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With the advent of highthroughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. The dissection of IG repertoires promises to transform our understanding of the adaptive immune system dynamics. Advances in sequencing technology now also allow the use of the Ion Torrent Personal Genome Machine (PGM) to cover the full length of IG mRNA transcripts. The applications of this benchtop scale HTS platform range from identification of new therapeutic antibodies to the deconvolution of malignant B cell tumors. In the context of this thesis, the usability of the PGM is assessed to investigate the IG heavy chain (IGH) repertoires of animal models. First, an innovate bioinformatics approach is presented to identify antigendriven IGH sequences from bulk sequenced bone marrow samples of transgenic humanized rats, expressing a human IG repertoire (OmniRatTM). We show, that these rats mount a convergent IGH CDR3 response towards measles virus hemagglutinin protein and tetanus toxoid, with high similarity to human counterparts. In the future, databases could contain all IGH CDR3 sequences with known specificity to mine IG repertoire datasets for past antigen exposures, ultimately reconstructing the immunological history of an individual. Second, a unique molecular identifier (UID) based HTS approach and network property analysis is used to characterize the CLLlike CD5+ B cell expansion of A20BKO mice overexpressing a natural short splice variant of the CYLD gene (A20BKOsCYLDBOE). We could determine, that in these mice, overexpression of sCYLD leads to unmutated subvariant of CLL (UCLL). Furthermore, we found that this short splice variant is also seen in human CLL patients highlighting it as important target for future investigations. Third, the UID based HTS approach is improved by adapting it to the PGM sequencing technology and applying a custommade data processing pipeline including the ImMunoGeneTics (IMGT) database error detection. Like this, we were able to obtain correct IGH sequences with over 99.5% confidence and correct CDR3 sequences with over 99.9% confidence. Taken together, the results, protocols and sample processing strategies described in this thesis will improve the usability of animal models and the Ion Torrent PGM HTS platform in the field if IG repertoire research.
Background: The growing production and use of engineered AgNP in industry and private households make increasing concentrations of AgNP in the environment unavoidable. Although we already know the harmful effects of AgNP on pivotal bacterial driven soil functions, information about the impact of silver nanoparticles (AgNP) on the soil bacterial community structure is rare. Hence, the aim of this study was to reveal the long-term effects of AgNP on major soil bacterial phyla in a loamy soil. The study was conducted as a laboratory incubation experiment over a period of 1 year using a loamy soil and AgNP concentrations ranging from 0.01 to 1 mg AgNP/kg soil. Effects were quantified using the taxon-specific 16S rRNA qPCR.
Results: The short-term exposure of AgNP at environmentally relevant concentration of 0.01 mg AgNP/kg caused significant positive effects on Acidobacteria (44.0%), Actinobacteria (21.1%) and Bacteroidetes (14.6%), whereas beta-Proteobacteria population was minimized by 14.2% relative to the control (p ≤ 0.05). After 1 year of exposure to 0.01 mg AgNP/kg diminished Acidobacteria (p = 0.007), Bacteroidetes (p = 0.005) and beta-Proteobacteria (p = 0.000) by 14.5, 10.1 and 13.9%, respectively. Actino- and alpha-Proteobacteria were statistically unaffected by AgNP treatments after 1-year exposure. Furthermore, a statistically significant regression and correlation analysis between silver toxicity and exposure time confirmed loamy soils as a sink for silver nanoparticles and their concomitant silver ions.
Conclusions: Even very low concentrations of AgNP may cause disadvantages for the autotrophic ammonia oxidation (nitrification), the organic carbon transformation and the chitin degradation in soils by exerting harmful effects on the liable bacterial phyla.
