2012-06-15, von Elert, Eric, Zitt, Anja, Schwarzenberger, Anke

Daphnia has been shown to acquire tolerance to cyanobacterial toxins within an animals' lifetime and to transfer this tolerance to the next generation. Here we used a strain of the cyanobacterium Microcystis aeruginosa, which contained two chymotrypsin inhibitors (BN920 and CP954), the green alga Scenedesmus obliquus as reference food and a clone of D. magna to investigate the physiological mechanism of acquired tolerance to these cyanobacterial toxins. The intracellular concentrations of CP954 and BN920 were 1550 and 120 μmol l^–1, respectively. When food suspensions of the green alga contained >60% M. aeruginosa, growth rates of D. magna were reduced. When grown on the green alga, three chymotrypsins ranging in mass from 16 to 22 kDa were distinguished in D. magna. Purified BN920 and CP954 specifically inhibited D. magna chymotrypsins. Feeding with encapsulated BN920 resulted in growth depression in D. magna and replacement of the chymotrypsins by three chymotrypsins with smaller molecular mass. With just 20% M. aeruginosa, the same changes in the chymotrypsin pattern as with the pure inhibitor were observed. IC₅₀ values for inhibition of chymotrypsins of D. magna growing on the green alga were 5.4 nmol l^–1 (BN920) and 7.4 nmol l^–1 (CP954). When D. magna was grown on 20% M. aeruginosa, 2.2-fold higher IC₅₀ values were observed. This indicated that increased tolerance to these dietary inhibitors was acquired within an animal's lifetime by remodelling the digestive chymotrypsins, which in turn serves as an intra-generational defence against these cyanobacterial inhibitors. This mechanism might be relevant for the transfer of tolerance to the next generation through maternal effects.

2010, Schwarzenberger, Anke, Zitt, Anja, Kroth, Peter G., Mueller, Stefan, Elert, Eric von

Background
The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.

Results
Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects.

Conclusion
Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might determine the degree of growth reduction in D. magna in the presence of cyanobacterial protease inhibitors. The rapid response of Daphnia to cyanobacterial protease inhibitors supports the assumption that dietary cyanobacterial protease inhibitors exert a strong selection pressure on Daphnia proteases themselves.