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Lawrence I. Gilbert - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: Evidence for mobilization and entry mechanisms
Insect biochemistry and molecular biology, 2005Co-Authors: Susan K. Fellner, Robert Rybczynski, Lawrence I. GilbertAbstract:Prothoracicotropic hormone (PTTH) stimulates Ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated Ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.
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Control and biochemical nature of the ecdysteroidogenic pathway.
Annual review of entomology, 2002Co-Authors: Lawrence I. Gilbert, Robert Rybczynski, James T. WarrenAbstract:Molting is elicited by a critical titer of ecdysteroids that includes the principal molting hormone, 20-hydroxyecdysone (20E), and ecdysone (E), which is the precursor of 20E but also has morphogenetic roles of its own. The prothoracic glands are the predominate source of ecdysteroids, and the rate of synthesis of these polyhydroxylated sterols is critical for molting and metamorphosis. This review concerns three aspects of Ecdysteroidogenesis: (a) how the brain neuropeptide prothoracicotropic hormone (PTTH) initiates a transductory cascade in cells of the prothoracic gland, which results in an increased rate of ecdysteroid biosynthesis (upregulation); (b) how the concentrations of 20E in the hemolymph feed back on the prothoracic gland to decrease rates of Ecdysteroidogenesis (downregulation); and (c) how the prothoracic gland cells convert cholesterol to the precursor of E and then 20E, a series of reactions only now being understood because of the use of a combination of classical biochemistry and molecular genetics.
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Dynamic regulation of prothoracic gland Ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects.
Insect biochemistry and molecular biology, 2000Co-Authors: Lawrence I. Gilbert, Akira Mizoguchi, Qisheng Song, Wendy A. Smith, Shinji Nagata, Robert Rybczynski, R Morreale, H Matubayashi, M Shionoya, Hiroshi KataokaAbstract:Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone (rPTTH) from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or are inconsistent with, previous studies over the past 20years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of cAMP (adenosine 3':5' cyclic monophosphate) synthesis; requirement for extracellular Ca(2+) in in vitro studies; Ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells in each brain hemisphere as the source of PTTH (the prothoracicotropes); the ability of antibodies to rPTTH to inhibit Ecdysteroidogenesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.
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Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland.
Insect biochemistry and molecular biology, 1998Co-Authors: Qisheng Song, Lawrence I. GilbertAbstract:Insect molting and metamorphosis are elicited by a class of ecdysteroids, mainly 20-hydroxyecdysone (20E), the precursor of which is synthesized in the prothoracic gland. 20E acts via the ecdysone receptor (EcR) and its heterodimer partner ultraspiracle (USP). Analysis of the prothoracic gland of Manduca sexta revealed that the developmental expression and phosphorylation of a specific USP form, p47, is positively correlated with Ecdysteroidogenesis and that 20E, but not ecdysone, is responsible for initiating the translational expression and phosphorylation of p47. The latter forms a functional complex with EcR and the ligand-complex interaction results in the down regulation of Ecdysteroidogenesis and the inhibition of prothoracicotropic hormone (PTTH)-stimulated Ecdysteroidogenesis. The composite data suggest that USP plays a key role in modulating PTTH-stimulated ecdysteroid biosynthesis through the selective expression and phosphorylation of the p47 USP isoform.
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Control of Ecdysteroidogenesis: Activation and inhibition of prothoracic gland activity
Invertebrate Neuroscience, 1997Co-Authors: Lawrence I. Gilbert, Qisheng Song, Robert RybczynskiAbstract:The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insect Manduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca^2+ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a β tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.
Patrizia Falabella - One of the best experts on this subject based on the ideXlab platform.
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Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression
Frontiers in physiology, 2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Ecdysteroidogenesis and development in heliothis virescens lepidoptera noctuidae focus on ptth stimulated pathways
Journal of Insect Physiology, 2018Co-Authors: Carmen Scieuzo, Marisa Nardiello, Marco Pezzi, Bradleich S Vinson, Marilena Leis, Heiko Vogel, Rosanna Salvia, Sabino Aurelio Bufo, Milvia Chicca, Patrizia FalabellaAbstract:Abstract Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two pathways are potentially involved in PTTH-stimulated Ecdysteroidogenesis, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated the potential roles of both these pathways in Heliothis virescens Ecdysteroidogenesis. We identified putative proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated Ecdysteroidogenesis. Finally, we analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin, in order to better understand the role of the TOR pathway in larval growth. When rapamycin was added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH signaling-stimulated Ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic growth.
