Drosophila melanogaster and the associated trade-offs. We document a correlationbetween variations in 20-hydroxyecdysone levels and the altered timing of metamorphic events during the lifecycle. Following desiccation, we observed an extension in the larval longevity whereas the duration of the pupal andadult stages was significantly shortened. Alternately, feeding of 20-hydroxyecdysone apparently led to the restorationof the normal temporal pattern of development in the desiccated group. In spite of the desiccation-responsiveheterochronic shifts in development, the overall lifespan post recovery remained almost unaltered among thedesiccated and undesiccated groups suggesting plasticity in developmental control. This observation reminisces ‘canalization-like’ phenomenon that buffers alterations in the overall lifespan. We thus identified a desiccation responsiveperiod in the lifespan of D. melanogaster during which variations in ecdysone levels are capable to alter thetemporal course of development.

  10. Endocrine regulation of predator-induced phenotypic plasticity.

    Science.gov (United States)

    Dennis, Stuart R; LeBlanc, Gerald A; Beckerman, Andrew P

    2014-11-01

    Elucidating the developmental and genetic control of phenotypic plasticity remains a central agenda in evolutionary ecology. Here, we investigate the physiological regulation of phenotypic plasticity induced by another organism, specifically predator-induced phenotypic plasticity in the model ecological and evolutionary organism Daphnia pulex. Our research centres on using molecular tools to test among alternative mechanisms of developmental control tied to hormone titres, receptors and their timing in the life cycle. First, we synthesize detail about predator-induced defenses and the physiological regulation of arthropod somatic growth and morphology, leading to a clear prediction that morphological defences are regulated by juvenile hormone and life-history plasticity by ecdysone and juvenile hormone. We then show how a small network of genes can differentiate phenotype expression between the two primary developmental control pathways in arthropods: juvenoid and ecdysteroid hormone signalling. Then, by applying an experimental gradient of predation risk, we show dose-dependent gene expression linking predator-induced plasticity to the juvenoid hormone pathway. Our data support three conclusions: (1) the juvenoid signalling pathway regulates predator-induced phenotypic plasticity; (2) the hormone titre (ligand), rather than receptor, regulates predator-induced developmental plasticity; (3) evolution has favoured the harnessing of a major, highly conserved endocrine pathway in arthropod development to regulate the response to cues about changing environments (risk) from another organism (predator).

  11. Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster

    Indian Academy of Sciences (India)

    Madhu G Tapadia; Naveen K Gautam

    2011-08-01

    Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of -tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

  12. Phytoecdysteroids of the East Asian Caryophyllaceae

    Directory of Open Access Journals (Sweden)

    Elena Novozhilova

    2015-01-01

    Full Text Available Background: Occurrence of integristerone A (1, 20-hydroxyecdysone (2, ecdysone (3, 2-deoxy-20-hydroxyecdysone (4 has been analyzed in 64 species of the East Asian Caryophyllaceae. Materials and Methods: Ecdysteroid content was determinate by high-performance liquid chromatography (HPLC. HPLC with a high-resolution mass spectrometry was performed on Shimadzu LCMS-IT-TOF (Japan system equipped with a LC-20A Prominence liquid chromatograph, a photodiode array detector SPD-M20A and ion-trap/time-of-flight mass spectrometer. Results: New sources of phytoecdysteroids: Melandrium sachalinense and Melandrium firmum have been revealed. It is the 1 st time that two has been identified in M. sachalinense and M. firmum; 1 in the species: Lychnis fulgens, Silene repens, Silene foliosa, Silene stenophylla, Silene jenisseensis and M. sachalinense; 3 in Lychnis cognata; 4 in L. fulgens, S. stenophylla and S. jenisseensis (the tribe Lychnideae, the subfamily Caryophylloideae. Ecdysteroid-negative taxa are Spergularia rubra of the tribe Sperguleae; species of the genera Minuartia, Honckenya, Eremogone, Arenaria, Moehringia, Pseudostellaria, Fimbripetalum, Stellaria and Cerastium of the tribe Alsineae; Scleranthus annuus of the tribe Sclerantheae, as well as the East Asian representatives of the genera Gypsophila, Psammophiliela, Dianthus and Saponaria of the tribe Diantheae; Oberna and Agrostemma of the tribe Lychnideae. Conclusion: This investigation shows the most promising sources of ecdysteriods are species of genera Silene and Lychnis.

  13. Cysteine Proteinase-1 and Cut Protein Isoform Control Dendritic Innervation of Two Distinct Sensory Fields by a Single Neuron

    Directory of Open Access Journals (Sweden)

    Gray R. Lyons

    2014-03-01

    Full Text Available Dendrites often exhibit structural changes in response to local inputs. Although mechanisms that pattern and maintain dendritic arbors are becoming clearer, processes regulating regrowth, during context-dependent plasticity or after injury, remain poorly understood. We found that a class of Drosophila sensory neurons, through complete pruning and regeneration, can elaborate two distinct dendritic trees, innervating independent sensory fields. An expression screen identified Cysteine proteinase-1 (Cp1 as a critical regulator of this process. Unlike known ecdysone effectors, Cp1-mutant ddaC neurons pruned larval dendrites normally but failed to regrow adult dendrites. Cp1 expression was upregulated/concentrated in the nucleus during metamorphosis, controlling production of a truncated Cut homeodomain transcription factor. This truncated Cut, but not the full-length protein, allowed Cp1-mutant ddaC neurons to regenerate higher-order adult dendrites. These results identify a molecular pathway needed for dendrite regrowth after pruning, which allows the same neuron to innervate distinct sensory fields.

  14. Temporal Coordination of Carbohydrate Metabolism during Mosquito Reproduction.

    Directory of Open Access Journals (Sweden)

    Yuan Hou

    2015-07-01

    Full Text Available Hematophagous mosquitoes serve as vectors of multiple devastating human diseases, and many unique physiological features contribute to the incredible evolutionary success of these insects. These functions place high-energy demands on a reproducing female mosquito, and carbohydrate metabolism (CM must be synchronized with these needs. Functional analysis of metabolic gene profiling showed that major CM pathways, including glycolysis, glycogen and sugar metabolism, and citrate cycle, are dramatically repressed at post eclosion (PE stage in mosquito fat body followed by a sharply increase at post-blood meal (PBM stage, which were also verified by Real-time RT-PCR. Consistent to the change of transcript and protein level of CM genes, the level of glycogen, glucose and trehalose and other secondary metabolites are also periodically accumulated and degraded during the reproductive cycle respectively. Levels of triacylglycerols (TAG, which represent another important energy storage form in the mosquito fat body, followed a similar tendency. On the other hand, ATP, which is generated by catabolism of these secondary metabolites, showed an opposite trend. Additionally, we used RNA interference studies for the juvenile hormone and ecdysone receptors, Met and EcR, coupled with transcriptomics and metabolomics analyses to show that these hormone receptors function as major regulatory switches coordinating CM with the differing energy requirements of the female mosquito throughout its reproductive cycle. Our study demonstrates how, by metabolic reprogramming, a multicellular organism adapts to drastic and rapid functional changes.

  15. Molecular characterization and developmental expression of the gene encoding the prothoracicotropic hormone in the beet armyworm,Spodoptera exigua

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Prothoracicotropic hormone (PTTH), a neuropeptide hormone stimulating the prothoracic glands to synthesize ecdysone, plays an important role in regulating postembryonic development in insects. The cDNA encoding PTTH was isolated and sequenced from the beet armyworm, Spodoptera exigua (Spe). The deduced a?mino acid sequence is composed of a signal peptide, a peptide (65 amino acids) of un-known function, and a mature PTTH molecule (111 amino acids). The Spe-PTTH shows similarities (45.5%―70.3%) to other known PTTHs reported in Lepidoptera species, but 7 cysteine r?esidues and the hydrophobic regions were conserved. Whole-mount immunocytochemistry by using an antiserum against recombinant Helicoverpa armigera PTTH showed that Spe-PTTH was synthesized in two pairs of neurosecretory cells in the S. exigua brain. Northern blot analysis demonstrates the presence of a 1.2-kb transcript in the brain. The Spe-PTTH mRNA is detectable at high levels at the wandering larval stage, early pupal stage, and pharate adult stage, suggesting that the Spe-PTTH gene might be corre-lated with molting, metamorphosis, and reproduction.