Species can show strong variation of local abundance across their ranges. Recent analyses suggested that variation in abundance can be related to environmental suitability, as the highest abundances are often observed in populations living in the most suitable areas. However, there is limited information on the mechanisms through which variation in environmental suitability determines abundance. We analysed populations of the microendemic salamander Hydromantes flavus, and tested several hypotheses on potential relationships linking environmental suitability to population parameters. For multiple populations across the whole species range, we assessed suitability using species distribution models, and measured density, activity level, food intake and body condition index. In high-suitability sites, the density of salamanders was up to 30-times higher than in the least suitable ones. Variation in activity levels and population performance can explain such variation of abundance. In high-suitability sites, salamanders were active close to the surface, and showed a low frequency of empty stomachs. Furthermore, when taking into account seasonal variation, body condition was better in the most suitable sites. Our results show that the strong relationship between environmental suitability and population abundance can be mediated by the variation of parameters strongly linked to individual performance and fitness.
The availability of data on the feeding habits of species of conservation value may be of great importance to develop analyses for both scientific and management purposes. Stomach flushing is a harmless technique that allowed us to collect extensive data on the feeding habits of six Hydromantes species. Here, we present two datasets originating from a three-year study performed in multiple seasons (spring and autumn) on 19 different populations of cave salamanders. The first dataset contains data of the stomach content of 1,250 salamanders, where 6,010 items were recognized; the second one reports the size of the intact prey items found in the stomachs. These datasets integrate considerably data already available on the diet of the European plethodontid salamanders, being also of potential use for large scale meta-analyses on amphibian diet.
Leeches can parasitize many vertebrate taxa. In amphibians, leech parasitism often has potential detrimental effects including population decline. Most of studies on the host-parasite interactions involving leeches and amphibians focus on freshwater environments, while they are very scarce for terrestrial amphibians. In this work, we studied the relationship between the leech Batracobdella algira and the European terrestrial salamanders of the genus Hydromantes, identifying environmental features related to the presence of the leeches and their possible effects on the hosts. We performed observation throughout Sardinia (Italy), covering the distribution area of all Hydromantes species endemic to this island. From September 2015 to May 2017, we conducted >150 surveys in 26 underground environments, collecting data on 2629 salamanders and 131 leeches. Water hardness was the only environmental feature correlated with the presence of B. algira, linking this leech to active karstic systems. Leeches were more frequently parasitizing salamanders with large body size. Body Condition Index was not significantly different between parasitized and non-parasitized salamanders. Our study shows the importance of abiotic environmental features for host-parasite interactions, and poses new questions on complex interspecific interactions between this ectoparasite and amphibians.
Global human population growth is associated with many problems, such asrnfood and water provision, political conflicts, spread of diseases, and environmental destruction. The mitigation of these problems is mirrored in several global conventions and programs, some of which, however, are conflicting. Here, we discuss the conflicts between biodiversity conservation and disease eradication. Numerous health programs aim at eradicating pathogens, and many focus on the eradication of vectors, such as mosquitos or other parasites. As a case study, we focus on the "Pan African Tsetse and Trypanosomiasis Eradication Campaign," which aims at eradicating a pathogen (Trypanosoma) as well as its vector, the entire group of tsetse flies (Glossinidae). As the distribution of tsetse flies largely overlaps with the African hotspots of freshwater biodiversity, we argue for a strong consideration of environmental issues when applying vector control measures, especially the aerial applications of insecticides.rnFurthermore, we want to stimulate discussions on the value of speciesrnand whether full eradication of a pathogen or vector is justified at all. Finally, we call for a stronger harmonization of international conventions. Proper environmental impact assessments need to be conducted before control or eradication programs are carried out to minimize negative effects on biodiversity.
