Eric von Elert

Life history consequences of sterol availability in the aquatic keystone species Daphnia

The absence of essential biochemical nutrients, such as polyunsaturated fatty acids or sterols, has been considered as a mechanism determining trophic interactions between the herbivore Daphnia and its phytoplankton food source. Here, we experimentally quantify the sensitivity of two Daphnia species to decreasing amounts of dietary sterols by measuring variations in life history traits. The two species Daphnia magna and D. galeata were fed different mixtures of the sterol-containing green alga Scenedesmus obliquus and the sterol-free cyanobacterium Synechococcus elongatus; a higher proportion of Synechococcus in the food is equivalent to a decrease in dietary sterols. To address the significance of sterol limitation, the Daphnia species were also fed Synechococcus supplemented with cholesterol. In both species, somatic and population growth rates, maternal dry mass, the number of viable offspring, and the probability of survival were significantly reduced with the lower availability of sterols. A high correlation between the sterol content of the mixed diet and the somatic and population growth rates was found, and growth on cholesterol-supplemented Synechococcus fitted well into this correlation. Somatic growth of first-clutch neonates grown on 100% Synechococcus exhibited a pattern similar to that of somatic growth of their mothers grown on the different food regimes, which demonstrated the significance of maternal effects for sterol-limited population growth. Daphnia galeata had a twofold higher incipient limiting sterol level than D. magna, which indicated interspecific differences in sterol requirements between the two Daphnia species. The results suggest a strong impact of dietary sterols on life history traits and therefore, population dynamics of the keystone species Daphnia.

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

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 Daphnias 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.

Impact of 10 dietary sterols on growth and reproduction of daphnia galeata

In crustaceans, cholesterol is anEssential nutrient, which they must directly obtain from their food or by bioconversion from other dietary sterols. Eukaryotic phytoplankton contain a great variety of sterols that differ from cholesterol in having additional substituents or different positions and/or number of double bonds in the side chain or in the sterol nucleus. In this study, we investigated to what extent these structural features affect the growth and reproduction of Daphnia galeata in standardized growth experiments with the cyanobacterium Synechococcus elongatus supplemented with single sterols as food source. The results indicated that Δ5 (sitosterol, stigmasterol,Desmosterol) and Δ5,7 (7-dehydrocholesterol, ergosterol) sterols meet the nutritional requirements of the daphnids, while the Δ7 sterol lathosterol supports somatic growth and reproduction to a significantly lower extent than cholesterol. Dihydrocholesterol (Δ0) and lanosterol (Δ8) did not improve the growth of D. galeata, and growth was adversely affected by the Δ4 sterol allocholesterol. Sterols seem to differ in their allocation to somatic growth and reproduction. Thus, structural differences of dietary sterols have pronounced effects on life-history traits of D. galeata.

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

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 approaches. However, fish kairomones caused an up-regulation, and Chaoborus kairomone caused a down-regulation of cyclophylin, 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 D. magna, i.e. glyceraldehyde-3-phosphate dehydrogenase and ubiquitin conjugating enzyme, which suggests that microcystins in cyanobacteria have more general effects on the metabolism of D. magna 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.

Ecological significance of sterols in aquatic food webs

Sterols are indispensable for a multitude of physiological processes in all eukaryotic organisms. In most eukaryotes, sterols are synthesized de novo from low molecular weight precursors. Some invertebrates (e.g., all arthropods examined to date), however, are incapable of synthesizing sterols de novo, and therefore have to acquire sterols from their diet. Here, we aim to demonstrate that such nutritional requirements not only affect the performance of an individual in its environment but may also have major consequences for the function of aquatic ecosystems. Starting from general patterns of occurrence and biosynthesis of sterols, we next explore the physiological properties and nutritional requirements of sterols. These aspects are then integrated into a more ecological perspective. We emphasize their effects on aquatic food webs in general and on herbivorous zooplankton in particular with the major aim to outline how the interplay of physiological capabilities of individual herbivores and trophic interactions in the food web will determine the effect of low dietary provision of sterols on structure and function of aquatic ecosystems.

