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Stephen S. Tobe - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the nmda receptor and its role in regulating reproduction in the cockroach Diploptera punctata
    2015
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. Tobe
    Abstract:

    The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternatively spliced variants in the cockroach Diploptera punctata . This is the first report of NR1 paralogs in insects. The tissue distributions and expression profiles of DpNR1A , DpNR1B and DpNR2 in different tissues were also investigated. Previous studies have demonstrated NMDA-stimulated biosynthesis of juvenile hormone (JH) in the corpora allata through the influx of extracellular Ca2+ in Diploptera punctata . However, our data show that the transcript levels of DpNR1A , DpNR1B and DpNR2 were low in the corpora allata. MK-801, a high-affinity antagonist of NMDAR, did not show any effect on JH biosynthesis in vitro . In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-801 resulted in any significant change in JH biosynthesis or basal oocyte growth. Injection of animals with a high dose of MK-801 (30 µg per animal per injection), which paralyzed the animals for 4–5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata .

  • characterization of the juvenile hormone pathway in the viviparous cockroach Diploptera punctata
    2015
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. Tobe
    Abstract:

    Juvenile hormones (JHs) are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase (HMGR) and Juvenile hormone acid methyltransferase (JHAMT). As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

  • mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach Diploptera punctata
    2014
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Sven Zels, Jozef Vanden Broeck, Stephen S. Tobe
    Abstract:

    The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca2+ and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.

  • Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development
    2013
    Co-Authors: Elisabeth Marchal, Barbara Stay, Juan Huang, Ekaterina F Hult, Stephen S. Tobe
    Abstract:

    The Pacific beetle cockroach, Diploptera punctata, has proven to be a valuable model insect in the study of the dynamics regulating juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymphal development and reproduction. This stems in part from its unusual mode of reproduction, adenotrophic viviparity, in which females give birth to live young that have been nourished throughout embryonic development by a protein-rich ‘milk’ secreted by the wall of the brood sac or uterus. In this animal, as in most insects, JH regulates both vitellogenin production and its uptake by developing oocytes. However, JH has an antagonistic effect on embryonic development and following oviposition of the fertilized oocytes into the brood sac, JH production halts, in part through the action of a peptide family, the FGLa allatostatins. JH production remains at a low level throughout pregnancy and is only reinstated at the end of gestation, at which time, the next wave of oocytes begins to develop and enter vitellogenesis. Thus, JH production in this species is precisely regulated, since the appearance of JH at inappropriate times would result in abortion of the embryos. Numerous factors are responsible for the regulation of JH biosynthesis, including peptides, biogenic amines, neurotransmitters, ecdysteroids and second messenger effectors. In this review, we discuss these factors and highlight potentially fruitful areas of future research. Although several of the enzymes of the biosynthetic pathway have been cloned, the precise points of rate limitation remain uncertain. The dissection of the biosynthetic pathway and its control awaits the completion of the genome and transcriptome of this important model insect.

  • sequencing and validation of housekeeping genes for quantitative real time pcr during the gonadotrophic cycle of Diploptera punctata
    2013
    Co-Authors: Elisabeth Marchal, Ekaterina F Hult, Juan Huang, Stephen S. Tobe
    Abstract:

    Background Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.

Barbara Stay - One of the best experts on this subject based on the ideXlab platform.

  • Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development
    2013
    Co-Authors: Elisabeth Marchal, Barbara Stay, Juan Huang, Ekaterina F Hult, Stephen S. Tobe
    Abstract:

    The Pacific beetle cockroach, Diploptera punctata, has proven to be a valuable model insect in the study of the dynamics regulating juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymphal development and reproduction. This stems in part from its unusual mode of reproduction, adenotrophic viviparity, in which females give birth to live young that have been nourished throughout embryonic development by a protein-rich ‘milk’ secreted by the wall of the brood sac or uterus. In this animal, as in most insects, JH regulates both vitellogenin production and its uptake by developing oocytes. However, JH has an antagonistic effect on embryonic development and following oviposition of the fertilized oocytes into the brood sac, JH production halts, in part through the action of a peptide family, the FGLa allatostatins. JH production remains at a low level throughout pregnancy and is only reinstated at the end of gestation, at which time, the next wave of oocytes begins to develop and enter vitellogenesis. Thus, JH production in this species is precisely regulated, since the appearance of JH at inappropriate times would result in abortion of the embryos. Numerous factors are responsible for the regulation of JH biosynthesis, including peptides, biogenic amines, neurotransmitters, ecdysteroids and second messenger effectors. In this review, we discuss these factors and highlight potentially fruitful areas of future research. Although several of the enzymes of the biosynthetic pathway have been cloned, the precise points of rate limitation remain uncertain. The dissection of the biosynthetic pathway and its control awaits the completion of the genome and transcriptome of this important model insect.

