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Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative movement disorder caused by expansion of CAG repeats in the ATXN2 gene beyond 33 units, while healthy individuals carry 22-23 repeats. First symptoms of SCA2 include uncoordinated movement, ataxic gait and slowing of the saccadic eye movements in line with the early pronounced atrophy of cerebellum, spinal cord and brainstem. Cerebellar Purkinje cells and spinal cord motor neurons are the most affected cells from ATXN2 expansions. Later on, patients manifest distal amyotrophy, problems in breathing and swallowing, depression and cognitive decline caused by widespread degeneration throughout the brain. The striking loss of mass in the brain, due to severe myelin fat atrophy, is accompanied by a similar reduction in the peripheral fat stores. After the devastating progression of disease, the severity and duration of which depends on the CAG repeat size, genetic background and environmental factors, patients succumb to SCA2 mostly because of respiratory failure at the terminal stage. Larger repeat sizes lead to an earlier manifestation of the disease and a more rapid progression. Aside from SCA2, intermediate-length and short pathogenic CAG expansions in ATXN2 between 26-39 repeats significantly increase the risk of developing other neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), fronto-temporal lobar dementia (FTLD) or Parkinson plus tauopathies like progressive supranuclear palsy (PSP) in various cohorts across the world.
Ataxin-2 (ATXN2) is a ubiquitously expressed cytosolic protein most famous for its involvement in neurodegenerative disease caused by the expanded poly-glutamine (polyQ) domain corresponding to a genomic (CAG)n tract. This N-terminal polyQ domain has no known function, other than increasing the aggregation propensity of mutant ATXN2 and facilitating interaction with other polyQ containing proteins, leading to their sequestration. The progressive accumulation of ATXN2 into cytosolic foci, and also that of its interaction partners over time, underlies the molecular pathomechanism. Next to polyQ domain, ATXN2 also contains a Like-Sm domain (Lsm), an Lsm-associated domain (LsmAD), multiple proline-rich domains (PRD) and a Poly(A)-Binding-Protein (PABP)-interacting motif (PAM2).
Through its Lsm/LsmAD domains, ATXN2 directly binds to a large number of transcripts, regulating their quality and translation rate. In a similar fashion, through its direct interaction with PABP via PAM2 motif, ATXN2 indirectly modifies the fate of even larger number of transcripts and global translation. Several PRDs scattered across the protein help ATXN2 associate with growth factor receptors and other endocytosis factors, modulating nutrient uptake and downstream signaling.
ATXN2 is a stress response factor. Therefore, its involvement in nutrient uptake plays a crucial part in cell’s capability to overcome non-permissive conditions. Upon nutrient deprivation, oxidative stress, proteotoxicity, heat stress or Ca2+ imbalance, ATXN2 relocalizes into cytosolic ribonucleoprotein particles known as stress granules (SGs), together with PABP, several eukaryotic translation initiation factors, many other RNA-binding proteins (RBP) with their target transcripts and the small ribosomal subunit. Collectively, they modulate the stability of the trapped transcripts, favoring the maturation and translation of IRES-dependent stress response proteins instead, according to the specific need. Many RBPs interact either directly or in an RNA-dependent manner in the SGs, and due to the large number of ALS-causing mutations identified in them (such as TDP-43, FUS, TIA-1, hnRNPA2/B1), SGs became a hot topic in neuropathology. Acute SGs serve to halt translation and growth, and to spend energy only for survival until stress disappears. However, chronic SG assembly eventually activates apoptotis leading to cell death. While the polyQ expansions in ATXN2 enhance SG stability, reduce their dissociation rate after stress, and lead to aberrant post-translational modifications of other SG components like TDP-43, complete loss of ATXN2 delays SG formation and results in easily dissolvable foci.
