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The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication.
Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature.
Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs).
The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day.
To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner.
However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock.
Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.
Das Secosterid Vitamin D3 wird durch die Nahrung aufgenommen oder im Organismus synthetisiert, wobei eine Reaktion in der Haut durch einen photochemischen Prozess katalysiert wird.Durch zwei Hydroxylierungsschritte in Leber und Niere wird Vitamin D3 über 25(OH) Vitamin D3 zum aktiven 1,25(OH)2 Vitamin D3-Hormon. 1,25(OH)2 Vitamin D3 hat eine wichtige Funktion im Knochenstoffwechsel, es reguliert die Ca2+-Resorption im Dünndarm. Die 1,25(OH)2 Vitamin D3-Synthese in der Niere wird durch Parathormon (PTH) kontrolliert. Ist die Serum Ca2+-Konzentration niedrig, wird PTH ausgeschüttet und die 1a-Hydroxylase, das 25(OH) Vitamin D3-aktivierende Enzym, stimuliert. Das Prinzip der (Seco)steroid-Aktivierung und -Inaktivierung in glandulären Organen, wie Leber und Niere mit anschließender Freisetzung der aktiven Hormone und Transport zu den jeweiligen Zielgeweben gilt heute nicht mehr uneingeschränkt. Auch Einzelzellen sind in der Lage Steroid-modifizierende Enzyme, die Hydroxylasen und Dehydrogenasen, zu exprimieren. Monozytäre Zellen exprimieren das 1,25(OH)2 Vitamin D3-aktivierende und das -inaktivierende Enzym, die 1a-Hydroxylase und die 24-Hydroxylase. Sie sind somit in der Lage, 1,25(OH)2 Vitamin D3 zu sezernieren, welches parakrin auf Nachbarzellen wirken kann. In diesem Zusammenhang wurde die Expression und Regulation der 1a-Hydroxylase in peripheren Blutmonozyten (PBM) und monozytären THP1-Zellen untersucht. Durch Supplementation der Zellen mit dem Substrat 25(OH) Vitamin D3 konnte die Produktion an aktivem 1,25(OH)2 Vitamin D3-Hormon in PBM signifikant gesteigert werden. In PBM konnte im Gegensatz zum systemischen Ca2+-Stoffwechsel nur ein geringer Einfluss auf die 1a-Hydroxylase-Aktivität beobachtet werden. Durch RT-PCR-Amplifikation konnte eine Expression des PTH Rezeptors Typ 1 (PTHR1) in PBM und Dendritischen Zellen nachgewiesen werden. Ein weiterer Ligand des PTHR1 ist PTH related Protein (PTHrP), ein Faktor der die Tumorhyperkalzämie propagiert. Durch Markierungsexperimente mit fluoreszenz-markiertem PTHrP konnte gezeigt werden, dass PTHrP an die Zellmembran von PBM und Dendritischen Zellen bindet und in den Zellkern von Dendritischen Zellen transportiert wird. Im Rahmen dieser Arbeit wurde die Expression 1,25(OH)2 Vitamin D3-responsive Gene in Monozyten/Makrophagen untersucht. Die Expression der 24-Hydroxylase wird innerhalb der Differenzierung von myeloischen THP1-Zellen zu Makrophagen- bzw. Osteoklasten-ähnlichen Zellen transient induziert. Als weiteres 1,25(OH)2 Vitamin D3-responsives Gen wurde die Expression von Osteopontin (OPN) untersucht. OPN ist ein vor allem in Knochen vorkommendes Matrixprotein, das wesentlich an der Zelladhäsion beteiligt ist. OPN wird in THP1-Zellen im Zuge der Differenzierung zunehmend exprimiert. Durch immunhistochemische Untersuchungen konnte OPN in Granulomen von Morbus Crohn- und Leberschnitten detektiert werden. Es spielt hier eine wesentliche Rolle bei der Granulomentstehung. Die Thioredoxin Reduktase 1 (TR1) ist ein Selenoenzym, welches maßgeblich an der Reduktion von Disulfidbindungen in Proteinen beteiligt ist. Es moduliert Protein/Protein- und Protein/DNA-Interaktionen wie die Bindung der Transkriptionsfaktoren AP1 und NFkB an DNA-responsive Elemente. Die Expression der TR1 wird in THP1-Zellen im Rahmen der Differenzierung induziert und ist in differenzierten Zellen maximal. Aktivitätsmessungen deckten sich mit dieser Beobachtung. In peripheren Blutmonozyten steigt die TR-Aktivität alleine durch Adhäsion der Zellen an das Kulturgefäß und nach Behandlung mit 1,25(OH)2 Vitamin D3. Die Untersuchungen der vorliegenden Arbeit zeigten eine Abhängigkeit der TR-Aktivität vom Differenzierungsgrad der Zellen und der Supplementation des Mediums mit dem Spurenelement Selen. Die Expression weiterer Selenoproteine in monozytären Zellen wurde nachgewiesen. So konnten durch 75Selenit-Markierungsexperimente neun Selenoproteine in THP1-Zellen detektiert werden, von