Abstract
BackgroundThe 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.ResultsNine 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.ConclusionHere 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.
Highlights
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
Many cyanobacterial protease inhibitors act against serine proteases, which represent the most important digestive proteases in the gut of Daphnia magna [19], and of which a surprisingly high number was found in the genome of D. pulex [20], a closely related species of D. magna
There was no difference between the protease pattern of the Daphnia homogenate and the gut Somatic growth rates on different food treatments When feeding on the green alga S. obliquus, D. magna grew at 0.47 d-1 (Fig. 1), whereas the growth rate on 20% of the cyanobacterium M. aeruginosa was significantly reduced in both cyanobacterial treatments
Summary
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. Increasing nutrient input has led to eutrophication in many lakes, which coincides with the increasing dominance of bloom-forming cyanobacteria in the phytoplankton assemblages [1,2] This increasing dominance of cyanobacteria has been claimed to be a major factor leading to the decline in Daphnia abundance across and within lakes [3,4,5]. Many cyanobacterial protease inhibitors act against serine proteases (i.e. trypsins and chymotrypsins), which represent the most important digestive proteases in the gut of Daphnia magna [19], and of which a surprisingly high number was found in the genome of D. pulex [20], a closely related species of D. magna
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