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Scorpions commonly are assumed to hunt on living prey. But under laboratory conditions they also respond very sensitively to dead insects lying on the substrate. In many cases the motionless prey is seized and consumed. It was investigated how this behavior can be elicited. The buthid scorpions Androctonus australis (L.) and Buthus occitanus (Am.) not only find motionless prey again which was stung but managed to escape before dying: They also respond to extracts of the cuticle of prey insects. After touching prey marks' either with the tips of the chelae fingers or the tarsi of the walking legs or the pectine organs specific responses (searching, seizing, feeding) are released at a high rate. Behavioral experiments demonstrate for the first time the chemosensitivity of the pectine organs for which only mechanosensitivity had been proofed formerly. Mechanical as well as contact chemical stimulation of these organs cause scorpions to orient towards the stimulus source which is grasped, retained and consumed or rejected depending on its quality. The probably responsible chemosensitive receptors are already described in the literature. The possible adaptive value and the biological significance of contact chemoreception in prey catching and in other aspects of the life of scorpions is discussed.
Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.
Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.
The src-gene family in mammals and birds consists of 9 closely related protein tyrosine kinases. We have cloned the c-yes and fyn bomologues of the src-family from the teleost fish Xiphophorus helleri. Both genes show a high degree of sequence conservation and exhibit all structural motifs diagnostic for functional src-like protein tyrosine kinases. Sequence comparisons revealed three domains (exon 2, exons 3--6, exons 7-12) which evolve at different rates. Both genes exhibit an identical expression pattern, with preferential expression in neural tissues. No transcripts of c-yes were found in liver wbich is contrary to the situation in higher vertebrales. In malignant melanoma, elevated Ieveis of c-yes andfyn were detected indicating a possible function during secondary steps of tumor progression for src-related tyrosine kinases.
Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.
The increase in intensively used areas and climate change are direct and indirect consequences of anthropogenic actions, caused by a growing population and increasing greenhouse gas emissions. The number of research studies, investigating the effects of land use and climate change on ecosystems, including flora, fauna, and ecosystem services, is steadily growing. This thesis contributes to this research area by investigating land-use and climate effects on decomposer communities (arthropods and microbes) and the ecosystem service ‘decomposition of dead material’.
Chapter II deals with consequences of intensified land use and climate change for the ecosystem service ‘decomposition of dead organic material’ (necromass). Considering the severe decline in insects, we experimentally excluded insects from half of the study objects. The decomposition of both dung and carrion was robust to land-use changes. Dung decomposition, moreover, was unaffected by temperature and the presence/ absence of insects. Along the altitudinal gradient, however, highest dung decomposition was observed at medium elevation between 600 and 700 m above sea level (although insignificant). As a consequence, we assume that at this elevation there is an ideal precipitation:temperature ratio for decomposing organisms, such as earthworms or collembolans. Carrion decomposition was accelerated by increasing elevation and by the presence of insects, indicating that increasing variability in climate and an ongoing decline in insects could modify decomposition processes and consequently natural nutrient cycles. Moreover, we show that different types of dead organic material respond differently to environmental factors and should be treated separately in future studies.
In Chapter III, we investigated land-use and climate effects on dung-visiting beetles and their resource specialization. Here, all beetles that are preferentially found on dung, carrion or other rotten material were included. Both α- and γ-diversity were strongly reduced in agricultural and urban areas. High precipitation reduced dung-visiting beetle abundance, whereas γ-diversity was lowest in the warmest regions. Resource specialization decreased with increasing temperatures. The results give evidence that land use as well as climate can alter dung-visiting beetle diversity and resource specialization and may hence influence the natural balance of beetle communities and their contribution to the ecosystem service ‘decomposition of dead material’.
The following chapter, Chapter IV, contributes to the findings in Chapter II. Here, carrion decomposition is not only explained by land-use intensity and climate but also by diversity and community composition of two taxonomic groups found on carrion, beetles and bacteria. The results revealed a strong correlation between bacteria diversity and community composition with temperature. Carrion decomposition was to a great extent directed by bacterial community composition and precipitation. The role of beetles was neglectable in carrion decomposition. With this study, I show that microbes, despite their microscopic size, direct carrion decomposition and may not be neglected in future decomposition studies.
In Chapter V a third necromass type is investigated, namely deadwood. The aim was to assess climate and land-use effects on deadwood-inhabiting fungi and bacteria. Main driver for microbial richness (measured as number of OTUs) was climate, including temperature and precipitation. Warmer climates promoted the diversity of bacteria, whereas fungi richness was unaffected by temperature. In turn, fungi richness was lower in urban landscapes compared to near-natural landscapes and bacteria richness was higher on meadows than on forest sites. Fungi were extremely specialized on their host tree, independent of land use and climate. Bacteria specialization, however, was strongly directed by land use and climate. These results underpin previous studies showing that fungi are highly specialized in contrast to bacteria and add new insights into the robustness of fungi specialization to climate and land use.
I summarize that climate as well as intensive land use influence biodiversity. Temperature and precipitation, however, had positive and negative effects on decomposer diversity, while anthropogenic land use had mostly negative effects on the diversity of decomposers.
Morphemes are the smallest meaningful parts of words and therefore represent a natural unit to study the evolution of words. To analyze the influence of language change on morphemes, we performed a large scale analysis of German and English vocabulary covering the last 200 years. Using a network approach from bioinformatics, we examined the historical dynamics of morphemes, the fixation of new morphemes and the emergence of words containing existing morphemes. We found that these processes are driven mainly by the number of different direct neighbors of a morpheme in words (connectivity, an equivalent to family size or type frequency) and not its frequency of usage (equivalent to token frequency). This contrasts words, whose survival is determined by their frequency of usage. We therefore identified features of morphemes which are not dictated by the statistical properties of words. As morphemes are also relevant for the mental representation of words, this result might enable establishing a link between an individual’s perception of language and historical language change.
Connecting lysosomes and mitochondria – a novel role for lipid metabolism in cancer cell death
(2019)
Background
The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood.
Methods
LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements.
Results
Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells.
Conclusion
This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.
To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.
Background: Heterococcus is a microalgal genus of Xanthophyceae (Stramenopiles) that is common and widespread in soils, especially from cold regions. Species are characterized by extensively branched filaments produced when grown on agarized culture medium. Despite the large number of species described exclusively using light microscopic morphology, the assessment of species diversity is hampered by extensive morphological plasticity.