  • cuticular hydrocarbon synthesis and its maternal provisioning to embryos in the viviparous cockroach Diploptera punctata
    2005
    Co-Authors: Elsa Youngsteadt, Barbara Stay, Yongliang Fan, Coby Schal
    Abstract:

    Embryos of the viviparous cockroach Diploptera punctata accumulate large amounts of hydrocarbon (HC) of either maternal or embryonic origin. HC synthesis and its accumulation in maternal and embryonic tissues were measured over the course of gestation. Female abdominal integument was the only tissue that synthesized appreciable amounts of HC in vitro, and did so at an increasing rate from the time of mating to mid-pregnancy, when rates of synthesis declined. The embryos synthesized HC at rates o1% those of the female, showing that the majority of HC detected in and on embryos was of maternal origin. The brood sac that houses the developing embryos did not synthesize HC in vitro, indicating that HC must be transported from the female abdominal integument to the embryos. The mass of female epicuticular HC was constant at � 183mg, while her internal HC increased fourfold frommating to mid-pregnancy, then declined until parturition. The decline in internal HC reflected both declining HC synthesis in the female and greater export to the embryos, as embryonic internal HC increased 250-fold prior to parturition. An external HC coating over the oothecal covering and chorion of the embryos increased to mid-pregnancy, then declined. Unlike oviparous cockroaches, D. punctata females fed throughout the reproductive cycle, reflecting the nutritional demands of continuously provisioning the developing embryos. r 2005 Elsevier Ltd. All rights reserved.

  • evolution of a novel function nutritive milk in the viviparous cockroach Diploptera punctata
    2004
    Co-Authors: Anna Williford, Barbara Stay, Debashish Bhattacharya
    Abstract:

    Cockroach species show different degrees of maternal contribution to the developing offspring. In this study, we identify a multigene family that encodes water-soluble proteins that are a major component of nutritive "Milk" in the cockroach, Diploptera punctata. This gene family is associated with the evolution of a new trait, viviparity, in which the offspring receive nutrition during the gestation period. Twenty-five distinct Milk complementary DNAs were cloned and partially characterized. These complementary DNAs encode 22 distinct Milk peptides, each of length 171 amino acids, including a 16-amino acid signal peptide sequence. Southern blot analysis confirms the presence of multiple copies of Milk genes in D. punctata. Northern analysis indicates tissue- and stage-specific Milk gene expression. Examination of the deduced amino acid sequences identifies the presence of structurally conserved regions diagnostic of the lipocalin protein family. The shared exon/intron structure of one of the Milk loci with lipocalin genes further supports a close evolutionary relationship between these sequences.

  • allatostatin in ovaries oviducts and young embryos in the cockroach Diploptera punctata
    2003
    Co-Authors: Andrea P. Woodhead, Kuen K Chan, M E Thompson, Barbara Stay
    Abstract:

    The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.

  • Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study
    2003
    Co-Authors: René Feyereisen, J. Koener, Barbara Stay, G Johnson, Stephen S. Tobe
    Abstract:

    Abstract Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oocyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C 16 juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C 16 juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro ; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C 16 juvenile hormone synthesis by the corpora allata. Inhibition of C 16 juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro . This inhibition does not appear to be related to the spontaneous activity of the glands in vitro , and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.

Ann-shyn Chiang - One of the best experts on this subject based on the ideXlab platform.

  • Cell Size Control by Cyclic GMP Regulates Juvenile Hormone Synthesis in Corpora Allata of the Cockroach, Diploptera punctata
    2015
    Co-Authors: Lingwen Chang, Chuanmei Tsai, Deming Yang, Ann-shyn Chiang
    Abstract:

    ABSTRACT The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates growth, metamorphosis and reproduction of insects. In the the corpus allatum (CA) of the cockroach Diploptera punctata adult females, cyclic rise and decline in rates of JH synthesis occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increases with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) was low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP contents. Consequently, elevated cGMP triggers massive and synchronous autophagic activities resulting in cell atrophy and reduction of protein contents. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis

  • glutamate gated chloride channels inhibit juvenile hormone biosynthesis in the cockroach Diploptera punctata
    2005
    Co-Authors: Hsin Ping Liu, Shuchen Lin, Chiyen Lin, Shihrung Yeh, Ann-shyn Chiang
    Abstract:

    Abstract Juvenile hormone (JH) synthesized and released from endocrine gland corpus allatum (CA) plays an important role in insect metamorphosis, vitellogenesis and reproduction. Glutamate is a major neurotransmitter in the nervous system and its activated receptors possess excitatory and inhibitory forms in muscle fibers of invertebrates. Previously, we have shown that the rise of intracellular calcium through excitatory glutamate receptors, N -methyl- d -aspartate (NMDA) and non-NMDA-type channels stimulates JH synthesis in the cockroach, Diploptera punctata . Here, we demonstrate the occurrence of inhibitory chloride permeable glutamate (GluCl) receptors on CA cell membranes. Application of the GluCl channel activators, ibotenic acid (Ibo) and ivermectin, but not γ-aminobutyric acid caused a decline in JH synthesis in glands of either high or low activity during the gonadotrophic cycle. Also, while recording the membrane potential of the isolated whole CA glands intracellularly, Ibo induced a hyperpolarizated response. Both changes in the membrane potential and inhibition of JH synthesis could be abolished by the application of the chloride channel blocker picrotoxin. Finally, we found both excitatory and inhibitory glutamate receptors cause antagonistic effects on rates of JH synthesis. These results indicate a novel function of GluCl channels in the inhibition of JH synthesis that could be a potential pathway for developing a new generation of insecticides.

  • cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata
    2005
    Co-Authors: Lingwen Chang, Chuanmei Tsai, Deming Yang, Ann-shyn Chiang
    Abstract:

    The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.

  • ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach Diploptera punctata
    2002
    Co-Authors: Ann-shyn Chiang, Maciej A Pszczolkowski, Hsin Ping Liu, Shuchen Lin
    Abstract:

    Abstract By monitoring changes in the cytosolic [Ca 2+ ] i and rates of juvenile hormone (JH) synthesis in response to l -glutamate agonists and antagonists, we identified and characterized glutamate receptor subtypes in corpus allatum (CA) cells of the cockroach, Diploptera punctata . During the first ovarian cycle, corpora allata exhibited a cycle of changes in sensitivity to l -glutamate correlated to cyclic changes in rates of JH synthesis. When exposed to 60 μM l -glutamate in vitro, the active corpora allata of day-4 mated females produced 60% more JH, while inactive corpora allata at other ages showed 10–20% stimulatory response. Pharmacological characterization using various l -glutamate receptor agonists and antagonists indicated that several ionotropic subtypes of l -glutamate receptors were present in the CA. The CA showed an increase in rates of JH synthesis in response to NMDA, kainate, and quisqualate, but not to AMPA in both L-15 medium and minimum incubation medium. In contrast, applications of the metabotropic receptor-specific agonist trans -ACPD failed to elicit a change in the cytosolic [Ca 2+ ] i and JH production. An elevation of cytosolic calcium concentration, followed by 20–30% rise in JH production, was observed when active CA cells were exposed to 10–40 μM kainate. Kainate had no stimulatory effect on JH synthesis in calcium-free medium. The kainate-induced JH synthesis was blocked by 20 μM CNQX but was not affected by 20 μM NBQX. Kainate-stimulated JH production was not suppressed by MK-801 (a specific blocker of NMDA-receptor channel), nor was NMDA-stimulated JH production affected by CNQX (a specific antagonist of kainate receptor). These data suggest that active CA cells are stimulated to synthesize more JH by a glutamate-induced calcium rise via NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic l -glutamate receptors. The metabotropic-subtype and ionotropic AMPA-subtype l -glutamate receptors are unlikely to be present on active CA cells.

