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

Lyngbyastatins 8–10, Elastase Inhibitors with Cyclic Depsipeptide Scaffolds Isolated from the Marine Cyanobacterium Lyngbya semiplena

Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8–10 (1–3). The structures of 1–3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1–3 are closely related to lyngbyastatins 4–7. Like the latter compounds, we found 1–3 to inhibit porcine pancreatic elastase, with IC50 values of 123 nM, 210 nM and 120 nM, respectively

Combined and single effects of pesticide carbaryl and toxic Microcystis aeruginosa on the life history of Daphnia pulicaria

The combined influence of a pesticide (carbaryl) and a cyanotoxin (microcystin LR) on the life history of Daphnia pulicaria was investigated. At the beginning of the experiments animals were pulse exposed to carbaryl for 24 h and microcystins were delivered bound in Microcystis’ cells at different, sub-lethal concentrations (chronic exposure). In order to determine the actual carbaryl concentrations in the water LC–MS/MS was used. For analyses of the cyanotoxin concentration in Daphnia’s body enzyme-linked immunosorbent assay (ELISA) was used. Individual daphnids were cultured in a flow-through system under constant light (16 h of light: 8 h of dark), temperature (20°C), and food conditions (Scenedesmus obliquus, 1 mg of C l−1). The results showed that in the treatments with carbaryl egg numbers per female did not differ significantly from controls, but the mortality of newborns increased significantly. Increasing microcystin concentrations significantly delayed maturation, reduced size at first reproduction, number of eggs, and newborns. The interaction between carbaryl and Microcystis was highly significant. Animals matured later and at a smaller size than in controls. The number of eggs per female was reduced as well. Moreover, combined stressors caused frequent premature delivery of offspring with body deformations such as dented carapax or an undeveloped heart. This effect is concluded to be synergistic and could not be predicted from the effects of the single stressors.

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.

Dietary Essential Amino Acids Affect the Reproduction of the Keystone Herbivore Daphnia pulex

Recent studies have indicated that nitrogen availability can be an important determinant of primary production in freshwater lakes and that herbivore growth can be limited by low dietary nitrogen availability. Furthermore, a lack of specific essential nitrogenous biochemicals (such as essential amino acids) might be another important constraint on the fitness of consumers. This might be of particular importance for cladoceran zooplankton, which can switch between two alternative reproductive strategies – the production of subitaneously developing and resting eggs. Here, we hypothesize that both the somatic growth and the type of reproduction of the aquatic keystone herbivore Daphnia is limited by the availability of specific essential amino acids in the diet. In laboratory experiments, we investigated this hypothesis by feeding a high quality phytoplankton organism (Cryptomonas) and a green alga of moderate nutritional quality (Chlamydomonas) to a clone of Daphnia pulex with and without the addition of essential amino acids. The somatic growth of D. pulex differed between the algae of different nutritional quality, but not dependent on the addition of dissolved amino acids. However, in reproduction experiments, where moderate crowding conditions at saturating food quantities were applied, addition of the essential amino acids arginine and histidine (but not lysine and threonine) increased the total number and the developmental stage of subitaneous eggs. While D. pulex did not produce resting eggs on Cryptomonas, relatively high numbers of resting eggs were released on Chlamydomonas. When arginine and histidine were added to the green algal diet, the production of resting eggs was effectively suppressed. This demonstrates the high, but previously overlooked importance of single essential amino acids for the reproductive strategy of the aquatic keystone herbivore Daphnia

Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S–23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains

Vertical distribution of zooplankton in a shallow peatland pond: the limiting role of dissolved oxygen

We investigated the diel vertical distribution patterns of microcrustacean zooplankton (Cladocera, Copepoda) in a shallow pond (max. depth: 70 cm) of the Öreg-turján peatland (Ócsa, Central Hungary) during three 24-h periods in July (19–20th), August (17–18th) and September (11–12th) 2011. Environmental variables showed remarkable vertical stratification. Oxygen concentration was close to zero in the entire water column from night until sunrise, while the lower strata (from 20 cm below the surface) were close to anoxic during all three diel cycles. It proved to be the main determinant of the vertical distribution of microcrustaceans. Accordingly, the highest proportion of individuals was present in the surface layer. Chlorophyll-a concentration and phytoplankton biomass were inversely distributed compared to zooplankton. Microcrustaceans (mainly Daphnia curvirostris) migrated to the middle layer only in August, which could be explained by a trade-off between food resources, dissolved oxygen (DO) and competition with littoral zooplankters. The diurnal density patterns of microcrustaceans suggested horizontal migration into the aquatic macrophytes during night, which could be a strategy to avoid Chaoborus predation. Our results show that strong vertical gradients of abiotic and biotic factors occur even in such shallow waterbodies. Among them, DO can maintain constant vertical aggregation of zooplankters by limiting their occurrence to the surface layers