Ability of Daphnia Cell-Free Extract to Damage Escherichia coli Cells

A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and β-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cels were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg²⁺ and Ca²⁺ ions augmented the activity of the Daphnia extract on E. coli cells.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Prevalence of kairomone-induced diapause in Daphnia magna from habitats with and without fish

The prevalence of the diapause response of the freshwater crustacean Daphnia magna to chemical cues on fish predation was evaluated in 35 clones originating from 14 European water bodies—inhabited or not inhabited by fish. Clonal lineages of experimental animals were cultured for 4 weeks in the presence or absence of water to which an extract of faeces of crucian carps (Carassius carassius), that were fed with Daphnia, was added. The proportion of females producing diapausing forms during the experimental period was used as a measure of the diapause response. A positive diapause response to fish predation cues was observed in 43% of investigated clones that originated from 71% of the water bodies tested. This indicated that the diapause response to fish scent is a common phenomenon in Daphnia magna from various locations in Europe. Surprisingly, no signs of a stronger diapause reaction to the tested cues were found in Daphnia originating from water bodies inhabited by fish compared with those from fish-free habitats.     

Food quality of mixed bacteria–algae diets for Daphnia magna

Bacteria can comprise a large fraction of seston in aquatic ecosystems and can therefore significantly contribute to diets of filter-feeding zooplankton. To assess the effect of three heterotrophic bacteria (Flavobacterium sp., Pseudomonas sp. and Escherichia coli) on survival, growth and egg production of juvenile Daphnia magna during six-day growth experiments, five ratios of bacteria–Scenedesmus obliquus mixtures were fed. Potential growth-limiting effects mediated by essential biochemicals were assessed upon supplementation of pure bacterial diets with a sterol (cholesterol) or a polyunsaturated fatty acid (EPA). Pure bacterial diets always had detrimental effects on Daphnia. However, cholesterol supplementation of Flavobacterium sp. enhanced growth rates of Daphnia. Diets containing Pseudomonas impaired Daphnia growth even at low dietary proportions (20%), indicating their toxicity. In contrast, Daphnia grew at relative high dietary proportions of Flavobacterium sp. and E. coli (80–50%). In fact, diets containing small proportions of these heterotrophic bacteria (Flavobacterium ≤50%, E. coli 20%) even significantly increased Daphnia growth rates compared to pure algal diets, indicating a nutritional upgrading by these bacteria. Our results suggest that the relative contribution of bacteria and phytoplankton to total dietary carbon as well as their phylogenetic composition strongly influence Daphnia fitness and potentially other filter-feeding zooplankton under field conditions.     

Seasonal dynamics of sestonic protease inhibition: impact on Daphnia populations

Daphnia populations often show rapid microevolutionary adaptation to environmental changes. Here, we investigated the possibility that microevolution of Daphnia populations could be driven by natural sestonic Protease Inhibition (PI). We hypothesized that PI changes seasonally, which might lead to concomitant changes in tolerance to PI in a co-occurring Daphnia magna population. In order to test this, seston from a eutrophic pond was sampled regularly over two successive years. Extracts of these freeze-dried samples were used to determine their Inhibitory Potential (IP) on D. magna gut proteases. In the summer seston the IP against chymotrypsins exceeded that of spring seston 200-fold. In order to test for possible impacts on the co-existing D. magna population, we isolated clones before (spring) and after (fall) the peak of the IP. Microsatellite analyses revealed that the two subpopulations were genetically distinct. Individual exposure of three clones from each population to varying concentrations of a cyanobacterium that contains chymotrypsin inhibitors revealed a decrease in population and somatic growth rate for each clone, but no seasonal effects on Daphnia’s tolerance. In order to include maternal effects, we conducted a multi-clonal competition experiment on various cyanobacterial concentrations. However, no evidence for seasonally increased tolerance of D. magna to dietary protease inhibitors could be found.     

Water fleas require microbiota for survival,growth and reproduction

Microbiota have diverse roles in the functioning of their hosts; experiments using model organisms have enabled investigations into these functions. In the model crustacean Daphnia, little knowledge exists about the effect of microbiota on host well being. We assessed the effect of microbiota on Daphnia magna by experimentally depriving animals of their microbiota and comparing their growth, survival and fecundity to that of their bacteria-bearing counterparts. We tested Daphnia coming from both lab-reared parthenogenetic eggs of a single genotype and from genetically diverse field-collected resting eggs. We showed that bacteria-free hosts are smaller, less fecund and have higher mortality than those with microbiota. We also manipulated the association by exposing bacteria-free Daphnia to a single bacterial strain of Aeromonas sp., and to laboratory environmental bacteria. These experiments further demonstrated that the Daphnia–microbiota system is amenable to manipulation under various experimental conditions. The results of this study have implications for studies of D. magna in ecotoxicology, ecology and environmental genomics.     

