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Institute
- Theodor-Boveri-Institut für Biowissenschaften (81) (remove)
Sonstige beteiligte Institutionen
ResearcherID
- J-8841-2015 (1)
Bees receive nectar and pollen as reward for pollinating plants. Pollen of different plant species varies widely in nutritional composition. In order to select pollen of appropriate nutritional quality, bees would benefit if they could distinguish different pollen types. Whether they rely on visual, olfactory and/or chemotactile cues to distinguish between different pollen types, has however been little studied. In this study, we examined whether and how Apis mellifera workers differentiate between almond and apple pollen. We used differential proboscis extension response conditioning with olfactory and chemotactile stimulation, in light and darkness, and in summer and winter bees. We found that honeybees were only able to differentiate between different pollen types, when they could use both chemotactile and olfactory cues. Visual cues further improved learning performance. Summer bees learned faster than winter bees. Our results thus highlight the importance of multisensory information for pollen discrimination.
The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.
Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin–Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli.
Due to intensive agriculture honeybees are threatened by various pesticides. The use of one group of them, the neonicotinoids, was recently restricted by the European Union. These chemicals bind to the nicotinic acetylcholine receptor (nAchR) in the honeybee brain. Recently, Bayer AG released a new pesticide by the name of “Sivanto” against sucking insects. It is assumed to be harmless for honeybees, although its active ingredient, flupyradifurone, binds nAchR similar to the neonicotinoids. We investigated if this pesticide affects the taste for sugar and cognitive performance in honeybee foragers. These bees are directly exposed to the pesticide while foraging for pollen or nectar. Our results demonstrate that flupyradifurone can reduce taste and appetitive learning performance in honeybees foraging for pollen and nectar, although only the highest concentration had significant effects. Most likely, honeybee foragers will not be exposed to these high concentrations. Therefore, the appropriate use of this pesticide is considered safe for honeybees, at least with respect to the behaviors studied here.
Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par
In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par
Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par
The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par
In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{WÜ} cells by using newly generated \textit{Mip\textsuperscript{WÜ}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{WÜ} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par
My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}$>$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par
In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par
After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par
Plant-associated fungi can affect the plants‘ interaction with herbivores and
other microorganisms. For example, many common forage grasses are infected
with Epichloë endophytes. The endophytes systemically colonize the aerial
parts of the plants. They produce bioprotective alkaloids that can negatively
affect insects and livestock feeding on the grasses, and interact with other
fungal species which living from the plants‘ nutrients. Environmental conditions
strongly influence Epichloë endophytes. Endophyte-mediated effects
on herbivores are more pronounced under increased temperatures and the
endophytes may benefit from land use in managed grasslands. Under the
framework of the large-scale German project “Biodiversity Exploratories”, I
investigated whether infection rates and alkaloid concentrations of Epichloë
festucae var. lolii in Lolium perenne (Chapter I) and Epichloë endophytes (E.
uncinata, E. siegelii) in Festuca pratensis (Chapter II) depend on land use and
season. Further I analysed, whether foliar fungal assemblages of L. perenne
are affected by the presence of Epichloë endophytes (Chapter IV).
Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -
(2018)
All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: Sönke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the
M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.
Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell.
This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology.
Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host.
Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis.
HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi
(2018)
The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
Aspergillus fumigatus is a saprophytic, cosmopolitan fungus that attacks patients with a weak immune system. A rational solution against fungal infection aims to manipulate fungal metabolism or to block enzymes essential for Aspergillus survival. Here we discuss and compare different bioinformatics approaches to analyze possible targeting strategies on fungal-unique pathways. For instance, phylogenetic analysis reveals fungal targets, while domain analysis allows us to spot minor differences in protein composition between the host and fungi. Moreover, protein networks between host and fungi can be systematically compared by looking at orthologs and exploiting information from host–pathogen interaction databases. Further data—such as knowledge of a three-dimensional structure, gene expression data, or information from calculated metabolic fluxes—refine the search and rapidly put a focus on the best targets for antimycotics. We analyzed several of the best targets for application to structure-based drug design. Finally, we discuss general advantages and limitations in identification of unique fungal pathways and protein targets when applying bioinformatics tools.