  • three dimensional mapping of brain neuropils in the cockroach Diploptera punctata
    2001
    Co-Authors: Ann-shyn Chiang, Shu Ling Chiu, Yeechien Liu, Chihying Huang, Changhuain Hsieh
    Abstract:

    Herein, we present a complete three-dimensional (3D) map of major neuropil structures in the central brain of the cockroach Diploptera punctata. The positions of the structures have been ascertained by confocal microscopy, which, until now—for reasons of tissue opacity and nonhomogeneity—has been thought impractical in imaging fluorescently labeled structures thicker than 150 μm. In this report, however, we have used digestive enzymes and microwave-aided fixation to stain, clear, and optically section, in its entirety, an intact central brain more than 500 μm thick. The central brain from an adult female cockroach was stained thoroughly with the membrane probe NBD-ceramide and the DNA probe propidium iodide. The central brain as well as such neuropil regions as mushroom bodies, central complex, antennal glomeruli, and lobus glomerulati were individually outlined, segmented, and reconstructed in three dimensions to a spatial resolution of approximately 1 μm in the X-Y plane and 3 μm in the Z plane. The volume and surface area of each neuropil compartment were determined, and Kenyon cells of the mushroom bodies were counted. We determined that each brain hemisphere contains about 230,000 Kenyon cells, 99 antennal lobe glomeruli, and 40 lobus glomerulatus glomeruli. Segmented compartments were assigned as separate channels and merged into a single data base to reconstruct a 3D central brain containing eight different channels. This is the first 3D map at submicron resolution of an entire animal's brain that measures more than 500 μm in thickness. J. Comp. Neurol. 440:1–11, 2001. © 2001 Wiley-Liss, Inc.

William G Bendena - One of the best experts on this subject based on the ideXlab platform.

  • quantification of allatostatin receptor mrna levels in the cockroach Diploptera punctata using real time pcr
    2008
    Co-Authors: Panida Lungchukiet, Stephen S. Tobe, Jinrui Zhang, William G Bendena
    Abstract:

    The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.

  • molecular cloning and characterization of an allatostatin like receptor in the cockroach Diploptera punctata
    2008
    Co-Authors: Panida Lungchukiet, Stephen S. Tobe, Jinrui Zhang, Cameron B Donly, William G Bendena
    Abstract:

    Abstract Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH 2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a λZAP-cDNA library prepared from the corpora allata of the D. punctata . As an approach to testing the function of this receptor in vivo , the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.

  • expression of allatostatin in the oviducts of the cockroach Diploptera punctata
    2002
    Co-Authors: Chris S. Garside, William G Bendena, P M Koladich, Stephen S. Tobe
    Abstract:

    The cockroach allatostatins (Y/FXFGL/Ia ASTs) are a ubiquitous family of peptides in the invertebrates. They affect numerous physiological processes including the inhibition of juvenile hormone III (JH) biosynthesis, inhibition of muscle contraction, inhibition of ovarian ecdysteroid biosynthesis and inhibition of vitellogenin (Vg) release from the fat body. We have developed and optimized a sensitive and specific quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Diploptera punctata AST (Dippu-AST) expression. Using this technique we show that tissues of both lateral and common oviducts and the ovary express message for Dippu-AST. Moreover, the pattern of expression observed in the oviducts and ovary is strikingly similar with significant changes occurring during the reproductive cycle. Specifically, expression of AST is drastically reduced during the time of maximal vitellogenin (Vg) uptake, with higher levels measured prior to and following vitellogenesis. Furthermore, using immunocytochemistry, we have shown Dippu-AST-like-immunoreactivity in the terminal abdominal ganglion, as well as in ventral nerve 7, some branches of which innervate the common and lateral oviducts with other branches innervating the bursa copulatrix and brood sac of mated female D. punctata. The pattern of Dippu-AST expression and immunocytochemical staining suggests that ASTs function, in part, to regulate the cycle of vitellogenesis in mated female D. punctata.

  • effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata
    1999
    Co-Authors: M Fuse, William G Bendena, J R Zhang, Emily A Partridge, Ronald J Nachman, Ian Orchard, Stephen S. Tobe
    Abstract:

    Abstract Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and α-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)φ2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18] . Thus, in this organ, AST and LMS have at least two distinct physiological effects.