Combined exposure to parasite and pesticide causes increased mortality in the water flea Daphnia

Organisms are exposed to multiple biotic and abiotic environmental stressors, which can influence the dynamics of individual populations and communities. Populations may also genetically adapt to both natural (e.g. disease) and anthropogenic (e.g. chemical pollution) stress. In the present study, we studied fitness consequences of exposure to both a parasite (i.e. biotic) and a pesticide (i.e. abiotic) for the water flea Daphnia. In addition, we investigated whether these fitness consequences change through time as a population evolves. Thus, we exposed Daphnia magna clones, hatched from dormant eggs isolated from different time layers of a natural dormant egg bank, to the parasite Pasteuria ramosa and the insecticide diazinon in a multifactorial experiment. While our experimental treatments for unknown reasons failed to induce disease symptoms in the Daphnia, we did observe a reduced survival of D. magna when simultaneously exposed to both the parasite and the pesticide. No increased mortality upon exposure to individual stressors was observed. We did not observe an evolutionary change in fitness response of the Daphnia clones hatched from different time horizons upon exposure to stressors.     

Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.     

Molecular genetic identification and phylogeny of Daphnia species (Crustacea, Cladocera) from water bodies of the Lake Chany basin

The data on the molecular genetic identification of Daphnia species from the water bodies of the Lake Chany basin are presented. Phylogenetic relationships between these species have been established. The fragments of the mitochondrial DNA genes were used as genetic markers. According to the data obtained, the water bodies examined were inhabited by five Daphnia species, including Daphnia (Daphnia) galeata Sars, D. (D.) longispina O. F. Müller, D. (D.) curvirostris Eylmann, D. (D.) pulex Leydig, and D. (Ctenodaphnia) magna Straus. In addition, longispina a-like individuals that form a separate phylogenetic lineage was identified.     

How<Emphasis Type="Italic"> Daphnia</Emphasis> copes with excess carbon in its food

Animals that maintain near homeostatic elemental ratios may get rid of excess ingested elements from their food in different ways. C regulation was studied in juveniles of Daphnia magna feeding on two Selenastrum capricornutum cultures contrasting in P content (400 and 80 C:P atomic ratios). Both cultures were labelled with ¹⁴C in order to measure Daphnia ingestion and assimilation rates. No significant difference in ingestion rates was observed between P-low and P-rich food, whereas the net assimilation of ¹⁴C was higher in the treatment with P-rich algae. Some Daphnia were also homogeneously labelled over 5 days on radioactive algae to estimate respiration rates and excretion rates of dissolved organic C (DOC). The respiration rate for Daphnia fed with high C:P algae (38.7% of body C day^-1) was significantly higher than for those feeding on low C:P algae (25.3% of body C day^-1). The DOC excretion rate was also higher when animals were fed on P-low algae (13.4% of body C day^-1) than on P-rich algae (5.7% of body C day^-1) . When corrected for respiratory losses, total assimilation of C did not differ significantly between treatments (around 60% of body C day^-1). Judging from these experiments, D. magna can maintain its stoichiometric balance when feeding on unbalanced diets (high C:P) primarily by disposing of excess dietary C via respiration and excretion of DOC.     

CRISPR/Cas-Mediated Targeted Mutagenesis in Daphnia magna

The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18–47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

Sperm number assessed by flow cytometry in species of Daphnia (Crustacea,Cladocera)

Daphnia magna and Daphnia pulex are two important model species in ecotoxicology. In daphniids, studies of the effects of contaminants have mostly focused on female life history traits, yet it would also be important to examine male reproductive traits, particularly in relation to endocrine disruptors. In this study, we developed a protocol that uses flow cytometry to measure sperm number in individual males of different species of Daphnia. We tested our protocol on 114 males from several clones of three common species of Daphnia. Sperm count varied widely among individuals and reached high numbers (up to 1.45 × 10⁵). Positive relationships between male length and sperm number were observed in D. pulex and Daphnia pulicaria, but not in D. magna. Important inter‐clonal differences in sperm production were observed in all species, with some clones producing very little sperm. Duplicated sperm samples showed on average only 6% difference in sperm counts. Sperm counts were stable at least over a 2‐hr period and up to 5 hr for most samples. This sperm isolation protocol and flow cytometric enumeration approach will be of major interest to ecotoxicologists.     