  16. Molecular characterization and developmental expression of the gene encoding the prothoracicotropic hormone in the beet armyworm, Spodoptera exigua

    Institute of Scientific and Technical Information of China (English)

    XU Jun; SU JianYa; SHEN JinLiang; XU WeiHua

    2007-01-01

    Prothoracicotropic hormone (PTTH), a neuropeptide hormone stimulating the prothoracic glands to synthesize ecdysone, plays an important role in regulating postembryonic development in insects. The cDNA encoding PTTH was isolated and sequenced from the beet armyworm, Spodoptera exigua (Spe).The deduced amino acid sequence is composed of a signal peptide, a peptide (65 amino acids) of unknown function, and a mature PTTH molecule (111 amino acids). The Spe-PTTH shows similarities(45.5%-70.3%) to other known PTTHs reported in Lepidoptera species, but 7 cysteine residues and the hydrophobic regions were conserved. Whole-mount immunocytochemistry by using an antiserum against recombinant Helicoverpa armigera PTTH showed that Spe-PTTH was synthesized in two pairs of neurosecretory cells in the S. exigua brain. Northern blot analysis demonstrates the presence of a 1.2-kb transcript in the brain. The Spe-PTTH mRNA is detectable at high levels at the wandering larval stage, early pupal stage, and pharate adult stage, suggesting that the Spe-PTTH gene might be correlated with molting, metamorphosis, and reproduction.

  17. A new antigenic marker specifically labels a subpopulation of the class II Kenyon cells in the brain of the European honeybee Apis mellifera.

    Science.gov (United States)

    Watanabe, Takayuki; Kubo, Takeo

    2015-01-01

    The mushroom bodies are the higher-order integration center in the insect brain and are involved in higher brain functions such as learning and memory. In the social hymenopteran insects such as honeybees, the mushroom bodies are the prominent brain structures. The mushroom bodies are composed of lobed neuropils formed by thousands of parallel-projecting axons of intrinsic neurons, and the lobes are divided into parallel subdivisions. In the present paper, we report a new antigenic marker to label a single layer in the vertical lobes of the European honeybee Apis mellifera. In the brain of A. mellifera, a monoclonal antibody (mAb) 15C3, which was originally developed against an insect ecdysone receptor (EcR) protein, immunolabels a single layer of the vertical lobes that correspond to the most dorsal layer of the γ-lobe. The 15C3 mAb recognizes a single ~200 kDa protein expressed in the adult honeybee brain. In addition, the 15C3 mAb immunoreactivity was also observed in the lobes of the developing pupal mushroom bodies. Since γ-lobe is well known to their extensive reorganization that occurs during metamorphosis in Drosophila, the novel antigenic marker for the honeybee γ-lobe allows us to investigate morphological changes of the mushroom bodies during metamorphosis.

  18. LC-MS(n) characterization of steroidal saponins in Helleborus niger L. roots and their conversion products during fermentation.

    Science.gov (United States)

    Duckstein, Sarina M; Stintzing, Florian C

    2015-01-01

    Steroidal saponins comprise a substantial part of the secondary metabolite spectrum in the medicinal plant Helleborus niger L. (black hellebore). The saponin fraction from the roots was investigated by LC-MS(n) resulting in 38 saponins and β-ecdysone. Nine diosgenyl-type glycosides, mainly furostanols consisting of the aglycones diosgenin, macranthogenin, sceptrumgenin, and sarsasapogenin were accompanied by 5 diosgenyl-type saponins exhibiting an aglycone with an additional OH group. However, the most relevant compounds were 24 acetylated polyhydroxy saponins including hellebosaponins A and D. The enzymes glucuronidase, β-glucosidase, and pectinase were used to obtain an idea on potential fermentative transformation reactions by incubation of the isolated model saponins macranthosid I and hellebosaponin A. In a second step, aqueous H. niger extracts containing a much greater range of saponins were monitored during fermentation and 12months of storage. The metabolites were examined and assigned by LC-MS(n) and targeted extracted ion current (EIC) scan analyses. Good agreement was found among the results from the model compounds and the whole aqueous fermented extracts. The native diosgenyl-type furostanol saponins were converted to spirostanols under scission of hexoses. Alteration of the acetylated polyhydroxy saponins, exclusively spirostanols, took place following cleavage of acetyl groups and terminal deoxyhexoses. Most interestingly, the pentoses of the sugar chain at C(1) were not affected. Conversion of acetylated polyhydroxy saponins resulted in a final structure type which was stable and detectable, even after 12months of fermentation and storage.

  19. Drosophila eggshell production: identification of new genes and coordination by Pxt.

    Directory of Open Access Journals (Sweden)

    Tina L Tootle

    Full Text Available Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals.

  20. Genomic sequence around butterfly wing development genes: annotation and comparative analysis.

    Directory of Open Access Journals (Sweden)

    Inês C Conceição

    conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation.

  1. Organic solvents impair life-traits and biomarkers in the New Zealand mudsnail Potamopyrgus antipodarum (Gray) at concentrations below OECD recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Lecomte, V.; Noury, P.; Tutundjian, R. [Irstea, UR MAEP, Laboratoire d’écotoxicologie, 5 rue de la Doua, CS70077, 69626 Villeurbanne Cedex (France); Buronfosse, T. [VetAgro-Sup, Campus vétérinaire, Endocrinology Laboratory, 69280 Marcy l’Etoile (France); Garric, J. [Irstea, UR MAEP, Laboratoire d’écotoxicologie, 5 rue de la Doua, CS70077, 69626 Villeurbanne Cedex (France); Gust, M., E-mail: marion.gust@irstea.fr [Irstea, UR MAEP, Laboratoire d’écotoxicologie, 5 rue de la Doua, CS70077, 69626 Villeurbanne Cedex (France)

    2013-09-15

    Highlights: •Acetone (20 μl l{sup −1}) accelerates embryonic development in Potamopyrgus antipodarum. •Ethanol (20 μl l{sup −1}) decreases growth in juvenile mudsnails. •Acetone, ethanol, methanol and DMSO increase E2 levels in snails. •Carrier solvents impair gene expression. •DMSO is to be preferred. -- Abstract: Potamopyrgus antipodarum is a gastropod mollusk proposed for use in the development of reproduction tests within the Organization for Economic Cooperation and Development (OECD). Numerous chemicals, including endocrine disrupters, are relatively water-insoluble, and water-miscible solvents are currently used for testing them. OECD recommends a maximum concentration of 100 μl l{sup −1}. As several studies highlighted effects of lower concentrations of solvents, this study assessed the effects of 20 μl l{sup −1} acetone, ethanol, methanol and dimethylsulfoxide (DMSO) on juvenile and adult snails during 42 days. Ethanol decreased juvenile growth, while acetone increased the rate of embryonic development. All solvents increased estradiol-like levels in adult snails. DMSO only increased mRNA expression of vitellogenin-like gene, while acetone, ethanol and methanol decreased mRNA expression of three nuclear receptor (estrogen receptor-like, ecdysone-induced protein and chicken ovalbumin upstream promoter transcription factor) genes as well as of genes encoding proteins involved in genomic (prohibitin-2) and non-genomic (striatin) pathways of estrogens activity in vertebrates. This study highlights the confounding effects of low concentrations of solvents and recommends avoiding their use. Where solvent use is inevitable, their concentrations and type should be investigated for suitability for the measured endpoints prior to use in chemical testing strategies.

  2. Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

    Science.gov (United States)

    Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A; Pan, Wei-Dong; Chen, Fa-Jun

    2015-01-01

    Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

  3. Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

    Directory of Open Access Journals (Sweden)

    Gui-Jun Wan

    Full Text Available Although there are considerable reports of magnetic field effects (MFE on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i CRY1 and CRY2 as putative magnetosensors, (ii JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii CYP307A1 in the ecdysone pathway, and (iv reproduction-related Vitellogenin (Vg. The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

  4. Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper

    Science.gov (United States)

    Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A.; Pan, Wei-Dong; Chen, Fa-Jun

    2015-01-01

    Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE. PMID:26173003

  5. Evaluation of halofenozide against prey mosquito larvae Culex pipiens and the predator fish Gambusia affinis: impact on growth and enzymatic activities.

