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.

BackgroundTwo major biological stressors of freshwater zooplankton of the genus Daphnia are predation and fluctuations in food quality. Here we use kairomones released from a planktivorous fish (Leucaspius delineatus) and from an invertebrate predator (larvae of Chaoborus flavicans) to simulate predation pressure; a microcystin-producing culture of the cyanobacterium Microcystis aeruginosa and a microcystin-deficient mutant are used to investigate effects of low food quality. Real-time quantitative polymerase chain reaction (QPCR) allows quantification of the impact of biotic stressors on differential gene activity. The draft genome sequence for Daphnia pulex facilitates the use of candidate genes by precisely identifying orthologs to functionally characterized genes in other model species. This information is obtained by constructing phylogenetic trees of candidate genes with the knowledge that the Daphnia genome is composed of many expanded gene families.ResultsWe evaluated seven candidate reference genes for QPCR in Daphnia magna after exposure to kairomones. As a robust approach, a combination normalisation factor (NF) was calculated based on the geometric mean of three of these seven reference genes: glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein and succinate dehydrogenase. Using this NF, expression of the target genes actin and alpha-tubulin were revealed to be unchanged in the presence of the tested kairomones. The presence of fish kairomone up-regulated one gene (cyclophilin) involved in the folding of proteins, whereas Chaoborus kairomone down-regulated the same gene.We evaluated the same set of candidate reference genes for QPCR in Daphnia magna after exposure to a microcystin-producing and a microcystin-free strain of the cyanobacterium Microcystis aeruginosa. The NF was calculated based on the reference genes 18S ribosomal RNA, alpha-tubulin and TATA-box binding protein. We found glyceraldehyde-3-phosphate dehydrogenase and ubiquitin conjugating enzyme to be up-regulated in the presence of microcystins in the food of D. magna. These findings demonstrate that certain enzymes of glycolysis and protein catabolism are significantly upgregulated when daphnids ingest microcystins. Each differentially regulated gene is a member of an expanded gene family in the D. pulex genome. The cyclophilin, GapDH and UBC genes show moderately large sequence divergence from their closest paralogs. Yet actin and alpha-tubulin genes targeteted by our study have nearly identical paralogs at the amino acid level.ConclusionGene expression analysis using a normalisation factor based on three reference genes showed that transcription levels of actin and alpha-tubulin were not substantially changed by predator-borne chemical cues from fishes or invertebrates, although changes in expression on the protein level were shown elsewhere. These changes in protein level could be caused by others than the investigated paralogs, showing the importance of the construction of phylogenetic trees for candidate gene approac...

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin-inhibitor-containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC(50) values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour-joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.

SUMMARYDaphnia has been shown to acquire tolerance to cyanobacterial toxins within an animalsʼ 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moll -1 (BN920) and 7.4nmoll -1 (CP954). When D. magna was grown on 20% M. aeruginosa, 2.2-fold higher IC 50 values were observed. This indicated that increased tolerance to these dietary inhibitors was acquired within an animalʼs 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.

BackgroundCyanobacteria constitute a serious threat to freshwater ecosystems by producing toxic secondary metabolites, e.g. microcystins. These microcystins have been shown to harm livestock, pets and humans and to affect ecosystem service and functioning. Cyanobacterial blooms are increasing worldwide in intensity and frequency due to eutrophication and global warming. However, Daphnia, the main grazer of planktonic algae and cyanobacteria, has been shown to be able to suppress bloom-forming cyanobacteria and to adapt to cyanobacteria that produce microcystins. Since Daphnia’s genome was published only recently, it is now possible to elucidate the underlying molecular mechanisms of microcystin tolerance of Daphnia.ResultsDaphnia magna was fed with either a cyanobacterial strain that produces microcystins or its genetically engineered microcystin knock-out mutant. Thus, it was possible to distinguish between effects due to the ingestion of cyanobacteria and effects caused specifically by microcystins. By using RNAseq the differentially expressed genes between the different treatments were analyzed and affected KOG-categories were calculated. Here we show that the expression of transporter genes in Daphnia was regulated as a specific response to microcystins. Subsequent qPCR and dietary supplementation with pure microcystin confirmed that the regulation of transporter gene expression was correlated with the tolerance of several Daphnia clones.ConclusionsHere, we were able to identify new candidate genes that specifically respond to microcystins by separating cyanobacterial effects from microcystin effects. The involvement of these candidate genes in tolerance to microcystins was validated by correlating the difference in transporter gene expression with clonal tolerance. Thus, the prevention of microcystin uptake most probably constitutes a key mechanism in the development of tolerance and adaptation of Daphnia. With the availability of clear candidate genes, future investigations examining the process of local adaptation of Daphnia populations to microcystins are now possible.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-776) contains supplementary material, which is available to authorized users.