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

The role of food quality in clonal succession in Daphnia: an experimental test

A high genetic variation and recurrent changes in the genetic structure have been found in many pelagic populations. However, evidence that directly links these changes to differences in the ecological performance of particular genotypes is scarce. We hypothesized that within Daphnia, the specialization of clones occurring in a particular season to the food quality specific for that time of the year is responsible for the observed changes in the genetic structure of a population. This hypothesis was tested by comparing the fitness of spring and summer clones of the Daphnia longispina group, given food of biochemical quality relevant to these seasons. We identified significant intraspecific differences between clones of Daphnia that are specific for particular seasons, but there was no evidence that clones are adapted to the food quality available at the respective times of year. Summer clones reproduce at smaller size, and have a lower juvenile specific growth rate as compared to spring clones, irrespective of food quality. Spring clones invest more energy in somatic growth at the cost of reproduction, whereas summer clones invest more energy in reproduction at the cost of somatic growth. On the basis of the observed differences between spring and summer clones in their patterns of energy allocation, we suggest that other factors, most likely predation, are the major forces driving phenotypic and genetic diversity in the investigated Daphnia population of a large lake

β-Cyclocitral, a Grazer Defence Signal Unique to the Cyanobacterium Microcystis

β-Cyclocitral is often present in eutrophic waters and is a well known source of airborne and drinking water malodor, but its production and functional ecology are unresolved. This volatile organic compound (VOC) is derived from the catalytic breakdown of β-carotene, and evidence indicates that it is produced by the activation of a specific carotene oxygenase by all species of the bloom-forming cyanobacterium Microcystis. Previous work has shown that β-cyclocitral affects grazer behavior, but the nature of this interaction and its influence on predator-prey dynamics was unresolved. The present study combined analytical and behavioral studies to evaluate this interaction by using Microcystis NRC-1 and Daphnia magna. Results showed that β-cyclocitral was undetectable in live Microcystis cells, or present only at extremely low concentrations (2.6 amol /cell). In contrast, cell rupture activated a rapid carotene oxygenase reaction, which produced high amounts (77 ± 5.5 amol β-cyclocitral/cell), corresponding to a calculated maximum intracellular concentration of 2.2mM. The behavioral response of Daphnia magna to β-cyclocitral was evaluated in a bbe© Daphnia toximeter, where β-cyclocitral treatments induced a marked increase in swimming velocity. Acclimation took place within a few minutes, when Daphnia returned to normal swimming velocity while still exposed to β-cyclocitral. The minimum VOC concentration (odor threshold) that elicited a significant grazer response was 750nM β-cyclocitral, some 2,900 times lower than the per capita yield of a growing Microcystis cell after activation. Under natural conditions, initial grazer-related or other mode of cell rupture would lead to the development of a robust β-cyclocitral microzone around Microcystis colonies, thus acting as both a powerful repellent and signal of poor quality food to grazer

Understanding the differential impacts of two antidepressants on locomotion of freshwater snails (Lymnaea stagnalis)

There is growing evidence of negative impacts of antidepressants on behavior of aquatic non-target organisms. Accurate environmental risk assessment requires an understanding of whether antidepressants with similar modes of action have consistent negative impacts. Here, we tested the effect of acute exposure to two antidepressants, fluoxetine and venlafaxine (0–50 µg/L), on the behavior of non-target organism, i.e., freshwater pond snail, Lymnaea stagnalis. As compounds interact with chemical cues in the aquatic ecosystems, we also tested whether the effects altered in the presence of bile extract containing 5α-cyprinol sulfate (5α-CPS), a characterized kairomone of a natural predator, common carp (Cyprinus carpio). Behavior was studied using automated tracking and analysis of various locomotion parameters of L. stagnalis. Our results suggest that there are differences in the effects on locomotion upon exposure to venlafaxine and fluoxetine. We found strong evidence for a non-monotonic dose response on venlafaxine exposure, whereas fluoxetine only showed weak evidence of altered locomotion for a specific concentration. Combined exposure to compounds and 5α-CPS reduced the intensity of effects observed in the absence of 5α-CPS, possibly due to reduced bioavailability of the compounds. The results highlight the need for acknowledging different mechanisms of action among antidepressants while investigating their environmental risks. In addition, our results underline the importance of reporting non-significant effects and acknowledging individual variation in behavior for environmental risk assessment. Graphical Abstract: (Figure presented.)</p

Light intensity controls anti-predator defences in Daphnia: the suppression of life-history changes

A huge variety of organisms respond to the presence of predators with inducible defences, each of which is associated with costs. Many genotypes have the potential to respond with more than one defence, and it has been argued that it would be maladaptive to exhibit all possible responses at the same time. Here, we test how a well-known anti-fish defence in Daphnia, life-history changes (LHC), is controlled by light. We show that the kairomone-mediated reduction in size at first reproduction is inversely coupled to the light intensity. A similar effect was found for the kairomonemediated expression of candidate genes in Daphnia. We argue that the light intensity an individual is exposed to determines the degree of LHC, which allows for plastic adjustment to fluctuating environments and simultaneously minimizes the associated costs of multiple alternately deployable defences. It is hypothesized that this allows for a coupling of multiple defences, i.e. LHC and diel vertical migration

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

Physiological interaction of Daphnia and Microcystis with regard to cyanobacterial secondary metabolites

