Gene expression in Daphnia magna: response to cyanotoxins and predators

Schwarzenberger, Anke (2010). Gene expression in Daphnia magna: response to cyanotoxins and predators. PhD thesis, Universität zu Köln. Open Access

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Thesis (PhD thesis)

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Daphnia represents the major herbivore of phytoplankton and cyanobacteria and is the most important food source for zooplanktivorous predators. In Daphnia several phenotypic traits have been shown to be plastic in response to kairomones. Little is known about the underlying molecular basis of this plasticity. Changes in the level of actin and alpha-tubulin proteins were shown in D. magna that were exposed to kairomones. Here, in order to analyse the level of gene expression of the actin and alpha-tubulin proteins and of selected genes of the basic metabolism of Daphnia after exposure to kairomones, I conducted real-time PCR analyses. Kairomones released from a fish and from an invertebrate predator did not substantially change the transcription levels of actin and alpha-tubulin. Kairomones of both predators caused changes in gene-expression of cyclophylin, indicating major effects on protein folding. Over the last decades, cyanobacterial blooms have become a common phenomenon, which have been claimed to be a major factor leading to the decline of Daphnia biomass, because of the low food quality of cyanobacteria, e.g. due to cyanotoxins. The most-investigated group of these cyanotoxins are microcystins. A microcystin-containing strain of M. aeruginosa and its microcystin-free mutant were fed to D. magna. Dietary microcystins led to an up-regulation of GapDH and UBC, which both are involved in the basic metabolism of D. magna. Protease inhibitors represent another important group of cyanotoxins. The specific targets of cyanobacterial protease inhibitors in Daphnia are trypsins and chymotrypsins. Feeding on mixtures of a reference food alga and one of two cyanobacterial strains, that either contained trypsin or chymotrypsin inhibitors, led to reduced somatic growth of D. magna. Either of the dietary protease inhibitor types had pronounced effects on digestive proteases of D. magna at the protein level. An in situ inhibition of the respective protease was observed, as well as an increase in protease activity of the non-inhibited protease type. In the case of dietary chymotrypsin inhibitors, also new protease isoforms were established. The digestive proteases visible on activity stained protein gels were assigned to six different protease genes via LC-MS/MS and subsequent database-search. Real-time PCR analysis with primers established from these genes revealed an increase in gene-expression due to dietary protease inhibitors. After exposure of either trypsin or chymotrypsin inhibitors 5 D. magna clones, strong intra-specific differences in sensitivity, measured as relative growth rate reduction, were revealed. The degree of sensitivity depended on the type of protease inhibitor as well as on the clone exposed to these inhibitors. The 5 different D. magna clones showed physiological responses to dietary protease inhibitors. Linear correlations suggested that the extent of relative growth rate reduction was due to the residual activity of the inhibited protease type. Higher residual protease activity that apparently led to lower sensitivity of the D. magna clone seemed partly to emerge from increased protease gene-expression of the corresponding protease. However, other factors might be involved, e.g. the establishment of more active protease isoforms. One D. magna clone proved to have a unique protease or protease isoform. I investigated, whether the increased gene expression of proteases was transferred from mothers that had experienced dietary chymotrypsin inhibitors to their offspring. The offspring of experienced mothers showed increased protease gene-expression in comparison to progenies of naïve mothers. The adaptive value of this maternal effect could not be demonstrated here. However, a here proposed maternal transfer of storage proteins to the offspring of naïve and experienced mothers allowed to temporarily compensate for the presence of protease inhibitors in the diet.

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