Molecular adaptation and thermal plasticity in a cold-adapted Antarctic fish

Evolutionary adaptation and the connected acclimation capacity of species to changing environmental conditions represents one of the key factors in the composition and dynamics of any ecosystem. In marine environments temperature is one of the major factors defining the aquatic fauna. In respect to progressing climate change the question arises how individual species react to higher temperatures and how this affects the entire ecosystem. Species that are highly specialized on stable environmental conditions seem to be especially vulnerable. This thesis characterizes the Antarctic eelpout Pachycara brachycephalum (Pappenheim, 1912) in regards to its adaptation to low habitat temperatures as well as its capacity to acclimate to higher temperatures at the molecular level. A cDNA library was established and subjected to high-throughput sequencing to provide a basis for sequence analyses. To characterize differences between a cold-adapted and a eurythermal species, an already existing cDNA library of the closely related congener Zoarces viviparus (Linnaeus, 1758) was included in large-scale comparisons. The repertoire of functional genes reflected cold-adaptation features of cellular metabolism in P. brachycephalum, e.g. through a higher ratio of ubiquitin–related genes. These genes are of great importance in cold-adaptation and counter the cold denaturation of proteins. Furthermore, the amino acid sequences of orthologous proteins displayed differences between the eury- and the stenotherm. The observed position-specific interchanges in P. brachycephalum highly conform with the flexibility hypothesis. According to this hypothesis a protein may be destabilized in its three-dimensional structure by minimal changes within the amino acid sequence. This destabilization sustains reaction kinetics in the cold. In addition, differences were noted in the encoding of amino acids at DNA level. Within homologous proteins of P. brachycephalum amino acids are encoded with a preference for AT-richer triplets on the third codon position. This trend promotes less stable transitional states at this level, too, as the base pairing of AT is less stable than that of GC. This may facilitate transcription and translation in the cold and thus constitute an adaptation to the habitat conditions of the Antarctic eelpout. For studying the plasticity of this species in more detail, various acclimation experiments were conducted. At first, the chronology of warm acclimation was established by means of enzyme measurements as well as through expression analyses of various candidate genes. Through this the mechanisms that account for metabolic changes in the warmth could be identified at the protein as well as the transcriptomic level. The observed shift from lipid-based to carbohydrate-based metabolism provided evidence that the Antarctic eelpout prepares for hypoxemic conditions. The shift to carbohydrate-fuels is advantageous under anaerobic conditions, which may be elicited by warmer temperatures, due to elevated oxygen demand insufficiently met by the limited capacity of the circulation/respiration system. Various transcriptomic factors of the PPAR family were identified as important mediators of the metabolic shift. These nuclear–located receptors „measure“ the energy status within a cell and regulate the expression of various genes involved in lipid- and carbohydrate metabolism. The expression profiles of all 26 genes examined, provided an acclimation time course that can be divided into 3 phases: acute, mid-term and long-term. Another experiment was dedicated to the compilation of expression profiles of long-term acclimated specimens held at 6 different temperatures ranging from -1 to 9°C over a time period of 2 months. A total of 664 temperature-sensitive transcript sequences could be identified by using a custom microarray design based on the sequence information of the cDNA library. The growth optimum of the Antarctic eelpout was characterized by the smallest transcriptomic changes, i.e. the least regulatory effort. The differently expressed functions revealed specific patterns for the temperature ranges „cold“, „intermediate“ and „warm“, with specific features in energy metabolism. Again, the aforementioned metabolic shift could be observed, with additional indicators for a potential use of amino acids as an energy reserve in the cold. Further differences between acclimation temperatures in processes like transcription, translation, protein degradation or the organization of the cytoskeleton are discussed with respect to their implications for metabolic energy consumption. Furthermore, regulatory mechanisms increasing the proliferation of blood vessels that obviously modify the cardio-vascular system to counter an oxygen limitation have been identified in the warmth. Beyond an acclimation temperature of 6°C an extreme energy deficit is indicated by a severe weight loss of the specimen. This was accompanied by a classic cellular stress response, signifying an essential threshold temperature of the thermal window of P. brachycephalum. Overall, by combining various experimental approaches covering the entire thermal tolerance window and beyond, the correlation between the molecular and the whole animal level was established. The Antarctic eelpout displays capacities to acclimate to temperatures above the range of habitat temperatures. This thesis highlights the complexity of molecular adaptation at DNA, RNA and protein levels as well as various functional shifts in metabolism enabling life at various acclimation temperatures. The present findings help to substantiate the framework of the oxygen and capacity limited thermal tolerance at the molecular level.