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No description available.

The study analyzes genealogy and coevolutionary relationships between the bloodsucking louse Polyplax serrata (Anoplura) and its host of the genus Apodemus (Rodentia). It uses the tools of molecular biology and phylogenetics for interpretation of the parasite distribution in respect to the georaphy and host specificity

Parasites represent one of the most common ecological strategies and host-parasite coevolution belongs among the major processes governing evolution of biodiversity on the global scale. Genetic structure and diversity of populations of parasites and their hosts, and their genetic connectivity are the key elements in long-term population survival and evolution. Host switches often disturb the parallel evolution of interacting taxa, even in highly host-specific parasites. Evaluation of importance of the degree of intimacy between parasites and hosts is not a trivial task, because evolutionary patterns observed today were formed by an interplay of many (sometimes previously unforeseen) historical and ecological factors. To reveal the mechanisms of coevolution between parasites and their hosts, inter- and intra-specific genealogical structures in three model systems were analyzed: namely, the sucking lice Polyplax serrata and Apodemus hosts, chewing lice of the genus Menacanthus and endoparasitic coccidean genus Eimeria from Apodemus mice.

This thesis deals with the chemical communication of wood mouse of genus Apodemus with a focus on the special sebaceous gland on the ventral side of the tail - the caudal organ. Role of the caudal organ in chemical communication is unclear. To illustrate the function of the sebaceous glands, I decided to use metods of 454 pyrosequencing, proteomic analysis and qPCR. I created a database of transcriptionally active genes of caudal organ using 454 pyrosequencing. In proteomic analysis, I focused on proteins with a molecular weight 14-18kDa to verify the presence of the lipocalins, proteins involved in chemical communication. I compared the relative mRNA abundance of selected genes between individuals of the opposite sex using qPCR. In my research, I found that the caudal organ is active organ and caudal organ could participate in chemical communication. I detected a different relative mRNA abundance of genes in males and females at the mRNA level, which could play a role in differences in development of caudal organ between individuals of the opposite sex. Finally, I suggested possible ways of using the caudal organ in the chemical communication in wood mouse genus Apodemus from the obtained results. Key words: caudal organ, Apodemus, chemical communication, FABP5, Scd1