Besides well-known positive aspects of conservation tillage combined with mulching, a drawback may be the survival of phytopathogenic fungi like Fusarium species on plant residues. This may endanger the health of the following crop by increasing the infection risk for specific plant diseases. In infected plant organs, these pathogens are able to produce mycotoxins like deoxynivalenol (DON). Mycotoxins like DON persist during storage, are heat resistant and of major concern for human and animal health after consumption of contaminated food and feed, respectively. Among fungivorous soil organisms, there are representatives of the soil fauna which are obviously antagonistic to a Fusarium infection and the contamination with mycotoxins. Earthworms (Lumbricus terrestris), collembolans (Folsomia candida) and nematodes (Aphelenchoides saprophilus) provide a wide range of ecosystem services including the stimulation of decomposition processes which may result in the regulation of plant pathogens and the degradation of environmental contaminants. Several investigations under laboratory conditions and in the field were conducted to test the following hypotheses: (1) Fusarium-infected and DON-contaminated wheat straw provides a more attractive food substrate than non-infected control straw (2) the introduced soil fauna reduce the biomass of F. culmorum and the content of DON in infected wheat straw under laboratory and field conditions (3) the species interaction of the introduced soil fauna enhances the degradation of Fusarium biomass and DON concentration in wheat straw; (4) the degradation efficiency of soil fauna is affected by soil texture. The results of the present thesis pointed out that the degradation performance of the introduced soil fauna must be considered as an important contribution to the biological control of plant diseases and environmental pollutants. As in particular L. terrestris revealed to be the driver of the degradation process, earthworms contribute to a sustainable control of fungal pathogens like Fusarium and its mycotoxins in wheat straw, thus reducing the risk of plant diseases and environmental pollution as ecosystem services.
Exposure to fine and ultra-fine environmental particles is still a problem of concern in many industrialized parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Since many years air pollution is recognized as a critical problem in western countries, which led to rigorous regulation of air quality and the introduction of strict guidelines. However, the upper thresholds for particulates in ambient air recommended by the world health organization are often exceeded several times in newly industrialized countries. Such high levels of air pollution have the potential to induce adverse effects on human health. The response triggered by air pollutants is not limited to local effects of the respiratory system but is often systemic, resulting in endothelial dysfunction or atherosclerotic malady. The link between air pollution and cardiovascular disease is now accepted by the scientific community but the underlying mechanisms responsible for the pro-atherogenic potential still need to be unraveled in detail. Based on the results from in- vivo and in vitro studies the production of reactive oxygen species due to exposure to particles is the most important mechanism to explain the observed adverse effects. However, the doses that were applied in many in vivo and in vitro studies are far beyond the range of what humans are exposed to and there is the need for more realistic exposure studies. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. One of the objectives of this PhD thesis was the establishment and further improvement of a complex coculture system initially described by Alfaro-Moreno et al. [1]. The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane to mimic the cell response of the alveolar surface in vitro in conjunction with native aerosol exposure (VitrocellTM chamber). The tetraculture system was carefully characterized to ensure its performance and repeatability of results. The spatial distribution of the cells in the tetraculture was analyzed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the Transwellâ„¢. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The latter cells formed colonies under submerged conditions, which disappeared at the air-liquid-interface (ALI). The VitrocellTM aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine nanoparticles (NPs) in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. Furthermore, all cell lines were found to be able to respond to xenobiotic model compounds, such as benzo[a]pyrene (B[a]P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the upregulation of CYP1 mRNA. With this tetraculture system the response of the endothelial part of the alveolar barrier was studied in- vitro in a still realistic exposure scenario representing the conditions for a polluted situation without direct exposure of endothelial cells. After exposure to diesel exhaust particulate matter (DEPM) the expression of different anti-oxidant target genes and inflammatory genes such as NAD(P)H dehydrogenase quinone 1 (NQO1), superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HMOX1), as well as the nuclear translocation nuclear factor erythroid-derived 2 (Nrf2) was evaluated. In addition, the potential of DEPM to induce the upregulation of CYP1A1 mRNA in the endothelium was analyzed. DEPM exposure led not to an upregulation of the anti-oxidant or inflammatory target genes, but to clear nuclear translocation of Nrf2. The endothelial cells responded to the DEPM treatment also with the upregulation of CYP1A1 mRNA and nuclear translocation of the aryl hydrocarbon receptor (AhR). Overall, DEPM triggered a response in the endothelial cells after indirect exposure of the tetraculture system to low doses of DEPM, underlining the sensitivity of ALI exposure systems. The use of the tetraculture together with the native aerosol exposure equipment may finally lead to a more realistic judgment regarding the hazard of new compounds and/or new nano-scaled materials in the future. For the first time, it was possible to study the response of the endothelial cells of the alveolar barrier in vitro in a realistic exposure scenario avoiding direct exposure of endothelial cells to high amounts of particulates.