Most of the stressors that induce SG formation eventually converge on energetic deficit. Therefore, it is logical that the ultimate task of SGs is to stop further growth when it cannot be afforded. In yeast, the molecular mechanism underlying this growth arrest was explained as sequestration of the master growth regulator complex, Target-of-Rapamycin Complex 1 (TORC1), into SGs in an ATXN2-dependent manner. The repressor effect of ATXN2 on mammalian TORC1 (mTORC1) and global protein translation had already been documented in earlier studies; complete loss of ATXN2 function in knock-out mouse (Atxn2-KO) resulted in mTORC1 hyperactivity and transcriptional upregulation of multiple ribosomal subunits indicating an increased need for these machines. ...
The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
Evidence is increasingly pointing towards a significant global decline in biodiversity. The drivers of this decline are numerous, including habitat change and overexploitation, rapid deforestation, pollution, exotic species and disease, and finally climate change as an emerging driver of biodiversity change (Nakamura, et al., 2013; Hancocks, 2001; Pereira, Navarro & Martins, 2012). Raising public awareness of the need to conserve biological diversity is essential to safeguard the richness of life forms all over the world (Lindemann-Matthies, 2002). In this regard, institutions such as science museums, zoos and aquariums have the potential to play an important role (Rennie & Stocklmayer, 2003). Especially, zoos can provide a productive learning environment (Miles & Tout, 1992), facilitating the promotion of public conservation awareness and the adoption of pro-environmental behaviours that would reduce negative human impacts on biodiversity (Barongi, et al., 2015).
Based on these concepts, my study contributes to the developing field of visitor studies. Taking as reference non-zoo visitors and zoo visitors, I have focused on reviewing some aspects of conservation education, such as people's awareness of conservation, people's interest in animals and people's feelings towards animals and attitudes towards zoos. The study identified differences between non-regular and regular zoo visitors in interests in animals, as well as visitor attitudes towards conservation issues and zoos. Therefore, the present study indicated that positive emotional reactions and, in particular, a perceived sense of connection to the animal were linked and depended on the frequency of zoo visits. It was as well remarkable, that conservation awareness was influenced by the interest in animals, the interest in visiting zoos, the attitudes towards these institutions, and the age and the country of origin. All these variables had a greater effect in the conservation consciousness of the participants. Additionally interestingly, the main reason for visiting zoos in every country was to learn something about animals. This highlights the educational role of zoos and broadly supports the idea that people want to visit zoos to learn something about animals, in turn facilitating pro-conservation learning and changes in attitude. They are uniquely positioned to interact with visitors, communities, and society and to contribute by providing an informative and entertaining environment. Visiting zoos could led to contribute to promoting animal connectedness and interest in species.
Background: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3–V4 region of the 16S rRNA gene using Illumina MiSeq.
Results: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones.
Conclusions: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Weiterentwicklung und Evaluation eines drei-dimensionalen Hautmodelles zur pharmakologischen Testung
(2008)
Orthopockenviren sind große DNA-Viren, die im Zytoplasma der Wirtszelle replizieren und für über 200 Proteine kodieren. Sie besitzen ein breites Wirtszellspektrum (host-range) und modulieren auf komplexe Art und Weise zelluläre Prozesse, um ihre Replikation zu gewährleisten. Zu diesem Genus der Familie der Pockenviren gehört auch das modifizierte Vacciniavirus Ankara (MVA). MVA ist ein hoch attenuiertes, replikationsdefizientes Impfvirus, dem im Vergleich zu ursprünglichen Vacciniavirus-Stämmen viele virale Genfunktionen fehlen. Zu diesen verlorengegangenen Genen zählen so genannte host-range-Gene, die für das breite Wirtszellspektrum des Vacciniavirus (VACV) verantwortlich