Cyanobacterial blooms in freshwater ecosystems are a matter of high concern with respect to human health and ecosystem services. Investigations on the role of cyanobacterial secondary metabolites have largely been confined to microcystins, although cyanobacteria produce a huge variety of toxic or inhibitory secondary metabolites. Mass occurrences of toxic cyanobacteria strongly impact freshwater zooplankton communities; especially the unselective filter feeder Daphnia. Daphnids have been shown to successfully suppress bloom formation. However, the opposite situation, i.e. the suppression of Daphnia populations by cyanobacteria can be observed as well. To understand these contradictory findings the elucidation of the underlying physiological mechanisms that help daphnids to cope with cyanotoxins is crucial. We fed Daphnia magna with the cyanobacterium Microcystis aeruginosa PCC7806 for 24 h and used high-resolution LCMS analytics to analyze the Microcystis cells, the Daphnia tissue and the surrounding medium in order to investigate the fate of seven investigated cyanobacterial compounds (cyanopeptolins A-C, microcyclamide 7806A and aerucyclamides B-D). For none of these bioactive compounds evidence for biotransformation or biodegradation by Daphnia were found. Instead feeding and subsequent release experiments point at the importance of transport mechanisms in Daphnia with regard to the cyanopeptolins A and C and microcyclamide 7806A. In addition we found hints for new inducible defense mechanism in Microcystis against predation by Daphnia. These putative defense mechanisms include the elevated production of toxic compounds other than microcystins, as could be demonstrated here for aerucyclamide 8 and D, cyanopoeptolin B and microcyclamide 7806A. Moreover, our data demonstrate the elevated active export of at least one cyanobacterial compound (microcyclamide 7806A) into the surrounding medium as a response to grazer presence, which might constitute an entirely new not yet described cyanobacterial defense mechanism. (C) 2014 Elsevier B.V. All rights reserved

Multiclonal study of Daphnia magna with respect to adaptation to toxic cyanobacteria

Mass developments of toxic cyanobacteria have increased in frequency due to global warming and eutrophication. Such cyanobacterial blooms impact whole freshwater ecosystems, especially reducing the abundance of herbivory species of the genus Daphnia. These negative effects on Daphnia have frequently been attributed to cyanobacterial secondary metabolites, among them hepatotoxic microcystins and protease inhibitors. Protease inhibitors inhibit major digestive proteases in the gut of Daphnia which results in reduced fitness, that is, population growth. To date evidence for local adaptation of Daphnia to cyanobacteria is confined to microcystin‐producing cyanobacteria and based on comparison of individual clones from different populations but lacks evidence from multiclone microcosm experiments. In the present study, D. magna clones from a Swedish lake where they coexist with the microcystin‐free Microcystis sp. strain BM25 were compared to clones from a Polish population without cyanobacteria, first in single‐clone experiments and subsequently in a multiclonal experimental population. The Swedish clones were assumed to be locally adapted to this protease inhibitor‐producing cyanobacterium and indeed showed higher population growth rates, a proxy for fitness, and dominated the population in the presence of dietary Microcystis sp. BM25, but not in the absence of this cyanobacterium. The results indicate an adaptive tolerance of the Swedish population and point at local adaptation to locally co‐occurring protease inhibitor‐producing cyanobacteria.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/50110000165

Physiological responses to stoichiometric constraints: nutrient limitation and compensatory feeding in a freshwater snail

Nitrogen (N) and phosphorus (P) are considered to be essential nutrients that control secondary production in various ecosystems; insufficient availability of N and P can limit herbivore growth. Here, data are presented from field samplings and from a laboratory experiment on the potential of primary producers low in P, N, or P and N to constrain growth of the freshwater gastropod Radix ovata. The filamentous green alga Ulothrix fimbriata was cultured under different nutrient regimes, resulting in algae with different C:N:P ratios. The pure algae were fed in high and low quantities to juvenile R. ovata. Low availability of N and especially P in the algae strongly constrained the biomass accrual of the herbivore. In accordance with theoretical predictions, these food quality differences were highly dependent on the food quantity. The snails' growth rate was significantly related to their body C:P ratio, thereby supporting the growth rate hypothesis. R. ovata displayed a pronounced compensatory feeding response to low-nutrient food that could partly dampen but not fully compensate the food quality effects on snail growth. Increased feeding of gastropods at low P and/or N availability leads to depletion of periphyton biomass; hence compensatory feeding would shift the benthic herbivore community from a P or N limitation to a C limitation and thus have whole-ecosystem effects

Predator evasion in zooplankton is suppressed by polyunsaturated fatty acid limitation

Herbivorous zooplankton avoid size-selective predation by vertical migration to a deep, cold water refuge. Adaptation to low temperatures in planktonic poikilotherms depends on essential dietary lipids; the availability of these lipids often limits growth and reproduction of zooplankton. We hypothesized that limitation by essential lipids may affect habitat preferences and predator avoidance behavior in planktonic poikilotherms. We used a liposome supplementation technique to enrich the green alga Scenedesmus obliquus and the cyanobacterium Synecchococcus elongatus with the essential lipids, cholesterol and eicosapentaenoic acid (EPA), and an indoor system with a stratified water-column (plankton organ) to test whether the absence of these selected dietary lipids constrains predator avoidance (habitat preferences) in four species of the key-stone pelagic freshwater grazer Daphnia. We found that the capability of avoiding fish predation through habitat shift to the deeper and colder environment was suppressed in Daphnia unless the diet was supplemented with EPA; however, the availability of cholesterol did not affect habitat preferences of the tested taxa. Thus, their ability to access a predator-free refuge and the outcome of predator-prey interactions depends upon food quality (i.e. the availability of an essential fatty acid). Our results suggest that biochemical food quality limitation, a bottom-up factor, may affect the top-down control of herbivorous zooplankton