The occurrence of cyclic heptapeptides, termed microcystins, is widespread in cyanobacteria and not restricted to the genus Microcystis; mass development of microcystin-producing cyanobacteria can constitute a high risk for intoxication of humans and livestock . Numerous studies have been carried out in order to determine the ecological significance of microcystin production in cyanobacteria. The availability of the mcy- mutant of PCC7806, which is genetically identical to the wild type except for its inability to synthesize microcystins , made it possible to more precisely analyse the role of microcystins in daphnid poisoning. Life-table experiments with the wild type and the mcy- mutant of PCC7806 with Daphnia galeata have shown that the wild type was poisonous to D. galeata, whereas the mutant strain had no lethal effects [17, 18]. These findings and similar results for D. magna  suggest that microcystins play a role in the defence of M. aeruginosa against zooplankton grazing.
Feeding on the cyanobacterium M. aeruginosa led to significantly reduced growth of D. magna compared to animals grown on the high quality food alga S. obliquus  or the non-toxic cyanobacterium S. elongatus . Although there was reduced growth in D. magna feeding on M. aeruginosa compared to the reference cyanobacterium, there was no difference between the wild type and the mcy- mutant treatment. However, a specific microcystin effect became evident on day five, when D. magna raised on the wild type strain died, whereas no mortality was observed in D. magna raised on the mutant strain.
In an in-vitro system, microcystin-LR has been shown to inhibit protein phosphatases 1 and 2A in crude extracts of Daphnia sp . However protein phosphatase 1 and 2A each comprise a family of protein serine/threonine phosphatases with a wide range of different specificities that are mediated by different interactors  and regulatory subunits [29, 30]. Hence it remains entirely unclear which specific physiological pathways in daphnids are affected by the binding of microcystin to protein phosphatases 1 and 2A.
Here for the first time in-situ effects of dietary microcystins on gene expression of daphnids were investigated. The experiments presented in this paper were designed to identify genes involved in the general metabolism in D. magna in which the expression level responds to the presence of microcystins. We therefore compared the effects of the microcystin-producing wild type M. aeruginosa PCC7806 and the mcy- mutant of this strain on the relative expression of genes involved in basic metabolism. We found substantial up-regulation of GapDH (Dappu-302823) and UBC (Dappu-120690) in response to the presence of microcystins in the food of D. magna, which demonstrates that certain enzymes of glycolysis and protein catabolism are significantly up-regulated when daphnids ingest microcystins. For the first time a specific gene regulation in response to dietary microcystins has been demonstrated in daphnids. This up-regulation might have enabled D. magna to avoid a microcystin-specific depression of growth until day four but could not prevent mortality on day five of the growth experiment.
Upon exposure to the microcystin-producing wild type of M. aeruginosa PCC7806, D. magna has been shown to develop a tolerance against this toxic strain within an individual's lifespan and to transfer this tolerance to the next generation through maternal effects, a fact that has been interpreted as an inducible defence against microcystin . It remains to be tested which role the observed up-regulation of GapDH and UBC plays in the inducible tolerance of D. magna to microcystins. Furthermore, clones of D. magna have been shown to differ in their tolerance to M. aeruginosa PCC7806 , which suggests a genetic basis for increased toxin tolerance. It remains to be investigated whether the up-regulation of GapDH and UBC contributes to the tolerance to M. aeruginosa PCC7806.
In addition to the microcystins in PCC7806 wild type, both the wild type and mcy- mutant PCC7806 produce other classes of secondary metabolites of unknown biological activity [32, 33]. D. magna feeding on either of these two strains revealed a substantial up-regulation of SucDH, and it remains to be seen which cyanobacterial compounds induce this up-regulation of a key enzyme of the tricarboxylic acid cycle. In order to account for possible general effects of cyanobacteria on expression of the investigated genes, we fed Synechococcus elongatus to D. magna. This cyanobacterium is easily ingested by daphnids and does not contain toxins or inhibitors . The effects of S. elongatus on GapDH, UBC and SucDH were negligible compared to the afore mentioned effects of M. aeruginosa, which indicates that the up-regulation of the tested loci of GapDH, UBC and SucDH in D. magna is a specific and not a general response to cyanobacterial secondary metabolites. It would be interesting to see, if this holds true for all different paralogs of the affected genes or if the up-regulation is restricted to specific clusters or single paralogs of these highly variable genes (Fig. 7E-F).
