Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

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.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways

“…To our knowledge, this is the first time that different trypsin isoforms have been shown to differ in their responsiveness to dietary proteins for a crustacean species. Differences in trypsin isoform expression have been found in Daphnia magna but in response to protease inhibitors in the diet (Schwarzenberger et al, 2010). It is noteworthy that in the present work, RNA extraction was carried out from tissue biopsies with no regard for the different cell types contained in the digestive gland.…”

Molecular and Chemical Analyses of Cyanobacterial Blooms in Tropical Lagoons from Southeast Brazil

“…These proteases production interferes with the grazing ability of some mesozooplankton, e.g. inhibiting Daphnia's digestive proteases [57], leading to the decline of freshwater herbivores in the environment.…”

Relationships among cyanobacteria, zooplankton and fish in sub-bloom conditions in the Sulejow Reservoir

“…Our results indicated that the biomass of both large and small filter feeders was significantly negatively correlated with microcystin concentrations, but contrary to our literature-based expectations (Hansson et al, 2007;Sun et al, 2012), it was not the large forms of zooplankton but the small filter feeders that were negatively correlated with the abundance of cyanobacteria (the concentration of Cya). Both daphniids and herbivorous copepods are potential consumers of cyanobacterial cells, but they generally avoid cyanobacteria, because it is a low-quality food item (MartinCreuzburg et al, 2008;Schwarzenberger et al, 2010). Copepods use multiple chemosensory signals in detecting toxins and avoiding their ingestion (Ger et al, 2011).…”

“…The cell shape and size of the colonies and the filaments formed by cyanobacteria disturb mechanical aspects of the filtering process, particularly for the large-bodied grazers of the genus Daphnia Cyanobacteria, zooplankton and fish in sub-bloom conditions (DeMott et al, 2001). The absence of sterols and polyunsaturated fatty acids (Martin-Creuzburg et al, 2008) and the production of protease inhibitors (Schwarzenberger et al, 2010) makes cyanobacteria a poor-quality food for zooplankton. Furthermore, the gelatinous or mucilaginous sheath produced by certain cyanobacteria species, e.g.…”