Antibiotics affect the growth responses of <Emphasis Type="Italic">Daphnia magna</Emphasis> to poor food quality

We tested the hypothesis that exposure to antibiotics alters the growth and reproductive responses of Daphnia magna to changing stoichiometric food quality. To do so, we measured growth and reproduction of differentially P-nourished Daphnia in the presence and absence of sublethal concentrations of antibiotics. We found that exposure to an antibiotic cocktail significantly reduced an index of the microbial load of Daphnia and altered its growth responses to changing dietary P-content. Growth rates of Daphnia consuming the most P-rich and P-poor food increased with antibiotic exposure but were negatively or not affected in animals eating mildly to moderately P-limiting food. Similar effects were found in a subsequent experiment where daphnid neonates were exposed to natural bacterial communities prior to receiving antibiotics and being fed different food C:P ratios. In contrast, antibiotic effects on Daphnia reproduction were either not detected (number and size of broods) or were relatively minor (day of first reproduction). We also found no evidence that gut flora provides defense against pathogenic bacterial infection; instead, infection rates in Daphnia by a bacterial microparasite, Pasteuria ramosa, decreased in animals that had experienced prior antibiotic exposure. Our results demonstrate that antibiotic exposure reduced the microbial load and altered growth rates of an important zooplankton herbivore. Given the mediating role of animal’s food C:P ratio, our results show that interactions between Daphnia and its microbial symbionts vary in strength and nature partly with the host’s nutritional state.     

Feeding behavior as an index of copper stress in Daphnia magna and Brachionus calyciflorus

1. The effect of short-term exposure to the fungicide copper sulfate, on the feeding behavior of two freshwater zooplankters, the rotifer Brachionus calyciflorus and the cladocera Daphnia magna, was studied.2. Both species were exposed to sublethal levels (0.01, 0.02, 0.05 and 0.08 mg/l) of copper sulfate during 24 hr in order to determine the effect of this toxicant on filtration and ingestion rates.3. The experiments were performed using the unicellular algae Nannochloris oculata in a density of 5 × 10⁵ cells/ml, as food for both organisms.4. Rates of filtration and ingestion declined on B. calyciflorus and D. magna with increasing toxicant concentrations.5. The effective copper sulfate concentration at which feeding rate was reduced to 50% of that in controls (ec₅₀) was 0.043 and 0.090 mg/1 for B. calyciflorus and D. magna, respectively.     

Experimental evidence of negative interference in Daphnia magna

Individuals of Daphnia magna Straus living together in groups were found to influence each other in a density-dependent manner. The effect appeared as an inhibition in offspring production and was demonstrated to be induced in part or completely by metabolic products released by Daphnia itself (negative interference). Negative interference was measurable even when there were only two individuals in 50 ml medium (40 animals · 1^–1) and increased up to a density of six animals per 50 ml (120 animals · 1^–1). Also, animals living singly in Daphnia-conditioned water showed a decrease in reproduction. For the measurements a cascade of vessels was developed in which flow-through conditions ensured a constant concentration of algae throughout the system.     

A comparison of the sensitivities of Daphnia magna and Daphnia pulex to six different cyanobacteria

Cyanobacteria have become an important environmental concern due to their ability to produce a wide range of natural toxins. At present, very few studies describe concentration response curves for cyanobacteria other than Microcystis. However, field evidence highlights that both cyanobacterial concentration as well as cyanobacterial species composition vary considerably with season and year. Therefore, the aim of this study was to investigate the effects of different cyanobacteria at various concentrations of these cyanobacteria in the diet on the reproduction of Daphnia pulex and Daphnia magna. Those two species were chosen to assess whether the cyanobacteria-daphnid dynamics could be generalized for the Daphnia genus. Results demonstrated that both slope and EC50 of the concentration response curves depend upon the Daphnia species, the cyanobacteria species and the potential interaction between the two. This has two major consequences. First, the differences in sensitivity to cyanobacteria between D. magna and D. pulex depend upon concentration of the specific cyanobacteria. Second, we noted different mechanisms of toxicity for the two zooplankton species, a more general mechanism of toxicity for D. pulex and a more specific one for D. magna. Our data therefore suggest that results of studies investigating effects of cyanobacteria at different concentrations cannot be generalized across species. Furthermore, mechanisms of toxicity are not only cyanobacteria specific, but also dependent on the exposed species, even for rather closely related species such as in the Daphnia genus. Whenever possible, we therefore propose to combine a multi-species approach together with a full concentration response analysis to reach more general conclusions concerning the effects of cyanobacteria on zooplankton.     

Combined effects of food shortage and oxygen deficiency on life history characteristics and filter screens of Daphnia

Daphnia magna were reared in environments with different concentrationsof food and dissolved oxygen, and their life history characteristicsand filter-screen areas were analyzed. Both low food concentrationand low oxygen concentration degraded Daphnia fitness (as measuredby individual growth and reproduction), and synergism was detectedstatistically for the effects of the two factors together. Lowfood concentration induced the development of large filter screensin Daphnia, but this development was suppressed by low oxygenconcentration, suggesting that low oxygen concentration reducesthe filtering rate of Daphnia in a food-limited environment.Therefore, energy uptake by Daphnia seems to be reduced by foodshortages and oxygen deficiencies jointly, and this reductionin energy uptake may be a possible mechanism causing the synergisticeffects of the two environmental stresses on the life historyof Daphnia.