Results: Two independent types of molecular data, the chloroplast-encoded psbA/rbcL spacer complemented by rbcL gene and the internal transcribed spacer 2 of the nuclear rDNA cistron (ITS2), congruently recovered a robust phylogenetic structure. With ITS2 considerable sequence and secondary structure divergence existed among the eight species, but a combined sequence and secondary structure phylogenetic analysis confined to helix II of ITS2 corroborated relationships as inferred from the rbcL gene phylogeny. Intra-genomic divergence of ITS2 sequences was revealed in many strains. The 'monophyletic species concept', appropriate for microalgae without known sexual reproduction, revealed eight different species. Species boundaries established using the molecular-based monophyletic species concept were more conservative than the traditional morphological species concept. Within a species, almost identical chloroplast marker sequences (genotypes) were repeatedly recovered from strains of different origins. At least two species had widespread geographical distributions; however, within a given species, genotypes recovered from Antarctic strains were distinct from those in temperate habitats. Furthermore, the sequence diversity may correspond to adaptation to different types of habitats or climates.
Conclusions: We established a method and a reference data base for the unambiguous identification of species of the common soil microalgal genus Heterococcus which uses DNA sequence variation in markers from plastid and nuclear genomes. The molecular data were more reliable and more conservative than morphological data.
Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.
Background
VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1.
Results
In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme.
Conclusions
The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
The human recombination activating gene 1 (RAGl) has previously been mapped to chromosomes 14q and 11 p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to llp13.
Here we describe a novel conditional mouse lung tumor model for investigation of the pathogenesis of human lung cancer. On the basis of the frequent involvement of the Ras-RAF-MEK-ERK signaling pathway in human non-small cell lung carcinoma (NSCLC), we have explored the target cell availability, reversibility, and cell type specificity of transformation by oncogenic C-RAF. Targeting expression to alveolar type II cells or to Clara cells, the two likely precursors of human NSCLC, revealed differential tumorigenicity between these cells. Whereas expression of oncogenic C-RAF in alveolar type II cells readily induced multifocal macroscopic lung tumors independent of the developmental state, few tumors with type II pneumocytes features and incomplete penetrance were found when targeted to Clara cells. Induced tumors did not progress and were strictly dependent on the initiating oncogene. Deinduction of mice resulted in tumor regression due to autophagy rather than apoptosis. Induction of autophagic cell death in regressing lung tumors suggests the use of autophagy enhancers as a treatment choice for patients with NSCLC.
Neuroanatomical data in fly brain research are mostly available as spatial gene expression patterns of genetically distinct fly strains. The Drosophila standard brain, which was developed in the past to provide a reference coordinate system, can be used to integrate these data. Working with the standard brain requires advanced image processing methods, including visualisation, segmentation and registration. The previously published VIB Protocol addressed the problem of image registration. Unfortunately, its usage was severely limited by the necessity of manually labelling a predefined set of neuropils in the brain images at hand. In this work I present novel tools to facilitate the work with the Drosophila standard brain. These tools are integrated in a well-known open-source image processing framework which can potentially serve as a common platform for image analysis in the neuroanatomical research community: ImageJ. In particular, a hardware-accelerated 3D visualisation framework was developed for ImageJ which extends its limited 3D visualisation capabilities. It is used for the development of a novel semi-automatic segmentation method, which implements automatic surface growing based on user-provided seed points. Template surfaces, incorporated with a modified variant of an active surface model, complement the segmentation. An automatic nonrigid warping algorithm is applied, based on point correspondences established through the extracted surfaces. Finally, I show how the individual steps can be fully automated, and demonstrate its application for the successful registration of fly brain images. The new tools are freely available as ImageJ plugins. I compare the results obtained by the introduced methods with the output of the VIB Protocol and conclude that our methods reduce the required effort five to ten fold. Furthermore, reproducibility and accuracy are enhanced using the proposed tools.
In this century new experimental and computational techniques are adding an enormous amount of information, revealing many biological mysteries. The complexities of biological systems still broach new questions. Till now the main approach to understand a system has been to divide it in components that can be studied. The upcoming new paradigm is to combine the pieces of information in order to understand it at a global level. In the present thesis we have tried to study infectious diseases with such a global ‘Systems Biology’ approach. In the first part the apoptosis pathway is analyzed. Apoptosis (Programmed cell death) is used as a counter measure in different infections, for example viral infections. The interactions between death domain containing proteins are studied to address the following questions: i) How specificity is maintained - showing that it is induced through adaptors, ii) how proliferation/ survival signals are induced during activation of apoptosis – suggesting the pivotal role of RIP. The model also allowed us to detect new possible interacting surfaces. The pathway is then studied at a global level in a time step simulation to understand the evolution of the topology of activators and inhibitors of the pathway. Signal processing is further modeled in detail for the apoptosis pathway in M. musculus to predict the concentration time course of effector caspases. Further, experimental measurements of caspase-3 and viability of cells validate the model. The second part focuses on the phagosome, an organelle which plays an essential role in removal of pathogens as exemplified by M. tuberculosis. Again the problem is addressed in two main sections: i) To understanding the processes that are inhibited by M. tuberculosis; we focused on the phospholipid network applying a time step simulation in section one, which plays an important role in inhibition or activation of actin polymerization on the phagosome membrane. ii) Furthermore, actin polymers are suggested to play a role in the fusion of the phagosome with lysosome. To check this hypothesis an in silico model was developed; we find that the search time is reduced by 5 fold in the presence of actin polymers. Further the effect of length of actin polymers, dimensions of lysosome, phagosome and other model parameter is analyzed. After studying a pathway and then an organelle, the next step was to move to the system. This was exemplified by the host pathogen interactions between Bordetella pertussis and Bordetella bronchiseptica. The limited availability of quantitative information was the crucial factor behind the choice of the model type. A Boolean model was developed which was used for a dynamic simulation. The results predict important factors playing a role in Bordetella pathology especially the importance of Th1 related responses and not Th2 related responses in the clearance of the pathogen. Some of the quantitative predictions have been counterchecked by experimental results such as the time course of infection in different mutants and wild type mice. All these computational models have been developed in presence of limited kinetic data. The success of these models has been validated by comparison with experimental observations. Comparative models studied in chapters 6 and 9 can be used to explore new host pathogen interactions. For example in chapter 6, the analysis of inhibitors and inhibitory paths in three organism leads to the identification of regulatory hotspots in complex organisms and in chapter 9 the identification of three phases in B. bronchiseptica and inhibition of IFN-γ by TTSS lead us to explore similar phases and inhibition of IFN-γ in B. pertussis. Further an important significance of these models is to identify new components playing an essential role in host-pathogen interactions. In silico deletions can point out such components which can be further analyzed by experimental mutations.
