19 search hits
-
Isolation and characterization of the B-type allatostatin gene of Gryllus bimaculatus de Geer (Ensifera, Gryllidae)
(2004)
-
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.
-
The signal transduction of the adipokinetic hormone and regulation of energy metabolism in the cricket, Gryllus bimaculatus de Geer (Ensifera: Gryllidae)
(2004)
-
Anurag N. Anand
- The fat body from the cricket, Gryllus bimaculatus incorporates acetate, glycerol or palmitate into lipid in vitro. Adipokinetic hormone (AKH) inhibits acetate incorporation into lipid by the fat body from adult crickets in vitro. AKH inhibits palmitate incorporation into lipid to a small extent, however, it does not influence the incorporation of palmitate into different lipid classes. In the presence of AKH, glycerol incorporation into lipid by the adult cricket fat body increases by several fold. AKH does not influence the incorporation of glycerol into different lipid classes. The fat body incorporates glycerol mainly into triacylglycerol (TAG) and almost exclusively into the backbone. AKH inhibits fatty acid (FA) synthesis but not the coupling of FAs with the glycerol backbone. Triacylglycerol lipase (TGL) from the fat body, in last larval instar and adult crickets, shows an age-dependent pattern of activity. AKH activates TGL, but does not regulate ACCase, ACL and FAS upon treatment of the fat body for a short period, under the assay conditions. The FAS and acetate incorporation activities are highly correlated in an age-dependent manner and are highest on day 2 of the adult stage. The activation of the formation of sn-1,2-DAG from TAG, in the fat body by AKH seems to be via the removal of FAs at positions 1 and 3, followed by reacylation of 2-MAG. The presence of calcium in the incubation medium is crucial for the inhibition of acetate incorporation by AKH; possibly it is essential for the binding of AKH to the receptor. The influx of acetate and calcium into and efflux of calcium from the cytosol are not affected by AKH. The release of calcium from intracellular calcium stores in the fat body, caused by thapsigargin, inhibits acetate incorporation irreversibly and shows a tendency for the activation of TGL. The calcium ionophore ionomycin inhibits acetate incorporation by the fat body reversibly and shows a tendency for TGL activation. Another ionophore, A23187 also inhibits acetate incorporation. Caffeine and theophylline inhibit acetate incorporation by the fat body in a reversible manner and tend to activate TGL. Caffeine seems to act via the release of calcium from intracellular stores and not via increase in the cellular cAMP concentrations. cAMP analogues and agonists do not influence acetate incorporation, however, the agonists, IBMX and forskolin cause a multifold increase in the cAMP concentrations in the fat body. AKH does not affect the cAMP concentrations in the fat body suggesting that cAMP is not involved in the signal transduction. As the activation of protein kinase C by PMA (a phorbol ester) does not affect acetate incorporation, diacylglycerol does not seem to be involved in the AKH signal transduction. The activation and/or translocation of TGL and inhibition of fatty acid synthesis by AKH seems to be via the release of calcium from intracellular calcium stores. Lipid and protein form a major part of the fat body in the penultimate larval instar crickets, while glycogen forms a minor part. However, in comparison with the adult (glycogen content of fat body about 1%) and last larval instar crickets (glycogen content about 3%), the penultimate larval instar crickets contain higher amounts of glycogen (about 9%). AKH inhibits acetate incorporation into lipid by the fat body from penultimate larval instar crickets. The patterns of acetate incorporation and inhibition by AKH are similar in both, males and females. The inhibition is dose-dependent with an EC50 of 79 pM AKH. AKH seems to play an important role in the development and reproduction of insects, in addition to its role during flight metabolism.
