1. Molecular physiology of Daphnia haemoglobin
Haemoglobin (Hb), the extracellular respiratory protein in the haemolymph of Daphnia, is a multi-subunit, multi-domain macromolecule with a molecular weight of approximately 500 kDa in D. magna. In this species, at least four Hb genes (dhb1 - dhb4) encode for at least seven different Hb subunit types (DHbA - DHbG) with molecular weights between 36 and 41 kDa. In response to changes of environmental factors as oxygen concentration or temperature, the Hb concentration in the haemolymph varies (up to 19fold). Additionally, the subunit composition of the macromolecules varies with concomitant changes of Hb oxygen affinity (up to 5fold). At present, we focus on the relationships between Hb genes and Hb subunits and on the Hb macromolecule structure in haemoglobin-poor ("pale") and haemoglobin-rich ("red") D. magna. Molecular biological methods (e.g. Southern blot analysis, quantitative RT-PCR), proteinbiochemical methods (e.g. 2-D gel electrophoresis and MALDI mass spectroscopy; chromatofocussing) and other techniques (e.g. in situ hybridization, computer modelling of macromolecule structure based on transmission electron micrographs ) are applied (partly in collaboration) to reveal the molecular reasons for the Hb subunit heterogeneity, to analyze composition and structure of the macromolecules and to relate structure to molecular function

2. Mechanisms, phenotypic plasticity and genotypic determination of thermal tolerance in Daphnia and Chaoborus larvae: consequences for fitness and biotic interactions (Project within the AQUASHIFT Priority program)
Global warming may have far-reaching effects on aquatic ecosystems through direct or indirect effects on the physiological systems of its members. The genus Daphnia plays a central role in the ecology of almost all standing freshwater, and Chaoborus larvae are prominent invertebrate predators of Daphnia. For a mechanistic understanding of thermal effects in Daphnia and in Chaoborus larvae, field data analysis, physiological and ecological experiments, biochemical and genetical investigations and retrospective studies will be brought together: investigation of the seasonal changes of phenotypic acclimatization and/or of population structure (clonal structure) and clone-specific thermal tolerance will allow to evaluate future ecological consequences of global warming. Both, mechanisms and degrees of thermal tolerance as well as traits related to physiological fitness will be analyzed and linked to environmental conditions.
    Answers to the following questions will be sought: What are the physiological and biochemical mechanisms causing differences in thermal tolerance? Which part of these mechanisms is genetically fixed (adaptation) and which is phenotypically plastic (acclimatization)? Which part is species- or clone-specific and which are characteristics common to all? What are the costs of an improved or the benefits of a reduced thermal performance and vice versa? Are there any seasonal changes concerning thermal tolerance and fitness due to phenotypic plasticity or due to the clonal structure of Daphnia populations? How near to the edges of their thermal tolerance ranges do species and clones live during the seasons? The necessary data base for a general view on Daphnia will come from a comparison of differently thermally adapted, yet closely related clones and species.

Lamkemeyer, T., Zeis, B., and Paul, R.J. 2003. Temperature acclimation influences temperature-related behaviour as well as oxygen transport physiology and biochemistry in the water flea Daphnia magna. Can. J. Zool. 81: 237-249.

Paul, RJ, Lamkemeyer, T, Maurer, J, Pinkhaus O, Pirow R, Seidl M, Zeis B. 2004. Thermal acclimation in the microcrustacean Daphnia: a survey of behavioural, physiological and biochemical mechanisms. J THERM BIOL 29: 655-662

Paul, RJ, Zeis, B, Lamkemeyer, T, Seidl M, Pirow R. 2004. Control of oxygen transport in the microcrustacean Daphnia: regulation of haemoglobin expression as central mechanism of  adaptation to different oxygen and temperature conditions. ACTA PHYSIOL SCAND 182: 259-275.

Zeis, B., Becher, B., Lamkemeyer, T., Rolf, S., Pirow, R., and Paul, R.J. 2003. The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties. Comp. Biochem. Physiol. B 134: 243-252.

Zeis, B., Becher, B., Goldmann, T., Clark, R., Vollmer, E., Bölke, B., Bredebusch, I., Lamkemeyer, T., Pinkhaus, O., Pirow, R., and Paul, R.J. 2003. Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures. Biol. Chem. 384: 1133-1145.

Zeis, B, Lamkemeyer, T, Paul, RJ. 2004. Molecular adaptation of Daphnia magna hemoglobin. MICRON 35: 47-49.

Zeis, B., Pinkhaus, O., Bredebusch, I., Paul, R.J., 2004. Oxygen preference of Daphnia magna is influenced by Po2-acclimation and biotic interactions. Physiol. Biochem. Zool. 78:384–393.

Zeis, B., Maurer, J., Pinkhaus, O., Bongartz, E., Paul, R.J., 2005. A swimming activity assay shows the thermal tolerance of Daphnia magna to be influenced by temperature acclimation. Can. J. Zool. 82: 1605–1613.

Lamkemeyer T, Paul RJ, Stöcker W, Yiallouros I, Zeis B (2005) Macromolecular isoforms of Daphnia magna haemoglobin. Biol Chem 386, 1087-1096

Lamkemeyer T, Zeis B, Decker H, Jaenicke E, Waschbüsch D, Gebauer W, Markl J, Meissner U, Rousselot M, Zal F, Paul RJ (2006) Molecular mass of macromolecules and subunits and quaternary structure of haemoglobin from the microcrustacean Daphnia magna. FEBS J 273: 3393-3410