Cryopreservation van genotypes bij Daphnia magna.

The water flea Daphnia magna is an important model organism in many research areas, such as ecology, ecotoxicology, evolutionary biology and eco-genomics and most of these applications use clonal lines produced via asexual reproduction. To date, maintenance of the clonal lines can only be achieved through continuous culturing. This is a labor-intensive process and entails the risk of losing important lines because of contamination, disease or accidents. In this PhD research, we tried to solve this problem by developing a cryopreservation protocol for the asexual eggs of Daphnia magna.

The dormant eggs of Daphnia, produced by sexual reproduction, are drought and freeze resistant and are thereby an excellent reference to determine what is needed for eggs to survive cryopreservation. We determined the main biochemical differences between sexual and asexual eggs. We found that asexual eggs have higher concentrations of fatty acids than sexual eggs, however a certain concentration of long-chain PUFA, especially EPA and ARA, is maintained in sexual eggs even when they are not provided by the food and this in both neutral lipids and phospholipids. Sugar content of sexual and asexual eggs was very distinct, sexual eggs contained high amounts of trehalose (4.15% of their dry weight), while asexual only contain 0.006% trehalose. Also polyamine analysis revealed some difference between the two egg types. Asexual eggs always contained higher amounts of the metabolite diaminopropane and lower amounts of putrescine and spermidine in comparison with sexual eggs.

In a second step we tried to modify the composition of asexual eggs in order to increase their stress resistance. Fatty acid composition of asexual is strongly influenced by the maternal food, so to boost the PUFA composition of these eggs we simply switch to a diet containing more long-chain PUFA. PUFA composition of the asexual eggs of D. magna is also strongly influence by the culturing temperature of the females, they allocate more unsaturated fatty acids to their eggs at colder temperatures. In addition, we tried to increase trehalose concentration of the asexual eggs by supplementation of the maternal diet with trehalose containing liposomes, but changes were only minor.

In parallel, the protocol for cryopreservation was optimized. To obtain sufficient dehydration we choose for a two-step pretreatment for the eggs before applying droplet vitrification. The pretreatment consisted of a glycerol loading in a 10% glycerol solution for 30 min. This was followed by osmotic dehydration in a vitrification solution. The vitrification solution with the lest mortality after exposure for 10 or 20 min contains 10% glycerol, 30% methanol and 0.5M trehalose.

In the last step, we combined the procedure to alter the biochemical properties of asexual eggs and our most suitable cryopreservation protocol. This stepwise approach led to successful cryopreservation of subitaneous Daphnia magna eggs. Although survival rates are still low, this is a large step forward in the process towards safe storage of clonal lines.