denen fünf sezerniert werden. Ein weiteres, in monozytären Zellen charakterisiertes Selenoprotein ist die zelluläre Glutathionperoxidase. Ihre Aktivität konnte in Selenit-supplementierten Zellen um das 70fache gesteigert werden. Die Kultivierung monozytärer Zellen unter Selenit-Supplementation beeinflusst die Funktion dieser Zellen wesentlich. So konnte beobachtet werden, dass die Anzahl an phagozytierenden, zu Makrophagen differenzierten THP1-Zellen nach Selenit-Supplementation abnahm, während die Phagozytoserate der einzelnen Zellen anstieg. Die erzielten Ergebnisse zeigen, dass monozytäre Zellen mit Komponenten des 1,25(OH)2 Vitamin D3 Stoffwechsels ausgestattet sind und aktives 1,25(OH)2 Vitamin D3-Hormon produzieren, sezernieren und inaktivieren können. Die lokale Kontrolle der 1,25(OH)2 Vitamin D3 Stoffwechsels ausgestattet sind und aktives 1,25(OH)2 Vitamin D3-responsiver Gene, wie die Expression des Selenoproteins TR1, das einen direkten Einfluss auf den Redoxstatus und den Abbau reaktiver Sauerstoffverbindungen in diesen und Nachbarzellen ausübt.
In the last decades, both the incidence and the severity of asthma have steadily increased. Furthermore, available therapies only treat the symptoms but do not cure the disease. Immune modulation induced by TLR agonists may be a promising novel approach to effectively treat asthma as it targets the underlying immunopathology directly rather than one mediator alone. The aim of this thesis was to investigate if the immunostimulatory properties of Toll-like receptor (TLR) agonists can be utilized to develop novel therapeutic intervention strategies for the treatment of asthma using murine models of allergic inflammation. For this purpose five different TLR agonists were tested in preclinical mouse models of acute and chronic asthma, both in preventive and therapeutic settings. Firstly, TLR-2, 3, 4, 7/8 and 9 agonists were delivered intratracheally at different doses before pulmonary allergen exposure in the asthma model of acute inflammation. TLR9 agonist CpG-containing oligodeoxynucleotides (CpG) > TLR7 agonist Resiquimod (R848) > TLR3 agonists poly(I:C) strongly reduced allergen induced airway eosinophilia and IL-4 levels in a dose-dependent manner. All TLR agonists increased neutrophil numbers, TLR4 agonist lipopolysaccharide (LPS) > TLR2 agonist lipoteichonic acid (LTA) > poly(I:C) > CpG > R848 and, with the exception of R848, the amount of pro-inflammatory cytokines in the airways. Suppressive effects were not dependent upon IFN-γ and IL-10 or associated with increased numbers of regulatory T cells in the airways. All TLR agonists, except LTA, similarly reduced airway eosinophilia and IL-4 levels when applied therapeutically after allergen challenge. These results show that the TLR agonists have different suppressive effects on TH2 responses in the airways which further depend on the dose and the experimental setup in which they were tested. Interestingly, all agonists induced airway neutrophilia, albeit to different degrees, raising the question if TLR ligands are safe for human use when applied directly into the lung. Different TLR agonists are also being developed for human use as adjuvants combined with allergen in specific immunotherapy. Recent clinical data suggest that this may be achieved by induction of allergen-specific TH1 responses. For this reason, the ability of different TLR agonists to induce allergen-specific TH1 and suppress allergen-specific TH2 responses in a preclinical setting was investigated in this thesis. Different doses of the TLR agonists were applied together with allergen, then mice were exposed to allergen aerosol. CpG > LPS >LTA dose-dependently strongly suppressed the development of airway eosinophilia with poly(I:C) and R848 having no effect. The decrease in eosinophilic numbers was associated withincreased neutrophils present in the airways. IL-4 and IL-5 levels in the bronchoalveolar lavage fluid were also decreased when poly(I:C), LPS, and CpG were used. All TLR agonists increased allergen-specific IgG2a, and with the exception of poly(I:C), reduced allergen-specific IgE levels in the serum. Cutaneous anaphylaxis to allergen was completely prevented when LPS or CpG were given as adjuvant. The strongest TH1 responses were induced by CpG and poly(I:C), characterized by the presence of IFN-γ in the bronchoalveolar lavage and the highest allergen-specific IgG2a levels in