-
Charakterisierung der reduktiven Aktivitäten der CO-Dehydrogenasen aus Oligotropha carboxidovorans und Streptomyces thermoautotrophicus
(2004)
-
Antonius Sarjiya
- Es wurde in dieser Arbeit entdeckt, dass die aerobe CO-Dehydrogenase in der Lage ist, alternative Substrate wie Azid, Nitrit und Nitrat zu reduzieren. CO-Dehydrogenase aus Oligotropha carboxidovorans und Streptomyces thermoautotrophicus führen die Reduktionsreaktion wie folgt durch: Azid wird zu Ammonium und molekularem Distickstoff reduziert. N3- + 2 e- + 3 H+ -> NH3 + N2 Nitrit wird zu Ammonium reduziert. NO2- + 6 e- + 7 H+ -> NH3 + 2H2O Nitrat wird zu Nitrit reduziert. NO3- + 2H+ + 2e- -> NO2- + H2O Das katalytische [CuSMoO2]-Zentrum der CO-Dehydrogenase ist verantwortlich für die Reduktion von Azid und Nitrit. Ohne einem aktiven katalytischen-Zentrum findet die Reduktion von Azid und Nitrit nicht statt. Die CO-Dehydrogenase katalysiert gleichzeitig die H2-Oxidation und die Azid-Reduktion. Die während der H2-Oxidation entstehenden Elektronen werden für die Azid-Reduktion verwendet. H2 + N3- + H+ -> NH3 + N2 Oxidation und Reduktion finden mit größter Wahrscheinlichkeit am katalytisch aktiven [CuSMoO2]-Cluster der CO-Dehydrogenase statt. Die gleichzeitige Anwesenheit von CO und Azid hemmt die CO-Oxidations- und Azid-Reduktionsaktivität. Die Kristallstruktur zeigt, dass in Gegenwart von CO und Azid aus dem [CuSMoO2]-Cluster das Cu-Atom und der Sulfidoligand entfernt sind. Die Nitrat-Reduktion der CO-Dehydrogenase ist unabhängig vom katalytisch aktiven [CuSMoO2]-Cluster. Die CO-Dehydrogenase aus S. thermoautotrophicus zeigt ähnliche Oxidations- und Reduktionsaktivitäten. Die CO-Dehydrogenase aus dem thermophilen, Gram-positiven Bakterium S. thermoautotrophicus reagiert sehr ähnlich wie die CO-Dehydrogenase aus dem mesophilen, Gram-neagtiven Bakterium O. carboxidovorans.
-
Funktionale Analyse des ATPase- und des Zn2+-Bindungs-Motivs der Bacillus subtilis Protease FtsH und deren Einfluss auf die Sporulation
(2004)
-
Matthias Kotschwar
- Die membrangebundene, ATP- und Zn2+- abhängige Metalloprotease FtsH ist in E. coli bereits gut untersucht. Es handelt sich um ein multifunktionales Protein mit ATPase- und Protease-Aktivität. Dort wurden eine Vielzahl von E. coli ftsH-Mutanten mit pleiotropen Phänotyp charakterisiert und die Auswirkung von gezielten Mutationen in vivo und in vitro analysiert (Akiyama et al., 1994; Akiyama et al., 1996). Über FtsH aus B. subtilis ist hingegen noch wenig bekannt. Bisher wurden erst die Phänotypen einer ftsH-Nullmutante und einer Insertionsmutante beschrieben, die zu einem pleiotropen Phänotyp führen. Die Rolle bestimmter Bereiche oder Aminosäuren von FtsH wurden hier nicht untersucht (Deuerling et al., 1995; Deuerling et al., 1997; Lysenko et al., 1997). In ihrer Diplomarbeit konstruierte Eva Harfst 1999 vier B. subtilis ftsH-Allele, indem sie gezielt Aminosäuren in der Walker-A-Box und im Zinkbindungsmotiv austauschte, wie sie bereits für E. coli und Hefe FtsH beschrieben wurden. Diese mutierten ftsH-Gene und ein wildtypisches zur Kontrolle brachte sie in den pX-Vector ein, wodurch sie unter die Kotrolle eines Xylose-induzierbaren Promotors gestellt wurden. In der vorliegenden Arbeit wurden die mutierten ftsH-Allele in das B. subtilis-Chromosom integriert und in einem zweiten Schritt das wildtypische ftsH deletiert. Die Phänotypen der so erzeugten Mutanten (sowohl die aus dem ersten Schritt, die noch das wildtypische ftsH tragen, als auch die aus dem zweiten Schritt, die nur noch das mutierte Allel tragen) wurden hinsichtlich des filamentösen Wachstums, der Sporulation, der Sensitivität gegenüber Hitzeschock und hyperosmotischen Bedingungen analysiert. Dabei stellte sich heraus, das die mutierten Allele keinen Einfluss auf den Phänotyp der Stämme haben, die das wildtypische ftsH noch tragen, und in den Knockout-Stämmen das wildtypische ftsH nicht ersetzen können, also den Phänotyp einer ftsH- Nullmutante zeigen. Des weiteren sind die mutierten ftsH-Allele