Blaues Blut unterm Eis : Funktion und Evolution von Cephalopod Haemocyanin entlang latitudinaler Gradienten.

The Southern Ocean hosts a rich and diverse fauna that required specialist adaptations to colonize and persist at temperatures close to freezing. While much has been revealed about key adaptations in Antarctic fishes little is known about evolutionary strategies of other Antarctic ectotherms, particularly the abundant benthic incirrate octopods. Their oxygen demand is largely fuelled by the blue oxygen transporter haemocyanin that however, due to its increasing functional failure towards colder temperatures, poses a prime target for cold-adaptive adjustment. While haemocyanin structure has been well understood it remains unclear which molecular features and evolutionary trajectories explain functional properties of octopod haemocyanin. This thesis thus aimed to unravel cold-adaptive features of octopod haemocyanin and the underlying molecular features that evolved to sustain oxygen supply at sub-zero temperatures. Haemocyanin function is best assessed by oxygen binding experiments, which however was challenged due to the minute haemolymph volumes non-model organisms like Antarctic octopods, yield. I thus upgraded an oxygen diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and tested the setup for Octopus vulgaris, the Antarctic eelpout and a Baikal amphipod. This technical advancement enabled simultaneous recordings of pH and oxygen dependent pigment absorbance in only 15 microliter of sample. Results were highly reproducible and accurate and provided detailed insight to the complex and dynamic spectral features of three diverse blood-types. To identify cold-adaptive functional traits of blood oxygen transport this study compared haemocyanin oxygen binding properties, oxygen carrying capacities and haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the temperate Octopus pallidus and the subtropical Eledone moschata. Compared to octopods from warmer climates, Pareledone charcoti showed incomplete but significantly reduced oxygen affinity, which together with increased haemocyanin concentrations and high physically dissolved oxygen levels supported oxygen supply at zero degree Celsisu. Therefore, unlike many Antarctic fishes Pareledone charcoti continued to rely on an oxygen transporting pigment. High temperature sensitivity of oxygen binding enabled Pareledone charcoti to utilise most of the oxygen bound by haemocyanin at 10 degree celsius. The concomitant relief for the circulatory system at warmer temperatures promotes warm tolerance and thus eurythermy in Pareledone charcoti. Underlying molecular mechanisms were studied by comparing 239 partial haemocyanin sequences of the functional unit f and g of 28 octopods species of polar, temperate, subtropical and tropical origin. Despite high conservation of these haemocyanin regions several sites were positively selected for their charge properties at the molecule's surface. Net surface charges were generally elevated in polar octopods suggesting that charge-charge interactions raise intrinsic pK values to stabilise quaternary structure against higher ambient pH present in cold waters. The presence of at least two haemocyanin isoforms and high allelic variation in polar octopods indicate sustained genetic diversity of haemocyanin and thus the genetic potential to regulate blood oxygen transport in the cold. Further, amino acid variability located within a potential metal binding site suggests regulation of blood oxygen transport in octopods via altered intrinsic sensitivity to allosteric ligands In conclusion, this study revealed significant adaptations of octopod haemocyanin at the functional and molecular level that support oxygen supply at near freezing temperatures. However, "imperfect" functional adaptation and ensuing reliance on high haemocyanin levels in Pareledone charcoti seems to add to the various design constraints of octopods compared to fishes. Hence, at second glance functional oxygen reserves indicate a higher capacity to sustain oxygen transport at warmer temperatures and together with a potential ability to regulate and reverse cold-adaptive molecular traits may be key to determine future winners and losers in an ecosystem facing radical environmental change