Climate change and habitat fragmentation modify the natural habitat of many wetland biota and lead to new compositions of biodiversity in these ecosystems. While the direct effects of climate are often well known, indirect effects due to biotic interactions remain poorly understood. The water meadow grasshopper, Chorthippus montanus, is a univoltine habitat specialist, which is adapted to permanently moist habitats. Land use change and drainage led to highly fragmented populations of this generally flightless species. In large parts of the Palaearctic Ch. montanus occurs sympatrically with its widespread congener, the meadow grasshopper Chorthippus parallelus. Due to their close relationship and their similar songs, hybridization is likely to occur in syntopic populations. Such a species pair of a habitat specialist and a habitat generalist represents an ideal model system to examine the role of ongoing climate change and an accumulation of extreme climatic events on the life history strategies, population dynamics and inter-specific interactions. In Chapter I a laboratory experiment was conducted to identify the impact of environmental factors on intra-specific life-history traits of Ch. montanus. Like other Orthoptera species, Ch. montanus follows a converse temperature size rule. In line with the dimorphic niche hypothesis, which states that sexual size dimorphism evolved in response to the different sexual reproductive roles, both sexes showed different responses to increasing density at lower temperatures. Males attained smaller body sizes at high densities, whereas females had a prolonged development time. This is the first evidence for a sex-specific phenotypic plasticity in Ch. montanus. Females benefit from the prolonged development as their reproductive success depends on the size and number of egg clutches they may produce. By contrast, the reproductive success of males depends on the chance to fertilize virgin females, which increases with faster development. This may become a disadvantage for Ch. montanus as an intraspecific phenology shift may increase hybridization risk with the sibling species. Despite the widespread assumption that hybridization between two sympatric species is rare due to complete reproductive barriers, the genetic analyses of 16 populations (Chapter II) provided evidence for wide prevalence of hybridization between both species in the wild. As no complete admixture was found in the examined population, it is assumed that hybridization only occurs in ecotones between wetlands and drier parts. Reproductive barriers (habitat isolation, behavior, phenology) seem to prevent the genetic swamping of Ch. montanus populations. Although a behavioral experiment showed that mate choice presents an important reproductive barrier between both species, the experiment also revealed that reproductive barriers could be altered by environmental change (e.g. increasing heterospecific frequency). Chapter III analyzes the impact of extreme climatic events on population dynamics and interspecific hybridization. A mark-recapture analysis combined with weather records over five years provides evidence that the embryonic development in Ch. montanus is vulnerable to extreme climatic events. Strong population declines in Ch. montanus lead to a disequilibrium between Ch. montanus and Ch. parallelus populations and increases the risk of hybridization. The highest hybridization risk was found in the first weeks of a season, when both species had an overlapping phenology. Furthermore, hybrids were generally localized at the edge of the Ch. montanus distribution with higher heterospecific encounter probabilities. The hybridization rate reached up to 19.6%. The genetic analyses in Chapter II and III show that hybridization differentially affects specialists and generalists. While generalists may benefit from hybridization by an increasing genetic diversity, such a positive correlation was not found for Ch. montanus. The results underline the importance of reproductive barriers for the co-existence of these sympatric species. However, climate change and other anthropogenic disturbances alter reproductive barriers and promote hybridization, which may threaten small populations by genetic displacement. As anthropogenic hybridization is recognized as a major threat to biodiversity, it should be considered in environmental law and policy. In Chapter IV the role of hybrids and hybridization in three levels of law and the historical backgrounds of hybrids becoming a part of legal instruments is analyzed. Due to legal uncertainties and the complexity of this topic a legal assessment of hybrids is challenging and argues for species-specific approaches. Nonetheless, existing legal norms provide a suitable basis, but need to be specified. Finally, this chapter discusses different opportunities for the management of hybrids and hybridization in a conservation perspective and their necessity.