ebenfalls nicht in der Lage, die Defizienz in der Subtilisin-Sekretion zu kompensieren. ATPase-Tests zeigten, dass die eine Mutation im Walker-A-Motiv die ATPase-Aktivität der beiden betroffenen Proteine ausschaltete. Protease-Tests ergaben, dass keines der mutierten FtsH-Proteine noch eine Protease-Aktivität aufweist. Das bedeutet, dass einerseits die Basenaustausche im Zinkbindungsmotiv die Protease-Aktivität ausschalten, andererseits aber auch keine Protease-Aktivität ohne ATPase- Aktivität möglich ist. Daraus folgt weiterhin, dass die ATPase-Aktivität des FtsH-Proteins allein nicht ausreicht, um einen der untersuchten pleiotropen Phänotypen einer ftsH--Mutante zu kompensieren. Die Funktionalität sowohl der ATPase- als auch der Protease-Domäne ist also für FtsH und somit für B. subtilis hinsichtlich des filamentösen Wachstums, der Sporulation, der Sensitivität gegenüber Hitzeschock und hyperosmotischen Bedingungen essentiell. Anhand von Northern-Blot-Hybridisierungsexperimenten konnte gezeigt werden, dass FtsH eine essentielle Rolle bei der Phosphorylierung von Spo0A haben muss, da ohne FtsH kein aktives Spo0A gebildet wird. Die daraus resultierende Sporulationsdefizienz kann aber durch eine Spo0A-Mutante, die ohne Phosphorylierung aktiv ist, fast vollständig kompensiert werden. FtsH ist also während der Sporulation nur für die Phosphorylierung von Spo0A essentiell.
-
Verbesserte Schätzungen von CO2- und N2O-Flussraten von Böden mitteleuropäischer Ökosysteme - Entwicklung konzeptioneller Neuerungen von Bodenemissionsmodellen
(2004)
-
Sascha Reth
- Im Zuge dreier Intensivmessperioden innerhalb des Projektverbundes VERTIKO (Vertikaltransporte von Energie und Spurenstoffen an Ankerstationen und ihre räumliche/zeitliche Extrapolation unter komplexen natürlichen Bedingungen) wurden in Melpitz (24.09.-12.10.2001), Lindenberg (03.06.-06.07.2002) und Tharandt (18.05.-23.05. und 08.06.-14.06.2003) simultane Messungen der N2O- und CO2-Emissionen aus Wiesen-, Acker- und Waldböden durchgeführt. Die Emissionsdaten wurden mit einem photoakustischen Infrarot-Monitor in geschlossenen Kammern erhoben. Die entwickelte automatische Steuereinrichtung erlaubte es 5 Kammern simultan als räumliche Replikate zu messen und ermöglichte damit den Einsatz unter geringem Personalaufwand. In Rahmen dieser Arbeit wurde an einer Kalibrierungskampagne in Hyytiälä / Finnland teilgenommen, in der sowohl das automatisierte Messsystem gegen einen Referenzgasfluss, als auch gegen 19 Kammersysteme anderer Forschungseinrichtungen verglichen wurde. Die Anwendbarkeit der Methode wurde durch den Vergleich bestätigt und im weiteren wurde für jedes System ein Kalibrierungsfaktor ermittelt, der einen Vergleich mit anderen Ergebnissen erlaubt. Die vorliegende Arbeit konnte weiterhin zeigen, dass verschiedene Messmethoden, Bodenkammermessungen und Eddy Kovarianz, auf einer Brache vergleichbare Resultate erzielen. Ein gewichtetes Footprintmodell ermöglichte den direkten Vergleich von CO2-Flüssen, die hauptsächlich aus dem Quellgebiet Brache emittiert wurden und daher wenig durch Senkenterme kontaminiert schienen. Zudem wurde die Einfluss der Nettoflüsse aus der benachbarten Wiesenfläche untersucht. Die Emissionshöhen von CO2 und N2O variierten stark zwischen verschiedenen Landnutzungstypen. Generell konnten für beide Gase auf Wiesen und landwirtschaftlich genutzten Böden höhere Emissionen als auf Waldböden gefunden werden. Auch innerhalb der Standorte war die zeitliche und räumliche Heterogenität hoch. Diese Arbeit entwickelte ein nichtlineares Regressionsmodell auf der Basis der gemessenen Flussraten zur Berechnung von CO2-Emissionen für verschiedene Landnutzungstypen, welches eine regionale Anwendung erlaubt. Neben Bodentemperatur und -wassergehalt als Parameter wurden der pH-Wert und die Wurzelmasse berücksichtigt. Als zusätzlicher und unerwarteter Parameter wurde der zeitlicher Abstand zu einem Regenereignis als Steuergröße identifiziert. Auch für N2O wurde ein nichtlineares Regressionsmodell (DenNit) für regionale Anwendungen entwickelt, das eine Abschätzung der N2O-Emissionen von Brachen, Wiesen und Wald mit lediglich 6 Faktoren (Bodentemperatur, Bodenwassergehalt, pH-Wert, Nitrat- und Ammonium-verfügbarkeit, zeitlicher Abstand zu einem Regenereignis) erlaubt. Im Anschluss wurden die entwickelten Modelle für den Zeitraum dreier Intensivmesskampagnen für die verschiedenen Landnutzungstypen angewendet und zeigten gute Übereinstimmungen mit publizierten Emissionsdaten anderer Arbeiten. Die Vorhersagbarkeit von Bodenatmungsraten und Lachgasemissionen mit den entwickelten Modellen konnte deutlich gesteigert werden.
-
Facultative butterfly-ant interactions - the role of variation in composition of nectar secretions
(2004)
-
Holger Daniels
- The significance of variation in nectar secretions of facultatively ant-associated lycaenid butterfly larvae was investigated. The strongly myrmecophilous European Polyommatus coridon, and two moderately myrmecophilous species, the Palaearctic P. icarus and the subtropical Zizeeria knysna were used. Both Polyommatus species are closely related, Z. knysna is a far more distant relative. To obtain high numbers of caterpillars a new method for breaking the egg diapause of the univoltine P. coridon was established, resulting in 65% subitaneously hatching larvae. Based on observations of ants tending caterpillars a new “artificial ant” was assembled, allowing further studies on the ants’ stimulatory antennation pattern. Nectar-harvesting with micro-capillaries from caterpillars attended by ants was optimised and allowed determination of individual secretion droplet size based on large samples. Mean droplet size was 3.7nl in P. coridon, 2nl in P. icarus and 1.4nl in Z. knysna, in the latter two species 65-79% smaller than previously reported. Comparative chemical analyses (HPLC) revealed sucrose as main sugar component in nectar of all three species. In P. coridon it was accompanied by glucose and rarely by further sugars, but never by melezitose. In P. icarus and Z. knysna melezitose was the second most-important component, followed by fructose and glucose. Total sugar contents were 43.6±14.8g/l for P. coridon, 74.2g/l for P. icarus and 68.3±22.6g/l for Z. knysna. P. coridon nectar contained up to 14 amino acids. Major component was always leucine (50% of total), further important were tyrosine, proline, arginine, and phenylalanine. P. icarus nectar comprised up to six amino acids, dominated by tyrosine and phenylalanine. Z. knysna nectar contained only alanine and proline. Total amino acid contents were 9.7±3.4g/l for P. coridon, 1.2g/l for P. icarus and 0.3±0.2g/l for Z. knysna. Nectar composition was considerably different from hemolymph composition. Larval food had minor influence on P. coridon nectar composition. Caterpillars fed with semi-synthetic diet secreted more sucrose, with a trend towards higher total sugar content, and produced nectar with a more homogeneous amino acid pattern than larvae reared on natural host plants. Bioassays with ants from three different subfamilies (Lasius niger (Formicinae), Myrmica rubra (Myrmicinae), Tapinoma melanocephalum (Dolichoderinae)) demonstrated a preference for sucrose (standard concentration 0.1mol/l, as in P. coridon nectar) over monosaccharides. Melezitose in nectar concentration (P. icarus) was not preferred to sucrose. Some single amino acids in sucrose solutions were preferred over pure sucrose, e.g. leucine by L. niger, or phenylalanine and tyrosine by M. rubra. In general, raising amino acid concentration did not enhance preferences and even reduced them in some cases. Mixtures of four or eleven amino acids in sucrose and complete nectar analoga were preferred to sucrose. L. niger preferred a balanced mixture over an energetically similar, less balanced mixture. Due to the tremendous variability in gustatory preferences exhibited by the broad range of largely unpredictable ant visitors, lycaenid caterpillars should either decide on a balanced mixture, containing possible amino acid ‘key compounds’ in moderate concentrations, or if this investment does not pay, should secrete sugar-rich nectars. Using the data from the HPLC analyses and the droplet size measurements combined with published studies on secretion rates allowed estimations of the lifetime energetic value of secretions. One P. coridon caterpillar would deliver 5.5-24.4J, one P. icarus 1.9-14.2J and one Z. knysna 0.7-2.8J. Dissection and gravimetric analysis allowed estimation of the caloric equivalent of larval biomass. One early P. coridon third instar (onset of nectar secretion) would yield 17.6J, and prepupae 656J. Z. knysna second instars would yield 0.95J, third instars 3.2J, and prepupae 83.1J. Thus preying on the caterpillars rather than harvesting their secretions would be of greater energetic benefit. Comparisons of this model with further reports show the rather low benefit accruing to ants from tending facultatively myrmecophilous lycaenids, underpinning that manipulation of ants (by means of still unknown chemicals) must also be involved. The model data also suggest that the mutualistic nature of facultative caterpillar-ant associations will not always be granted, and be strongly conditional. Nectar composition data support the view that in myrmecophilous lycaenids secretions rich in amino acids are related to intimate, often obligate ant-associations, whereas facultative and unspecific myrmecophiles rely more on sugars. Degree of myrmecophily seems to be a better predictor of secretion content than taxonomic relatedness. The low investment costs of the caterpillars into nectar secretions well explain the enormous taxonomic and geographical distribution of facultative myrmecophily.
-
Importance of floral scent compounds for the interaction between Silene latifolia (Caryophyllaceae) and the nursery pollinator Hadena bicruris (Lepidoptera: Noctuidae)
(2004)
-
Stefan Dötterl
- In the present study, the role of floral volatiles for the interaction between the nocturnal Caryophyllaceae Silene latifolia, and the noctuid moth Hadena bicruris was determined. This insect-plant relationship is one of the known nursery pollination systems, where pollinators reproduce within the flowers they pollinate. Silene latifolia is a dioecious weed, native to Europe and formerly introduced to North America. It is the main larval host plant of H. bicruris, which is distributed in Europe and North Africa. Especially night-active moths, among them H. bicruris, which are attracted by the flower scent, pollinate S. latifolia. However, until now, nothing was known about the role of single flower scent compounds for the attraction of the moths. This thesis describes the chemical composition and the geographical variability in the flower scent of S. latifolia. Furthermore, electrophysiological and behavioural tests with floral scent extracts and single authentic standard compounds were carried out in H. bicruris to identify the attractive compounds of the complex floral scent. To get an insight into the role of floral scent in guiding potential pollinators on flowers, the spatial fragrance pattern within the flowers of S. latifolia was determined, additionally. The variability in floral scent was very high, especially between different populations, and different chemotypes were characterised.Typical compounds in floral scent of S. latifolia were lilac aldehyde isomers, trans-beta-ocimene, benzaldehyde, phenyl acetaldehyde, or veratrole. Some of these compounds are known to attract nocturnal Lepidoptera species. To characterise antennal and behavioural responses of H. bicruris to various floral scent chemotypes of S. latifolia, and to S. vulgaris (which is rarely also used as host plant), different S. latifolia extracts, and a S. vulgaris extract were analysed using GC-MS methods. These extracts were further used in GC-FID/EAG and GC-MS/EAG detections, respectively. Main compounds in the tested extracts often elicited main signals in the antennae (e.g. lilac aldehydes, phenyl acetaldehyde). Some compounds elicited main signals in the antennae, though they were only minor components in the extracts (e.g. 3-methyl-butyl-aldoxime, benzaldehyde). Other compounds elicited only weak signals in the antennae, though they were abundant in the extracts (e.g. myrcene, methyl benzoate). The compounds of the most common chemotypes of S. latifolia were very sensitively detected by Hadena bicruris, whereas compounds of less abundant chemotypes were less sensitively detected. Floral scent blends that were dominated by lilac aldehydes or phenyl acetaldehyde effectively attracted moths. Hadena bicruris can electrophysiologically and behaviourally distinguish between its main host plant, S. latifolia, and the similarly scented S. vulgaris, another rarely used larval host plant, only by their floral scent. To identify floral scent compounds of S. latifolia that are important for the attraction of H. bicruris, the GC-FID/EAD or the GC-MS/EAD method was used in a first step to identify compounds that elicit signals in the antennae of the moth. Electrophysiologically very active compounds were tested in wind tunnel bioassays, and the attractivity of these compounds was compared to the attractivity of the natural scent of whole flowers of S. latifolia. The antennae of H. bicruris detected substances of several compound classes such as monoterpenoids, benzenoids, fatty acid derivatives, and nitrogen-bearing compounds. Lilac aldehydes were the most attractive compounds in wind tunnel bioassays, and attracted 90% of the tested moths, as did the scent of single flowers. Some compounds did not attract any moth, though they elicited significant signals in the antennae. To determine the parts of the female and male flowers responsible for scent emission, volatiles from attached intact flowers were sampled and then single flower parts were progressively removed. After each preparation step, volatiles were collected from the remaining “flower”. Especially the petals and the anthophore emitted the typical flower volatiles of S. latifolia; and compounds emitted from the petals differed from the compounds emitted by the anthophore. The anthophore emitted only lilac aldehydes and alcohols. Lilac aldehydes are known to be behaviourally very attractive for noctuid Lepidoptera such as Autographa gamma and Hadena bicruris, and they may serve as nectar guides in S. latifolia.
-
Measuring juvenile hormone and ecdysteroid titers in insect haemolymph simultaneously by LC-MS: The basis for determining the effectiveness of plant-derived alkaloids as insect growth regulators
(2004)
-
Stephanie Westerlund
- Aim of this thesis was to develop a liquid chromatography-mass spectrometry (LC-MS) method to monitor hormones and their metabolites in the haemolymph of insects simultaneously. Furthermore, some plant-derived alkaloids were structurally elucidated, which may be used as insect growth regulators thus affecting haemolymph hormone titers in putative pest species. Juvenile hormones (JHs), JH diols and ecdysteroids were easily separated by high performance-liquid chromatography (HPLC) in less than 20 min using a reversed-phase C18 column and a methanol-water gradient. Subjecting the JH-JHBP (juvenile hormone binding protein) complex to HPLC was sufficient in releasing JH from the JHBP. In order to prevent JH from binding to glass surfaces, it was necessary to include a carrier in the solvent. JHs have a high affinity to polypropylene vials and should therefore be avoided, if no carrier is being used, such as triton X-100. The darkening of the haemolymph due to eumelanin production by phenol oxidases served as a visual indicator of general enzyme activity in the haemolymph. Isooctane:MeOH (1:1, v/v) inactivated the phenol oxidase system when used at a solvent-haemolymph ratio of 10. An isooctane:MeOH extract of haemolymph was most efficient in keeping JH distributed evenly in solution and prevented JH from adhering to the glass vessel. JH concentration in standard solutions was reduced with increasing sonication time. Highest ionization of JH was achieved in MeOH for MS compared to ACN or by using formic acid as an additive. In the positive ESI (electrospray ionization) mode the most abundant ions formed in haemolymph extract of Gryllus bimaculatus was the sodium adduct for JHs, JH diols and JH acids. At higher JH concentrations, the potassium adduct was also observed. The sodium and the potassium adducts were present in ecdysteroid analysis. The same ionization pattern was observed in Spodoptera frugiperda, Myrmicaria eumenoides and Acyrthosiphon pisum haemolymph. Method validation of the LC-MS method confirmed reproducibility and repeatability. The LODs for JHs and JH diols were between 6 to 12 pg, and 93 pg for ecdysteroids. 72 haemolymph samples can be processed per day by the LC-ESI-MS method using an autosampler. JH and ecdysteroid titer measurements showed good agreement between haemolymph titers and developmental events in Spodoptera frugiperda and Gryllus bimaculatus larvae and Gryllus bimaculatus adults. In Gryllus bimaculatus female and male last instar larvae, the JH titers were low and steady until day 6. The ecdysone peak maximum shifted from day 3 to a 20-hydroxyecdysone maximum peak on day 5, coinciding with the onset of adult ecdysis. JH III titers increased on day 3 in paired Gryllus bimaculatus males, occurring simultaneously with spermatophore maturation and deposition. A similar response was seen with ecdysone titers. Mated female crickets experienced a JH III titer increase on day 4 which coincides with egg deposit on day 4. Ecdysone titers reach a maximum on day 3 and ovary weights on day 4. Besides JH III, JH I was found in 5 to 8-day old female adult crickets, and in 6 and 7-day old male adult crickets. 20-Hydroxyecdysone was found neither in female nor in male Gryllus bimaculatus mated adults. The “classical” interplay between JHs and ecdysteroids was observed in 5th instar Spodoptera frugiperda larvae. JH titers decreased towards the end of the larval stadium and 20-hydroxyecdysone gave a sharp peak on the last day of the 5th instar. Extremely low levels of JH were measured in 6th instar larvae. 20-Hydroxyecdysone and ecdysone titers increased simultaneously in prepupae. The already known alkaloids arborinine and arborine, and the for the first time isolated 4-methoxy-1-methyl-2(1H)-quinolinone from Glycosmis pentaphylla, were extracted from Glycosmis pentaphylla leaves and are discussed as possible insect growth regulators affecting hormone titers in the haemolymph of insect pest species.
-
Tritrophische Interaktionen zwischen transgenem, insektenresistentem Bacillus thuringiensis-Mais, dem Herbivoren Chilo partellus (Lepidoptera: Crambidae) und dem Parasitoiden Cotesia flavipes (Hymenoptera: Braconidae)
(2004)
-
Gernot Prütz
- Die Untersuchung der Wirkung transgener insektenresistenter Pflanzen auf entomophage Insekten ist von Bedeutung, denn Räuber und Parasitoide können eine wichtige Rolle als Gegenspieler von phytophagen Schädlingen spielen. In der vorliegenden Arbeit wurde exemplarisch die wirtsvermittelte Wirkung von transgenem insektenresistentem Bacillus thuringiensis-Mais („B.t.-Mais“) auf den gregären koinobionten larvalen Endoparasitoiden Cotesia flavipes (Hymenoptera: Braconidae) unter Laborbedingungen untersucht. Als Wirt diente Chilo partellus (Lepidoptera: Crambidae). C. partellus ist einer der wirtschaftlich bedeutendsten Schädlinge an Mais und Hirse in Afrika, der mittlerweile in Kenia von C. flavipes wirksam dezimiert wird. B.t.-Mais synthetisiert ein bakterielles Protein mit insektizider Wirkung, das B.t. delta-Endotoxin. Das B.t.-Toxin schädigt das für die Verdauung wichtige Mitteldarmepithel von Insekten, daher wurden die Waldbauerschen Verdaungsparameter von C. partellus nach Aufnahme von B.t.- bzw. Kontrollmais bestimmt. Ferner wurden life history Parameter von C. flavipes ermittelt. Da C. partellus-Larven nach Aufnahme von B.t.