4 search hits
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Isolation and characterization of the B-type allatostatin gene of Gryllus bimaculatus de Geer (Ensifera, Gryllidae)
(2004)
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Junling Wang
- 1. Cricket B type allatostatins, which belong to a neuropeptide family sharing the conserved W(X)6Wamide structure, exhibited inhibitory functions on the biosynthesis of juvenile hormones (JH) in vitro in the corpora allata (CA) as well as on ecdysteroid biosynthesis in the ovary of adult crickets (Gryllus bimaculatus). To understand the mechanisms of function of the pleiotropic cricket B type allatostatins, it is necessary to characterize their gene (preprohormone) and study the spatial and temporal expression patterns of the gene. 2. By PCR screening of a random primer cDNA library and by RACE (Rapid Amplification of cDNA Ends), a 535 bp 3´cDNA sequence of the cricket B type allatostatin gene was yielded. This 3´cDNA fragment encodes a putative translation product of 85 amino acids with potential dibasic endoproteolytic cleavage sites, which may allow processing into six peptides including three copies of Grybi-AS B1 (GWQDLNGGWGa) and single copies of Grybi-AS B2 (GWRDLNGGWGa), Grybi-AS B3 (AWRDLSGGWGa), and Grybi-AS B6 (AWNNLGSAWGa), respectively. Three of these deduced peptides were previously isolated from cricket brain extracts by conventional chromatographic techniques and were designated as Grybi-AS B1, Grybi-AS B2, and Grybi-AS B3. The Grybi-AS B6 neuropeptide represents a novel member of the B type allatostatins. 3. The nucleotide sequences encoding the type B allatostatins are high in GC-content and show strong homology. The highest GC-content was found for Grybi-AS B3 with 83.3%. The similarity of the nucleotide sequences encoding Grybi-AS B2 and Grybi-AS B1 is 93.3%, whereas Grybi-AS B2 and B3 share 90% nucleotide identity. 4. By Southern blot analyses, it was proven that the Grybi-AS type B gene is present as a single copy per haploid genome of G. bimaculatus. 5. By RT in situ PCR technique, it could be demonstrated that the Grybi-AS B gene is expressed in various tissues of 1 day old female adult crickets: In the central nervous system the Grybi-AS B gene expression was detected in the brain. In the protocerebrum, strong positive signals were found in the median neurosecretory cells, and to a lesser extent in lateral neurosecretory cells and in neurons. Gene expression was also found in the neurosecretory cells of the deuterocerebrum and the tritocerebrum. Furthermore, Grybi - AS B gene expression was localized in neurosecretory cells of the suboesophageal ganglion (SOG), the thoracic ganglia, and the abdominal ganglia. In the germarium and in primary oocytes of the ovary, Grybi-AS B gene expression was detected as condensed signals in the nuclei, but not in the prefollicular cells or the cytoplasm. With ongoing development of the oocytes, the signals in the nuclei (germinal vesicles) appeared as separated granules with weaker intensity, which finally disappeared, whereas in the follicular cells strong signals became apparent. Grybi-AS B gene expression was also detected in the epithelial cells of the accessory reproductive glands of female crickets. In the caecum and midgut, Grybi-AS B gene expression was found in endocrine secretory and epithelial cells, whereas in the hindgut, positive RT in situ PCR signals were detected in both longitudinal and circular muscles and in the gut epithelial cells. Grybi-AS B gene expression was also found in cells of the fat body and in thoracic (flight) muscles. 6. The results on Grybi-AS B gene expression as obtained by RT in situ PCR were confirmed by RT-PCR and RNA dot blot analyses. The expression of the Grybi-AS B gene in various tissues of adult females varied in an age-dependent manner. In brains of virgin females gene expression increased from the day of emergence until day 8 of adult life. In the ovary of virgin females gene expression showed a maximum at day 4 after ecdysis, whereas in mated females gene expression was high during the first two days and at days 6 to 7, but low inbetween. In caecum and midgut of virgin females gene expression was low during the first 5 days after ecdysis, but peaked at days 6 and 7, whereas in the hindgut gene expression was highest at day 3 of adult life. In the fat body, gene expression showed highest values on day 1 and days 6 to 7 after ecdysis. 7. Gene expression in brain, testes, and accessory reproductive glands of 0 to 3 days old male crickets was also demonstrated by RT-PCR and RNA dot blot analyses.
