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.

O horário de atividade é um importante aspecto da vida de um animal, refletindo sua fisiologia, interações com o ambiente e com outros animais. Dentre os sinais exógenos que ajudam a determinar o início e fim de um dia para um animal estão a luz, temperatura, precipitação e disponibilidade de alimento. Em nosso estudo buscamos determinar o horário de atividade de duas espécies de roedores cricetídeos, Akodon montensis e Delomys dorsalis, verificando como este é influenciado por variáveis ambientais (temperatura e umidade) e pela disponibilidade da semente da araucária (pinhão). Para isso, utilizamos 21 armadilhas acopladas com relógios para determinar o horário de captura dos animais em oito grades de floresta com araucária nativa. As armadilhas permaneceram funcionando por cinco noites consecutivas em excursões bimestrais no período de março de 2010 a fevereiro de 2011. Para determinar a influência do pinhão no horário de atividade comparamos a época dentro e fora de sua produção. Também removemos o pinhão, experimentalmente, de quatro das grades para determinar como a ausência desse recurso sazonal afeta o comportamento dos animais (grades remoção). Encontramos um padrão de atividade bimodal para A. montensis e unimodal para D. dorsalis, sendo as duas espécies predominantemente noturnas e apresentando padrões de atividade noturnos distintos. Em A. montensis, verificamos diferenciações no horário de atividade entre o inverno e o verão, com a atividade nos meses mais frios apresentando um pico no início na noite e, nos meses mais quentes, apresentando um pico no fim da noite. A. montensis também apresentou horários médios de atividade diferentes entre as grades controle (21h35min) e remoção (23h51min). Em D. dorsalis verificamos uma relação positiva entre o horário de atividade e a temperatura ambiente. O efeito da remoção do pinhão sobre a atividade de A. montensis reforça a importância dessa espécie para a fauna de pequenos mamíferos. / An important aspect of an animal’s life is its activity pattern, which reflects its physiology, interactions with the environment and with other animals. Among the exogenous signs that help determine the beginning and end of a day to an animal are light, temperature, precipitation and food availability. In our study we aimed to determine the daily activity patterns of two species of cricetid rodents, Akodon montensis and Delomys dorsalis, analyzing how these are influenced by the availability of an important seasonal resource, the seed of the araucaria tree (Araucaria angustifolia), and by environmental variables (temperature and humidity). To do so, we used 21 live-traps equipped with clocks that determined the time an individual was captured in eight grids placed in native araucaria forest. The traps were set on the grids for five consecutive nights during six excursions, which took place between March 2010 and February 2011. To determine the influence of the araucaria seed on the daily activity of the two species, we compared the times of capture registered during the production season of the seed and outside of the production period. We also experimentally removed the araucaria seeds from four of the grids, to determine how the absence of this seasonal resource affects the behavior of the studied species (removal grids). We found a bimodal daily activity pattern for A. montensis and a unimodal pattern for D. dorsalis, and both species were considered predominantly nocturnal and presented distinct nocturnal activity patterns. A. montensis showed a differentiation in its daily activity pattern between winter and summer, presenting a peak of activity in the beginning of the night in the colder months and at the end of night during the warmer months. A. montensis also presented different average activity hours between the control grids (9:35 p.m.) and removal grids (11:51 p.m.). We found a positive relationship between the daily activity time of D. dorsalis and ambient temperature. The effect of the removal of the araucaria seed on the daily activity of A. montensis reinforces the importance of this tree species to the small mammal fauna.