To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.
Background:
Small animal models of human diseases are an indispensable aspect of pre-clinical research. Being dynamic, most pathologies demand extensive longitudinal monitoring to understand disease mechanisms, drug efficacy and side effects. These considerations often demand the concomitant development of monitoring systems with sufficient temporal and spatial resolution.
Methodology and Results:
This study attempts to configure and optimize a clinical 3 Tesla magnetic resonance scanner to facilitate imaging of small animal central nervous system pathologies. The hardware of the scanner was complemented by a custom-built, 4-channel phased array coil system. Extensive modification of standard sequence protocols was carried out based on tissue relaxometric calculations. Proton density differences between the gray and white matter of the rodent spinal cord along with transverse relaxation due to magnetic susceptibility differences at the cortex and striatum of both rats and mice demonstrated statistically significant differences. The employed parallel imaging reconstruction algorithms had distinct properties dependent on the sequence type and in the presence of the contrast agent. The attempt to morphologically phenotype a normal healthy rat brain in multiple planes delineated a number of anatomical regions, and all the clinically relevant sequels following acute cerebral ischemia could be adequately characterized. Changes in blood-brain-barrier permeability following ischemia-reperfusion were also apparent at a later time. Typical characteristics of intracerebral haemorrhage at acute and chronic stages were also visualized up to one month. Two models of rodent spinal cord injury were adequately characterized and closely mimicked the results of histological studies. In the employed rodent animal handling system a mouse model of glioblastoma was also studied with unequivocal results.
Conclusions:
The implemented customizations including extensive sequence protocol modifications resulted in images of high diagnostic quality. These results prove that lack of dedicated animal scanners shouldn't discourage conventional small animal imaging studies.
Background: Males in some species of the genus Xiphophorus, small freshwater fishes from Meso-America, have an extended caudal fin, or sword - hence their common name "swordtails". Longer swords are preferred by females from both sworded and - surprisingly also, non-sworded (platyfish) species that belong to the same genus. Swordtails have been studied widely as models in research on sexual selection. Specifically, the pre-existing bias hypothesis was interpreted to best explain the observed bias of females in presumed ancestral lineages of swordless species that show a preference for assumed derived males with swords over their conspecific swordless males. However, many of the phylogenetic relationships within this genus still remained unresolved. Here we construct a comprehensive molecular phylogeny of all 26 known Xiphophorus species, including the four recently described species (X. kallmani, X. mayae, X. mixei and X. monticolus). We use two mitochondrial and six new nuclear markers in an effort to increase the understanding of the evolutionary relationships among the species in this genus. Based on the phylogeny, the evolutionary history and character state evolution of the sword was reconstructed and found to have originated in the common ancestral lineage of the genus Xiphophorus and that it was lost again secondarily.
Results: We estimated the evolutionary relationships among all known species of the genus Xiphophorus based on the largest set of DNA markers so far. The phylogeny indicates that one of the newly described swordtail species, Xiphophorus monticolus, is likely to have arisen through hybridization since it is placed with the southern platyfish in the mitochondrial phylogeny, but with the southern swordtails in the nuclear phylogeny. Such discordance between these two types of markers is a strong indication for a hybrid origin. Additionally, by using a maximum likelihood approach the possession of the sexually selected sword trait is shown to be the most likely ancestral state for the genus Xiphophorus. Further, we provide a well supported estimation of the phylogenetic relationships between the previously unresolved northern swordtail groups.
Conclusions: This comprehensive molecular phylogeny of the entire genus Xiphophorus provides evidence that a second swordtail species, X. monticolus, arose through hybridization. Previously, we demonstrated that X. clemenciae, another southern swordtail species, arose via hybridization. These findings highlight the potential key role of hybridization in the evolution of this genus and suggest the need for further investigations into how hybridization contributes to speciation more generally.
Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies.
Comprehensive bioinformatics identifies key microRNA players in ATG7-deficient lung fibroblasts
(2020)
Background: Deficient autophagy has been recently implicated as a driver of pulmonary fibrosis, yet bioinformatics approaches to study this cellular process are lacking. Autophagy-related 5 and 7 (ATG5/ATG7) are critical elements of macro-autophagy. However, an alternative ATG5/ATG7-independent macro-autophagy pathway was recently discovered, its regulation being unknown. Using a bioinformatics proteome profiling analysis of ATG7-deficient human fibroblasts, we aimed to identify key microRNA (miR) regulators in autophagy. Method: We have generated ATG7-knockout MRC-5 fibroblasts and performed mass spectrometry to generate a large-scale proteomics dataset. We further quantified the interactions between various proteins combining bioinformatics molecular network reconstruction and functional enrichment analysis. The predicted key regulatory miRs were validated via quantitative polymerase chain reaction. Results: The functional enrichment analysis of the 26 deregulated proteins showed decreased cellular trafficking, increased mitophagy and senescence as the major overarching processes in ATG7-deficient lung fibroblasts. The 26 proteins reconstitute a protein interactome of 46 nodes and miR-regulated interactome of 834 nodes. The miR network shows three functional cluster modules around miR-16-5p, miR-17-5p and let-7a-5p related to multiple deregulated proteins. Confirming these results in a biological setting, serially passaged wild-type and autophagy-deficient fibroblasts displayed senescence-dependent expression profiles of miR-16-5p and miR-17-5p. Conclusions: We have developed a bioinformatics proteome profiling approach that successfully identifies biologically relevant miR regulators from a proteomics dataset of the ATG-7-deficient milieu in lung fibroblasts, and thus may be used to elucidate key molecular players in complex fibrotic pathological processes. The approach is not limited to a specific cell-type and disease, thus highlighting its high relevance in proteome and non-coding RNA research.