MHC genes encode proteins that are responsible for the recognition of foreign antigens and the triggering of a subsequent, adequate immune response of the organism. Thus they hold a key position in the immune system of vertebrates. It is believed that the extraordinary genetic diversity of MHC genes is shaped by adaptive selectional processes in response to the reoccurring adaptations of parasites and pathogens. A large number of MHC studies were performed in a wide range of wildlife species aiming to understand the role of immune gene diversity in parasite resistance under natural selection conditions. Methodically, most of this work with very few exceptions has focussed only upon the structural, i.e. sequence diversity of regions responsible for antigen binding and presentation. Most of these studies found evidence that MHC gene variation did indeed underlie adaptive processes and that an individual’s allelic diversity explains parasite and pathogen resistance to a large extent. Nevertheless, our understanding of the effective mechanisms is incomplete. A neglected, but potentially highly relevant component concerns the transcriptional differences of MHC alleles. Indeed, differences in the expression levels MHC alleles and their potential functional importance have remained unstudied. The idea that also transcriptional differences might play an important role relies on the fact that lower MHC gene expression is tantamount with reduced induction of CD4+ T helper cells and thus with a reduced immune response. Hence, I studied the expression of MHC genes and of immune regulative cytokines as additional factors to reveal the functional importance of MHC diversity in two free-ranging rodent species (Delomys sublineatus, Apodemus flavicollis) in association with their gastrointestinal helminths under natural selection conditions. I established the method of relative quantification of mRNA on liver and spleen samples of both species in our laboratory. As there was no available information on nucleic sequences of potential reference genes in both species, PCR primer systems that were established in laboratory mice have to be tested and adapted for both non-model organisms. In the due course, sets of stable reference genes for both species were found and thus the preconditions for reliable measurements of mRNA levels established. For D. sublineatus it could be demonstrated that helminth infection elicits aspects of a typical Th2 immune response. Whereas mRNA levels of the cytokine interleukin Il4 increased with infection intensity by strongyle nematodes neither MHC nor cytokine expression played a significant role in D. sublineatus. For A. flavicollis I found a negative association between the parasitic nematode Heligmosomoides polygyrus and hepatic MHC mRNA levels. As a lower MHC expression entails a lower immune response, this could be evidence for an immune evasive strategy of the nematode, as it has been suggested for many micro-parasites. This implies that H. polygyrus is capable to interfere actively with the MHC transcription. Indeed, this parasite species has long been suspected to be immunosuppressive, e.g. by induction of regulatory T-helper cells that respond with a higher interleukin Il10 and tumor necrosis factor Tgfb production. Both cytokines in turn cause an abated MHC expression. By disabling recognition by the MHC molecule H. polygyrus might be able to prevent an activation of the immune system. Indeed, I found a strong tendency in animals carrying the allele Apfl-DRB*23 to have an increased infection intensity with H. polygyrus. Furthermore, I found positive and negative associations between specific MHC alleles and other helminth species, as well as typical signs of positive selection acting on the nucleic sequences of the MHC. The latter was evident by an elevated rate of non-synonymous to synonymous substitutions in the MHC sequences of exon 2 encoding the functionally important antigen binding sites whereas the first and third exons of the MHC DRB gene were highly conserved. In conclusion, the studies in this thesis demonstrate that valid procedures to quantify expression of immune relevant genes are also feasible in non-model wildlife organisms. In addition to structural MHC diversity, also MHC gene expression should be considered to obtain a more complete picture on host-pathogen coevolutionary selection processes. This is especially true if parasites are able to interfere with systemic MHC expression. In this case advantageous or disadvantageous effects of allelic binding motifs are abated. The studies could not define the role of MHC gene expression in antagonistic coevolution as such but the results suggest that it depends strongly on the specific parasite species that is involved. / Die Hauptaufgabe von MHC-kodierten Proteinen ist die Erkennung von körperfremden Molekülen sowie das Einleiten einer adäquaten Immunantwort, womit sie eine Schlüsselrolle im Immunsystem der Wirbeltiere einnehmen. Man nimmt an, dass ihre außergewöhnliche Vielfalt eine Antwort auf die sich ständig anpassenden Parasiten und Krankheitserreger ist, durch adaptive Selektion erhalten wird und dass die individuelle Allelausstattung einen Großteil der Parasitenbelastung erklärt, wofür bereits zahlreiche MHC-Studien Hinweise gefunden haben. Trotzdem ist unser Verständnis über die wirkenden Mechanismen teilweise noch lückenhaft. Ein stark vernachlässigter Aspekt hierbei sind z.B. eventuelle Unterschiede in der Genexpression der MHC-Allele und eine geringere Expression wäre gleichbedeutend mit einer geringeren Aktivierung des Immunsystems. Ich habe hierzu zwei frei lebende Kleinsäugerarten (Delomys sublineatus, Apodemus flavicollis) unter natürlichen Selektionsbedingungen untersucht. Dabei habe ich neben der genotypischen Diversität von MHC-Genen auch deren Expression, sowie die Genexpression immunregulativer Zytokine mit in Betracht gezogen und in Relation zur individuellen Belastung mit gastrointestinalen Helminthen gesetzt. Anhand von Leber und Milzproben beider Arten habe ich die Methode der ‚real-time PCR‘ zur relativen Quantifizierung von mRNA im Labor etabliert. Bereits für die Labormaus etablierte PCR-Primersysteme wurden an beiden Arten getestet und so konnten stabile Referenzgene gefunden werden, die Grundvoraussetzung für zuverlässige Genexpressionsmessungen. Für D. sublineatus konnte gezeigt werden, dass Helminthenbefall eine typische Th2 Immunantwort induziert, und dass der Zytokin Il4 Gehalt mit Befallsintensität strongyler Nematoden zunimmt. Es wurde für D. sublineatus kein signifikanter Zusammenhang zwischen MHC Expression oder anderen Zytokinen mit Helminthenbefall gefunden. In A. flavicollis wurde ein negativer Zusammenhang zwischen haptischer MHC-Expression und dem parasitären Nematoden Heligmosomoides polygyrus festgestellt, was auf eine Immunvermeidungsstrategie des Nematoden hindeutet. Ich fand typische positive und negative Assoziationen zwischen MHC-Allelen und anderen Helminthenarten, sowie Zeichen eines positiven Selektionsdruckes auf den MHC-Sequenzen, was sich durch eine erhöhte Rate aminosäureverändernder Mutationen zeigte. Diese nicht-synonymen Veränderungen waren auf Positionen innerhalb des zweiten Exons des DRB-Genes beschränkt, wohingegen die untersuchten Bereiche des ersten und dritten Exons stark konserviert vorlagen. Diese variablen Positionen kodieren Schlüsselstellen im Bereich der Antigenbindungsstelle im MHC Molekül. Zusammenfassend zeigt diese Arbeit, dass Genexpressionsstudien auch an Wildtieren durchgeführt und verlässliche Daten erzeugt werden können. Zusätzlich zur strukturellen Vielfalt sollten zukünftig auch mögliche Genexpressionsunterschiede bei MHC-Studien berücksichtigt werden, um ein kompletteres Bild der koevolutiven Wirt-Parasiten-Beziehungen zeichnen zu können. Dies ist vor allem dann von evolutiver Bedeutung, wenn die Parasiten in der Lage sind die MHC Expression aktiv zu beeinflussen. Die Studien konnten nicht die exakte Bedeutung von MHC-Genexpression in der antagonistischen Koevolution definieren, aber sie konnten zeigen dass diese Bedeutung stark von den jeweils beteiligten Partnern abzuhängen vermag.