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The Allatoregulatory Neuropeptides and their Genes in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)
(2004)
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Mohatmed Abdel-latief
- The genes encoding the S. frugiperda allatotropin (Spofr-AT), allatostatin (Spofr-AST), allatostatin type-A (Spofr-AST A) peptide family and allatotropin 2 (Spofr-AT 2) peptides were isolated from S. frugiperda brain cDNA. The Spofr-AT gene is expressed in three mRNA isoforms with 134, 171, and 200 amino acids, respectively, differing from each other by alternative splicing. The Spofr-AST cDNA encodes 125 amino acid residues including one copy of the Manse-AST mature peptide (type-C allatostatin). The deduced precursor sequence of Spofr-AST A gene contains 231 amino acids and allowed unambiguous identification of nine (or ten) peptides of YFXFGL-a peptide family, which are tandemly arranged in three blocks. A cDNA that encodes 53 amino acids was cloned from S. frugiperda brain cDNA, including one copy of a non-amidated decapeptide (Arg-Val-Arg-Gly-Asn-Pro-Ile-Ser-Cys-Phe-OH). This peptide strongly stimulates the synthesis and release of juvenile hormone (JH) in vitro by the corpora allata (CA) of S. frugiperda adult females and was code-named Spofr-AT 2. The primary structure of Spofr-AT 2 is identical at its C-terminus (-NPISCF) with the M. sexta type-C allatostatin (Manse-AST). One-step RT-PCR for semi-quantification of the gene expression, it is demonstrated that both genes (Spofr-AT and Spofr-AST) are expressed in brain, digestive tract, and reproductive organs of larvae, pupae, and adults of S. frugiperda in a time-, tissue-, and sex specific manner. The tissue-specific localization of the prohormone expression, as demonstrated by whole-mount in situ hybridization, confirms the overall tissue distribution of the prohormones as shown by RT-PCR and supports the pleiotropic functions of the peptides. Spofr-AST type-A gene is expressed in the brain, midgut, and reproductive organs of S. frugiperda larvae and adults in a time- and tissue-specific manner. Data confirm the nature of the allatostatin type-A peptides as brain/gut myoregulatory hormones. Northern blotting and RT-PCR analyses revealed that the Spofr-AT 2 gene is expressed as one transcript in the brain, midgut, and ovary in a tissue- and developmental-specific manner. Treating the CA with the synthetic peptide caused an up to tenfold increase in the release of JH. The stimulation of JH release was dose-dependent with an apparent EC50 of ca. 10-7 M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of synthetic Manse-AST. In conclusion, the presented date strengthen the hypothesis that “allatoregulating” neuropeptides are diverse in structure, widely distributed and exhibit multiple functions. The functions may be tissue-specific as well as specific to particular developmental stages of insects. Knowledge of the various peptide precursor sequences has opened the way for synthesis of these peptides for detailed physiological and functional studies. Further quantitative experiments formulated in context of the life history of the animals will certainly yield a more detailed understanding of the mode of action of these peptides in S. frugiperda. Other major challenges in the future will be to clone the receptors for these peptides and to study the receptor distribution in the fall armyworm.