the serum. This data supports approaches to use TLR9 or TLR4 agonists for human therapy as adjuvant in combination with allergen in novel specific immunotherapy formulations. In the last part of the thesis, it was investigated if TLR activation can also affect the pathology of severe chronic asthma. Therapeutic administration of R848 or CpG reduced features of inflammation and remodeling. Both agonists showed superior effects to dexamethasone, with CpG being more efficient than R848. This result again supports a TLR9-based therapy as a viable option for the treatment of severe chronic asthma which may present a potential alternative for anti-inflammatory therapy with steroids. Taken together, the results of this thesis support the use of TLR agonists to treat asthma. The most favorable efficacy/safety ratio is to be expected from TLR-based therapies combining TLR4 or TLR9 agonists with allergen in specific immunotherapy. In regard to TLR agonist monotherapy, R848 and CpG showed the most promising profiles, CpG particularly in a model of severe chronic asthma. However, since all TLR agonists used in this study also showed pro-inflammatory potential, the safety aspect of such an approach needs to be taken into account.
Upon oncogenic stress, the tumor suppressor Arf can induce irreversible cell cycle arrest or apoptosis, depending on the oncogenic insult. In this study, it could be shown that Arf interacts with Myc and the Myc-associated zinc-finger protein Miz1 to facilitate repression of genes involved in cell adhesion. Formation of a DNA-binding Arf/Myc/Miz1 complex disrupts interaction of Miz1 with its coactivator nucleophosmin and induces local heterochromatinisation, causing cells to lose attachment and undergo anoikis. The assembly of the complex relies on Myc, which might explain why high Myc levels trigger apoptosis and not cell cycle arrest in the Arf response. This mechanism could play an important role in eliminating cells harboring an oncogenic mutation. Arf furthermore induces sumoylation of Miz1 at a specific lysine by repressing the desumoylating enzyme Senp3. A sumoylation-deficient mutant of Miz1 however does not show phenotypic differences under the chosen experimental conditions. Myc can also be modified by Sumo by multisumoylation at many different lysines, which is unaffected by Arf. The exact mechanism and effect of this modification however stays unsolved.
Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Veränderungen ihrer Umwelt anzupassen. Während letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System täglich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltveränderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abhängigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gestörten biologischen Tagesrhythmen, wie zum Beispiel Schlafstörungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern kürzlich durchgeführte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine mögliche Verbindung zwischen Stress-induzierten und regulären rhythmischen Anpassungen des Organismus an Umweltveränderungen an. Molekulare und zelluläre Mechanismen dieser Verknüpfung sind bisher noch nicht bekannt.
Während die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine mögliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antikörperfärbungen sowie Studien mit Reporterlinien zeigen deutliche Färbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis für p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivität von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zusätzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht führen zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivität offenbaren zusätzlich die Notwendigkeit von p38 MAPK für wildtypisches Timing der Abendaktivität sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verzögerten Beginn der Abendaktivität und stark verlängerte Freilaufperioden. In Übereinstimmung mit Effekten auf das Laufverhalten scheint darüber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila’s wichtigsten Uhrneuronen eine verspätete nukleäre Translokation von Period zur Folge zu haben. Westernblots legen zusätzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen mögliche Erklärung für den verspäteten Kerneintritt des Uhrproteins. Abschließende Stützung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase“ hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren könnte.
Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die Möglichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.
Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
Anthropogenic activities are causing air pollution. Amongst air pollutants, tropospheric ozone is a major threat to human health and ecosystem functioning. In this dissertation, I present three studies that aimed at increasing our knowledge on how plant exposure to ozone affects its reproduction and its interactions with insect herbivores and pollinators.
For this purpose, a new fumigation system was built and placed in a greenhouse. The annual plant Sinapis arvensis (wild mustard) was used as the model plant.
Plants were exposed to either 0 ppb (control) or 120 ppb of ozone, for variable amounts of time and at different points of their life cycle. After fumigation, plants were exposed to herbivores or pollinators in the greenhouse, or to both groups of insects in the field.
My research shows that ozone affected reproductive performance differently, depending on the timing of exposure: plants exposed at earlier ages had their reproductive fitness increased, while plants exposed later in their life cycle showed a tendency for reduced reproductive fitness. Plant phenology was a key factor influencing reproductive fitness: ozone accelerated flowering and increased the number of flowers produced by plants exposed at early ages, while plants exposed to ozone at later ages tended to have fewer flowers. On the other hand, the ozone-mediated changes in plant-insect interactions had little impact on plant reproductive success.
The strongest effect of ozone on plant-pollinator interactions was the change in the number of flower visits received per plant, which was strongly linked to the number of open flowers. This means that, as a rule, exposure of plants to ozone early in the life cycle resulted in a higher number of pollinator visits, while exposure later in the life cycle resulted in fewer flower visits by potential pollinators. An exception was observed: the higher number of visits performed by large syrphid flies to young ozone-exposed plants than to the respective control plants went beyond the increase in the number of open flowers in those plants. Also, honeybees spent more time per flower in plants exposed to ozone than on control plants, while other pollinators spent similar amounts of time in control and ozone-exposed plants. This guild-dependent preference for ozone-exposed plants may be due to species-specific preferences related to changes in the quality and quantity of floral rewards.
In the field, ozone-exposed plants showed only a tendency for increased colonization by sucking herbivores and slightly more damage by chewing herbivores than control plants. On the other hand, in the greenhouse experiment, Pieris brassicae butterflies preferred control plants over ozone-exposed plants as oviposition sites. Eggs laid on ozone-exposed plants took longer to hatch, but the chances of survival were higher. Caterpillars performed better in control plants than in ozone-exposed plants, particularly when the temperature was high.
Most of the described effects were dependent on the duration and timing of the ozone exposure and the observed temperature, with the strongest effects being observed for longer exposures and higher temperatures. Furthermore, the timing of exposure altered the direction of the effects.
The expected climate change provides ideal conditions for further increases in tropospheric ozone concentrations, therefore for stronger effects on plants and plant-insect interactions. Acceleration of flowering caused by plant exposure to ozone may put plant-pollinator interactions at risk by promoting desynchronization between plant and pollinator activities. Reduced performance of caterpillars feeding on ozone-exposed plants may weaken herbivore populations. On the other hand, the increased plant reproduction that results from exposing young plants to ozone may be a source of good news in the field of horticulture, when similar results would be achieved in high-value crops. However, plant response to ozone is highly species-specific. In fact, Sinapis arvensis is considered a weed and the advantage conferred by ozone exposure may increase its competitiveness, with negative consequences for crops or plant communities in general. Overall, plant exposure to ozone might constitute a threat for the balance of natural and agro-ecosystems.