A large proportion of the earth's surface is dedicated to food production, and agriculture is widely acknowledged to influence local biodiversity via habitat loss and degradation. Landscape genetics is an emerging field which can provide detailed understanding of how wildlife populations are influenced by landscape configuration and composition but the approach is yet to be fully integrated with agroecology. When addressing landscape genetics questions, small mammals may provide insight; they may act as model organisms, they are abundant, they are relatively easy to sample and they may have important ecological roles within arable ecosystems. This thesis merged the study of arable landscapes, landscape genetics and small mammals, to develop what is known about the landscape genetics of wild species in this dynamic habitat type. To decide upon a study organism, small mammals were surveyed at an example arable field site. Wood mice (Apodemus sylvaticus) were found to be the most abundant species and a microsatellite marker multiplex was developed for genotyping individuals. Two aspects of their landscape genetics in arable habitat were investigated. First, the possibility of temporal patterns in fine scale genetic structure of arable populations was explored, since this had not been investigated previously. Next, inter-population genetic differentiation was examined to determine whether arable habitat acted as a barrier to gene flow for this species. At the fine scale, three genetically distinct clusters of wood mice were identified and temporal variation in the spatial pattern was confirmed. There was no evidence that arable habitat acted as a barrier to gene flow for this species in comparison to populations in urban habitat, which showed significant differentiation. It is hoped that the landscape genetic insights provided by this thesis will encourage greater momentum for conducting landscape genetics studies in agricultural habitat.

Proteins from the lipocalin family play significant roles during the transport of chemical signals. These globular proteins (molecular mass: 17,7 - 21,7 kDa) are able to bind a wide spectrum of volatile ligands. Since they decay continuously, the scent impact is prolonged. Major urinary proteins (MUPs) are lipocalins, typically found in the urine and used during olfactory communication in the house mouse. But the urine is not the only source of chemical signals. Many species of mammals perform selfgrooming - a technique in which saliva is transported into the fur. The goal of the thesis is to identify main proteins that are involved in chemical communication by selected species of the genus Apodemus, and to discover the source of production (i.e. saliva or urine). The Experion capillary electrophoresis system was used to confirm the absence of lipocalins in the urine in two Apodemus species (A. uralensis, A. sylvaticus). Furthermore, a lot of lipocalins is present in the saliva of above-mentioned species. The quantity and types of salivary lipocalins was identified by 2D electrophoresis along with mass spectrometry analyses (MALDI MS/MS) in three species of the genus Apodemus (A. uralensis, A. sylvaticus and A. flavicollis). The conclusion of this inter-specific comparison suggests, that sociality...

Wood mice of the genus Apodemus rank among the most common extant mammals throughout Europe. A history of its species and their distributional ranges is to be looked upon as a serious topic relevant also for general concepts on current setting of European biotic diversity. Nevertheless, despite several phylogeographic studies on that subject and a rich fossil record of the genus, many details of that issue remains only very poorly comprehended. This state is essentially caused by enormous complications with phenotype discrimination of individual species, their extensive variation and broad between-species overlaps in all phenotype traits. The present project was intended to establish the techniques enabling reliable species identification applicable to fragmentary fossil materials and to use them for retrieving information on range dynamics and phylogenetic history of individual species during the Quaternary past. It begun with detailed morphometric analyses of extant populations, using the characters available in fossils, and with establishing a concept of alternative parataxonomic classification covering the complete phenotype span of the genus. The respective techniques were applied onto an extensive set of well-dated records from the Late Pleistocene-Holocene sedimentary series from diverse...

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