    Science.gov (United States)

    Soltani, N; Chouahda, S; Smagghe, G

    2008-01-01

    Culex pipiens (Diptera: Culicidae) is the most widely distributed mosquito species in Algeria and many other countries in the world. Mosquitoes are generally controlled by conventional insecticides but these may pose strong secondary effects on the environment. In this context, the insect growth regulators (IGRs) have shown promise in controlling pest insects. Halofenozide (23% EC) is a novel IGRs belonging to the class of non-steroidal ecdysone agonists, and it was found toxic for larvae of C. pipiens. In addition biological methods constitute an alternative to chemical control. Several fish species have been tested against mosquitoes, and Gambusia affinis was found very efficient. In the present study we evaluated the impact of this new potent insecticide (halofenozide) on growth and metric indexes in the larvivorous fish G. affinis under laboratory conditions. In addition, the effects were evaluated on the enzymatic activities of acetyl cholinesterase (AChE) and glutathione S-transferase (GST). The insecticide was added in water at two concentrations (12.6 and 28.6 microg/L) corresponding to the LC50 and LC90 obtained against fourth instar larvae of C. pipiens, and adult females of G. affinis were exposed to halofenozide for 30 days. At different exposure times we measured the length and weight of fishes, the index of condition (K), the gonado-somatic ratio (GSR) and the hepato-somatic ratio (HSR). The results showed that halofenozide had no significant (p>0.05) effects on growth, metric indexes and AChE activities. However, treatment caused a significant induction (p<0.05) in GST activities at days 15 and 30 with the highest dose. Our results indicate that this ecdysteroid agonist presented only minor secondary effects on the non-target fish species, and so it has potential for controlling of mosquitoes in an integrated manner.

  6. Ovicidal activity of three insect growth regulators against Aedes and Culex mosquitoes.

    Science.gov (United States)

    Suman, Devi S; Wang, Yi; Bilgrami, Anwar L; Gaugler, Randy

    2013-10-01

    Interspecific variations in the susceptibility of freshly and embryonated eggs of Aedes albopictus, Ae. aegypti, Ae. atropalpus and Culex pipiens were tested against three classes of insect growth regulators (IGRs) including ecdysone agonist (azadirachtin), chitin synthesis inhibitor (diflubenzuron) and juvenile hormone analog (pyriproxyfen) at 0.001, 0.01, 0.1 and 1.0ppm concentrations. Egg hatching inhibition was dose dependent, the highest being at 1.0ppm concentration for freshly laid eggs of Ae. albopictus (pyriproxyfen: 80.6%, azadirachtin: 42.9% and diflubenzuron: 35.8%). Aedes aegypti showed lower egg hatching inhibition when exposed to pyriproxyfen (47.3%), azadirachtin (15.7%) and diflubenzuron (25.5%). Freshly laid eggs of Cx. pipiens were most susceptible to diflubenzuron. Aedes atropalpus eggs were tolerant to all three classes of IGRs. Embryonated eggs of Ae. albopictus, Ae. aegypti, Ae. atropalpus and Cx. pipiens were resistant to pyriproxyfen, azadirachtin and diflubenzuron than freshly laid eggs. The median desiccation time (DT50) of Ae. atropalpus eggs was maximum (5.1h) as compared to Ae. aegypti (4.9h), Ae. albopictus (3.9h) or Cx. pipiens (1.7h) eggs. Insignificant relationship between the rates of desiccation and egg hatching inhibition suggests other factors than physical providing eggs the ability to tolerate exposures to various IGRs. Egg hatching inhibition was due to the alteration in embryonic development caused by IGRs. Changes in the egg shell morphology and abnormal egg hatching from the side of the egg wall instead of operculum, was observed at higher concentrations of diflubenzuron. Morphological and physiological variations in eggs may be the key factor to influence the ovicidal efficacy of IGRs. The present data provide a base line for the improvement of the ovicidal efficacy of the insecticide and its formulation.

  7. Effects of juvenile hormone analogue on ecdysis prevention induced by precocene in Rhodnius prolixus (Hemiptera: Reduviidae

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    Patricia de Azambuja

    1984-12-01

    Full Text Available Precocene II, added to the meal of fourth-instar larvae of Rhodnius prolixus (25 mug/ml of blood, induced an in crease in the duration of the molting cycle. This effect was related to the decrease of both the nuclear area of the prothoracic gland cells and the mitotic activity in epidermal cellS. juvenile hormone analogue applied topically (60 mug/insect together with Precocene II treatment avoided atrophy of the prothoracic glands and induced a higher number of epidermal mitosis accelerating the time of subsequent ecdysis. A possible relationship between juvenile hormone and production of ecdysone is discussed.Adicionado ao sangue alimentar na dose de 25 mug/ml o precoceno II causou um aumento no período de intermuda em ninfas de 4o. estadio de Rhodnius prolixus. Este atraso da muda foi relacionado com a diminuição da área dos núcleos das celulas das glandulas protoracicas e com a queda da atividade mitotica das células da epiderme do inseto. Um análogo de hormônio juvenil aplicado topicamente (60 mug/inseto junto com o tratamento oral com precoceno II preveniu a atrofia das glândulas protorácicas e induziu um aumento no número de mitoses nas células da epiderme, diminuindo o período de intermuda nestes insetos.A possivel relação entre a ação do hormônio juvenil e a producao de ecdisona pelas glândulas protorácicas e discutida.

  8. The effects of binary UV filter mixtures on the midge Chironomus riparius.

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    Ozáez, Irene; Morcillo, Gloria; Martínez-Guitarte, José-Luis

    2016-06-15

    Organic ultraviolet (UV) filters are used in a wide variety of products, including cosmetics, to prevent damage from UV light in tissues and industrial materials. Their extensive use has raised concerns about potential adverse effects in human health and aquatic ecosystems that accumulate these pollutants. To increase sun radiation protection, UV filters are commonly used in mixtures. Here, we studied the toxicity of binary mixtures of 4-methylbenzylidene camphor (4MBC), octyl-methoxycinnamate (OMC), and benzophenone-3 (BP-3), by evaluating the larval mortality of Chironomus riparius. Also molecular endpoints have been analyzed, including alterations in the expression levels of a gene related with the endocrine system (EcR, ecdysone receptor) and a gene related with the stress response (hsp70, heat shock protein 70). The results showed that the mortality caused by binary mixtures was similar to that observed for each compound alone; however, some differences in LC50 were observed between groups. Gene expression analysis showed that EcR mRNA levels increased in the presence of 0.1mg/L 4MBC but returned to normal levels after exposure to mixtures of 4MBC with 0.1, 1, and 10mg/L of BP-3 or OMC. In contrast, the hsp70 mRNA levels increased after exposure to the combinations tested of 4MBC and BP-3 or OMC mixtures. These data suggest that 4MBC, BP-3, and OMC may have antagonist effects on EcR gene transcription and a synergistic effect on hsp70 gene activation. This is the first experimental study to show the complex patterned effects of UV filter mixtures on invertebrates. The data suggest that the interactions within these chemicals mixtures are complex and show diverse effects on various endpoints.

  9. Introduction In Vitro Culture Withania Somnifera for Obtain Secondary Metabolites and Further Study

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    R.E. PAVLIKOV

    2014-06-01

    Full Text Available Ashwagandha is a branching erect shrub height from 30 to 150 cm covered with thick vegetation down (tomentosa. The leaves are oval, up to 10 cm in length and 2-2.5 cm in width.Green or yellow flowers of which then grow red fruit. Ashwagandha roots fleshy, cylindrical, white or light brown in color. Ashwagandha is found in arid parts of India, Asia and Northern Africa.Ashwagandha contains steroidal compounds are of great interest for researchers, is: steroidal lactones ergostanovogo type, in particular vitanolidy A-Y, degidrovitanolid -R, vitasomniferin -A, vitasomidienon, vitasomniferols A-C, vitaferin A, vitanon and etc. In addition, Ashwagandha contains fitosterolysitoindozidy VII - X and beta-sitosterol, alkaloids (ashvagandin, kuskohigrin, Tropin, psevdotropin, izopellitierin, anaferin, a variety of amino acids, including tryptophan, as well as large amounts of iron.Vitanolidy (withanolides - fitosteroidov group representing unsaturated steroidal lactones polioksisteroidy. The first compound of this class - vitaferin A - has been allocated in the 60s. the last century of the Indian plant Withania somnifera (fam. Solanaceae. Currently, there are several rows of this class of compounds (somniferin, vitanon. Used as biologically active food supplements. Found in Physalis have anti-inflammatory, analgesic and anti-cancer effects.Interest steroidal lactone acting opposite ecdysone (molting hormones on insects by inhibiting the molting.Ashwagandha has: antioxidant, toning, anti-aging, antiseptic, blood purifier, anti-viral, anti-inflammatory, immunomodulatory, healing, anabolic properties.An aqueous extract of Ashwagandha root to prevent the development of lipid peroxidation caused deliberately for experimental purposes in rats and mice (Dhuley 1998a. Withania somnifera extract with an equimolar concentration sitoindozidov VII - X and vitaferina A, caused an increase in the level of superoxide dismutase, catalase, glutathione peroxidase and in

  10. Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae).

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    Candido-Silva, Juliana Aparecida; Machado, Maiaro Cabral Rosa; Hartfelder, Klaus Hartmann; de Almeida, Jorge Cury; Paçó-Larson, Maria Luisa; Monesi, Nadia

    2015-03-01

    The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.

  11. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

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    Li Xianchun

    2007-03-01

    Full Text Available Abstract Background Transposons, i.e. transposable elements (TEs, are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements, SINEs (short interspersed nuclear elements, MITEs (miniature inverted-repeat transposable elements, one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1 implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1 involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes.

  12. Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics.

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    Giraudo, Maeva; Audant, Pascaline; Feyereisen, René; Le Goff, Gaëlle

    2013-05-01

    The fall armyworm Spodoptera frugiperda is a major polyphagous pest in agriculture and little is known on how this insect can adapt to the diverse and potentially toxic plant allelochemicals that they ingest or to insecticides. To investigate the involvement of nuclear receptors in the response of S. frugiperda to its chemical environment, we cloned SfHR96, a nuclear receptor orthologous to the mammalian xenobiotic receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also cloned ultraspiracle (USP), the ortholog of retinoid X receptor (RXR) that serves as partner of dimerization of PXR and CAR. Cloning of SfUSP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. The expression of these receptors as well as the ecdysone receptor was studied during specific steps of development in different tissues. SfHR96 was constitutively expressed in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupal stage, as well as in Sf9 cells. EcR and SfUSP-2 showed peaks of expression before larval moults and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Receptor induction was followed after exposure of larvae or cells to 11 chemical compounds. SfHR96 was not inducible by the tested compounds. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene. The cloning of these nuclear receptors is a first step in understanding the important capacities of adaptation of this insect pest.

  13. Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata.

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    Poupardin, Rodolphe; Schöttner, Konrad; Korbelová, Jaroslava; Provazník, Jan; Doležel, David; Pavlinic, Dinko; Beneš, Vladimír; Koštál, Vladimír

    2015-09-21

    Diapause is a developmental alternative to direct ontogeny in many invertebrates. Its primary adaptive meaning is to secure survival over unfavourable seasons in a state of developmental arrest usually accompanied by metabolic suppression and enhanced tolerance to environmental stressors. During photoperiodically triggered diapause of insects, the ontogeny is centrally turned off under hormonal control, the molecular details of this transition being poorly understood. Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction in the larvae of the drosophilid fly Chymomyza costata with the goal of identifying candidate genes and processes linked to upstream regulatory events that eventually lead to a complex phenotypic change. Short day photoperiod triggering diapause was associated to inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development. The mRNA levels of several key genes involved in 20-HE biosynthesis, perception, and signalling were significantly downregulated under short days. Hormonal change was translated into downregulation of a series of other transcripts with broad influence on gene expression, protein translation, alternative histone marking by methylation and alternative splicing. These changes probably resulted in blockade of direct development and deep restructuring of metabolic pathways indicated by differential expression of genes involved in cell cycle regulation, metabolism, detoxification, redox balance, protection against oxidative stress, cuticle formation and synthesis of larval storage proteins. This highly complex alteration of gene transcription was expressed already during first extended night, within the first four hours after the change of the photoperiodic signal from long days to short days. We validated our RNAseq differential gene expression results in an independent qRT-PCR experiment involving wild

  14. Genome-wide tissue-specific gene expression, co-expression and regulation of co-expressed genes in adult nematode Ascaris suum.

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    Bruce A Rosa

    2014-02-01

    Full Text Available BACKGROUND: Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus. We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. METHODOLOGY/PRINCIPAL FINDINGS: Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. CONCLUSIONS/SIGNIFICANCE: The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis

  15. Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

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    Techa, Sirinart; Chung, J Sook

    2015-01-01

    Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

  16. Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH expression in the blue crab, Callinectes sapidus.

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    Sirinart Techa

    Full Text Available Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH and crustacean hyperglycemic hormone (CHH. Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w ratio. Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

  17. Ras-like family small GTPases genes in Nilaparvata lugens: Identification, phylogenetic analysis, gene expression and function in nymphal development

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    Wang, Weixia; Li, Kailong; Wan, Pinjun; Lai, Fengxiang; Fu, Qiang; Zhu, Tingheng

    2017-01-01

    Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control. PMID:28241066

  18. Neverland regulates embryonic moltings through the regulation of ecdysteroid synthesis in the water flea Daphnia magna, and may thus act as a target for chemical disruption of molting.

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    Sumiya, Eri; Ogino, Yukiko; Toyota, Kenji; Miyakawa, Hitoshi; Miyagawa, Shinichi; Iguchi, Taisen

    2016-11-01

    Embryo development in arthropods is accompanied by a series of moltings. A cladoceran crustacean Daphnia magna molts three times before reaching first instar neonate during embryogenesis. Previous studies argued ecdysteroids might regulate D. magna embryogenesis. However, no direct evidence between innate ecdysteroids fluctuation and functions has been forthcoming. Recently, we identified genes involved in ecdysteroid synthesis called, neverland (neverland1 and neverland 2) and shade and in the ecdysteroid degradation (Cyp18a1). To understand the physiological roles of ecdysteroids in D. magna embryos, we performed expression and functional analyzes of those genes. Examining innate ecdysteroids titer during embryogenesis showed two surges of ecdysteroids titer at 41 and 61 h after oviposition. The first and second embryonic moltings occurred at each ecdysteroid surge. Expression of neverland1 and shade began to increase before the first peak in ecdysteroid. Knockdown of neverland1 or shade by RNAi technique caused defects in embryonic moltings and subsequent development. The ecdysteroids titer seemingly decreased in nvd1-knowckdown embryos. Knockdown of Cyp18a1 resulted in early embryonic lethality before the first molting. Our in situ hybridization analysis revealed that nvd1 was prominently expressed in embryonic gut epithelium suggesting the site for an initial step of ecdysteroidgenesis, a conversion of cholesterol to 7-dehydrocholesterol and possibly for ecdysone production. Taken together, de novo ecdysteroid synthesis by nvd1 in the gut epithelial cells stimulates molting, which is indispensable for D. magna embryo development. These findings identify neverland as a possible target for chemicals, including various pesticides that are known to disrupt molting, development and reproduction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  19. [Impact of microsporidia on hormonal balance in insect hosts].

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    Issi, I V; Tokarev, Iu S

    2002-01-01

    Microsporidia (M) is a phylum of protists parasitizing obligatory in animal cells. Long way of adaptation of M to intracellular parasitism resulted in establishment of quite close relationships between the parasite and its host. Different species of M induce in their hosts symptoms similar to those caused by misbalance of juvenile hormone (JH) and ecdysone. M infection leads to pathology of different hormone-dependent functions such as cell differentiation and specialization, molting, metamorphosis, diapause and reproduction of insects. The signs of hormonal dysfunction evidence for elevated titer of JH in M-infected insects. Two possible explanation of this could be offered: JH secretion by M or specific influence of the parasites on the insect endocrine systems. Impact on insect endogenous JH titer by M could be mediated by affection of secretory activity of corpora allata or by suppression of enzymatic degradation of JH. According to different hypotheses, insect hormonal status during microsporidiosis could be modified by a) insect host stress-reaction, b) exhaustion of insect host reserves, characteristic for acute phase of the disease, c) destruction of infected insect cells and tissues during mass sporogenesis of M. Data found in literature and provided by our experiments evidence for presence of JH analogues or juvenilizing substance in the extracts of M spores. From detailed examination of pathological process it is also seen that juvenilizing effect of M infection is usually restricted to the invaded regions of tissues (i.e. expressed locally) but not a systemic one. Ability of M to modify morpho-functional features of infected tissues at the level of hormonal regulation is undoubtfully a prominent adaptation for stabilizing "microsporidia-insect" parasite-host systems.

  20. The effects of fipronil and the photodegradation product fipronil desulfinyl on growth and gene expression in juvenile blue crabs, Callinectes sapidus, at different salinities.

    Science.gov (United States)

    Goff, Andrew D; Saranjampour, Parichehr; Ryan, Lauren M; Hladik, Michelle L; Covi, Joseph A; Armbrust, Kevin L; Brander, Susanne M

    2017-05-01

    Endocrine disrupting compounds (EDCs) are now widely established to be present in the environment at concentrations capable of affecting wild organisms. Although many studies have been conducted in fish, less is known about effects in invertebrates such as decapod crustaceans. Decapods are exposed to low concentrations of EDCs that may cause infertility, decreased growth, and developmental abnormalities. The objective herein was to evaluate effects of fipronil and its photodegradation product fipronil desulfinyl. Fipronil desulfinyl was detected in the eggs of the decapod Callinectes sapidus sampled off the coast of South Carolina. As such, to examine specific effects on C. sapidus exposed in early life, we exposed laboratory-reared juveniles to fipronil and fipronil desulfinyl for 96h at three nominal concentrations (0.01, 0.1, 0.5μg/l) and two different salinities (10, 30ppt). The size of individual crabs (weight, carapace width) and the expression of several genes critical to growth and reproduction were evaluated. Exposure to fipronil and fipronil desulfinyl resulted in significant size increases in all treatments compared to controls. Levels of expression for vitellogenin (Vtg), an egg yolk precursor, and the ecdysone receptor (EcR), which binds to ecdysteroids that control molting, were inversely correlated with increasing fipronil and fipronil desulfinyl concentrations. Effects on overall growth and on the expression of EcR and Vtg differ depending on the exposure salinity. The solubility of fipronil is demonstrated to decrease considerably at higher salinities. This suggests that fipronil and its photodegradation products may be more bioavailable to benthic organisms as salinity increases, as more chemical would partition to tissues. Our findings suggest that endocrine disruption is occurring through alterations to gene expression in C. sapidus populations exposed to environmental levels of fipronil, and that effects may be dependent upon the salinity at

  1. Binary cell fate decisions and fate transformation in the Drosophila larval eye.

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    Abhishek Kumar Mishra

    Full Text Available The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5 or the green-sensitive Rhodopsin 6 (Rh6. Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.

  2. The effects of fipronil and the photodegradation product fipronil desulfinyl on growth and gene expression in juvenile blue crabs, Callinectes sapidus, at different salinities

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    Goff, Andrew D.; Saranjampour, Parichehr; Ryan, Lauren M.; Hladik, Michelle; Covi, Joseph A.; Armbrust, Kevin L.; Brander, Susanne M.

    2017-01-01

    Endocrine disrupting compounds (EDCs) are now widely established to be present in the environment at concentrations capable of affecting wild organisms. Although many studies have been conducted in fish, less is known about effects in invertebrates such as decapod crustaceans. Decapods are exposed to low concentrations of EDCs that may cause infertility, decreased growth, and developmental abnormalities. The objective herein was to evaluate effects of fipronil and its photodegradation product fipronil desulfinyl. Fipronil desulfinyl was detected in the eggs of the decapod Callinectes sapidus sampled off the coast of South Carolina. As such, to examine specific effects on C. sapidus exposed in early life, we exposed laboratory-reared juveniles to fipronil and fipronil desulfinyl for 96 hours at three nominal concentrations (0.01, 0.1, 0.5 μg/L) and two different salinities (10, 30 ppt). The size of individual crabs (weight, carapace width) and the expression of several genes critical to growth and reproduction were evaluated. Exposure to fipronil and fipronil desulfinyl resulted in significant size increases in all treatments compared to controls. Levels of expression for vitellogenin (Vtg), an egg yolk precursor, and the ecdysone receptor (EcR), which binds to ecdysteroids that control molting, were inversely correlated with increasing fipronil and fipronil desulfinyl concentrations. Effects on overall growth and on the expression of EcR and Vtg differ depending on the exposure salinity. The solubility of fipronil is demonstrated to decrease considerably at higher salinities. This suggests that fipronil and its photodegradation products may be more bioavailable to benthic organisms as salinity increases, as more chemical would partition to tissues. Our findings suggest that endocrine disruption is occurring through alterations to gene expression in C. sapidus populations exposed to environmental levels of fipronil, and that effects may be dependent upon the

  3. Genome-Wide Responses of Female Fruit Flies Subjected to Divergent Mating Regimes.

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    Dave T Gerrard

    Full Text Available Elevated rates of mating and reproduction cause decreased female survival and lifetime reproductive success across a wide range of taxa from flies to humans. These costs are fundamentally important to the evolution of life histories. Here we investigate the potential mechanistic basis of this classic life history component. We conducted 4 independent replicated experiments in which female Drosophila melanogaster were subjected to 'high' and 'low' mating regimes, resulting in highly significant differences in lifespan. We sampled females for transcriptomic analysis at day 10 of life, before the visible onset of ageing, and used Tiling expression arrays to detect differential gene expression in two body parts (abdomen versus head+thorax. The divergent mating regimes were associated with significant differential expression in a network of genes showing evidence for interactions with ecdysone receptor. Preliminary experimental manipulation of two genes in that network with roles in post-transcriptional modification (CG11486, eyegone tended to enhance sensitivity to mating costs. However, the subtle nature of those effects suggests substantial functional redundancy or parallelism in this gene network, which could buffer females against excessive responses. There was also evidence for differential expression in genes involved in germline maintenance, cell proliferation and in gustation / odorant reception. Interestingly, we detected differential expression in three specific genes (EcR, keap1, lbk1 and one class of genes (gustation / odorant receptors with previously reported roles in determining lifespan. Our results suggest that high and low mating regimes that lead to divergence in lifespan are associated with changes in the expression of genes such as reproductive hormones, that influence resource allocation to the germ line, and that may modify post-translational gene expression. This predicts that the correct signalling of nutrient levels to the

  4. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

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    Ana Talamillo

    2013-04-01

    Full Text Available SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor, the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

  5. Protein tyrosine phosphatase encoded in Cotesia plutellae bracovirus suppresses a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Kim, Jiwan; Hepat, Rahul; Lee, Daeweon; Kim, Yonggyun

    2013-09-01

    Parasitization by an endoparasitoid wasp, Cotesia plutellae, inhibits a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella. This study tested an inhibitory effect of C. plutellae bracovirus (CpBV) on the metamorphosis of P. xylostella. Parasitized P. xylostella exhibited significantly reduced prothoracic gland (PTG) development at the last instar compared to nonparasitized larvae. Expression of the ecdysone receptor (EcR) was markedly suppressed during the last instar larvae parasitized by C. plutellae. By contrast, expression of the insulin receptor (InR) significantly increased in the parasitized larvae. Microinjection of CpBV significantly inhibited the larva-to-pupa metamorphosis of nonparasitized larvae in a dose-dependent manner. Injection of CpBV also inhibited the expression of the EcR and increased the expression of the InR. Individual CpBV segments were transiently expressed in its encoded genes in nonparasitized larvae and screened to determine antimetamorphic viral gene(s). Out of 21 CpBV segments, two viral segments (CpBV-S22 and CpBV-S27) were proved to inhibit larva-to-pupa metamorphosis by transient expression assay. RNA interference of each gene encoded in the viral segments was applied to determine antimetamorphic gene(s). Protein tyrosine phosphatase, early expressed gene, and four hypothetical genes were selected to be associated with the antimetamorphic activity of CpBV. These results suggest that antimetamorphosis of P. xylostella parasitized by C. plutellae is induced by inhibiting PTG development and subsequent ecdysteroid signaling with viral factors of CpBV.

  6. Genome-Wide Responses of Female Fruit Flies Subjected to Divergent Mating Regimes

    Science.gov (United States)

    Gerrard, Dave T.; Fricke, Claudia; Edward, Dominic A.; Edwards, Dylan R.; Chapman, Tracey

    2013-01-01

    Elevated rates of mating and reproduction cause decreased female survival and lifetime reproductive success across a wide range of taxa from flies to humans. These costs are fundamentally important to the evolution of life histories. Here we investigate the potential mechanistic basis of this classic life history component. We conducted 4 independent replicated experiments in which female Drosophila melanogaster were subjected to ‘high’ and ‘low’ mating regimes, resulting in highly significant differences in lifespan. We sampled females for transcriptomic analysis at day 10 of life, before the visible onset of ageing, and used Tiling expression arrays to detect differential gene expression in two body parts (abdomen versus head+thorax). The divergent mating regimes were associated with significant differential expression in a network of genes showing evidence for interactions with ecdysone receptor. Preliminary experimental manipulation of two genes in that network with roles in post-transcriptional modification (CG11486, eyegone) tended to enhance sensitivity to mating costs. However, the subtle nature of those effects suggests substantial functional redundancy or parallelism in this gene network, which could buffer females against excessive responses. There was also evidence for differential expression in genes involved in germline maintenance, cell proliferation and in gustation / odorant reception. Interestingly, we detected differential expression in three specific genes (EcR, keap1, lbk1) and one class of genes (gustation / odorant receptors) with previously reported roles in determining lifespan. Our results suggest that high and low mating regimes that lead to divergence in lifespan are associated with changes in the expression of genes such as reproductive hormones, that influence resource allocation to the germ line, and that may modify post-translational gene expression. This predicts that the correct signalling of nutrient levels to the

  7. Molecular mechanisms of juvenile hormone action%保幼激素的分子作用机制研究

    Institute of Scientific and Technical Information of China (English)

    周树堂; 郭伟; 宋佳晟

    2012-01-01

    Insect development, metamorphosis and reproduction are coordinated and regulated by juvenile hormone (JH) and ecdysone (20-hydroxyecdysone, 20E). However, the molecular mechanisms of JH are poorly understood compared with those of 20E. There has been much research undertaken to identify the bona fide JH nuclear receptor, to elucidate the molecular mechanisms of JH in insect metamorphosis and reproduction, and to elucidate the crosstalk between JH and 20E, using the red flour beetle Tribolium castaneum, fruit fly Drosophila melanogaster, tobacco hornworm Manduca sexta and other insects as model systems. This review highlights the most recent progress in these areas.%保幼激素( juvenile hormone,JH)和蜕皮激素(20-hydroxyecdysone,20E)是协同调控昆虫发育、变态与生殖的两个重要激素.由于20E的主要分子作用机制已经比较明了,揭示JH的分子作用机制成为过去20多年来昆虫学领域研究的一个重点和难点.国内外多个研究团队利用赤拟谷盗Tribolium castaneum、果蝇Drosophila melanogaster、烟草天蛾 Manduca sexta 等为模式,在JH受体的鉴定、JH在昆虫发育变态和生殖中的分子调控机制以及JH与20E在分子水平上的交互作用等方面开展了大量的研究工作,本文就近几年在这些方面取得的主要研究进展作一个综述.

  8. 20-Hydroxyecdysone activates PGRP-SA mediated immune response in Locusta migratoria.

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    Han, Pengfei; Han, Jiao; Fan, Jiqiao; Zhang, Min; Ma, Enbo; Li, Sheng; Fan, Renjun; Zhang, Jianzhen

    2017-02-27

    20-hydroxyecdysone (20E) has been implicated in regulating the immune response in insects. Conflicting conclusions on 20E regulating immunity have been reported in model holometabolous species. However, in hemimetabolous insects, the role of 20E as an immune-suppressor or activator and the mechanism remains unclear. The migratory locust Locusta migratoria is a representative member of hemimetabolous insects. Here, digital gene expression (DGE) profiles of Locusta migratoria treated with 20E were analyzed. Pattern recognition receptors [peptidoglycan recognition protein (PGRP-SA), PGRP-LE, and gram-negative binding protein (GNBP3)] and antimicrobial peptides (defensin, diptericin, and i-type lysozyme) were significantly induced by 20E in fat body. These immune-related genes significantly increased their mRNA levels during the high-20E stage. Antibacterial activities in plasma were enhanced after 20E injection and during the high-20E developmental stage. Conversely, when 20E signal was suppressed by RNAi of EcR (ecdysone receptor), the expression levels of these genes and antibacterial activities failed to be increased by 20E injection and during the high-20E developmental stage, and the mortality increased after being infected by entomogenous fungus. The knockdown of PGRP-SA inhibited the expression level of defensin, diptericin and i-type lysozyme in fat body and reduced antibacterial activities in plasma. 20E injection could not significantly induce the expression of antimicrobial peptides after RNAi of PGRP-SA. These results demonstrated that 20E enhanced the immune response by activating PGRP-SA in L. migratoria.

  9. Two chitinase 5 genes from Locusta migratoria: molecular characteristics and functional differentiation.

    Science.gov (United States)

    Li, Daqi; Zhang, Jianqin; Wang, Yan; Liu, Xiaojian; Ma, Enbo; Sun, Yi; Li, Sheng; Zhu, Kun Yan; Zhang, Jianzhen

    2015-03-01

    The duplication of chitinase 5 (Cht5) into two to five different genes has been reported only in mosquito species to date. Here, we report the duplication of Cht5 genes (LmCht5-1 and LmCht5-2) in the migratory locust (Locusta migratoria). Both LmCht5-1 (505 aa) and LmCht5-2 (492 aa) possess a signal peptide and a catalytic domain with four conserved motifs, but only LmCht5-1 contains a chitin-binding domain. Structural and phylogenetic analyses suggest that LmCht5-1 is orthologous to other insect Cht5 genes, whereas LmCht5-2 might be newly duplicated. Both LmCht5 genes were expressed in all tested tissues with LmCht5-1 highly expressed in hindgut and LmCht5-2 highly expressed in integument, foregut, hindgut and fat bodies. From the fourth-instar nymphs to the adults, LmCht5-1 and LmCht5-2 showed similar developmental expression patterns with transcript peaks prior to each nymphal molting, suggesting that their expression levels are similarly regulated. Treatment with 20-hydroxyecdysone (20E; the most active molting hormone) and reducing expression of EcR (ecdysone receptor gene) by RNAi increased and decreased expression of both LmCht5 genes, respectively, indicating that both genes are responsive to 20E. Although transcript level of LmCht5-2 is generally 10-fold higher than that of LmCht5-1, RNAi-mediated suppression of LmCht5-1 transcript led to severe molting defects and lethality, but such effects were not seen with RNAi of LmCht5-2, suggesting that the newly duplicated LmCht5-2 is not essential for development and survivorship of the locust.

  10. Comparative characterization of two intracellular Ca²⁺-release channels from the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Liu, Yaping; Li, Chengjun; Gao, Jingkun; Wang, Wenlong; Huang, Li; Guo, Xuezhu; Li, Bin; Wang, Jianjun

    2014-10-21

    Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are members of a family of tetrameric intracellular Ca(2+)-release channels (CRCs). While it is well known in mammals that RyRs and IP3Rs modulate multiple physiological processes, the roles of these two CRCs in the development and physiology of insects remain poorly understood. In this study, we cloned and functionally characterized RyR and IP3R cDNAs (named TcRyR and TcIP3R) from the red flour beetle, Tribolium castaneum. The composite TcRyR gene contains an ORF of 15,285 bp encoding a protein of 5,094 amino acid residues. The TcIP3R contains an 8,175 bp ORF encoding a protein of 2,724 amino acids. Expression analysis of TcRyR and TcIP3R revealed significant differences in mRNA expression levels among T. castaneum during different developmental stages. When the transcript levels of TcRyR were suppressed by RNA interference (RNAi), an abnormal folding of the adult hind wings was observed, while the RNAi-mediated knockdown of TcIP3R resulted in defective larval-pupal and pupal-adult metamorphosis. These results suggested that TcRyR is required for muscle excitation-contraction (E-C) coupling in T. castaneum, and that calcium release via IP3R might play an important role in regulating ecdysone synthesis and release during molting and metamorphosis in insects.