sind, deren molekulare Funktion aber größtenteils unbekannt ist. Diese Arbeit befasste sich zum einen mit der Untersuchung der Rolle der host-range-Gene K1L und C7L in der MVA-Infektion. Zum anderen sollte geprüft werden, ob der im MVA-Genom unvollständige Leserahmen F11L durch Wiederherstellung seiner Funktionalität den Wirtstropismus von MVA erweitern kann. Das Fehlen von K1L und C7L in MVA ist mit dem Verlust der späten viralen Genexpression verbunden. Als mögliche Ursache hierfür wurde in dieser Arbeit die Phosphorylierung des eukaryotischen Translationsinitiationsfaktors 2alpha (eIF2alpha) entdeckt, welche zum Abbruch der Proteinsynthese in der infizierten Zelle führt. Unter den möglichen Kinasen wurde die Proteinkinase R (PKR) als das verantwortliche Schlüsselenzym identifiziert und somit gezeigt, dass das K1- und C7-Protein den anti-viralen PKR-eIF2alpha-Signalweg inhibieren. Es stellte sich heraus, dass die eIF2alpha-Phosphorylierung alleine jedoch nicht für das Fehlen der späten Genexpression verantwortlich ist. Neben dem inhibitorischen Einfluss auf den PKR-eIF2alpha-Signalweg zeigte sich, dass C7 die Aktivierung des NFKB-Signalwegs reduziert, welcher für eine anti-virale Antwort der Wirtszelle wichtig ist. Ein weiterer Ansatz zur Aufklärung der K1- und C7-Funktion bestand darin, zelluläre Interaktionspartner zu identifizieren. Hierbei konnte das heterogene nukleäre Ribonukleoprotein K (HNRPK) als möglicher Interaktionspartner von C7 entdeckt werden. Neben den bisher bekannten host-range-Genen gibt es vermutlich weitere Gene, die den Wirtsbereich des VACV definieren. Das im MVA-Genom defekte F11L-Gen war ein guter Kandidat für eine solche Genfunktion, da es bei der Virionenmorphogenese, Virusausbreitung und der Migration VACV-infizierter Zellen eine Rolle zu spielen scheint. Deshalb wurde ein rekombinantes MVA mit vollständiger F11L-Gensequenz konstruiert und das Wirtszellspektrum dieses Virus untersucht. Die Reparatur des F11L-Gens ermöglichte MVA die Induktion von Zellbewegung nach Infektion, jedoch blieben seine unvollständige Morphogenese und eingeschränkte Vermehrungsfähigkeit in Säugetierzellen unbeeinflusst. F11L hat daher zumindest keine selbstständige Funktion als VACV host-range-Gen. Die Ergebnisse dieser Arbeit sind ein Beitrag zum besseren Verständnis der komplexen Virus-Wirts-Interaktionen des VACV sowie des eingeschränkten Wirtstropismus des Impfvirus MVA.
In the insect brain, the mushroom body is a higher order brain area that is key to memory formation and sensory processing. Mushroom body (MB) extrinsic neurons leaving the output region of the MB, the lobes and the peduncle, are thought to be especially important in these processes. In the honeybee brain, a distinct class of MB extrinsic neurons, A3 neurons, are implicated in playing a role in learning. Their MB arborisations are either restricted to the lobes and the peduncle, here called A3 lobe connecting neurons, or they provide feedback information from the lobes to the input region of the MB, the calyces, here called A3 feedback neurons. In this study, we analyzed the morphology of individual A3 lobe connecting and feedback neurons using confocal imaging. A3 feedback neurons were previously assumed to innervate each lip compartment homogenously. We demonstrate here that A3 feedback neurons do not innervate whole subcompartments, but rather innervate zones of varying sizes in the MB lip, collar, and basal ring. We describe for the first time the anatomical details of A3 lobe connecting neurons and show that their connection pattern in the lobes resemble those of A3 feedback cells. Previous studies showed that A3 feedback neurons mostly connect zones of the vertical lobe that receive input from Kenyon cells of distinct calycal subcompartments with the corresponding subcompartments of the calyces. We can show that this also applies to the neck of the peduncle and the medial lobe, where both types of A3 neurons arborize only in corresponding zones in the calycal subcompartments. Some A3 lobe connecting neurons however connect multiple vertical lobe areas. Contrarily, in the medial lobe, the A3 neurons only innervate one division. We found evidence for both input and output areas in the vertical lobe. Thus, A3 neurons are more diverse than previously thought. The understanding of their detailed anatomy might enable us to derive circuit models for learning and memory and test physiological data.