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Stephen S. Tobe - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of the nmda receptor and its role in regulating reproduction in the cockroach Diploptera punctata
The Journal of Experimental Biology, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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 .
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characterization of the juvenile hormone pathway in the viviparous cockroach Diploptera punctata
PLOS ONE, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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.
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mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach Diploptera punctata
Insect Biochemistry and Molecular Biology, 2014Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Sven Zels, Jozef Vanden Broeck, Stephen S. TobeAbstract: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.
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sequencing and validation of housekeeping genes for quantitative real time pcr during the gonadotrophic cycle of Diploptera punctata
BMC Research Notes, 2013Co-Authors: Elisabeth Marchal, Ekaterina F Hult, Juan Huang, Stephen S. TobeAbstract: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.
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the synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata
Peptides, 2009Co-Authors: Charoula Kaskani, Jinrui Zhang, Constantine Poulos, Stephen S. TobeAbstract:Abstract We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH 46 and Dippu-DH 31 , on fluid secretion by Malpighian tubules of male Diploptera punctata . We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH 46 , or slight inhibitory activity, in the case of DH 31 .
Ann-shyn Chiang - One of the best experts on this subject based on the ideXlab platform.
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Cell Size Control by Cyclic GMP Regulates Juvenile Hormone Synthesis in Corpora Allata of the Cockroach, Diploptera punctata
2015Co-Authors: Lingwen Chang, Chuanmei Tsai, Deming Yang, Ann-shyn ChiangAbstract: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
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cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata
Insect Biochemistry and Molecular Biology, 2005Co-Authors: Lingwen Chang, Chuanmei Tsai, Deming Yang, Ann-shyn ChiangAbstract: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.
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ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach Diploptera punctata
Insect Biochemistry and Molecular Biology, 2002Co-Authors: Ann-shyn Chiang, Maciej A Pszczolkowski, Hsin Ping Liu, Shuchen LinAbstract: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.
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insect nmda receptors mediate juvenile hormone biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Ann-shyn Chiang, Maciej A Pszczolkowski, Wei Yong Lin, Hsin Ping Liu, Shu Ling Chiu, Glenn L HolbrookAbstract:Abstract In vertebrates, the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. We have now demonstrated immunohistochemically glutamatergic nerve terminals in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibited elevated cytosolic [Ca2+], but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg2+. Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 μM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg2+. Having obtained substantive evidence of functioning NMDAR in insect corpora allata, we used reverse transcription PCR to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, we used the same primers for DNMDAR1 to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. Our results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution.
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three dimensional mapping of brain neuropils in the cockroach Diploptera punctata
The Journal of Comparative Neurology, 2001Co-Authors: Ann-shyn Chiang, Shu Ling Chiu, Yeechien Liu, Chihying Huang, Changhuain HsiehAbstract: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.
Barbara Stay - One of the best experts on this subject based on the ideXlab platform.
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evolution of a novel function nutritive milk in the viviparous cockroach Diploptera punctata
Evolution & Development, 2004Co-Authors: Anna Williford, Barbara Stay, Debashish BhattacharyaAbstract: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.
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allatostatin in ovaries oviducts and young embryos in the cockroach Diploptera punctata
Journal of Insect Physiology, 2003Co-Authors: Andrea P. Woodhead, Kuen K. Chan, M E Thompson, Barbara StayAbstract: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.
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localization and physiological effects of rfamides in the corpora allata of the cockroach Diploptera punctata in relation to allatostatins
Peptides, 2003Co-Authors: Barbara Stay, J R Zhang, Rodney Kwok, Stephen S. TobeAbstract:The distribution of FMRFamide immunoreactivity in the brain-retrocerebral complex of adult female Diploptera punctata was examined. Immunoreactivity was observed in the brain and corpus allatum as well as in the corpus cardiacum. Immunoreactivity co-localized with allatostatin immunoreactivity within several lateral neurosecretory cells of the brain and in their endings within the corpus allatum. By in vitro radiochemical assay of juvenile hormone release, the effect of two native D. punctata RFamides, an FLRFamide (Leucomyosuppressin) and an FIRFamide were examined. The latter, for which the sequence (SKPANFIRFamide) is reported here, stimulated juvenile hormone release but acted only on corpora allata from females at the end of vitellogenesis (day 6). The interaction of these two RFamides and three D. punctata allatostatins, Dippu-AST 2, 5, and 7 were similarly examined. Only Dippu-AST 2 stimulated release of RFamides from the corpora allata and only on day 6 whereas both RFamides were able to attenuate the inhibitory activity of Dippu-AST 2.
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methyl farnesoate and juvenile hormone production in embryos of Diploptera punctata in relation to innervation of corpora allata and their sensitivity to allatostatin
Peptides, 2002Co-Authors: Barbara Stay, J R Zhang, Stephen S. TobeAbstract:Corpora allata (CA) of embryos of Diploptera punctata have been previously shown to produce JH III. We have re-examined sesquiterpenoid biosynthesis throughout embryonic development and have found that early embryos produce both methyl farnesoate (MF) and JH III; as development proceeds, less MF and more JH is produced. The cockroach allatostatin peptide Dippu-allatostatin (AST) 7 inhibits sesquiterpenoid production by CA of mid to late embryos whereas it exerts a dose-dependent stimulatory effect in early embryos. This stimulatory effect is particularly apparent on MF biosynthesis. CA become innervated by allatostatin-containing nerves in early embryos (35% development). Shortly thereafter, the allatostatin-containing innervation of the CA appears complete.
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allatostatin in hemocytes of the cockroach Diploptera punctata
Cell and Tissue Research, 1997Co-Authors: James R Skinner, Andrea P. Woodhead, William G Bendena, Steve E Fairbairn, Barbara StayAbstract: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.
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identification and characterization of the nmda receptor and its role in regulating reproduction in the cockroach Diploptera punctata
The Journal of Experimental Biology, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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 .
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characterization of the juvenile hormone pathway in the viviparous cockroach Diploptera punctata
PLOS ONE, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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.
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mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach Diploptera punctata
Insect Biochemistry and Molecular Biology, 2014Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Sven Zels, Jozef Vanden Broeck, Stephen S. TobeAbstract: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.
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sequencing and validation of housekeeping genes for quantitative real time pcr during the gonadotrophic cycle of Diploptera punctata
BMC Research Notes, 2013Co-Authors: Elisabeth Marchal, Ekaterina F Hult, Juan Huang, Stephen S. TobeAbstract: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.
Juan Huang - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of the nmda receptor and its role in regulating reproduction in the cockroach Diploptera punctata
The Journal of Experimental Biology, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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 .
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characterization of the juvenile hormone pathway in the viviparous cockroach Diploptera punctata
PLOS ONE, 2015Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Stephen S. TobeAbstract: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.
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mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach Diploptera punctata
Insect Biochemistry and Molecular Biology, 2014Co-Authors: Juan Huang, Ekaterina F Hult, Elisabeth Marchal, Sven Zels, Jozef Vanden Broeck, Stephen S. TobeAbstract: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.
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sequencing and validation of housekeeping genes for quantitative real time pcr during the gonadotrophic cycle of Diploptera punctata
BMC Research Notes, 2013Co-Authors: Elisabeth Marchal, Ekaterina F Hult, Juan Huang, Stephen S. TobeAbstract: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.