  11. MOLT-INHIBITING HORMONE LEVELS AND ECDYSTEROID TITER DURING A MOLT CYCLE OF PORTUNUS TRITUBERCULATUS%三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化

    Institute of Scientific and Technical Information of China (English)

    汪春建; 朱冬发; 亓一舟; 胡则辉; 谢熙; 沈洁

    2013-01-01

    Portunus trituberculatus as a popular table delicacy is one of the most important fishery and aquaculture species of crab around the coast of China. In crustaceans, molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ-sinus gland (XO-SG) of the eyestalks, had been proposed to regulate molting by inhibiting the synthesis of ecdysteroids from Y-organs (YO). The method for determining the levels of MIH mRNA in the swimming crab had been developed using relative quantification of quantitative real-time PCR (qRT-PCR). We found the expression level of MIH mRNA was the highest in the XO-SG. By taking surstage D0 as the control group, the levels of MIH mRNA were analyzed by 2-ΔΔct in a molt cycle, and the results showed that MIH transcripts down-regulated 0.42±0.08, increased (1.09±0.09, increased 1.35±0.16 fold in stage A, B, C, respectively, and down-regulated 0.78±0.07, down-regulated 0.27±0.08, down-regulated 0.20±0.04 fold in surstage D1, D2, D3/4, respectively. In addition, we used the method of high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) to complete the process of measuring the consistency of portunus molting ecdysteroid (20-hydroxyecdysone, 20E) in hemolymph. The results showed that the consistency of ecdysone was below the instrument detection limit of 0.33 pg in the post molt stage (A/B). In the inter-molt period (C), the consistency of ecdysone gradually returned to (1.666±0.762) ng/mL. In the pre-molt ecdysteroid titer increased gradually to (4.047±1.5133), (6.756±4.928) and (8.609±3.827) ng/mL in surstage D0, D1 and D2, respectively. The ecdysteroid titer increased steadily to a peak of (19.534±4.799) ng/mL in the surstage D3, then dropped to 11.616 ng/mL in surstage D4. These stage-specific expression changes in MIH mRNA levels were accompanied by significant fluctuations in hemolymph ecdysteroid titer. During a molt cycle of the swimming crab, the expression of MIH exhibited

  12. Regulation of Gene Expression Patterns in Mosquito Reproduction.

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    Sourav Roy

    2015-08-01

    Full Text Available In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM. During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E and the Ecdysone-Receptor (EcR. Between 36 h and 48 h, the third wave of gene activation-regulated mainly by HR3-occurs. Juvenile Hormone (JH and its receptor Methoprene-Tolerant (Met are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the

  13. Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

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    Daojun Cheng

    Full Text Available The transcription factor Broad Complex (BR-C is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1, a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

  14. Molecular markers of endocrine disruption in aquatic organisms.

    Science.gov (United States)

    Rotchell, Jeanette M; Ostrander, Gary K

    2003-01-01

    A wide range of organic contaminant compounds prevalent in the aquatic environment has been shown to exhibit hormone-disrupting activity. The actual potency of such compounds are low compared with endogenous hormones, such as 17beta-estradiol, but may still produce detrimental biological effects. Induced hormone levels are routinely measured using commercial testing kits, though these fail to relate to actual effects. Field and laboratory studies on the biological effects of environmental estrogens have, in the past, largely relied on assays of vitellogenin (vtg) induction in male fish, reduced growth in testes formation, and intersex incidence. Here, we critically review the current and potential application of molecular techniques in assessing the adverse biological reproductive effects of endocrine-disrupting chemicals in aquatic organisms. The role of fish (estrogen, androgen, and progestogen) hormone receptors and invertebrate (ecdysone) hormone receptor, egg production (vtg and chorion) proteins, steroid biosynthesis enzymes (aromatase, sulfotransferase, and hydroxysteroid dehydrogenase), DNA damage, apoptosis, and their potential development as biomarkers are discussed in turn. In each case, the sequences characterized are presented and homologies across species are highlighted. Molecular methods of gauging vtg and zona radiata (ZR) expression and protein concentrations have included immunoassay and reverse transcription polymerase chain reaction (RT-PCR). Suggestions for the isolation for key gene expression products (aromatase, ZR, and vtg, for instance), from a wider range of fish species using degenerate primers, are given. Endocrine disruption in invertebrates has received less attention compared with fish, partly because the knowledge regarding invertebrate endocrinology is limited. Here we review and suggest alternate isolation strategies for key players in the imposex induction process: vitellin (Vn), aromatase, and Ala-Pro-Gly-Trp (APGW) amide

  15. A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

    Science.gov (United States)

    Yeh, Maw-Sheng; Lai, Ching-Yi; Liu, Chun-Hung; Kuo, Ching-Ming; Cheng, Winton

    2009-01-01

    Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual pro

  16. Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus.

    Science.gov (United States)

    Techa, Sirinart; Alvarez, Javier V; Sook Chung, J

    2015-04-01

    Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

    Science.gov (United States)

    Mohamed, Ahmed A M; Wang, Qiushi; Bembenek, Jadwiga; Ichihara, Naoyuki; Hiragaki, Susumu; Suzuki, Takeshi; Takeda, Makio

    2014-01-01

    Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

  18. Effects of anti-ecdysteroid quaternary derivatives of azole analogues of metyrapone on the post-embryonic development of the red cotton bug (Dysdercus cingulatus F).

    Science.gov (United States)

    Bélai, Iván; Fekete, Gábor

    2003-04-01

    In order to improve the larvicidal activity of the azole analogues of metyrapone, previously found to have a strong inhibitory activity on ecdysone 20-monooxygenase (E-20-M) from the fleshfly Neobellieria bullata Parker, soft-alkylated compounds (3-(1,1-dimethyl-2-oxo-2-phenylethyl)-1-dodecanoyloxymethyl-1H-imidazolium chloride, sPIM) and (1-(1,1-dimethyl-2-oxo-2-phenylethyl)-4-dodecanoyloxymethyl-1H-1,2,4-triazolium chloride, sPTM), derivatives of phenyl-imidazolyl-metyrapone (PIM) and phenyl-1,2,4-triazolyl-metyrapone (PTM), respectively, were synthesized. Both sPIM and sPTM, designed as propesticides, inhibited E-20-M in vitro at 10(-4) M concentration, which was unexpected since they had been expected to be inactive in vitro and to gain activity only within the organism. sPTM significantly delayed the pupariation of N. bullata larvae and this effect could be reversed by the simultaneous application of 20-hydroxyecdysone (20E), supporting the hypothesis that sPTM can act by interfering with the moulting hormone system. Due to this in vitro activity, sPTM and sPIM cannot be considered to be simple drug precursors, and their structure should contain structural elements (pharmacophores) responsible for the observed biological effects. In order to examine this hypothesis, derivatives of sPTM and sPIM were synthesised in which the hydrolytically labile N(+)-CH2O(CO)- moiety was changed to the more stable N(+)-CH2CH2(CO)-group. In three new stable derivatives, a dodecylamino or a phenyl group, respectively, is attached to the carbonyl group to obtain PTM and PIM derivatives quaternised with a 2-dodecylcarbamoylethyl or a 3-oxo-3-phenylpropyl group. In one derivative, the 2-oxo-2-phenylethyl quaternising group has one fewer carbon atom. In addition to their moderate activity (LC50 = 10(-6)-10(-5) M) against the red cotton bug Dysdercus cingulatus F, they delayed development and caused developmental abnormalities, including mortality in the pharate phase, mortality

  19. Molecular evolution of ultraspiracle protein (USP/RXR in insects.

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    Ekaterina F Hult

    Full Text Available Ultraspiracle protein/retinoid X receptor (USP/RXR is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR. In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d(N/d(S, suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d(S is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP

  20. Survival strategies of freshwater insects in cold environments

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    Valeria LENCIONI

    2004-09-01

    Full Text Available At high latitudes and altitudes, ice formation is a major variable affecting survival of freshwater fauna and hence the abundance and composition of invertebrate communities. Freezing, but also desiccation and anoxia, are lethal threats to all life stages of aquatic insects, from the eggs to the adults. During cold periods, the aquatic stages commonly remain in or move to a portion of the water body that will not freeze or dry (e.g., deep waters of lakes, springs and hyporheic zone where they can remain active. Less frequently they migrate to habitats that will freeze at the onset of winter. Insects have developed a complex of strategies to survive at their physiological temperature minimum, comprising (a morphological (melanism, reduction in size, hairiness/pubescence, brachyptery and aptery, (b behavioural (basking in the sun, changes in feeding and mating habit, parthenogenesis, polyploidy, ovoviviparity, habitat selection and cocoon building, (c ecological (extension of development to several years by quiescence or diapause and reduction of the number of generations per year, (d physiological and biochemical (freezing tolerance and freezing avoidance adaptations. Most species develop a combination of these survival strategies that can be different in the aquatic and terrestrial phase. Freezing avoidance and freezing tolerance may be accompanied by diapause. Both cold hardiness and diapause manifest during the unfavourable season and: (i involve storage of food resources (commonly glycogen and lipids; (ii are under hormonal control (ecdysone and juvenile hormone; (iii involve a depression or suppression of the oxidative metabolism with mitochondrial degradation. However, where the growing season is reduced to a few weeks, insects may develop cold hardiness without entering diapause, maintaining in the haemolymph a high concentration of Thermal Hysteris Proteins (THPs for the entire year and a slow but continuous growth. A synthesis of

  1. N-acetyltransferase (nat is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

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    Ahmed A M Mohamed

    Full Text Available Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD and LD12:12 (SD, and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT caused dysfunction of photoperiodism. dsRNA(PER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT decreased melatonin while dsRNA(PER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

  2. The effects of binary UV filter mixtures on the midge Chironomus riparius

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    Ozáez, Irene; Morcillo, Gloria; Martínez-Guitarte, José-Luis, E-mail: jlmartinez@ccia.uned.es

    2016-06-15

    Organic ultraviolet (UV) filters are used in a wide variety of products, including cosmetics, to prevent damage from UV light in tissues and industrial materials. Their extensive use has raised concerns about potential adverse effects in human health and aquatic ecosystems that accumulate these pollutants. To increase sun radiation protection, UV filters are commonly used in mixtures. Here, we studied the toxicity of binary mixtures of 4-methylbenzylidene camphor (4MBC), octyl-methoxycinnamate (OMC), and benzophenone-3 (BP-3), by evaluating the larval mortality of Chironomus riparius. Also molecular endpoints have been analyzed, including alterations in the expression levels of a gene related with the endocrine system (EcR, ecdysone receptor) and a gene related with the stress response (hsp70, heat shock protein 70). The results showed that the mortality caused by binary mixtures was similar to that observed for each compound alone; however, some differences in LC50 were observed between groups. Gene expression analysis showed that EcR mRNA levels increased in the presence of 0.1 mg/L 4MBC but returned to normal levels after exposure to mixtures of 4MBC with 0.1, 1, and 10 mg/L of BP-3 or OMC. In contrast, the hsp70 mRNA levels increased after exposure to the combinations tested of 4MBC and BP-3 or OMC mixtures. These data suggest that 4MBC, BP-3, and OMC may have antagonist effects on EcR gene transcription and a synergistic effect on hsp70 gene activation. This is the first experimental study to show the complex patterned effects of UV filter mixtures on invertebrates. The data suggest that the interactions within these chemicals mixtures are complex and show diverse effects on various endpoints. - Highlights: • Chironomus riparius is sensitive to UV filter binary mixtures. • UV filters binary mixtures show antagonism on survival of 4th instar larvae. • BP-3 and OMC antagonize the stimulatory effect of 4MBC on EcR gene. • 4MBC, OMC, and BP-3 induce hsp70

  3. 甲氧虫酰肼对舞毒蛾幼虫解毒酶及其体内蛋白质表达的影响%Effect of Methoxyfenozide on Activities of Detoxifying Enzymes and Expression of Proteins in Lymantria dispar larvae

    Institute of Scientific and Technical Information of China (English)

    廖月枝; 严善春; 曹传旺; 刘丹

    2012-01-01

    In order to study the insecticidal activity of methoxyfenozide ( RH-2485 ) against the larvae of Lymantria dispar ,the pesticide bioactivity of the chemical against the different instar larvae of L. dispar and its effect on the activities of detoxifying enzymes of the insect were assayed using leaf film method, and the expression of proteins in different tissues of the larvae were detected with SDS-PAGE. The results showed that methoxyfenozide had a high toxicity against the larvae,especially 2nd instar and 3rd instar, and the toxicity was obviously different with different larva instars. The activities of detoxifying enzymes, such as carboxylesterase ( CarE ) , MFO O-demethylase ( MFOD ) and glutathione S-transfer (GST) , in the 2nd , 4th , 6th instar larvae were significantly induced or inhibited by the methoxyfenozide treatment. The impact of methoxyfenozide on these enzymes was significantly different at different treatment time. After the 4th instar larvae were fed with methoxyfenozide, the protein expression pattern in the hemolymph ,midgut and epidermis was different from that in the control. The effect of methoxyfenozide on proteins in the hemolymph and midgut was obvious in 12 h and 24 h, whereas the effect on protein expression in epidermis tissue was more significant in 48 h. These results indicated that methoxyfenozide as a non-steroidal ecdysone had a higher biological activity against L. dispar, and the major detoxifying enzymes in the insect body were significantly interfered, showing that methoxyfenozide had a high toxic effect against L. dispar. The specific proteins were produced in the hemolymph , midgut and epidermal tissue, which might interfere with the normal physiological metabolism of insects and epidermal formation.%为研究甲氧虫酰肼(RH-2485)对舞毒蛾幼虫的杀虫活性,采用叶片药膜法测定该药剂对舞毒蛾不同龄期幼虫的生物活性及对其体内解毒酶活性的影响,并通过SDS-PAGE对舞毒

  4. Structure and function of sterol carrier proteins in insects%昆虫固醇转运蛋白的结构与功能

    Institute of Scientific and Technical Information of China (English)

    张丽丽; 郭兴荣; 冯启理; 郑思春

    2011-01-01

    In insects, cholesterol is not only one of the main components of cell membranes, but also a precursor of ecdysone biosynthesis. However, because insects lack two key enzymes for cholesterol biosynthesis, they can not autonomously synthesize cholesterol from simple compounds and therefore have to obtain sterols from their diet. Insects must convert food sterols into cholesterol to meet the requirements of growth, development and reproduction. Sterol carrier proteins (SCPs) are main transport proteins for sterol absorption and transport in insects. It is critical to study the relationship between structure and function of SCPs for understanding the roles of SCPs in sterol transport. In this review, recent progress in the study of the structure, expression and distribution of SCP genes and proteins, post-translation modification, crystal structure, ligand-binding specificity and possible absorption and transport pathways of insect SCPs was summarized and the potential of using SCPs as a molecular target for pest insect control was also discussed.Studies indicate that transcript expression of SCP genes and post-translation modifications of SCP proteins vary depending on different species. In dipteran insects such as Aedes aegypti and Drosophila melangoster SCP-x gene encodes SCP-x and SCP-2 proteins, while there are additional SCP-2 genes and SCP-2-1ike genes encoding SCP-2 and SCP-2-1ike proteins, respectively. In lepidopteran insects such as Spodoptera littoralis,Spodoptera litura and Bombyx mori, the transcript expression and translation processes of SCP-x gene are similar to those in vertebrates, in which SCP-2 protein is produced after post transcription and translation modifications of a unique SCP-x gene. SCP-x and SCP-2 proteins are localized in peroxisomes. SCP-2 protein consists of 5 αt-helixes and 5 β-sheets and the αS-helix appears to impact the binding of the protein to substrates. SCP-2 protein can bind with different affinity to cholesterol