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- DNA Analytics Core Facility, Biocenter, University of Würzburg, Würzburg, Germany (1)
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The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis
(2014)
Background
It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal.
Results
We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells.
Conclusions
In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.
Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis.
Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock.
In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats.
We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD.
It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity.
Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication.
Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.
Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species' threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project - and avert - future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups - including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems - ). We make site-level summary data available alongside this article. The full database will be publicly available in 2015.
Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAFV600E–mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAFV600E-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis.
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
The synaptonemal complex (SC) is a highly conserved structure in sexually reproducing organism. It has a tripartite, ladder-like organization and mediates the stable pairing, called synapsis, of the homologous chromosomes during prophase of meiosis I. Failure in homolog synapsis result in aneuploidy and/or apoptosis of the developing germ cells.
Since 1956, the SC is subject of intense research and its presence was described in various species from yeast to human. Its structure was maintained during millions of years of evolution consist-ing of two parallel lateral elements (LEs), joined by numerous transverse filaments (TFs) which run perpendicular to the LEs and an electron dense central element (CE) in the middle of the SC. Individual protein components, however, were characterized only in few available model organ-isms, as for example Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Ceanorhabditis elegans and Mus musculus. Rather unexpectedly, these characterizations failed to detect an evolutionary homology between the protein components of the different SCs. This fact challenged the general idea of a single origin of the SC in the evolution of meiosis and sexual reproduction.
This thesis now addressed itself to the task to unravel the discrepancy between the high conser-vation of the SC structure and its diverse and apparently non-homologous protein composition, focusing on the animal kingdom. It is the first study dealing with the evolution of the SC in Meta-zoa and demonstrates the monophyly of the mammalian SC components in metazoan species. The thesis demonstrates that at least four out of seven murine SC proteins emerged in Eumeta-zoa at the latest and have been likewise part of an ancient SC as it can be found in the present-day cnidarian species Hydra. This SC displays the common organization and already possesses the minimal protein kit corresponding to the three different structural domains: LEs, TFs and the CE. Additionally, the individual phylogenies of the murine SC proteins revealed the dynamic evolu-tionary history of the ancient SC. Further components were added during the diversification of Bilateria and vertebrates while ancestral proteins likely duplicated in the vertebrate lineage and diversified or got lost in the branch leading to ecdysozoan species. It is hypothesized that the apparently non-homologous SC proteins in D. melanogaster and C. elegans actually do derive from the ancient SC proteins but diversified beyond recognition during the fast evolution of Ar-thropoda and Nematoda.
The study proposes Hydra as an alternative invertebrate model system for meiosis and SC re-search to the standard organisms D. melanogaster and C. elegans. Recent results about the cni-darian SC as well as the possible application of standard methods is discussed and summarized in the concluding section.
Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71–110 and HlyAΔ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158–167 and HlyAΔ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71–110 and HlyAΔ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71–110, and HlyAΔ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.
The consequences of habitat change for human well-being are assumed to be especially extreme in Burkina Faso. The country is located in a highly drought-sensitive zone of West Africa, and small‐scale subsistence farmers may be especially affected if losses of biodiversity lead to changes in ecosystem functioning; many depend on more or less degraded lands for agricultural production.
The overall aim of the present thesis consequently was to characterize the functional traits of soil-organisms which are crucial for a productive and balanced soil environment in the study region – termites and ants. They are true ecosystem engineers whose activity alters the habitat. Through soil-turnover in the course of constructing biogenic structures of varying size and nature (mounds, nests, galleries, soil-sheetings, foraging-holes), they bioturbate huge amounts of soil masses and exert massive effects on soil structure, positively influencing the fertility, stability, aeration and water infiltration rate into soils; and they provide habitats for other species. In sub-Saharan Africa, ants and termites are the only active soil macrofauna during the long dry season; in the sub-Sahel zone of Burkina Faso, termites even represent the only active, quantitatively remarkable decomposers all year round. Since no information was available about the actual diversity of the focal arthropods, I divided the thesis in two main parts: In the first part, a baseline study, I assessed the local termite and ant fauna, and investigated their quantitative and qualitative response to changing habitat parameters resulting from increasing human impact (‘functional response traits’). In the second and applied part, I addressed the impact of the biogenic structures which are important for the restoration of degraded soils (‘functional effect traits’).
Two traditional agricultural systems characteristic for the study region were selected. Each system represented a land-use intensification gradient comprising four distinct habitats now differing in the magnitude of human intervention but formerly having the same initial state. The first disturbance gradient, the temporal cross-section of a traditional soil water conservation technique to restore degraded heavily encrusted, barren soil named Zaï in Ouahigouya (Yatenga province, sub-Sahel zone); the second disturbance gradient, an agriculture type using crop rotation and fallow as nutrient management techniques near Fada N’Gourma (Gourma province, North-Sudanese zone).