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Auswirkungen der RNA-Interferenz auf allatoregulierende Neuropeptide und Lokalisation der B-Typ Allatostatine bei Spodoptera frugiperda
(2009)
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Manuela Griebler
- Zusammenfassung Bei Insekten regulieren Peptidhormone (Neuropeptide) viele physiologische und entwicklungsrelevante Prozesse, wie Wachstum, Häutung, Metamorphose, Reproduktion, Diapause, Fressverhalten und den Metabolismus. Allatoregulierende Neuropeptide hemmen (Allatostatine, AS) oder fördern (Allatotropine, AT) die Juvenilhormonbiosynthese in den Corpora allata, können aber auch andere Funktionen aufweisen, wie z.B. eine myoregulatorische Aktivität. In dieser Arbeit wurde zunächst die Lokalisation der B-Typ Allatostatine [W(X)6W-amide] aus Spodoptera frugiperda immunologisch untersucht. In Gehirnen adulter, 2 Tage alter Weibchen trat eine deutliche Immunfärbung in medianen neurosekretorischen Zellen des Protocerebrums, im oberen Bereich des Zentralkörpers und in den Antennalloben (Glomeruli) auf. In den Thorakal- und Abdominalganglien von Larven des letzten Larvenstadiums wurde eine intensive Färbung im Bereich des Neuropils und eine schwächere Immunreaktion im Bereich der lateral gelegenen neurosekretorischen Zellen beobachtet. Starke Färbung war auch in den Malpighischen Gefäßen sichtbar, die wahrscheinlich der apikalen Membran zwischen den Hauptzellen zuzuordnen ist, sowie im Mitteldarm und in larvalen Hoden. Anschließend wurden die physiologischen Funktionen der bereits charakterisierten allatoregulierenden Neuropeptide AT 2 und AS-C-Typ aus S. frugiperda nach einer Hemmung der Genexpression mittels der RNA-Interferenz Methode untersucht. Es wurden Expressionsstudien durchgeführt, um die Wirksamkeit und die Spezifität der RNA-Interferenz in verschiedenen Geweben zu überprüfen. Zudem wurden die Auswirkungen der RNA-Interferenz auf verschiedene physiologische Parameter untersucht. Dabei lag der Schwerpunkt der Experimente auf der Regulierung der Juvenilhormonbiosynthese, der Reproduktion, der Verpaarung und der Metamorphose. Nach Injektion und auch nach Fütterung von AS-C- bzw. AT 2-doppelsträngiger RNA (dsRNA) war die Expression der mRNA im Gehirn und Darm von Larven reduziert. Zudem führte die Injektion von AS-C- bzw. AT 2-dsRNA auch bei adulten Weibchen und Männchen unterschiedlicher Altersstufen zu einer Reduktion der Expression im Gehirn. Die Reduktion der Transkription bei Larven ging mit einem erhöhten Juvenilhormon(JH)-Titer in der Hämolymphe der Tiere einher. Dies lässt eine allatostate Aktivität der beiden Peptide zu diesem Zeitpunkt vermuten. Die Folge des erhöhten JH-Titers war eine Verlängerung des letzten Larvenstadiums. Bei adulten Weibchen war der Effekt der Injektion von AS-C- bzw. AT 2-dsRNA auf den JH-Titer vom Alter der Tiere abhängig und variierte innerhalb der JH-Homologen (JH I bis JH III). Diese Ergebnisse lassen vermuten, dass beide Peptide bei adulten Weibchen unterschiedliche Wirkungen auf unterschiedliche JH-Homologe in spezifischen Stadien der Entwicklung haben. Adulte Männchen hatten nach einer Injektion von AS-C- bzw. AT 2-dsRNA an bestimmten Tagen einen erhöhten JH-Titer. Bei der Verpaarung werden mit der Spermatophore verschiedene Substanzen, darunter auch JH, vom Männchen auf das Weibchen übertragen. Bei mit Ringer injizierten Männchen konnte vor der Verpaarung JH in den akzessorischen Drüsen (AD) detektiert werden. Nach der Verpaarung nahm der JH-Titer in den männlichen AD stark ab. Bei unverpaarten Weibchen war kein JH in der Bursa copulatrix nachweisbar. Wurden die Weibchen jedoch verpaart, konnten große Mengen JH in der Bursa copulatrix gefunden werden. Mit AS-C-dsRNA injizierte Männchen zeigten vor der Verpaarung einen erhöhten JH-Titer in ihren AD, der nach der Verpaarung wieder niedriger war. Die mit diesen Männchen verpaarten Weibchen hatten einen erhöhten JH-Titer in der Bursa copulatrix im Vergleich zu mit Ringer injizierten verpaarten Weibchen. Die Ergebnisse lassen auf eine allatostate Wirkung der AS-C-Peptide auf den JH-Titer in den AD schließen. Bei mit AT 2-dsRNA injizierten Männchen war der JH-Titer in den AD, im Vergleich zu den Kontrollen, sowohl vor als auch nach der Verpaarung erniedrigt, ebenso wie in der Bursa copulatrix der mit diesen Männchen verpaarten Weibchen. Weibchen, die mit AS-C- bzw. AT 2-dsRNA injiziert worden waren, zeigten eine reduzierte Eiablage (Oviposition), ebenso wie Ringer injizierte Weibchen, die mit AS-C- bzw. AT 2-dsRNA injizierten Männchen verpaart worden waren. Diese Beobachtungen deuten darauf hin, dass die Fertilität der Männchen und Weibchen durch Neuropeptide unterschiedlich beeinflusst wird, und damit auch die Eiproduktion und Eiablage der Weibchen. Insektenneuropeptide eignen sich als (Bio)Insektizide, da sie die Physiologie und das Verhalten der Insekten während der Entwicklung und Reproduktion beeinflussen bzw. regulieren. Eine Expression von spezifischen dsRNAs in transgenen Pflanzen, gerichtet gegen allatoregulierende Neuropeptidgene, könnte die gezielte Bekämpfung von Schadinsekten, wie z. B. des Agrarschädlings S. frugiperda, ermöglichen.
