Target gene approaches: Gene expression in Daphnia magna exposed to predator-borne kairomones or to microcystin-producing and microcystin-free Microcystis aeruginosa

<p>Abstract</p> <p>Background</p> <p>Two major biological stressors of freshwater zooplankton of the genus <it>Daphnia </it>are predation and fluctuations in food quality. Here we use kairomones released from a planktivorous fish (<it>Leucaspius delineatus</it>) and from an invertebrate predator (larvae of <it>Chaoborus flavicans</it>) to simulate predation pressure; a microcystin-producing culture of the cyanobacterium <it>Microcystis aeruginosa </it>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 <it>Daphnia pulex </it>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 <it>Daphnia </it>genome is composed of many expanded gene families.</p> <p>Results</p> <p>We evaluated seven candidate reference genes for QPCR in <it>Daphnia magna </it>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: <it>glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein </it>and <it>succinate dehydrogenase</it>. Using this NF, expression of the target genes <it>actin </it>and <it>alpha-tubulin </it>were revealed to be unchanged in the presence of the tested kairomones. The presence of fish kairomone up-regulated one gene (<it>cyclophilin</it>) involved in the folding of proteins, whereas <it>Chaoborus </it>kairomone down-regulated the same gene.</p> <p>We evaluated the same set of candidate reference genes for QPCR in <it>Daphnia magna </it>after exposure to a microcystin-producing and a microcystin-free strain of the cyanobacterium <it>Microcystis aeruginosa</it>. The NF was calculated based on the reference genes <it>18S ribosomal RNA</it>, <it>alpha-tubulin </it>and <it>TATA-box binding protein</it>. We found <it>glyceraldehyde-3-phosphate dehydrogenase </it>and <it>ubiquitin conjugating enzyme </it>to be up-regulated in the presence of microcystins in the food of <it>D. magna</it>. 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 <it>D. pulex </it>genome. The <it>cyclophilin</it>, <it>GapDH </it>and <it>UBC </it>genes show moderately large sequence divergence from their closest paralogs. Yet <it>actin </it>and <it>alpha-tubulin </it>genes targeteted by our study have nearly identical paralogs at the amino acid level.</p> <p>Conclusion</p> <p>Gene expression analysis using a normalisation factor based on three reference genes showed that transcription levels of <it>actin </it>and <it>alpha-tubulin </it>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 approaches. However, fish kairomones caused an up-regulation, and <it>Chaoborus </it>kairomone caused a down-regulation of <it>cyclophylin</it>, which proved to be a potential target gene for further analysis of kairomone effects on the life history of daphnids. Changes in food quality required a different set of reference genes compared to the kairomone experiment. The presence of dietary microcystins led to an up-regulation of two genes involved in the basic metabolism of <it>D. magna</it>, i.e. <it>glyceraldehyde-3-phosphate dehydrogenase </it>and <it>ubiquitin conjugating enzyme</it>, which suggests that microcystins in cyanobacteria have more general effects on the metabolism of <it>D. magna </it>than previously thought. Phylogenetic trees resolving relationships among paralogs that share the same gene name are shown to be important for determining the identity of the candidate genes under investigation.</p

Negative Effects of Cyanotoxins and Adaptative Responses of Daphnia

The plethora of cyanobacterial toxins are an enormous threat to whole ecosystems and humans. Due to eutrophication and increases in lake temperatures from global warming, changes in the distribution of cyanobacterial toxins and selection of few highly toxic species/ strains are likely. Globally, one of the most important grazers that controls cyanobacterial blooms is Daphnia, a freshwater model organism in ecology and (eco)toxicology. Daphnia–cyanobacteria interactions have been studied extensively, often focusing on the interference of filamentous cyanobacteria with Daphnia’s filtering apparatus, or on different nutritional constraints (the lack of essential amino acids or lipids) and grazer toxicity. For a long time, this toxicity only referred to microcystins. Currently, the focus shifts toward other deleterious cyanotoxins. Still, less than 10% of the total scientific output deals with cyanotoxins that are not microcystins; although these other cyanotoxins can occur just as frequently and at similar concentrations as microcystins in surface water. This review discusses the effects of different cyanobacterial toxins (hepatotoxins, digestive inhibitors, neurotoxins, and cytotoxins) on Daphnia and provides an elaborate and up-to-date overview of specific responses and adaptations of Daphnia. Furthermore, scenarios of what we can expect for the future of Daphnia–cyanobacteria interactions are described by comprising anthropogenic threats that might further increase toxin stress in Daphnia.publishe

Gene expression and activity of digestive enzymes of Daphnia pulex in response to food quality differences

Food quality is an important factor influencing organisms' well-being. In freshwater ecosystems, food quality has been studied extensively for the keystone herbivore genus Daphnia, as they form the critical trophic link between primary producers and higher order consumers such as fish. For Daphnia, the edible fraction of phytoplankton in lakes (consisting mostly of unicellular algae and cyanobacteria) is extraordinarily diverse. To be able to digest different food particles, Daphnia possess a set of digestive enzymes that metabolize carbohydrates, lipids and proteins. Recent studies have found a connection between gene expression and activity of single digestive enzyme types of Daphnia, i.e. lipases and proteases, and transcriptome studies have shown that a variety of genes coding for gut enzymes are differentially expressed in response to different food algae. However, never before has a set of digestive enzymes been studied simultaneously both on the gene expression and the enzyme activity level in Daphnia. Here, we investigated several digestive enzymes of Daphnia pulex in a comparison between a high-quality (green algal) and a low-quality (cyanobacterial) diet. Diet significantly affected the expression of all investigated digestive enzyme genes and enzyme activity was altered between treatments. Furthermore, we found that gene expression and enzyme activity were significantly correlated in cellulase, triacylglycerol lipase and beta-glucosidase when switched from high to low-quality food. We conclude that one of the factors causing the often observed low biomass and energy transfer efficiency from cyanobacteria to Daphnia is probably the switch to a cost-effective overall increase of gene expression and activity of digestive enzymes of this herbivore