The Ecology and Population structure of the invasive Yelllow Crazy Ant Anoplolepis gracilipes
(2011)
The invasive Yellow Crazy Ant Anoplolepis gracilipes is a widespread tropical ant species which is particularly common in anthropogenically disturbed habitats in South-East Asia and the Indopacific region. Its native range is unknown, and there is little information concerning its social structure as a potential mechanism facilitating invasion as well as its ecology in one of the putative native ranges, South-East Asia. Using mitochondrial DNA sequences, I demonstrated that the majority of the current Indopacific colonies were likely introduced from South-East Asian populations, which in turn may have been introduced much earlier from a yet unidentified native range. By conducting behavioral, genetic and chemical analyses, I found that A. gracilipes supercolonies contain closely related individuals, thus resembling enlarged versions of monogynous, polydomous colonies of other ant species. Furthermore, mutually aggressive A. gracilipes supercolonies were highly differentiated both genetically and chemically, suggesting limited or even absent gene flow between supercolonies. Intranidal mating and colony-budding are most likely the predominant, if not the exclusive mode of reproduction and dispersal strategy of A. gracilipes. Consequently, a positive feedback between genetic, chemical and behavioral traits may further enhance supercolony differentiation though genetic drift and neutral evolution. This potential scenario led to the hypothesis that absent gene flow between different A. gracilipes supercolonies may drive them towards different evolutionary pathways, possibly including speciation. Thus, I examined one potential way by which gene flow between supercolonies of an ant species without nuptial flights may be maintained, i.e. the immigration of sexuals into foreign supercolonies. The results suggest that this option of maintaining gene flow between different supercolonies is likely impaired by severe aggression of workers towards allocolonial sexuals. Moreover, breeding experiments involving males and queens from different supercolonies suggest that A. gracilipes supercolonies may already be on the verge of reproductive isolation, which might lead to the diversification of A. gracilipes into different species. Regarding the ecological consequences of its potential introduction to NE-Borneo, I could show that A. gracilipes supercolonies may affect the local ant fauna. The ant community within supercolonies was less diverse and differed in species composition from areas outside supercolonies. My data suggest that the ecological dominance of A. gracilipes within local ant communities was facilitated by monopolization of food sources within its supercolony territory, achieved by a combination of rapid recruitment, numerical dominance and pronounced interspecific aggression. A. gracilipes’ distribution is almost exclusively limited to anthropogenically altered habitat, such as residential and agricultural areas. The rate at which habitat conversion takes place in NE-Borneo will provide A. gracilipes with a rapidly increasing abundance of suitable habitats, thus potentially entailing significant population growth. An potentially increasing population size and ecological dominance, however, are not features that are limited to invasive alien species, but may also occur in native species that become ‘pests’ in an increasing abundance of anthropogenically altered habitat. Lastly, I detected several ant guests in supercolonies of A. gracilipes. I subsequently describe the relationship between one of them (the cricket Myrmecophilus pallidithorax) and its ant host. By conducting behavioral bioassays and analyses of cuticular hydrocarbon (CHC) profiles, I revealed that although M. pallidithorax is attacked and consumed by A. gracilipes whenever possible, it may evade aggression from its host by a combination of supreme agility and, possibly, chemical deception. This thesis adds to our general understanding of biological invasions by contributing species-specific data on a previously understudied invasive organism, the Yellow Crazy Ant Anoplolepis gracilipes. Introductions which may have occurred a long time ago may make it difficult to determine whether a given species is an introduced invader or a native pest species, as both may have pronounced ecological effects in native species communities. Furthermore, this thesis suggests that supercolonialism in invasive ants may not be an evolutionary dead end, but that it may possibly give rise to new species due to reproductive boundaries between supercolonies evoked by peculiar mating and dispersal strategies.