Geographic ranges of species and their determinants are of great interest in the field of biogeography and are often studied in terms of the species" ecological niches. In this context, the range of a species is defined by the accessibility of an area, abiotic factors and biotic interactions, which affect a species" distributions with different intensities across spatial scales. Parapatry describes a distributional pattern in which the ranges of two species meet along sharp range limits with narrow contact zones. Such parapatric range limits are determined by changing abiotic conditions along sharp environmental gradients or can result from interspecific resource competition. However, it has been shown that often the interplay of abiotic conditions and species interactions determine parapatry. The geographic ranges of the land salamanders, Salamandra salamandra and S. atra, narrowly overlap in the European Alps with only few syntopic localities and to date, the cause of parapatry is unknown. The goal of this thesis was thus to identify the importance of abiotic and biotic factors for their parapatric range limits at different spatial scales. On a broad spatial scale, the role of climate for the parapatric range limits of the species was investigated within three contact zones in Switzerland. Climatic conditions at species" records were analysed and species distribution modelling techniques were used to explore the species" climatic niches and to quantify the interspecific niche overlap. Furthermore, it was tested whether the parapatric range limit coincides with a strong climatic gradient. The results revealed distinct niches for the species as well as the presence of strong climatic gradients which could explain the parapatric range limits of the species. Yet, there was a moderate interspecific niche overlap in all contact zones indicating that the species may co-occur and interact with each other in areas where they both find adequate conditions. Comparison among contact zones revealed geographic variation in the species" niches as well as in the climatic conditions at their records suggesting that the species can occur in a much wider range of conditions than they actually do. These findings imply that climate represents a main factor for the species" parapatric range limits. Yet, interspecific niche overlap and the geographic variation provide indirect evidence that interspecific interaction may also affect their spatial distribution. To test whether competition restricts the species" ranges on the habitat scale and to understand local syntopic co-occurrence of the salamanders within their contact zones, site-occupancy modelling was used. This approach allowed to find the habitat predictors that best explain the species" local distribution. While the slope of the site positively affected the occupancy probability of S. salamandra, no tested predictor explained that of S. atra. Also, there was no effect of the occurrence of one species on the occupancy probability of the other providing no evidence for competition. Should competition occur, it does not lead to spatial segregation of the species on this scale. Because biotic interactions most significantly affect the ranges of species on small spatial scales, the microhabitat conditions at locations of the species within syntopic contact zones were compared and a null model analysis was applied to determine their niche overlap. Resource selection probability function models were used to assess those attributes that affect the species" habitat selections. The results revealed species-specific microhabitat preferences related to leaf litter cover, tree number and that the species were active at different temperatures as well as times of the day. The high degree of diurnal activity of S. atra may be due to its preference of forest floor microhabitats that long remain suitable during daytime. Besides, there was a great niche overlap for shelters indicating that the species may compete for this resource. Differential habitat selection and the use of the available shelters at different times of the day may minimize species interactions and allow their local co-occurrence within contact zones. To identify whether the potential infection with the pathogenic chytrid fungus could serve as an alternative biotic explanation for the range margins of S. atra, several populations throughout its range were screened for infection. Since the occurrence of this pathogen was detected mostly at lower altitudes of the Alps, it may confine the range of S. atra to higher elevations. Because chytrid was not detected in any of the samples, the pathogen unlikely plays a role in determining its range limits. Overall, these findings underline the complexity of mechanisms that determine the range margins of parapatric species and provide an important basis for subsequent studies regarding the determinants of the parapatric distribution of the two salamander species.