Crowding‐induced changes in growth,reproduction and morphology of Daphnia

• 1 Daphnia may reach high population densities seasonally, or in patches, in lakes. To test the effects of chemicals released by high daphniid densities on their life‐history traits, nine species of Daphnia, D. magna, D. pulicaria, D. pulex, D. hyalina, D. galeata, D. laevis, D. lumholtzi, D. ambigua and D. cucullata, were grown in water from crowded Daphnia cultures in a flow‐through system in the presence of abundant food.
• 2 Water from Daphnia at 85 L^‐1 depressed growth rate, and lowered body size and clutch at first reproduction of six species of small‐bodied Daphnia (adult body length < 1.8 mm), but had no significant effects on larger species. Two clones of D. pulex differed in their growth rate in response to crowding, indicating that response patterns may vary within species.
• 3 Chemicals released by crowded D. magna reduced tail spine length in D. lumholtzi and D. cucullata by 37% and 11%, respectively, and induced changes in carapace morphology in D. lumholtzi and D. ambigua.
• 4 Chemicals released by crowded conspecifics may provide an additional, density‐dependent mechanism of population regulation; when large species of Daphnia coexist at a high population density with small species, these chemicals may reinforce the competitive advantage of large species.

     

Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient‐rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin‐inhibitor‐containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC₅₀ values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour‐joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.     

Effects of zebra mussels on cladoceran communities under eutrophic conditions

The purpose of this study was to determine how zebra mussels affected cladoceran community structure under eutrophic conditions. We conducted a mesocosm study where we manipulated the presence of zebra mussels and the presence of large-bodied Daphnia (Daphnia magna and Daphnia pulicaria). We also conducted a complimentary life-table experiment to determine how water from the zebra mussel treatment affected the life history characteristics of the cladoceran species. We anticipated that small- and large-bodied cladoceran species would respond differently to changes in algal quality and quantity under the effects of zebra mussels. Large-bodied Daphnia successfully established in the zebra mussel treatment but failed to grow in the control. We did not observe positive relationships between food concentrations and cladoceran abundances. However, the phosphorus content in the seston indicated that food quality was below the threshold level for large-bodied cladocerans at the beginning of the experiment. We believe that zebra mussels quickly enhanced the phosphorus content in the seston due to the excretion of inorganic phosphorus, thus facilitating the development of large-bodied Daphnia. In conclusion, our results suggest that zebra mussels can alter the phosphorus content of seston in lakes and this can affect the dynamics of crustacean zooplankton.     

Interclonal variation in diel horizontal migration behaviour of the water flea <Emphasis Type="Italic">Daphnia magna</Emphasis>—searching for a signature of adaptive evolution

In shallow temperate lakes, zooplankton populations may exhibit diel horizontal migration (DHM) and move towards macrophytes during the day to avoid fish. Using a natural Daphnia magna population, we undertook an experimental investigation aimed to describe the genetic variation for DHM and to study whether an adaptive micro-evolutionary response occurred to changes in macrophyte coverage and fish predation pressure through time. Twenty-seven D. magna clones were hatched from ephippia in the sediment of shallow Lake Ring, Denmark. This lake was eutrophied during the 20th century and was subject to restoration measures in the 1970s. The DHM behaviour of the clones was observed both in the presence and absence of fish kairomone. Significant interclonal variation in DHM behaviour occurred in both treatments. To study the micro-evolutionary response of the Lake Ring D. magna population, two approaches were used. First, we compared the DHM behaviour of clones derived from ephippia collected at different depths. A comparison was conducted between clones resurrected from the period of eutrophication (1960–1980) and from the period of recovery (1986–2000). A significant treatment (presence and absence of fish kairomone) × period interaction effect was identified, suggesting a significant micro-evolutionary response for DHM behaviour. The D. magna clones exhibited a significantly stronger horizontal migration response during the period of eutrophication than in the recovery phase. Second, clonal means, representing the influence of the genotype on the trait, were correlated with environmental conditions (macrophyte cover, fish predation pressure and Secchi depth). The results of this analysis also suggest that a micro-evolutionary response by Daphnia has occurred in reaction to changes in fish predation pressure. In periods with high fish predation pressure, Daphnia migrated more strongly towards the plants. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

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.     

Seston composition and the potential for Daphnia growth

A field survey was conducted to study the relationships amongst the composition of the seston, the nutritional value of the seston for herbivorous zooplankton (Daphnia), and selected water clarity parameters. Sixteen ponds in a wetland area and seven larger lakes, all located in south central Saskatchewan, Canada, were sampled for seston. The phytoplankton species were identified, and various biochemical seston variables were measured. A biotest using the zooplankter Daphnia magna, was employed to assess the nutritional value of the seston. The best seston variable to explain Daphnia growth was the phospholipid content (simple linear regression analysis: R ² _adj = 0.50). The water absorbance ratio A250/A365 was a good predictor of lipid content of the seston. Both the absorbance ratio A250/A365 and the dissolved organic carbon (DOC) concentration were negatively correlated with Daphnia growth. We hypothesize that the penetration of visible and ultraviolet radiation is an important determinant of seston quality, especially the phospholipid content, and that this has important implications for determining ultimate growth rates of herbivorous zooplankton.     