Many arthropods and vertebrates can cling to surfaces using adhesive pads on their legs. These pads are either smooth and characterised by a specialised, soft cuticle or they are hairy, i.e. densely covered with flexible adhesive setae. Animals climbing with adhesive organs are able to control attachment and detachment dynamically while running. The detailed mechanisms of how tarsal pads generate adhesive and frictional forces and how forces are controlled during locomotion are still largely unclear. The aim of this study was to clarify the attachment mechanism of smooth adhesive pads as present in many insects and tree frogs. To understand the function of these fluid-based adhesive systems, I characterized their performance under standardized conditions. To this end, experiments were conducted by simultaneously measuring adhesion, friction, and contact area in single adhesive pads. The first result of this study showed that friction in stick insect attachment pads is anisotropic: Attachment pads regularly detached when slid away from the body. Further analyses of "immobilized" arolia revealed that this anisotropy is not caused by an increased shear stress in the proximal direction, but by the instability of the tarsus when pushed distally. In the second part of this study, I analysed the role of the pad secretion present in insects and tree frogs. In stick insects, shear stress was largely independent of normal force and increased with velocity, seemingly consistent with the viscosity effect of a continuous fluid film. However, measurements of the remaining force two minutes after a sliding movement showed that adhesive pads could sustain considerable static friction in insects and tree frogs. Repeated sliding movements and multiple consecutive pull-offs of stick insect single legs to deplete adhesive secretion showed that on a smooth surface, friction and adhesion strongly increased with decreasing amount of fluid in insects. In contrast, stick insect pull-off forces significantly decreased on a rough substrate. Thus, the secretion does not generally increase attachment but does so only on rough substrates, where it helps to maximize contact area. When slides with stick insect arolia were repeated at one position so that secretion could accumulate, sliding shear stress decreased but static friction remained clearly present. This suggests that static friction in stick insects, which is biologically important to prevent sliding, is based on non-Newtonian properties of the adhesive emulsion rather than on a direct contact between the cuticle and the substrate. % Analogous measurements in toe pads of tree frogs showed that they are also able to generate static friction, even though their pads are wetted by mucus. In contrast to the mechanism proposed for insects, static friction in tree frogs apparently results from the very close contact of toe pads to the substrate and boundary lubrication. In the last section of this study, I investigated adhesive forces and the mode of detachment by performing pull-off measurements at different velocities and preloads. These experiments showed that preload has only an increasing effect on adhesion for faster pull-offs. This can be explained by the viscoelastic material properties of the stick insect arolium, which introduce a strong rate-dependence of detachment. During fast pull-offs, forces can spread over the complete area of contact, leading to forces scaling with area. In contrast, the pad material has sufficient time to withdraw elastically and peel during slow detachments. Under these conditions the adhesive force will concentrate on the circumference of the contact area, therefore scaling with a length, supporting models such as the peeling theory. The scaling of single-pad forces supported these conclusions, but large variation between pads of different stick insects did not allow statistically significant conclusions. In contrast, when detachment forces were quantified for whole insects using a centrifuge, forces scaled with pad contact area and not with length.
In the various groups of social bees, different systems of communication about food sources occur. These communication systems are different solutions to a common problem of social insects: efficiently allocating the necessary number of workers first to the task of foraging and second to the most profitable food sources. The solution chosen by each species depends on the particular ecological circumstances as well as the evolutionary history of that species. For example, the outstanding difference between the bumble bee and the honey bee system is that honey bees can communicate the location of profitable food sources to nestmates, which bumble bees cannot. To identify possible selection pressures that could explain this difference, I have quantified the benefits of communicating location in honey bees. I show that these strongly depend on the habitat, and that communicating location might not benefit bees in temperate habitats. This could be due to the differing spatial distributions of resources in different habitats, in particular between temperate and tropical regions. These distributions may be the reason why the mostly temperate-living bumble bees have never evolved a communication system that allows them to transfer information on location of food sources, whereas most tropical social bees (all honey bees and many stingless bees) are able to recruit nestmates to specific points in their foraging range. Nevertheless, I show that in bumble bees the allocation of workers to foraging is also regulated by communication. Successful foragers distribute in the nest a pheromone which alerts other bees to the presence of food. This pheromone stems from a tergite gland, the function of which had not been identified previously. Usage of a pheromone in the nest to alert other individuals to forage has not been described in other social insects, and might constitute a new mode of communicating about food sources. The signal might be modulated depending on the quality of the food source. Bees in the nest sample the nectar that has been brought into the nest. Their decision whether to go out and forage depends not only on the pheromone signal, but also on the quality of the nectar they have sampled. In this way, foraging activity of a bumble bee colony is adjusted to foraging conditions, which means most bees are allocated to foraging only if high-quality food sources are available. In addition, foraging activity is adjusted to the amount of food already stored. In a colony with full honeypots, no new bees are allocated to foraging. These results help us understand how the allocation of workers to the task of food collection is regulated according to external and internal nest conditions in bumble bees.