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Control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda
(2010)
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Digali Lwalaba
- This thesis investigates control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda. 1. Control of enzyme release in the cricket G. bimaculatus. Using flat-sheet preparations of the caecum, digestive enzyme release was investigated. More trypsin, aminopeptidase and amylase are secreted in the caecum of fed crickets than in unfed crickets, but basal levels of certain enzymes are released continuously even in unfed animals. A variable ratio of nutrients in ingested food leads to a different ratio of digestive enzyme release, but a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Maltose and glucose elevate amylase release from the tissues into the incubation medium, but starch does not. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase, and only low concentrations of peptone increase trypsin release. In crickets, the continuous release of proteases is sufficient to meet the needs for growth, and only moderate stimulation of trypsin results from feeding. Carbohydrates are used for energy, and the release of amylase is adjusted to the amount of food ingested. The neuropeptide allatostatin Grybi-AS 5 elevates the release of amylase in fed females, but not of trypsin or aminopeptidase, however, both amylase and trypsin release are stimulated by AS 5 in unfed crickets. Fed crickets have sufficient trypsin to obtain needed amino acids, but unfed do not, therefore the AS stimulation of trypsin release in unfed crickets makes sense. 2. Control of enzyme release in the larvae of S. frugiperda. A flat-sheet preparation of the ventriculus was used to test the release of amylase, trypsin and aminopeptidase in response to specific nutrients in the food and to specific neuropeptides. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. Dietary carbohydrates are used for energy, and larvae adjust amylase activity to carbohydrate ingestion. Amylase activity is 5-times higher in fed compared to unfed larvae, and sugars in the incubation medium induces more than a 300 % increase in amylase release. Plants contain a low level of protein, but larvae need proteins for growth, thus the larvae can not afford to lose proteins by egestion. Therefore, trypsin activity remains high even in unfed larvae. As a result, proteins and amino acids have little or no effect on trypsin or aminopeptidase release in incubated tissues. The control of enzymes release in response to food is most likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin release, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse- AT stimulates myoactivity. 3. Inhibition of enzyme release in the larvae of S. frugiperda. Exogenous inhibitors are produced by plants, and are ingested by the insect. Endogenous inhibitors are produced by the gut epithelial cells themselves. A dose-dependent inhibition of endogenous enzymes occur in the lumen after feeding L6 larvae with the exogenous serine protease inhibitor from soybean (SBTI), the specific trypsin inhibitor TLCK, an aminopeptidase inhibitor (bestatin), and an amylase inhibitor from wheat. Inhibition in tissue extracts is seen only with higher doses of SBTI and TLCK. Inhibition of enzyme release into the incubation medium is apparent only with very high doses of SBTI. Inhibition in the tissues and inhibition of release indicate a direct cellular response to an inhibitor present in the lumen. The elevation of aminopeptidase activity in response to ingested trypsin inhibitors indicates a cellular synthesis in response to the product of a digestive enzyme. The enzymes investigated are irreversibly inactivated by 10 min at 90°C, but the corresponding inhibitors are not, therefore endogenous inhibitors could be identified. Endogenous inhibitors are present in the ventricular cells and in the lumen. We suggest that the endogenous protease inhibitors may protect the epithelium by inactivation of the trypsin in underfed larvae. This is the first explanation of how insects are able to secrete an active trypsin.