No standard protocol existed for the assessment of termite and ant diversity in semi-arid (agro-) ecosystems; two widely accepted standard protocols provided the basis for the newly revised and combined rapid assessment protocol ‘RAP’: the ALL protocol for leaf litter ants of Agosti and Alonso (2000), and the transect protocol for termites in tropical forests of Jones and Eggleton (2000). In each study site, three to four replicate transects were conducted during the rainy seasons (2004—2008).
The RAP-protocol turned out to be very effective to characterize, compare and monitor the taxonomic and functional diversity of termites and ants; between 70% and 90% of the estimated total species richness were collected on all levels (transects, habitats, regions). Together in both regions, 65 ant species (25 genera) and 39 termite species (13 genera) were collected. These findings represent the first records for Burkina Faso. The data indicate a high sensitivity of termites and ants to land-use intensification. The diversity strongly decreased with increasing anthropogenic impact in the North-Sudan region. In total, 53 ant species (23 genera) and 31 termite species (12 genera) were found. Very promising results concerning the recovery potential of the soil-arthropods’ diversity were gathered in the Zaï system. The diversity of both taxa strongly increased with increasing habitat rehabilitation – in total, 41 ant species (16 genera) and 33 termite species (11 genera) were collected. For both taxa significant differences could be noted in the shape of the density variations along the gradient. For instance termites: Fungus-growers showed the greatest adaptability to different management practices. The greatest variations between the habitats were observed in soil and grass-feeding termites. Whole functional groups were missing in heavily impacted habitats, e.g. soil-, grass-, and wood-feeders were absent in the degraded site in the sub-Sahel zone. Several environmental parameters could be identified which significantly explained a great part of the variations in the composition of the arthropods’ communities; they indicate the importance of the habitats’ structural complexity (vegetation structure) and concomitant effects on diurnal temperature and moisture fluctuations, the availability of food sources, and the soil-structure. The diversity of termites in the sub-Sahel region was strongly correlated with the crown-cover percentages, the topsoils’ sand-content, and the availability of litter; in the North-Sudan region with the cumulated woody plant basal area, the topsoils’ clay- and organic matter-content. The parameters identified for ant communities in the Zaï system, were the height of trees, the topsoils’ clay-content and air humidity; in the North-Sudan region the habitats’ crown-cover percentages, the quantity of litter and again the height of trees.
In the second part of the thesis, I first rapidly assessed the (natural) variations in the amount of epigeal soil-structures along the two disturbance gradients in order to judge the relative importance of termites and ants for soil-turnover. The results illustrated impressively that a) in all study sites, termites were the main bioturbators while ant structures were of minor importance for soil turn-over; b) earthworms and grass-feeding termites contributed significantly to soil turn-over in the more humid North-Sudan region; and c) the bioturbated soil mass varied between seasons and years, however, the relative importance of the different taxa seemed to be fairly constant. In the sub-Sahel zone, fungus-growing Odontotermes and Macrotermes species fully take over the important function of bioturbation, leading to the transport of huge amounts of fine-textured soil material to the surface; with increasing habitat restoration, coarse fragments decreased in the upper horizons and became concentrated deeper along the soil profile.
Consequently, in the applied part, I concentrated on the bioturbation activity of fungus-growing termites in the four main stages of the Zaï system: crusted bare soil (initial stage), millet field, young and old forest. In each of the four Zaï sites nine experimental blocks (each comprising four plots of 1m2) were used to stimulate the foraging activity of fungus-growing termites with different, locally available organic materials (Aristida kerstingii hay, Bombax costatum wooden blocks, compost and a control without any organic amendment). The experiment was conducted twice for the duration of four weeks (rainy season 2005, dry season 2006). The plots were regularly checked and the increase of the area covered by sheetings chronologically followed. After four weeks a) all sheeting-soil was collected, air dried and separately weighed according to the different genera, and b) the foraging-holes were counted and their diameter measured. Additionally, c) ponded water infiltration was measured in selected plots, and d) the physicochemical properties of sheeting-soil were analyzed. In case of complete consumption of the offered hay during the experimental 4-weeks-duration, the same procedure (a, b) was followed before adding new hay to the respective plot.