  • allatostatin in hemocytes of the cockroach Diploptera punctata
    1997
    Co-Authors: James R Skinner, Andrea P. Woodhead, William G Bendena, Steve E Fairbairn, Barbara Stay
    Abstract:

    Allatostatins are neuropeptides that inhibit the production, by the corpora allata, of a major insect hormone, juvenile hormone. These peptides are produced by cells of the brain and ganglia as well as by midgut endocrine cells. Transport from these sites may contribute to the allatostatin content in the hemolymph (insect blood). Using a monoclonal antibody against Diploptera punctata allatostatin I (A-P-S-G-A-Q-R-L-Y-G-F-G-L-NH2) and in situ hybridization with a digoxigenin-labeled cRNA probe generated from a portion of the allatostatin gene, it is demonstrated that allatostatin is present in and synthesized by granular hemocytes of D. punctata. About 5% of the hemocytes react with anti-allatostatin antibody and a similar number hybridize with a cRNA probe that detects allatostatin-specific mRNA. Electron micrographs showed that allatostatin-immunoreactive material occurs in membrane-bound, uniformly dense granules that frequently fill fusiform-shaped cells. Allatostatin in cell and plasma fractions of hemolymph quantified by enzyme-linked immunosorbent assay and by bioassay for inhibition of juvenile hormone synthesis in vitro indicated that about equal quantities (0.1–0.2 fmol/μl) are present in cell and plasma fractions. The production of allatostatin by hemocytes suggests that allatostatins may function as regulatory peptides in hemolymph activities in addition to their other known functions.

Elisabeth Marchal - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the nmda receptor and its role in regulating reproduction in the cockroach Diploptera punctata
    2015
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. Tobe
    Abstract:

    The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternatively spliced variants in the cockroach Diploptera punctata . This is the first report of NR1 paralogs in insects. The tissue distributions and expression profiles of DpNR1A , DpNR1B and DpNR2 in different tissues were also investigated. Previous studies have demonstrated NMDA-stimulated biosynthesis of juvenile hormone (JH) in the corpora allata through the influx of extracellular Ca2+ in Diploptera punctata . However, our data show that the transcript levels of DpNR1A , DpNR1B and DpNR2 were low in the corpora allata. MK-801, a high-affinity antagonist of NMDAR, did not show any effect on JH biosynthesis in vitro . In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-801 resulted in any significant change in JH biosynthesis or basal oocyte growth. Injection of animals with a high dose of MK-801 (30 µg per animal per injection), which paralyzed the animals for 4–5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata .

  • characterization of the juvenile hormone pathway in the viviparous cockroach Diploptera punctata
    2015
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. Tobe
    Abstract:

    Juvenile hormones (JHs) are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase (HMGR) and Juvenile hormone acid methyltransferase (JHAMT). As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

  • mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach Diploptera punctata
    2014
    Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Sven Zels, Jozef Vanden Broeck, Stephen S. Tobe
    Abstract:

    The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca2+ and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.

  • Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development
    2013
    Co-Authors: Elisabeth Marchal, Barbara Stay, Juan Huang, Ekaterina F Hult, Stephen S. Tobe
    Abstract:

    The Pacific beetle cockroach, Diploptera punctata, has proven to be a valuable model insect in the study of the dynamics regulating juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymphal development and reproduction. This stems in part from its unusual mode of reproduction, adenotrophic viviparity, in which females give birth to live young that have been nourished throughout embryonic development by a protein-rich ‘milk’ secreted by the wall of the brood sac or uterus. In this animal, as in most insects, JH regulates both vitellogenin production and its uptake by developing oocytes. However, JH has an antagonistic effect on embryonic development and following oviposition of the fertilized oocytes into the brood sac, JH production halts, in part through the action of a peptide family, the FGLa allatostatins. JH production remains at a low level throughout pregnancy and is only reinstated at the end of gestation, at which time, the next wave of oocytes begins to develop and enter vitellogenesis. Thus, JH production in this species is precisely regulated, since the appearance of JH at inappropriate times would result in abortion of the embryos. Numerous factors are responsible for the regulation of JH biosynthesis, including peptides, biogenic amines, neurotransmitters, ecdysteroids and second messenger effectors. In this review, we discuss these factors and highlight potentially fruitful areas of future research. Although several of the enzymes of the biosynthetic pathway have been cloned, the precise points of rate limitation remain uncertain. The dissection of the biosynthetic pathway and its control awaits the completion of the genome and transcriptome of this important model insect.

  • sequencing and validation of housekeeping genes for quantitative real time pcr during the gonadotrophic cycle of Diploptera punctata
    2013
    Co-Authors: Elisabeth Marchal, Ekaterina F Hult, Juan Huang, Stephen S. Tobe
    Abstract:

    Background Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.