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Image_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.TIFF
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Table_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.DOCX
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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regulation of heliothis virescens prothoracic glands by cardiochiles nigriceps polydnavirus
Archives of Insect Biochemistry and Physiology, 1998Co-Authors: Francesco Pennacchio, Patrizia Falabella, Bradleich S VinsonAbstract:Heliothis virescens (F.) Larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage. This developmental alteration is caused by both an inactivation of prothoracic glands of last-instar larvae and an altered ecdysone metabolism. Decrease in Ecdysteroidogenesis in vitro was already evident in glands explanted from larvae that have attained the early cell formation stage (day 4 of fifth instar), 6 h after parasitoid oviposition. Ecdysteroidogenesis nearly ceased by 24 h after parasitoid oviposition. The degree of this biosynthetic depression increased as the time between parasitization and gland dissection increased. A time-course study allowed us to determine if both the degree of phosphorylation of regulatory target proteins, the rate of general protein synthesis and Ecdysteroidogenesis decreased in concert over time. The results provide further evidence in support of the hypothesis that these cellular activities in prothoracic gland cells are functionally correlated in steroidogenic responses. Treatment with calyx fluid and venom of C. nigriceps duplicates the parasitism-induced inactivation of host prothoracic glands. A 6-h conditioning in vitro of pupally committed host prothoracic glands with these parasitoid female reproductive secretions resulted in a significant depression of their ecdysteroid production. However, glands lost their sensitivity to calyx fluid and venom treatment when explanted from hosts that had already attained the cell formation stage. This was further supported by the fact that nearly all the host larvae parasitized on day 4 of fifth instar (cell formation stage) pupated, while parasitization on day 3 resulted in only 11% pupation. The coupled trioxsalen/UV irradiation treatment of C. nigriceps calyx fluid and venom eliminated their negative effect on biosynthetic activity in vitro by host prothoracic glands. This result indirectly demonstrates that C. nigriceps polydnavirus is the major regulating factor involved in the host prothoracic gland inactivation. Arch. Insect Biochem. Physiol. 38:1–10, 1998. © 1998 Wiley-Liss, Inc.
Hiroshi Kataoka - One of the best experts on this subject based on the ideXlab platform.
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Twenty-hydroxyecdysone produced by dephosphorylation and Ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.
Insect biochemistry and molecular biology, 2020Co-Authors: Daiki Fujinaga, Mika Takeshima, Mari H. Ogihara, Hajime Kawahara, Ikumi Kojima, Hiroshi KataokaAbstract:Abstract Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through Ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of Ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by Ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.
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Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.
Insect biochemistry and molecular biology, 2020Co-Authors: Xiang Zhou, Mika Takeshima, Mari H. Ogihara, Hiroshi Kataoka, Daiki Fujinaga, Cheng-wen Liu, Zhen Zhu, Yan-yuan BaoAbstract:Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (Ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for Ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, Ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require Ecdysteroidogenesis for ovarian development.
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Steroid hormones in Acari, their functions and synthesis
Applied Entomology and Zoology, 2019Co-Authors: Mari H. Ogihara, Demar Taylor, Hiroshi KataokaAbstract:Acari are arthropods with the conserved features of an exoskeleton that require the shedding of cuticle for growth. Steroid hormones, ecdysteroids, regulate the mechanisms necessary for growth and development. Acari lack juvenile hormones, so they utilize ecdysteroids as the solo lipophilic hormone throughout their life for molting as well as for reproduction. Although some research on Ecdysteroidogenesis has been conducted in the Acari, two important questions remain unclear: the active form of ecdysteroid and sites for Ecdysteroidogenesis. The active form of ecdysteroids in Acari is controversial. Analysis by mass spectrometry confirms that most Acari use 20-hydroxyecdysone (20E), but the spider mite Tetranychus urticae (Koch) (Acari: Tetranychidae) uses Ponasterone A (25-deoxy-20-hydroxyecdysone). Analyses of ecdysteoridogenic genes provide clear evidence that the ovary is the primary site of Ecdysteroidogenesis in both immature and mature ticks. In this review, the known functions of ecdysteroids are summarized and recent progress on Ecdysteroidogenesis is introduced.