Conventional anticancer chemotherapy is limited because of severe side effects as well as a quickly evolving multidrug resistance of the tumor cells. To address this problem, we have explored a C\(_{60}\) fullerene-based nanosized system as a carrier for anticancer drugs for an optimized drug delivery to leukemic cells.Here, we studied the physicochemical properties and anticancer activity of C\(_{60}\) fullerene noncovalent complexes with the commonly used anticancer drug doxorubicin. C\(_{60}\)-Doxorubicin complexes in a ratio 1:1 and 2:1 were characterized with UV/Vis spectrometry, dynamic light scattering, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The obtained analytical data indicated that the 140-nm complexes were stable and could be used for biological applications. In leukemic cell lines (CCRF-CEM, Jurkat, THP1 and Molt-16), the nanocomplexes revealed 3.5 higher cytotoxic potential in comparison with the free drug in a range of nanomolar concentrations. Also, the intracellular drug's level evidenced C\(_{60}\) fullerene considerable nanocarrier function.The results of this study indicated that C\(_{60}\) fullerene-based delivery nanocomplexes had a potential value for optimization of doxorubicin efficiency against leukemic cells.
Incubation of the colicinogenic Escherichia coli strain JC 411 (ColE1) at elevated temperatures (47-49°) leads to the accumulation of catenated molecules and replicative intermediates of this plasmid. Mature supercoiled OolE1 DNA molecules synthesized under these conditions have an increased number of tertiary turns as shown by electron microscopy. The monomeric tightly supercoiled molecules possess a slightly slower sedimentation rate and a higher binding capacity for ethidium bromide than supercoiJed monomers synthesized at lower temperatures. Recombination deficient mutants of E. coli recA, recB and recC, which carry the ColE1 plasmid, form about the same amount of catenated molecules at the elevated temperature as a rec+ strain. In addition, we have observed by electron microscopy a small percentage (.--.5% of the circular DNA molecules) of minicircular DNA molecules in all preparations of JC 411 (CoIE1). They are homogenous in size, with a molecular weight of 1.4 X 106 daltons. Addition of chloramphenicol to a culture of Proteus mirabilis (ColE1) leads to an increased amount of higher multiple circular oligomers and to a stimulated accumulation of catenated OolE1 DNA molecules of varying sizes. ColE1 DNA synthesis is more thermosensitive than chromosomal DNA replication in P. mirabili8. Plasmid replication stops completely at temperatures above 43°C.
Yersinia enterocolitica subsp. palearctica serobiotype O:3/4 comprises about 80-90 % of all human patient isolates in Germany and Europe and is responsible for sporadic cases worldwide. Even though this serobiotype is low pathogenic, Y. enterocolitica subsp. palearctica serobiotype O:3/4 is involved in gastroenteritis, lymphadenitis and various extraintestinal sequelae as reactive arthritis. The main animal reservoir of this serobiotype are pigs, causing a high rate of O:3/4 contaminations of raw pork in butcher shops in Germany (e.g. Bavaria 25 %) and countries in north-east Europe. As Y. enterocolitica O:3/4 is geographically and phylogenetically distinct from the so far sequenced mouse-virulent O:8/1B strain, complete genome sequencing has been performed for the European serobiotype O:3/4 DSMZ reference strain Y11, which has been isolated from a patient stool. To gain greater insight into the Y. enterocolitica subspecies palearctica group, also draft genome sequences of two other human O:3/4 isolates (strains Y8265, patient isolate, and Y5307, patient isolate associated with reactive arthritis), a closely related Y. enterocolitica palearctica serobiotype O:5,27/3 (strain Y527P), and two biotype 1A strains (a nosocomial strain of serogroup O:5 and an environmental serogroup O:36 isolate) have been performed. Those strains were compared to the high-pathogenic Y. enterocolitica subsp. enterocolitica serobiotype O:8/1B strain 8081 to address the peculiarities of the strain Y11 and the Y. enterocolitica subspecies palearctica group. The main focus was to unravel the pathogenic potential of strain Y11 and thus to identify novel putative virulence genes and fitness factors, especially those that may constitute host specificity of serobiotype O:3/4. Y. enterocolitica subspecies palearctica serobiotype O:3/4 strains lack most of the mouse-virulence-associated determinants of Y. enterocolitica subsp. enterocolitica serotype O:8, for example the HPI, Yts1 type 2 and Ysa type three secretion systems. In comparison, serobiotype O:3/4 strains obviously acquired a different set of genes and genomic islands for virulence and fitness such as the Ysp type three secretion system, an RtxA-like putative toxin, insecticidal toxins and a functional PTS system for N-acetyl-galactosamine uptake, named aga-operon. The aga-operon is able to support the growth of the Y. enterocolitica subsp. enterocolitica O:8/1B on N-acetyl-galactosamine after transformation with the aga operon. Besides these genes, also two prophages, PhiYep-2 and PhiYep-3, and a asn tRNA-associated GIYep-01 genomic island might influence the Y. enterocolitica subsp. palearctica serobiotype O:3/4 pathoadaptation. The PhiYep-3 prophage and the GIYep-01 island show recombination activity and PhiYep-3 was not found in all O:3/4 strains of a small strain collection tested. Y. enterocolitica subsp. palearctica serobiotype O:5,27/3 strain Y527P was found to be closely related to all serobiotype O:3/4 strains, whereas the biotype 1A isolates have more mosaic-segmented genomes and share putative virulence genes both with serobiotypes O:8/1B and O:3/4, which implies their common descent. Besides the pYV virulence plasmid, biotype 1A strains lack classical virulence markers as the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. Interestingly, there are no notable differences between the known virulence factors present in nosocomial and environmental strains, except the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital serogroup O:5 isolate.
Natural enemies have been shown to be effective agents for controlling insect pests in crops. However, it remains unclear how different natural enemy guilds contribute to the regulation of pests and how this might be modulated by landscape context. In a field exclusion experiment in oilseed rape (OSR), we found that parasitoids and ground-dwelling predators acted in a complementary way to suppress pollen beetles, suggesting that pest control by multiple enemies attacking a pest during different periods of its occurrence in the field improves biological control efficacy. The density of pollen beetle significantly decreased with an increased proportion of non-crop habitats in the landscape. Parasitism had a strong effect on pollen beetle numbers in landscapes with a low or intermediate proportion of non-crop habitats, but not in complex landscapes. Our results underline the importance of different natural enemy guilds to pest regulation in crops, and demonstrate how biological control can be strengthened by complementarity among natural enemies. The optimization of natural pest control by adoption of specific management practices at local and landscape scales, such as establishing non-crop areas, low-impact tillage, and temporal crop rotation, could significantly reduce dependence on pesticides and foster yield stability through ecological intensification in agriculture.
Compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) rDNA secondary structures correlate with Ernst Mayr’s biological species concept. This hypothesis also referred to as the CBC species concept recently was subjected to large-scale testing, indicating two distinct probabilities. (1) If there is a CBC then there are two different species with a probability of ~0.93. (2) If there is no CBC then there is the same species with a probability of ~0.76. In ITS2 research, however, the main problem is the multicopy nature of ITS2 sequences. Most recently, 454 pyrosequencing data have been used to characterize more than 5000 intragenomic variations of ITS2 regions from 178 plant species, demonstrating that mutation of ITS2 is frequent, with a mean of 35 variants per species, respectively per individual organism. In this study, using those 454 data, the CBC criterion is reconsidered in the light of intragenomic variability, a proof of concept, a necessary criterion, expecting no intragenomic CBCs in variant ITS2 copies. In accordance with the CBC species concept, we could demonstrate that the probability that there is no intragenomic CBC is ~0.99.
The nuclear paraspeckle assembly transcript 1 (NEAT1) locus encodes two long non-coding (lnc)RNA isoforms that are upregulated in many tumours and dynamically expressed in response to stress. NEAT1 transcripts form ribonucleoprotein complexes with numerous RNA-binding proteins (RBPs) to assemble paraspeckles and modulate the localisation and activity of gene regulatory enzymes as well as a subset of messenger (m)RNA transcripts. The investigation of the dynamic composition of NEAT1-associated proteins and mRNAs is critical to understand the function of NEAT1. Interestingly, a growing number of biochemical and genetic tools to assess NEAT1 interactomes has been reported. Here, we discuss the Hybridisation Proximity (HyPro) labeling technique in the context of NEAT1. HyPro labeling is a recently developed method to detect spatially ordered interactions of RNA-containing nuclear compartments in cultured human cells. After introducing NEAT1 and paraspeckles, we describe the advantages of the HyPro technology in the context of other methods to study RNA interactomes, and review the key findings in mapping NEAT1-associated RNA transcripts and protein binding partners. We further discuss the limitations and potential improvements of HyPro labeling, and conclude by delineating its applicability in paraspeckles-related cancer research.
Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
Comparing the appetitive learning performance of six European honeybee subspecies in a common apiary
(2021)
The Western honeybee (Apis mellifera L.) is one of the most widespread insects with numerous subspecies in its native range. How far adaptation to local habitats has affected the cognitive skills of the different subspecies is an intriguing question that we investigate in this study. Naturally mated queens of the following five subspecies from different parts of Europe were transferred to Southern Germany: A. m. iberiensis from Portugal, A. m. mellifera from Belgium, A. m. macedonica from Greece, A. m. ligustica from Italy, and A. m. ruttneri from Malta. We also included the local subspecies A. m. carnica in our study. New colonies were built up in a common apiary where the respective queens were introduced. Worker offspring from the different subspecies were compared in classical olfactory learning performance using the proboscis extension response. Prior to conditioning, we measured individual sucrose responsiveness to investigate whether possible differences in learning performances were due to differential responsiveness to the sugar water reward. Most subspecies did not differ in their appetitive learning performance. However, foragers of the Iberian honeybee, A. m. iberiensis, performed significantly more poorly, despite having a similar sucrose responsiveness. We discuss possible causes for the poor performance of the Iberian honeybees, which may have been shaped by adaptation to the local habitat.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the,plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing iocal differences in frequency and distribution patterns in the tumor. lntercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.
Behavioural adaptations have made the desert isopod Hemilepistus reaumuri the most successful herbivore and detritivore of the macrofauna of many arid areas in North Africa and Asia Minor. For survival and reproduction Hemilepistus is dependent on burrows. New burrows can only be dug during spring. With the time-consuming digging of a burrow, Hemilepistus has only made the first step towards solving its ecological problems. The burrows are vital and have to be continuously defended against competitors. This requirement is met by co-operation of individuals within the framework of a highly developed social behaviour. In spring adults form monogamous pairs in which partners recognize each other individually and later form, with their progeny, strictly closed family communities. Hemilepistus is compared with a Porcellio' sp. which has developed, convergently, a social behaviour which resembles that of Hemilepistus in many respects, but differs essentially in some aspects, partly reflecting differences in ecological requirements. This and a few other Porcellio species demonstrate some possible steps in the evolution of the social behaviour of Hemilepistus. The female Hemilepistus is-in contrast to Porcellio sp. - semelparous and the selective advantages of monogamy in its environment are not difficult to recognize. This chapter discusses how this mating system could have evolved and especially why monogamous behaviour is also the best method for the Hemilepistus male to maximize its reproductive success. The cohesion of pairs and of family communities in Hemilepistus is based on a highly developed chemical communication system. Individual- and family-specific badges owe their specificity to genetically determined discriminating substances. The nature of the badges raises a series of questions: e.g. since alien badges release aggression, how do parents avoid cannibalizing their young? Similar problems arise from the fact that family badges are mixtures of chemical compounds of very low volatility with the consequence that they can only be transferred by direct contact and that during moulting all substances are lost which an individual does not produce itself. It is shown that in solving these problems inhibiting properties (presumably substances) and learning play a dominant role.
Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
The human gut is home for thousands of microbes that are important for human life. As most of these cannot be cultivated, metagenomics is an important means to understand this important community. To perform comparative metagenomic analysis of the human gut microbiome, I have developed SMASH (Simple metagenomic analysis shell), a computational pipeline. SMASH can also be used to assemble and analyze single genomes, and has been successfully applied to the bacterium Mycoplasma pneumoniae and the fungus Chaetomium thermophilum. In the context of the MetaHIT (Metagenomics of the human intestinal tract) consortium our group is participating in, I used SMASH to validate the assembly and to estimate the assembly error rate of 576.7 Gb metagenome sequence obtained using Illumina Solexa technology from fecal DNA of 124 European individuals. I also estimated the completeness of the gene catalogue containing 3.3 million open reading frames obtained from these metagenomes. Finally, I used SMASH to analyze human gut metagenomes of 39 individuals from 6 countries encompassing a wide range of host properties such as age, body mass index and disease states. We find that the variation in the gut microbiome is not continuous but stratified into enterotypes. Enterotypes are complex host-microbial symbiotic states that are not explained by host properties, nutritional habits or possible technical biases. The concept of enterotypes might have far reaching implications, for example, to explain different responses to diet or drug intake. We also find several functional markers in the human gut microbiome that correlate with a number of host properties such as body mass index, highlighting the need for functional analysis and raising hopes for the application of microbial markers as diagnostic or even prognostic tools for microbiota-associated human disorders.
Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo.
Commuting to work: Nucleolar long non-coding RNA control ribosome biogenesis from near and far
(2021)
Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.
Insects exhibit complex systems of communication with chemical signalling being the most important mode. Although there are many studies on chemical communication in insects, the evolution of chemical signals is not well understood. Due to the conflict of interests between individuals, different selective pressures might act on sender and receiver. In this thesis I investigate different types of communication where either the sender, the receiver or both parties yield benefits. These studies were conducted with one digger wasp species, honeybees, one chrysidid wasp, and three ant species. Senders might benefit by exploiting existing preferences of receivers. Such sensory exploitation might influence the evolution of male signals that are designed to attract females. The sex pheromone of male European beewolves Philanthus triangulum (Hymenoptera, Crabronidae) might have evolved according to the sensory exploitation hypothesis. A three-step scenario is supported by our studies. First, a major component of the honeybee alarm pheromone, (Z)-11-eicosen-1-ol, is also found on the cuticles and in the air surrounding foraging honeybees. Second, it could be shown, that (Z)-11- eicosen-1-ol plays a crucial role as kairomone for prey identification of honeybees by beewolf females. Third, a reanalysis of the beewolf male sex pheromone shows a remarkable similarity of compounds between the pheromone and the honeybee cuticle, besides the co-occurrence of (Z)-11-eisosen-ol. The majority of the cuticular hydrocarbons of honeybees occur also in the headspace of foraging workers. These results strongly support the hypothesis that beewolf males evolved a pheromone that exploits the females’ pre-existing sensory sensitivity. In addition, the male sex pheromone shows a significantly higher similarity among brothers than among non-related individuals, which might enable beewolf females to discriminate against brothers and avoid detrimental effects of breeding. Together with the studies on the possible sensory exploitation this result shows that both, male and female beewolves probably gain more benefits than costs from the pheromone communication and, thus, the communication system as a whole can be regarded as cooperative. To maintain the reproductive division of labour in eusocial colonies, queens have to signal their presence and fecundity. In the ant Camponotus floridanus (Hymenoptera, Formicidae) queens mark their own eggs with a distinctive pattern of cuticular hydrocarbons. Two different hypotheses have been developed. One suggests a form of worker manipulation by the queen. The alternative hypothesis assumes a cooperative signal that provides information on the condition of the queen. The results of our investigation clearly favour the latter hypothesis. Chemical mimicry is a form of non-cooperative communication that benefits predominantly the sender. We provided conclusive evidence that the cockoo wasp, Hedychrum rutilans (Hymenoptera, Chrysididae), the primary brood parasitoid of Philanthus triangulum, evades recognition by beewolf females most probably by chemical mimicry of the odour of its host. Furthermore, the adaptation of the chemical signature in the social ant parasite Protomognathus americanus (Hymenoptera, Formicidae) to its Leptothorax (Hymenoptera, Formicidae) hosts was investigated. Although this parasite is principally adapted to its hosts’ cuticular hydrocarbon profile, there are still pronounced differences between the profiles of parasites and hosts. This might be explained by the trade-off, which the parasites faces when confronted locally with two host species with different cuticular hydrocarbon profiles. Non-cooperative communication in the sense that only receivers benefit was discovered in the exploitation of honeybees volatile cuticular hydrocarbons by beewolf females. By using emitted (Z)-11-eicosen-1-ol as a kairomone, the receiver, the beewolf female, yields the benefits and the sender, the honeybee prey, bears all the costs. The results of these studies contribute to the understanding of the evolution of cooperative and non-cooperative communication with chemical signals taking into account differential benefits for sender and/or receiver.
The instructive component of waggle dance communication has been shown to increase resource uptake of Apis mellifera colonies in highly heterogeneous resource environments, but an assessment of its relevance in temperate landscapes with different levels of resource heterogeneity is currently lacking. We hypothesized that the advertisement of resource locations via dance communication would be most relevant in highly heterogeneous landscapes with large spatial variation of floral resources. To test our hypothesis, we placed 24 Apis mellifera colonies with either disrupted or unimpaired instructive component of dance communication in eight Central European agricultural landscapes that differed in heterogeneity and resource availability. We monitored colony weight change and pollen harvest as measure of foraging success. Dance disruption did not significantly alter colony weight change, but decreased pollen harvest compared to the communicating colonies by 40%. There was no general effect of resource availability on nectar or pollen foraging success, but the effect of landscape heterogeneity on nectar uptake was stronger when resource availability was high. In contrast to our hypothesis, the effects of disrupted bee communication on nectar and pollen foraging success were not stronger in landscapes with heterogeneous compared to homogenous resource environments. Our results indicate that in temperate regions intra-colonial communication of resource locations benefits pollen foraging more than nectar foraging, irrespective of landscape heterogeneity. We conclude that the so far largely unexplored role of dance communication in pollen foraging requires further consideration as pollen is a crucial resource for colony development and health.
Climatic extreme events can cause the shift or disruption of plant-insect interactions due to altered plant quality, e.g. leaf carbon to nitrogen ratios, and phenology. However, the response of plant-herbivore interactions to extreme events and climatic gradients has been rarely studied, although climatic extremes will increase in frequency and intensity in the future and insect herbivores represent a highly diverse and functionally important group. We set up a replicated climate change experiment along elevational gradients in the German Alps to study the responses of three plant guilds and their herbivory by insects to extreme events (extreme drought, advanced and delayed snowmelt) versus control plots under different climatic conditions on 15 grassland sites. Our results indicate that elevational shifts in CN (carbon to nitrogen) ratios and herbivory depend on plant guild and season. CN ratios increased with altitude for grasses, but decreased for legumes and other forbs. In contrast to our hypotheses, extreme climatic events did not significantly affect CN ratios and herbivory. Thus, our study indicates that nutritional quality of plants and antagonistic interactions with insect herbivores are robust against seasonal climatic extremes. Across the three functional plant guilds, herbivory increased with nitrogen concentrations. Further, increased CN ratios indicate a reduction in nutritional plant quality with advancing season. Although our results revealed no direct effects of extreme climatic events, the opposing responses of plant guilds along elevation imply that competitive interactions within plant communities might change under future climates, with unknown consequences for plant-herbivore interactions and plant community composition.