Good food versus bad food : the role of sterols and polyunsaturated fatty acids in determining growth and reproduction of Daphnia magna

The absence of dietary sterols and polyunsaturated fatty acids (PUFAs) has been shown to affect the performance of the freshwater herbivore Daphnia. Here, we compared somatic growth rates and clutch sizes of Daphnia magna reared on a diet of low food quality (Synechococcus elongatus) and of high food quality (Cryptomonas sp.) and investigated if and to what extent the absence of sterols or PUFAs in the cyanobacterium S. elongatus accounts for the observed differences in food quality. The supplementation of S. elongatus with cell-free lipid extracts (fatty acids, sterols, total lipids) obtained from the flagellate Cryptomonas sp. suggested that the superior food quality of Cryptomonas sp. is predominantly, but not completely, a combined effect of its sterol and PUFA composition. Our laboratory study suggests that somatic growth of D. magna feeding on S. elongatus is primarily constrained by the absence of sterols, whereas egg production is primarily limited by the absence of long chain PUFAs.

Food quality triggers the reproductive mode in the cyclical parthenogen Daphnia (Cladocera)

Cyclic parthenogenesis (heterogony) is a widespread reproductive mode found in diverse taxa such as digenean trematodes, gall wasps, gall midges, aphids, cladocerans and rotifers. It is of particular interest as it combines the advantages of asexual reproduction (rapid population growth) and sexual reproduction (recombination). Usually sexual reproduction is initiated when, or slightly before, environmental conditions deteriorate, and often results in the production of resting stages. The optimal timing of diapause induction must thus be under strong natural selection. Using the cladoceran Daphnia as a model system, we show here for the first time that the switch from parthenogenetic to sexual reproduction in a cyclical parthenogenetic organism can be influenced by the chemical composition of food. Under crowding conditions Daphnia reproduced parthenogenetically with subitaneous eggs when fed the algal species Cryptomonas sp., but started the production of resting eggs when fed with the green algal species Scenedesmus obliquus. Supplementation experiments with lipids and especially proteins showed that the induction of resting egg production in two clones of different Daphnia species was due to a dietary deficiency in the green alga. Hence, the low food quality induced a switch in the reproductive mode that may contribute to optimal timing of the sexual reproduction of Daphnia in nature. Furthermore, our results have two other major implications: first, they suggest that protein compounds should be added to the list of diet constituents potentially limiting or influencing Daphnia reproduction. Second, we show that the role of food quality goes far beyond the up to now documented effects of food quality on somatic growth and trophic transfer efficiency of herbivores: due to its effects on sexual reproduction and the production of resting eggs, food quality might influence genetic diversity and long-term persistence of Daphnia in lakes.

Deciphering the genetic basis of microcystin tolerance

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.

Settlement pattern of the zebra mussel, Dreissena polymorpha, as a function of depth in Lake Constance

Settlement on suitable substrata under favourable environmental conditions is an important factor for a successful recruitment of adult populations of Dreissena polymorpha. Therefore, the pattern of settlement of zebra mussel (Dreissena polymorpha) larvae at different depths was studied in Lake Constance. Maximum densities of larvae and newly settled juvenile mussels were observed at 4 m depth, while only single settlement episodes were recorded at greater depths (15 m and 30 m). Temperature fluctuation was used as a surrogate parameter for internal seiches. Biotic and abiotic parameters were subjected to a principal component analysis (PCA). The tight coupling of the internal seiches, larval abundance and settlement at 4-m depth vs. the lack of coupling of the latter two variables at greater depths indicated that water currents transported settling larvae to the substrata at greater depth. Our data suggest that physical factors, such as boundary mixing and internal seiches, should be considered as sources of variability in settlement.

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 kairomone-mediated 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.