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identification of ecdysteroidogenic enzyme genes and their expression during pupal diapause in the cabbage armyworm mamestra brassicae
Insect Molecular Biology, 2017Co-Authors: Mari H. Ogihara, Takayoshi Nakaoka, Yutaka Suzuki, H Ikeda, Nobuto Yamada, Juri Hikiba, Yoshinori Fujimoto, Kazuki Saito, Akira Mizoguchi, Hiroshi KataokaAbstract:In this study, we identified ecdysteroidogenic enzymes in the cabbage armyworm, Mamestra brassicae, and demonstrated reduced expression of these genes during diapause. Some insects employ a temporary developmental arrest, diapause, to survive in severe environments. The titres of the moulting hormone ecdysteroid were reduced in diapause pupae of M. brassicae; therefore, Ecdysteroidogenesis might be suppressed by a diapause-specific mechanism. To clarify expression changes of ecdysteroidogenic enzyme genes during diapause in M. brassicae, we first identified the genes for seven ecdysteroidogenic enzymes: Neverland, Non-molting glossy (Nm-g), CYP307A1 (Spook), CYP306A1 (Phantom), CYP302A1 (Disembodied), CYP315A1 (Shadow) and CYP314A1 (Shade). Enzymatic assays using heterologous expression in Drosophila Schneider 2 (S2) cells and analysis of mRNA distribution indicated that the identified genes were ecdysteroidogenic enzymes of M. brassicae. Expression levels of these ecdysteroidogenic enzyme genes were compared between prothoracic glands in different pupal stages throughout diapause. Immediately after pupation, diapause-destined pupae showed similar expression levels of ecdysteroidogenic enzyme genes to those of nondiapause pupae. All of these genes showed reduced gene expression after diapause initiation. Expression was immediately increased in diapause-destined pupae at the postdiapause quiescence phase. These results indicate that reduced expression of ecdysteroidogenic enzyme genes suppresses Ecdysteroidogenesis and maintains developmental arrest during diapause.
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Deep sequencing of the prothoracic gland transcriptome reveals new players in insect Ecdysteroidogenesis.
PLOS ONE, 2017Co-Authors: Takayoshi Nakaoka, Ikumi Koujima, Mika Takeshima, Xiangying Zhou, Mari H. Ogihara, Tetsuya Yamada, Yutaka Suzuki, Hiroshi KataokaAbstract:: 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.
Rosanna Salvia - One of the best experts on this subject based on the ideXlab platform.
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Ecdysteroidogenesis in heliothis virescens lepidoptera noctuidae recombinant prothoracicotropic hormone and brain extract show comparable effects
Journal of Insect Science, 2019Co-Authors: Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Rosanna Salvia, Sabino Aurelio Bufo, Andrea Scala, Antonio Franco, Angela Di Somma, Angela DuilioAbstract:: Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning Ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated Ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate Ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.
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Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression
Frontiers in physiology, 2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Ecdysteroidogenesis and development in heliothis virescens lepidoptera noctuidae focus on ptth stimulated pathways
Journal of Insect Physiology, 2018Co-Authors: Carmen Scieuzo, Marisa Nardiello, Marco Pezzi, Bradleich S Vinson, Marilena Leis, Heiko Vogel, Rosanna Salvia, Sabino Aurelio Bufo, Milvia Chicca, Patrizia FalabellaAbstract:Abstract Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two pathways are potentially involved in PTTH-stimulated Ecdysteroidogenesis, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated the potential roles of both these pathways in Heliothis virescens Ecdysteroidogenesis. We identified putative proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated Ecdysteroidogenesis. Finally, we analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin, in order to better understand the role of the TOR pathway in larval growth. When rapamycin was added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH signaling-stimulated Ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic growth.
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Image_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.TIFF
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Table_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.DOCX
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Ecdysteroidogenesis in heliothis virescens lepidoptera noctuidae recombinant prothoracicotropic hormone and brain extract show comparable effects
Journal of Insect Science, 2019Co-Authors: Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Rosanna Salvia, Sabino Aurelio Bufo, Andrea Scala, Antonio Franco, Angela Di Somma, Angela DuilioAbstract:: Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning Ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated Ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate Ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.
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Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression
Frontiers in physiology, 2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Ecdysteroidogenesis and development in heliothis virescens lepidoptera noctuidae focus on ptth stimulated pathways
Journal of Insect Physiology, 2018Co-Authors: Carmen Scieuzo, Marisa Nardiello, Marco Pezzi, Bradleich S Vinson, Marilena Leis, Heiko Vogel, Rosanna Salvia, Sabino Aurelio Bufo, Milvia Chicca, Patrizia FalabellaAbstract:Abstract Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two pathways are potentially involved in PTTH-stimulated Ecdysteroidogenesis, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated the potential roles of both these pathways in Heliothis virescens Ecdysteroidogenesis. We identified putative proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated Ecdysteroidogenesis. Finally, we analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin, in order to better understand the role of the TOR pathway in larval growth. When rapamycin was added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH signaling-stimulated Ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic growth.
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Image_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.TIFF
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.
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Table_1_Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.DOCX
2018Co-Authors: Rosanna Salvia, Marisa Nardiello, Carmen Scieuzo, Heiko Vogel, Sabino Aurelio Bufo, Andrea Scala, Asha Rao, Patrizia FalabellaAbstract:Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of Ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG Ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits Ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking Ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.