Predation is an important stressor in aquatic communities, and many studies using Daphnia sp. have contributed to an understanding of the adaptive value of inducible anti-predator defences in the genus Daphnia. Achieving a better understanding of the mechanisms and constraints of the evolution of inducible anti-predator defences requires more research on the mechanisms of inducible defences at the molecular level. Only recently has this field been started to be explored. Our work was stimulated by the paper of Pijanowska & Kloc, (2004) , who used a clone of D. magna which has been shown to be plastic with regard to life-history traits and behaviour [10, 11, 13, 14, 34] in response to kairomones from fish and Chaoborus. Pijanowska & Kloc (2004)  have shown a dramatic decrease of the proteins actin and alpha-tubulin in this clone of D. magna when it was exposed to kairomones from planktivorous fish or the larvae of Chaoborus water midges. These identical effects of vertebrate and invertebrate kairomones suggested that actin might play a major role in anti-predator responses in D. magna in general. Using the same clone of D. magna, we here demonstrate that an exposure to chemical cues from both invertebrate and vertebrate predators results in a change in actin expression. However, although significant, the 1.75-fold (fish) increase and 0.94-fold (invertebrate) decrease in actin expression was rather moderate and did not reflect the dramatic decrease of the protein actin reported by Pijanowska & Kloc . The same holds true for the weak although significant increase in the gene alpha-tubulin in the fish (1.71) and the Chaoborus treatments (1.07). Since we found two possible alpha-tubulin orthologous protein sequences in D. pulex, which were very similar to each other (Fig. 7C), we concluded that the effect on the expression holds true for all paralogs in their cluster. Therefore, the substantial decrease of actin and alpha-tubulin on the protein level reported by Pijanowska & Kloc  could be a posttranslational process, e.g. miRNA-mediated regulation or increased degradation, as has been suggested by the authors . We conclude that these loci of actin and alpha-tubulin are no strong target genes for anti-predator defences. However, construction of phylogenetic trees reveals very high variability between the different paralogs of actin and alpha-tubulin (Fig. 7A-C). It remains to be tested if the decrease of actin and alpha-tubulin on the protein level reported by Pijanowska & Kloc  is caused by another paralogous sequence sharing the same gene name.
Following normalisation to NF, it turned out that the expression of 28S, UBC, 18S and cyclophilin was affected by the type of kairomone. Genes involved in protein biosynthesis (18S, 28S) and protein catabolism (UBC) were up-regulated by kairomone. These effects were considerably stronger for fish kairomone. The expression of cyclophylin (Dappu-92663), a gene involved in protein folding, was up-regulated in the presence of kairomones from vertebrate and down-regulated by kairomones from invertebrate predators. The finding that the two kairomones differ in their effect on cyclophylin in D. magna is in accord with the observation that the life-history response of this clone of D. magna differs between kairomones released from fish or Chaoborus . Cyclophilin, could serve as a potential target gene for further analysis of kairomone effects on daphnids. It remains to be seen how cyclophilin is involved in mediating kairomone effects on life history of daphnids and if this is specific to the orthologous sequence and to related paralogous sequences of cyclophilin.
Our study is the first detailed study that investigates effects of kairomones from vertebrate and invertebrate predators and of microcystin on gene expression of genes involved in different basic metabolic processes in D. magna. Kairomones from both vertebrate and invertebrate predators led to the well-established adaptive shifts in SFR in D. magna giving evidence for biologically active incubation water from either predator. Similarly, evidence for specific effects of microcystin comes from the higher mortality of D. magna on the wild type strain than on the mutant of M. aeruginosa PCC 7806. Calculating a combination normalisation factor based on the geometric mean of three genes for the kairomone experiment and for the growth experiment, stressor-specific regulation of some of the genes involved in basic metabolism is demonstrated.
All target genes in Daphnia show a surprisingly high variability between paralogs. If such a high variability holds true for other genes in D. magna, this could hint at a highly plastic genome, which might be adaptive for an animal that living in a very complex aquatic environment and therefore has to maintain a high potential for adaptations.