Species and clone-dependent effects of tilapia fish (Cichlidae) on the morphology and life-history of temperate and tropical <Emphasis Type="Italic">Daphnia</Emphasis>

In aquatic systems, tilapia introductions may result in marked changes in the structure of prey communities. In this study, we experimentally examined the effects of tilapia-mediated water at the individual and population levels of prey by exposing three Daphnia species to predation cues. We hypothesized that tilapia-mediated water determines reduced age and size at primipara, greater and faster reproduction, enhanced intrinsic rates of population increase (r), and longer tail spines in Daphnia; but that the magnitude of these changes would be species and clone-dependent. When three tropical D. laevis and one temperate D. similis clones were exposed to predation cues, adaptive changes were observed in some of the aforementioned parameters for each clone. The three D. laevis clones exhibited changes in all life-history and morphological measures. Temperate Daphnia spinulata displayed no changes but decreased r values in the presence of predators. The observed changes in the species and clones tested here suggest that, overall, both temperate and tropical Daphnia can detect and adaptively react to the risk of tilapia predation. However, only a fraction of the possible defenses may be displayed by individual clones. In contrast, D. spinulata seems more vulnerable to tilapia predation, given its long body length and absence of adaptive changes. Our study indicates that Daphnia can respond to tilapia-mediated water, and that interspecific and clonal variation exists between temperate and tropical species.     

An evolutionary perspective on the resistance of Daphnia to the epizoic rotifer Brachionus rubens

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Patterns and sources of variation in Daphnia phosphorus content in nature

It has recently been shown that Daphnia can vary in the phosphorus (P)-content of their body tissues, but the relative importance of genetic versus environmental causes for this variation is unexplored. We measured variation in P-content (as % body mass) of Daphnia from eight lakes and conducted experiments to contrast three sources of variation: interspecific variation, clonal variation and phenotypic plasticity. Daphnia P-content decreased with increasing seston C:P ratio across lakes. This relationship reflected both inter- and intraspecific variation. Daphnia parvula and D. dubia exhibited high P-content and were found in shallow lakes with low C:P seston, whereas D. pulicaria had low P-content and was found in deep, stratified lakes having high C:P seston. Populations of D. dentifera spanned this lake gradient and exhibited P-content that was negatively related to seston C:P. Evidence for phenotypic plasticity came from experiments with D. pulicaria and D. dentifera collected from a lake with P-deficient seston and fed a P-sufficient diet in the laboratory. In addition, populations of D. dentifera differed in P-content even after 7 d of feeding on P-sufficient resources, suggesting within-species clonal variation. However, mesocosm experiments revealed broad and surprisingly continuous variation in the P-content of individual clones of D. pulex (range 1.54–1.05%) and D. mendotae (1.51–1.07%) over a gradient in dietary C:P. The broad range in P-content exhibited by individual clones, acclimated for generations, suggests that variation in Daphnia P-content from laboratory experiments needs to be interpreted with caution. These results also show that phenotypic variation in response to environment can be a larger source of variation in P-content than genetic differences within or among species.     

Locally adapted gut microbiomes mediate host stress tolerance

While evidence for the role of the microbiome in shaping host stress tolerance is becoming well-established, to what extent this depends on the interaction between the host and its local microbiome is less clear. Therefore, we investigated whether locally adapted gut microbiomes affect host stress tolerance. In the water flea Daphnia magna, we studied if the host performs better when receiving a microbiome from their source region than from another region when facing a stressful condition, more in particular exposure to the toxic cyanobacteria Microcystis aeruginosa. Therefore, a reciprocal transplant experiment was performed in which recipient, germ-free D. magna, isolated from different ponds, received a donor microbiome from sympatric or allopatric D. magna that were pre-exposed to toxic cyanobacteria or not. We tested for effects on host life history traits and gut microbiome composition. Our data indicate that Daphnia interact with particular microbial strains mediating local adaptation in host stress tolerance. Most recipient D. magna individuals performed better when inoculated with sympatric than with allopatric microbiomes. This effect was most pronounced when the donors were pre-exposed to the toxic cyanobacteria, but this effect was also pond and genotype dependent. We discuss how this host fitness benefit is associated with microbiome diversity patterns.Subject terms: Microbial ecology, Microbial ecology, Freshwater ecology, Microbiome     