-Mais starben, bevor der Parasitoid seine Larvalentwicklung vollenden konnte, wurde mit verdünnten B.t.-Mais-Suspensionen gearbeitet, die auf Blätter von Kontrollmaispflanzen aufgetragen bzw. in Stängelstückchen von Kontrollmaispflanzen injiziert wurden. Jüngere C. partellus-Larven fressen an Blättern, ältere in Stängeln, deswegen wurde mit beiden Pflanzenteilen experimentiert. Nicht parasitierte C. partellus-Larven verschiedener Stadien nahmen in der B.t.-Gruppe weniger Nahrung auf als in der Kontrolle. Die Ursache der verringerten Nahrungsaufnahme lag möglicherweise in einer gegenüber der Kontrolle verlangsamten Darmpassage der Nahrung. Auch das Wachstum war gegenüber der Kontrolle verringert. Dies könnte die Folge der verringerten Nahrungsaufnahme, eines Mangels an Wasser oder eines Mangels an Proteinen sein, denn das B.t.-Toxin kann die Absorption von Aminosäuren blockieren und zu Flüssigkeitsverlust führen. Ferner war der Anteil der verdauten Nahrung, der in Körpermasse umgewandelt wurde, gegenüber der Kontrolle reduziert. Ein Grund dafür könnte in dem gegenüber der Kontrolle erhöhten energetischen Aufwand für die Regeneration des Mitteldarmepithels liegen. Die Wirkungen der B.t.-Mais-Suspension auf die Verdauungsparameter von C. partellus waren bei Gabe von Blatt- und Stängelfutter ähnlich und stimmen mit Literaturdaten überein. Wurden jedoch parasitierte C. partellus-Larven mit Maisblättern gefüttert, konnte kein einziger Parasitoid seine Lavalentwicklung im Wirt vollenden und sich verpuppen. Daher wurden parasitierte C. partellus-Larven im folgenden nur noch mit Stängelstückchen gefüttert. Diese sind möglicherweise aufgrund ihres gegenüber Blättern erhöhten Proteingehaltes für ältere C. partellus-Larvalstadien ernährungsphysiologisch geeigneter als Blätter. Parasitierte C. partellus-Larven nahmen während der ersten 4 Tage des Versuchs in der B.t.-Gruppe weniger Nahrung auf als in der Kontrolle. Ab dem fünften Tag war der Unterschied jedoch nicht mehr signifikant. Dies lag möglicherweise am Schlupf der Parasitoid-L1-Larven im Wirt, denn Parasitoiden können die Nahrungsaufnahme des Wirtes in Abhängigkeit von ihrem Larvalstadium beeinflussen. Verschiedene life history Parameter von C. flavipes waren gegenüber der Kontrolle beeinträchtigt. So konnte der Parasitoid nur in wenigen Wirten seine Entwicklung erfolgreich beenden, wenn der Wirt B.t.-Mais-Suspension aufgenommen hatte. Ferner war das Gewicht von Puppen, Kokons, Kokonspinnseide und adulten Parasitoiden gegenüber der Kontrolle verringert. Das Gewicht von Parasitoidweibchen ist ein Maß für Fitness, denn größere Weibchen haben oft eine längere Lebensdauer und produzieren mehr Eier als kleinere. Nur in der B.t.-Gruppe ergaben sich hochsignifikant negative Korrelationen zwischen der vom Wirt aufgenommenen Nahrungsmenge und der Anzahl an Parasitoid-Kokons pro Wirt. Darüberhinaus war die vom Wirt aufgenommene Nahrungsmenge nur in der B.t.-Gruppe mit der Entwicklungsdauer des Parasitoids positiv korreliert. Wahrscheinlich nahm die Menge des aufgenommenen B.t.-Toxins mit der aufgenommenen Nahrungsmenge zu. Höhere Toxinmengen führten dabei zu einer Erhöhung der Mortalität der Parasitoidlarven im Wirt bzw. verlangsamten die Entwicklung des Parasitoids. Die anhand des tritrophischen Modellsystems der vorliegenden Studie gezeigten Methoden könnten auch auf andere tritrophische Systeme mit verschiedenen Arten von transgenen Pflanzen übertragen werden, die insektizide Proteine synthetisieren. Da diese Proteine auf das Verdauungssystem wirken, wäre eine Bestimmung der Verdauungsparameter bei Herbivoren und Prädatoren sinnvoll. Wie in der vorliegenden Studie angedeutet, kann die Bestimmung der Verdauungsparameter zu einem genaueren Verständnis der Faktoren beitragen, die die Entwicklung des Parasitoids (oder Prädators) bestimmen.
-
The Allatoregulatory Neuropeptides and their Genes in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)
(2004)
-
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.