The comparison between the different plots, sites and seasons revealed clearly that hay was the most attractive bait; for each gram of hay removed, Odontotermes brought about 12 g soil to the surface, Macrotermes 4 g. Odontotermes was the only genus attracted by organic material to the degraded area, and was therefore the decisive primary physical ecosystem engineer in the Zaï system, initiating the restoration process. The mass of soil bioturbated in the course of foraging increased strongly from the degraded, barren towards the most rehabilitated reforested site. Combining all 36 experimental plots per Zaï stage, Odontotermes bioturbated 31.8 tons of soil per hectare and month dry season in the degraded area, and 32.4 tons ha-1 mon-1 in the millet fields; both genera moved 138.9 tons ha-1 mon-1 in the young and 215.5 tons ha-1 mon-1 in the old Zaï forest. Few comparable figures were found in the literature. In northern Burkina Faso, both genera constructed 20 tons of sheetings ha-1 mon-1 after mulching with a straw-wood mixture (Mando & Miedema 1997), and in Senegal, around 10 tons ha-1 mon-1 were moved in heavily foraged plots (Rouland et al. 2003). Within a site, soil turn-over and the number of foraging holes created was always highest in hay, followed by compost, then by wood and in the end control. The fungus-growers’ foraging-activity was leading to an enormous increase in surface pore space – after one month of induced foraging activity in hay-plots, the median number of foraging-holes increased from 142 m-2 in the degraded site up to 921 m-2 in the old Zaï forest. The creation of subterranean galleries and macropores significantly increased the water infiltration rate by a mean factor 2–4.
Laboratory analyses revealed that sheeting-soil differed strongly from the respective control soil as well as between the seasons, the food-type covered, and the two genera. Odontotermes-sheetings differed in more parameters than Macrotermes-sheetings, and dry season sheetings differed in more parameters (and more strongly) than rainy season sheetings. In the present study, soil organic matter, carbon and nitrogen contents were significantly increased in all dry season sheetings; in the rainy season mainly in those built on compost. Texture analysis pointed out that both genera used topsoil and soil from deeper horizons in varying mixture ratios, thereby supporting findings of Jouquet et al. (2006).
To summarize, the present thesis contributes to a better understanding of the functional response traits of termites and ants to changing environmental parameters resulting from increasing human impact. The RAP-protocol represents an easy-to-learn and very effective method to representatively characterize, compare and monitor the taxonomic and functional diversity of termites and ants. The experiment has provided conclusive evidence of the importance of the consideration of fungus-growing termites (particularly Odontotermes and Macrotermes species) when aiming to restore infertile, degraded and crusted soils and to maintain a sustainable agricultural production in the Sahel‐Sudanese zone of West Africa.
Intricate mechanisms discriminate between friends and foes in plants. Plant organs deploy overlapping and distinct protection strategies. Despite vulnerability to a plethora of pathogens, the growing tips of plants grow bacteria free. The shoot apical meristem (SAM) is among three stem cells niches, a self-renewable reservoir for the future organogenesis of leaf, stem, and flowers. How plants safeguard this high value growth target from infections was not known until now. Recent reports find the stem cell secreted 12-amino acid peptide CLV3p (CLAVATA3 peptide) is perceived by FLS2 (FLAGELLIN SENSING 2) receptor and activates the transcription of immunity and defense marker genes. No infection in the SAM of wild type plants and bacterial infection in clv3 and fls2 mutants illustrate this natural protection against infections. Cytokinins (CKs) are enriched in the SAM and regulate meristem activities by their involvement in stem cell signaling networks. Auxin mediates plant susceptibility to pathogen infections while CKs boost plant immunity. Here, in addition to the stem-cell-triggered immunity we also highlight a potential link between CK signaling and CLV3p mediated immune response in the SAM.
The Chaco leaf-cutting ant Atta vollenweideri is native to the clay-heavy soils of the Gran Chaco region in South America. Because of seasonal floods, colonies are regularly exposed to varying moisture across the soil profile, a factor that not only strongly influences workers' digging performance during nest building, but also determines the suitability of the soil for the rearing of the colony's symbiotic fungus. In this study, we investigated the effects of varying soil moisture on behaviours associated with underground nest building in A. vollenweideri. This was done in a series of laboratory experiments using standardised, plastic clay-water mixtures with gravimetric water contents ranging from relatively brittle material to mixtures close to the liquid limit. Our experiments showed that preference and group-level digging rate increased with increasing water content, but then dropped considerably for extremely moist materials. The production of vibrational recruitment signals during digging showed, on the contrary, a slightly negative linear correlation with soil moisture. Workers formed and carried clay pellets at higher rates in moist clay, even at the highest water content tested. Hence, their weak preference and low group-level excavation rate observed for that mixture cannot be explained by any inability to work with the material. More likely, extremely high moistures may indicate locations unsuitable for nest building. To test this hypothesis, we simulated a situation in which workers excavated an upward tunnel below accumulated surface water. The ants stopped digging about 12 mm below the interface soil/water, a behaviour representing a possible adaptation to the threat of water inflow field colonies are exposed to while digging under seasonally flooded soils. Possible roles of soil water in the temporal and spatial pattern of nest growth are discussed.