Combined effects of climate change and extreme events on plants, arthropods and their interactions
(2013)
I. Global climate change directly and indirectly influences biotic and abiotic components of ecosystems. Changes in abiotic ecosystem components caused by climate change comprise temperature increases, precipitation changes and more frequently occurring extreme events. Mediated by these abiotic changes, biotic ecosystem components including all living organisms will also change. Expected changes of plants and animals are advanced phenologies and range shifts towards higher latitudes and altitudes which presumably induce changes in species interactions and composition. Altitudinal gradients provide an optimal opportunity for climate change studies, because they serve as natural experiments due to fast changing climatic conditions within short distances. In this dissertation two different approaches were conducted to reveal species and community responses to climate change. First, species richness and community trait analyses along an altitudinal gradient in the Bavarian Alps (chapters II, III) and second, climate change manipulation experiments under different climatic contexts (chapters IV, V, IV). II. We performed biodiversity surveys of butterfly and diurnal moth species on 34 grassland sites along an altitudinal gradient in the National Park Berchtesgaden. Additionally, we analysed the dominance structure of life-history traits in butterfly assemblages along altitude. Species richness of butterflies and diurnal moths decreased with increasing altitude. The dominance of certain life-history-traits changed along the altitudinal gradient with a higher proportion of larger-winged species and species with higher egg numbers towards higher altitudes. However, the mean egg maturation time, population density and geographic distribution within butterfly assemblages decreased with increasing altitude. Our results indicate that butterfly assemblages were mainly shaped by environmental filtering. We conclude that butterfly assemblages at higher altitudes will presumably lack adaptive capacity to future climatic conditions, because of specific trait combinations. III. In addition to butterfly and diurnal moth species richness we also studied plant species richness in combination with pollination type analyses along the altitudinal gradient. The management type of the alpine grasslands was also integrated in the analyses to detect combined effects of climate and management on plant diversity and pollination type. Plant species richness was highest at intermediate altitudes, whereby the management type influenced the plant diversity with more plant species at grazed compared to mown or non-managed grasslands. The pollination type was affected by both the changing climate along the gradient and the management type. These results suggest that extensive grazing can maintain high plant diversity along the whole altitudinal gradient. With ongoing climate change the diversity peak of plants may shift upwards, which can cause a decrease in biodiversity due to reduced grassland area but also changes in species composition and adaptive potential of pollination types. IV. We set up manipulation experiments on 15 grassland sites along the altitudinal gradient in order to determine the combined effects of extreme climatic events (extreme drought, advanced and delayed snowmelt) and elevation on the nutritional quality and herbivory rates of alpine plants. The leaf CN (carbon to nitrogen) ratio and the plant damage through herbivores were not significantly affected by the simulated extreme events. However, elevation influenced the CN ratios and herbivory rates of alpine plants with contrasting responses between plant guilds. Furthermore, we found differences in nitrogen concentrations and herbivory rates between grasses, legumes and forbs, whereas legumes had the highest nitrogen concentrations and were damaged most. Additionally, CN ratios and herbivory rates increased during the growing season, indicating a decrease of food plant quality during the growing season. Contrasting altitudinal responses of grasses, legumes and forbs presumably can change the dominance structure among these plant guilds with ongoing climate change. V. In this study we analysed the phenological responses of grassland species to an extreme drought event, advanced and delayed snowmelt along the altitudinal gradient. Advanced snowmelt caused an advanced beginning of flowering, whereas this effect was more pronounced at higher than at lower altitudes. Extreme drought and delayed snowmelt had rather low effects on the flower phenology and the responses did not differ between higher and lower sites. The strongest effect influencing flower phenology was altitude, with a declining effect through the season. The length of flowering duration was not significantly influenced by treatments. Our data suggest that plant species at higher altitudes may be more affected by changes in snowmelt timing in contrast to lowland species, as at higher altitudes more severe changes are expected. However, the risk of extreme drought events on flowering phenology seems to be low. VI. We established soil-emergence traps on the advanced snowmelt and control treatment plots in order to detect possible changes in abundances and emergence phenologies of five arthropod orders due to elevation and treatment. Additionally, we analysed the responses of Coleoptera species richness to elevation and treatment. We found that the abundance and species richness of Coleoptera increased with elevation as well as the abundance of Diptera. However, the abundance of Hemiptera decreased with elevation and the abundances of Araneae and Hymenoptera showed no elevational patterns. The advanced snowmelt treatment increased the abundances of Araneae and Hymenoptera. The emergence of soil-hibernating arthropods was delayed up to seven weeks at higher elevations, whereas advanced snowmelt did not influence the emergence phenology of arthropods immediately after snowmelt. With climate change earlier snowmelt will occur more often, which especially will affect soil-hibernating arthropods in alpine regions and may cause desynchronisations between species interactions. VII. In conclusion, we showed that alpine ecosystems are sensitive towards changing climate conditions and extreme events and that many alpine species in the Bavarian Alps are endangered. Many alpine species could exist under warmer climatic conditions, however they are expected to be outcompeted by more competitive lowland species. Furthermore, host-parasite or predator-prey interactions can be disrupted due to different responses of certain guilds to climate change. Understanding and predicting the complex dynamics and potential risks of future climate change remains a great challenge and therefore further studies analysing species and community responses to climate change are needed.
Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia.
Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor.
Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific.
We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus.
In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus.
In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus.
Background
Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs.
Methods
Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC).
Results
RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease.
Conclusions
A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.
Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing
(2016)
Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing.
Background
The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment.
Methods
Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student’s t test was used for comparison between groups in all of the experiments.
Results
Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7–10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments.
Conclusions
We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.