Positive selection of digestive proteases in Daphnia: A mechanism for local adaptation to cyanobacterial protease inhibitors

Due to the combined effects of global warming and eutrophication, the frequency of deleterious cyanobacterial blooms in freshwater ecosystems has increased. In line with this, local adaptation of the aquatic keystone herbivore Daphnia to cyanobacteria has received major attention. Besides microcystins, the most frequent cyanobacterial secondary metabolites in such blooms are protease inhibitors (PIs). Recently, it has been shown that a protease gene showed copy number variation between four D. magna populations that differed in tolerance to PIs. From that study, we chose two distinct populations of D. magna which had or had not coexisted with cyanobacteria in the past. By calculating F_ST values, we found that the two populations were genetically more distant in the protease loci than in neutral loci. Population genetic tests applied to the tolerant population revealed that positive selection was most probably acting on the gene loci of the digestive protease CT448 and CT802. We conclude that the selection of digestive proteases and subsequent reduction in copy number is the molecular basis of evolutionary changes leading to local adaptation to PIs.     

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.     

Population-Genomic Analysis Identifies a Low Rate of Global Adaptive Fixation in the Proteins of the Cyclical Parthenogen Daphnia magna

Daphnia are well-established ecological and evolutionary models, and the interaction between D. magna and its microparasites is widely considered a paragon of the host-parasite coevolutionary process. Like other well-studied arthropods such as Drosophila melanogaster and Anopheles gambiae, D. magna is a small, widespread, and abundant species that is therefore expected to display a large long-term population size and high rates of adaptive protein evolution. However, unlike these other species, D. magna is cyclically asexual and lives in a highly structured environment (ponds and lakes) with moderate levels of dispersal, both of which are predicted to impact upon long-term effective population size and adaptive protein evolution. To investigate patterns of adaptive protein fixation, we produced the complete coding genomes of 36 D. magna clones sampled from across the European range (Western Palaearctic), along with draft sequences for the close relatives D. similis and D. lumholtzi, used as outgroups. We analyzed genome-wide patterns of adaptive fixation, with a particular focus on genes that have an a priori expectation of high rates, such as those likely to mediate immune responses, RNA interference against viruses and transposable elements, and those with a strongly male-biased expression pattern. We find that, as expected, D. magna displays high levels of diversity and that this is highly structured among populations. However, compared with Drosophila, we find that D. magna proteins appear to have a high proportion of weakly deleterious variants and do not show evidence of pervasive adaptive fixation across its entire range. This is true of the genome as a whole, and also of putative ‘arms race’ genes that often show elevated levels of adaptive substitution in other species. In addition to the likely impact of extensive, and previously documented, local adaptation, we speculate that these findings may reflect reduced efficacy of selection associated with cyclical asexual reproduction.     

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.     

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.     

An SNP-based second-generation genetic map of Daphnia magna and its application to QTL analysis of phenotypic traits

Background

Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories.

Results

Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures.

Conclusions

The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1033) contains supplementary material, which is available to authorized users.     

Does trimethylamine induce life-history reactions in Daphnia?

Laboratory experiments were carried out to investigate life-history reactions of Daphnia to trimethylamine (TMA), a substance, which has recently been found to induce a similar phototactic reaction in Daphnia as fish kairomones. The effects of different treatments (control, fish kairomone and TMA) on the life-history traits of five clones of D. magna and one clone of D. galeata and D. hyalina, respectively, were studied. Only D. magna exhibited significant reactions to TMA and to fish kairomone. D. galeata showed no significant responses to either TMA or to fish kairomone. D. hyalina reacted to the fish treatment with two traits (size and number of eggs at maturity). The comparison of the reaction norms to TMA and fish kairomone demonstrated that the directions of some responses to both factors were similar but were different for others such as for size at maturity. TMA resulted in a larger size at maturity in D. magna, whereas fish kairomone had no significant influence on this trait. We, therefore, conclude that trimethylamine is not the primary causative chemical agent in fish induced life-history adaptations in Daphnia.     

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.     

Depth-selection behavior and longevity in Daphnia: an evolutionary test for the predation-avoidance hypothesis

The classic evolutionary theory of senescence predicts that long lifespan evolves under low risk of extrinsic mortality. As lakes present planktonic animals with vertical gradients of mortality risk associated with fish predation, we expected the individual lifespan of Daphnia of the “hypolymnetic” clones to be longer than that of “epilimnetic” Daphnia. In order to test this prediction, we performed a laboratory study on 14 clones from the D. longispina species complex, taken during the daytime either from epilimnion or hypolimnion of three mesotrophic lakes. “Epilimnetic” Daphnia started reproduction earlier, aged faster, and lived shorter than their “hypolimnetic” conspecifics. Our results indirectly support the predation-avoidance hypothesis as the ultimate explanation for depth-selection behavior in Daphnia.