O horário de atividade é um importante aspecto da vida de um animal, refletindo sua fisiologia, interações com o ambiente e com outros animais. Dentre os sinais exógenos que ajudam a determinar o início e fim de um dia para um animal estão a luz, temperatura, precipitação e disponibilidade de alimento. Em nosso estudo buscamos determinar o horário de atividade de duas espécies de roedores cricetídeos, Akodon montensis e Delomys dorsalis, verificando como este é influenciado por variáveis ambientais (temperatura e umidade) e pela disponibilidade da semente da araucária (pinhão). Para isso, utilizamos 21 armadilhas acopladas com relógios para determinar o horário de captura dos animais em oito grades de floresta com araucária nativa. As armadilhas permaneceram funcionando por cinco noites consecutivas em excursões bimestrais no período de março de 2010 a fevereiro de 2011. Para determinar a influência do pinhão no horário de atividade comparamos a época dentro e fora de sua produção. Também removemos o pinhão, experimentalmente, de quatro das grades para determinar como a ausência desse recurso sazonal afeta o comportamento dos animais (grades remoção). Encontramos um padrão de atividade bimodal para A. montensis e unimodal para D. dorsalis, sendo as duas espécies predominantemente noturnas e apresentando padrões de atividade noturnos distintos. Em A. montensis, verificamos diferenciações no horário de atividade entre o inverno e o verão, com a atividade nos meses mais frios apresentando um pico no início na noite e, nos meses mais quentes, apresentando um pico no fim da noite. A. montensis também apresentou horários médios de atividade diferentes entre as grades controle (21h35min) e remoção (23h51min). Em D. dorsalis verificamos uma relação positiva entre o horário de atividade e a temperatura ambiente. O efeito da remoção do pinhão sobre a atividade de A. montensis reforça a importância dessa espécie para a fauna de pequenos mamíferos. / An important aspect of an animal’s life is its activity pattern, which reflects its physiology, interactions with the environment and with other animals. Among the exogenous signs that help determine the beginning and end of a day to an animal are light, temperature, precipitation and food availability. In our study we aimed to determine the daily activity patterns of two species of cricetid rodents, Akodon montensis and Delomys dorsalis, analyzing how these are influenced by the availability of an important seasonal resource, the seed of the araucaria tree (Araucaria angustifolia), and by environmental variables (temperature and humidity). To do so, we used 21 live-traps equipped with clocks that determined the time an individual was captured in eight grids placed in native araucaria forest. The traps were set on the grids for five consecutive nights during six excursions, which took place between March 2010 and February 2011. To determine the influence of the araucaria seed on the daily activity of the two species, we compared the times of capture registered during the production season of the seed and outside of the production period. We also experimentally removed the araucaria seeds from four of the grids, to determine how the absence of this seasonal resource affects the behavior of the studied species (removal grids). We found a bimodal daily activity pattern for A. montensis and a unimodal pattern for D. dorsalis, and both species were considered predominantly nocturnal and presented distinct nocturnal activity patterns. A. montensis showed a differentiation in its daily activity pattern between winter and summer, presenting a peak of activity in the beginning of the night in the colder months and at the end of night during the warmer months. A. montensis also presented different average activity hours between the control grids (9:35 p.m.) and removal grids (11:51 p.m.). We found a positive relationship between the daily activity time of D. dorsalis and ambient temperature. The effect of the removal of the araucaria seed on the daily activity of A. montensis reinforces the importance of this tree species to the small mammal fauna.

O horário de atividade é um importante aspecto da vida de um animal, refletindo sua fisiologia, interações com o ambiente e com outros animais. Dentre os sinais exógenos que ajudam a determinar o início e fim de um dia para um animal estão a luz, temperatura, precipitação e disponibilidade de alimento. Em nosso estudo buscamos determinar o horário de atividade de duas espécies de roedores cricetídeos, Akodon montensis e Delomys dorsalis, verificando como este é influenciado por variáveis ambientais (temperatura e umidade) e pela disponibilidade da semente da araucária (pinhão). Para isso, utilizamos 21 armadilhas acopladas com relógios para determinar o horário de captura dos animais em oito grades de floresta com araucária nativa. As armadilhas permaneceram funcionando por cinco noites consecutivas em excursões bimestrais no período de março de 2010 a fevereiro de 2011. Para determinar a influência do pinhão no horário de atividade comparamos a época dentro e fora de sua produção. Também removemos o pinhão, experimentalmente, de quatro das grades para determinar como a ausência desse recurso sazonal afeta o comportamento dos animais (grades remoção). Encontramos um padrão de atividade bimodal para A. montensis e unimodal para D. dorsalis, sendo as duas espécies predominantemente noturnas e apresentando padrões de atividade noturnos distintos. Em A. montensis, verificamos diferenciações no horário de atividade entre o inverno e o verão, com a atividade nos meses mais frios apresentando um pico no início na noite e, nos meses mais quentes, apresentando um pico no fim da noite. A. montensis também apresentou horários médios de atividade diferentes entre as grades controle (21h35min) e remoção (23h51min). Em D. dorsalis verificamos uma relação positiva entre o horário de atividade e a temperatura ambiente. O efeito da remoção do pinhão sobre a atividade de A. montensis reforça a importância dessa espécie para a fauna de pequenos mamíferos. / An important aspect of an animal’s life is its activity pattern, which reflects its physiology, interactions with the environment and with other animals. Among the exogenous signs that help determine the beginning and end of a day to an animal are light, temperature, precipitation and food availability. In our study we aimed to determine the daily activity patterns of two species of cricetid rodents, Akodon montensis and Delomys dorsalis, analyzing how these are influenced by the availability of an important seasonal resource, the seed of the araucaria tree (Araucaria angustifolia), and by environmental variables (temperature and humidity). To do so, we used 21 live-traps equipped with clocks that determined the time an individual was captured in eight grids placed in native araucaria forest. The traps were set on the grids for five consecutive nights during six excursions, which took place between March 2010 and February 2011. To determine the influence of the araucaria seed on the daily activity of the two species, we compared the times of capture registered during the production season of the seed and outside of the production period. We also experimentally removed the araucaria seeds from four of the grids, to determine how the absence of this seasonal resource affects the behavior of the studied species (removal grids). We found a bimodal daily activity pattern for A. montensis and a unimodal pattern for D. dorsalis, and both species were considered predominantly nocturnal and presented distinct nocturnal activity patterns. A. montensis showed a differentiation in its daily activity pattern between winter and summer, presenting a peak of activity in the beginning of the night in the colder months and at the end of night during the warmer months. A. montensis also presented different average activity hours between the control grids (9:35 p.m.) and removal grids (11:51 p.m.). We found a positive relationship between the daily activity time of D. dorsalis and ambient temperature. The effect of the removal of the araucaria seed on the daily activity of A. montensis reinforces the importance of this tree species to the small mammal fauna.