Cofilin
(1999)
This study has identified cofilin, an actin binding protein, as a control element in the reorganization of the actin cytoskeleton which is highly relevant for T lymphocyte activation. Cofilin is regulated in its activity by reversible phosphorylation which is inducible by stimulation through accessory receptors such as CD2 and CD28. First it could be demonstrated that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton and that only the dephosphorylated form of cofilin possesses the capacity to bind cytoskeletal actin in vivo. PI3-kinase inhibitors block both the dephosphorylation of cofilin and its association with the actin cytoskeleton. Importantly, cofilin, actin, PI3-kinase and one of its substrates, namely phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) which can bind to cofilin, co-localize within CD2-receptor caps. The cofilin/F-actin interaction has been identified as a crucial regulatory element for receptor cap formation and the strength of signal transduction. To this end, appropriately designed cell permeable non-toxic peptides that are homologous to actin binding motifs of the human cofilin sequence were introduced into untransformed human peripheral blood T lymphocytes. These peptides competitively and dose dependently inhibit the activation induced interaction of cofilin with the actin cytoskeleton in vivo. By this approach it was possible to study, for the first time, the functional consequences of this interaction in immunocompetent T cells. The present data demonstrate that inhibition of the actin/cofilin interaction in human T lymphocytes by means of these cofilin derived peptides abolishes receptor cap formation and strongly modulates functional T cell responses such as T cell proliferation, interleukin-2 production, cell surface expression of CD69, gIFN production, and CD95L expression. Importantly, receptor independent activation by PMA and calcium ionophore circumvents these peptide produced inhibitory effects on lymphocyte stimulation and places the cofilin/actin interaction to a proximal step in the cascade of signaling events following T cell activation via surface signals. The present results are novel since as yet no information existed regarding the molecular elements which link cell surface receptor stimulation directly to the resulting reorganization of the actin cytoskeleton.
Tropical rain forests and coral reefs are usually regarded as the epitome of complexity and diversity. The mechanisms, however, that allow so many species to coexist continuously, still need to be unraveled. Earlier equilibrium models explain community organization with a strict niche separation and specialization of the single species, achieved mainly by interspecific competition and consecutive resource partitioning. Recent non-equilibrium or stochastic models see stochastic factors ("intermediate disturbances") as more important. Such systems are characterized by broad niche overlaps and an unpredictable species composition. Mechanisms of coexistence are most interesting where species interactions are strongest and species packing is highest. This is the case within a functional group or guild where species use similar resources. In this project a community of seven closely related leaf beetle species (Chrysomelidae: Cassidinae) was investigated which coexist on a common host plant system (fam. Convovulaceae) in a tropical moist savanna (Ivory Coast, Comoé-Nationalpark). A broad overlap in the seasonal phenology of the leaf beetle species stood in contrast to a distinct spatial niche differentiation. The beetle community could be separated in a savanna-group (host plant: Ipomoea) and in a river side group (host plant: Merremia). According to a correspondence analysis the five species at the river side, using a common host plant, Merremia hederacea, proved to be predictable in their species composition. They showed a small scale niche differentiation along the light gradient (microhabitats). Laboratory studies confirmed differences in the tolerance towards high temperatures (up to 50°C in the field). Physiological trade-offs between phenology, microclimate and food quality seem best to describe patterns of resource use of the beetle species. Further a phylogeny based on mt-DNA sequencing of the beetle community was compared to its ecological resource use and the evolution of host plant use was reconstructed
A significant relatedness is of fundamental importance for the evolution and maintenance of social life (kin selection theory, Hamilton 1964a,b). Not only kin selection itself, but also more complex evolutionary theories make predictions on the occurrence of conflict and co-operation in animal societies. They all depend on the genetic relationships among individuals. Therefore, the study of unrelated, co-operating individuals provides a unique opportunity to critically test predictions based on these evolutionary theories. Using allozyme electrophoresis, the study species Pachycondyla villosa was found to represent three different species. Young queens in one of these species, provisionally called Pachycondyla cf. inversa, may co-operate during colony founding (pleometrosis). Approximately 50 per cent of all founding colonies collected near Itabuna, Brazil, consisted of two to five founding queens. Queens of P. cf. inversa have to forage for food (semi-claustral founding), and in founding associations only one queen specialised for this risky task. A microsatellite study showed that nestmate queens were typically not related. How can a division of labour be achieved, where one individual performs risky tasks to the favour of another individual to which it is not related? In contrast to the predictions made by group selectionists, this study provided clear evidence that the division of labour among co-foundresses of P. cf. inversa results from social competition: Co-foundresses displayed aggressive interactions and formed dominance hierarchies which predominantly served to force subordinates to forage. The frequency of queen antagonism increased with the duration since food was last added to the foraging arena. The social status was not, or only weakly associated with the reproductive status: As predicted by the reproductive skew theory, all foundresses laid eggs at similar rates, though the subordinate may be harassed during egg laying and occasionally, some of her eggs may be eaten by the dominant. The differential oophagy presumably was also reflected in a microsatellite study of foundress associations, which was conducted shortly after the first workers emerged: Here, the co-foundresses occasionally contributed unequally to the colony’s workers. Conflicts among workers or between workers and queens, e.g. over the division of labour or sex ratio, strongly depend on the genetic relationships among members of a colony. The number of two to five co-founding queens in polygynous colonies of P. cf. inversa, and the lack of relatedness among them, should lead to a decrease in the relatedness of workers. However, nestmate workers were closely related. Furthermore, worker relatedness may decrease as several queens were found to be multiply inseminated. Inbreeding coefficients were significantly different from zero in both queens and workers. No evidence for a geographical substructuring of the population was found. The deviation from random mating presumably was probably due to small, localised nuptial flights. Virgin queens do not mate near their natal nest and disperse before founding colonies. The analysis of cuticular hydrocarbons obtained from live queens revealed consistent differences between the patterns of cuticular hydrocarbons of queens with high vs. low rank: only high-ranking queens showed considerable amounts of cuticular pentadecane (n-C15) and heptadecene (n-C17:1). The presence of the two substances apparently was not associated with reproductive status. It is not yet known, if the two substances indeed serve to communicate high social status in P. cf. inversa. In experimentally assembled associations of two founding queens, queens engaged in aggressive interactions which already within one to twenty minutes resulted in stable dominance hierarchies. The queens attacking first usually won the contest and became dominant. Nest ownership at least for a couple of days did not influence the outcome of dominance interactions in the laboratory experiments, whereas queen body size apparently played an important role: In all eight trials, the larger queen became dominant. However, dominant queens from natural foundress associations were on average not larger than subordinates, suggesting that in the field, resident asymmetries might override size asymmetries only after a more prolonged period of nest ownership. Sequencing of the COI/COII region of mitochondrial DNA displayed sufficient variability for the study of the sociogenetic structure of the secondarily polygynous ant Pachycondyla obscuricornis: Six different haplotypes could be distinguished among six workers of different colonies from one study population in Costa Rica. The variability of other methods which were established (RFLPs, microsatellites, allozymes, and multilocus DNA fingerprinting) was too low for a further study on the genetic structure in P. obscuricornis.
A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of tbe L. seeligeri catalase gene.