What makes a man a man? Prenatal antennapedia expression is involved in the formation of the male phenotype in Daphnia

Cyclic parthenogenetic organisms show a switch in reproductive strategy from asexual to sexual reproduction upon the occurrence of unfavourable environmental conditions. The sexual reproductive mode involves the production of ameiotic diploid males and the fertilization of meiotic haploid eggs. One beautiful example for this switch between parthenogenesis and sexual reproduction is Daphnia. Male and female Daphnia from the same clone are genetically identical. Morphological differences should therefore only be due to differential gene expression. This differential gene expression leads to sexually dimorphic phenotypes with elongated and moveable (i.e. leg-like) first antennae in males in comparison to females. For other arthropods, it has been demonstrated that the formation of differential morphology of legs and antennae involves the regulation of the Hox gene antennapedia (antp). Here, we show that antp is expressed during the embryogenesis of Daphnia, and that adults contain much lower amounts of antp mRNA than eggs. The eggs of mothers that were treated with the juvenile hormone methyl farnesoate (responsible for the production of male offspring) showed lower expression of antp than parthenogenetically produced female eggs. We therefore conclude that differential antp expression is involved in the molecular pathways inducing the male phenotype of Daphnia

Daphnia's Adaptive Molecular Responses to the Cyanobacterial Neurotoxin Anatoxin-α Are Maternally Transferred

Cyanobacterial blooms are an omnipresent and well-known result of eutrophication and climate change in aquatic systems. Cyanobacteria produce a plethora of toxic secondary metabolites that affect humans, animals and ecosystems. Many cyanotoxins primarily affect the grazers of phytoplankton, e.g., Daphnia. The neurotoxin anatoxin-α has been reported world-wide; despite its potency, anatoxin-α and its effects on Daphnia have not been thoroughly investigated. Here, we investigated the effects of the anatoxin-α-producing Tychonema on life-history parameters and gene expression of nicotine-acetylcholine receptors (NAR), the direct targets of anatoxin-α, using several D. magna clones. We used juvenile somatic growth rates as a measure of fitness and analyzed gene expression by qPCR. Exposure to 100% Tychonema reduced the clones’ growth rates and caused an up-regulation of NAR gene expression. When 50% of the food consisted of Tychonema, none of the clones were reduced in growth and only one of them showed an increase in NAR gene expression. We demonstrate that this increased NAR gene expression can be maternally transferred and that offspring from experienced mothers show a higher growth rate when treated with 50% Tychonema compared with control offspring. However, the addition of further (anthropogenic) stressors might impair Daphnia’s adaptive responses to anatoxin-α. Especially the presence of certain pollutants (i.e., neonicotinoids), which also target NARs, might reduce Daphnia’s capability to cope with anatoxin-α.publishe

More Light Please : Daphnia Benefit From Light Pollution by Increased Tolerance Toward Cyanobacterial Chymotrypsin Inhibitors

Cryptochromes are evolutionary ancient blue-light photoreceptors that are part of the circadian clock in the nervous system of many organisms. Cryptochromes transfer information of the predominant light regime to the clock which results in the fast adjustment to photoperiod. Therefore, the clock is sensitive to light changes and can be affected by anthropogenic Artificial Light At Night (ALAN). This in turn has consequences for clock associated behavioral processes, e.g., diel vertical migration (DVM) of zooplankton. In freshwater ecosystems, the zooplankton genus Daphnia performs DVM in order to escape optically hunting predators and to avoid UV light. Concomitantly, Daphnia experience circadian changes in food-supply during DVM. Daphnia play the keystone role in the carbon-transfer to the next trophic level. Therefore, the whole ecosystem is affected during the occurrence of cyanobacteria blooms as cyanobacteria reduce food quality due to their production of digestive inhibitors (e.g., protease inhibitors). In other organisms, digestion is linked to the circadian clock. If this is also the case for Daphnia, the expression of protease genes should show a rhythmic expression following circadian expression of clock genes (e.g., cryptochrome 2). We tested this hypothesis and demonstrated that gene expression of the clock and of proteases was affected by ALAN. Contrary to our expectations, the activity of one type of proteases (chymotrypsins) was increased by ALAN. This indicates that higher protease activity might improve the diet utilization. Therefore, we treated D. magna with a chymotrypsin-inhibitor producing cyanobacterium and found that ALAN actually led to an increase in Daphnia’s growth rate in comparison to growth on the same cyanobacterium in control light conditions. We conclude that this increased tolerance to protease inhibitors putatively enables Daphnia populations to better control cyanobacterial blooms that produce chymotrypsin inhibitors in the Anthropocene, which is defined by light pollution and by an increase of cyanobacterial blooms due to eutrophication.publishe

Diet quality determines lipase gene expression and lipase/esterase activity in Daphnia pulex

We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments