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A Sampling of DGC Projects

Studies of the neural control of movement
Tanscriptomics in mode of toxicity and physiological condition studies
The Red Queen in action
Eco-evolutionary dynamics and adaptation
Chemical stress ecology and ecotoxicogenomics
Neuroendocrinology and peptidergic systems
Ecological genomics of body size variation
Functional genomics of endocrine disruption
Evolutionary genetics of reproduction
Motor proteins in insects and beyond
Quantitative proteomics
Inducible defenses
Evolutionary genomics
The evolution and function of Wnt signaling
Ecological developmental biology on polyphenisms
Adaptive control systems in response to environmental changes
Epigenetic inheritance and the control of offspring development
Transposable element annotation
Causes and phenotypic consequences of gene copy number variation
Physiological responses to climate change
C2H2 Zinc Finger genes
ABC proteins
Mechanism of gene regulation in response to environmental stimuli
Molecular mechanisms of inducible defenses
Using metabolomics approaches to assess toxicity
Genes involved in responding to predator odors and in adaptation to cyanobacterial toxins
Genetic engineering of Daphnia
Resurrection ecology and environmental genomics
Comparative analysis of nervous system development in arthropods

Studies of the neural control of movement in Daphnia

PROJECT DESCRIPTION: The broad goal of our work is to study neural development, neural control of movement, and neurodegeneration using the model organism, Daphnia, the water flea. We have created tools to reliably record and quantify swimming behavior and the movement of the post-abdominal claw. Current work aims to define the role of dopamine in Daphnia movement and to discover the effect on Daphnia movement of pharmacologically manipulating or destroying dopamine neurons. Further, this project aims to clone and characterize key genes involved in dopamine neuron function in Daphnia. Initial studies show that perturbation of the dopamine neurotransmitter system has a robust effect on Daphnia swimming behavior. Our data suggest that Daphnia is a useful model system to evaluate the pathophysiology of dopamine neuron degeneration as well as potential neuroprotective agents.

PROJECT LEADER: Matthew L. Beckman
INVESTIGATORS: Undergraduate students

Daphnia transcriptomics in mode of toxicity and physiological condition studies

PROJECT DESCRIPTION: Several thousands of chemicals are released into the environment and result in exposure of plants, animals and man. In most cases there is very little or no information concerning the mode of toxic action of these compounds. We use transcriptomics and proteomics approaches to create gene and protein expression level signatures of the molecular modes of action which are responsible for the toxicity of the compounds. This information is used to identify the targeted reaction pathways and cluster compounds in structural and functional toxicity classes. An intended application is the use of this information in the early detection of potential toxicity targets in the development of new chemicals and the molecular design of the compounds. Within this context we use Daphnia as one of the model species to generate molecular level signatures. These results are related to higher level effects such as effects on condition, growth and development which are of direct relevance for risk assessment purposes. The sensitivity of organisms to toxicants also depends on their overall condition, which in turn depends on a number of external and internal factors including aquatic exposure conditions, nutritional status and aging. Using the molecular approaches outlined above we explore in which ways these factors influence homeostatic regulation and alter the capacity of organisms to respond to natural and chemical stressors.

PROJECT LEADERS: Ronny Blust & Dries Knapen
INVESTIGATORS: Nathalie Dom, Melissa Penninck & Kris Laukens

The Red Queen in action in Daphnia: host-parasite coevolution revisited in a changing world

PROJECT DESCRIPTION: Our research project aims at obtaining insight into host-parasite Red Queen dynamics through time and across environments. We capitalize on a unique model system: archived sediments of the water flea Daphnia and its micro-parasites to document these dynamics in a changing world using a molecular and genomics perspective. We unite archived sediment samples with functional genomics to discover the genetic basis of coevolution in the wild and to investigate how changing environments modify host-parasite coevolutionary dynamics with a focus on species translocation and changes in ecosystem quality. Focus lays on (1) the reconstruction of the genomic underpinning of host-parasite coevolutionary dynamics; (2) the integration of Daphnia-parasite coevolutionary dynamics in a broader perspective, taking into account changing environments; (3) studying food quality effects on host-parasite interactions combining a biochemical with an eco- evolutionary approach. Our research will contribute to a better understanding of the influence of the environment on host-parasite coevolution and will allow to evaluate the general impact of environmental variation in coevolutionary dynamics on biodiversity.

PROJECT LEADER: Ellen Decaestecker
INVESTIGATORS: Marlies Coopman, Benjamin Lange, Lien Reyserhove, Isabel Vanoverberghe (in collaboration with Koenraad Muylaert and Imogen Foubert), Francisco Bonachela, Kuchi Srikeerthana (in collaboration with Patrick De Causmaecker) & Kevin Pauwels, Dino Verreydt (in collaboration with Luc De Meester)

Eco-evolutionary dynamics and adaptation genomics using Daphnia as model system

PROJECT DESCRIPTION: Our research group has heavily invested in the study of micro-evolutionary responses to environmental change, using the water flea Daphnia as a model system. Since the group established 15 years ago, the research has gradually built in complexity, taking a multi-stressor approach, and using layered dormant egg banks to reconstruct evolutionary responses in natural populations along extended time axis. Much attention has always been directed towards antagonistic biotic interactions such as predation and parasitism, because of the strong impact these stressors have on the dynamics of zooplankton populations and communities. Recently, we explicitly include responses to anthropogenic stressors (climate change, land use). Our overall aim is to understand how natural populations and communities adapt to environmental changes. We take a multifaceted approach, in which we combine genomics with studies on trait variation, and relate these to environmental gradients in space and time and community and ecosystem characteristics. We use a genome scan approach to identify signatures of evolution in wild populations along well-characterized environmental gradients, and validate the observed responses using paleogenomics (layered dormant egg banks) and experimental evolution. In this way we aim at an understanding of the genetic basis underpinning adaptation of Daphnia to natural environmental gradients, a key objective in biology and of the rapidly developing field of ecological and environmental genomics of which Daphnia is a key model species. We are in the process of identifying candidate genes responsible of adaptive responses to key selection pressures and their correlated fitness differences in the wild. The most important asset of our research group is the established ecological tradition that informs the newly emerging genomics studies, allowing the identification of ecologically relevant adaptive genomic responses in the wild.
K.U.Leuven is coordinator of the ESF collaborative project STRESSFLEA, focusing on ecological genomics of Daphnia and hosted the Daphnia Genomics Consortium 2010.

INVESTIGATORS: Luisa Orsini, Kevin Pauwels, Joost Vanoverbeke, Mieke Jansen, Cathy Duvivier, Aurora Geerts, Sarah Rousseaux, Katina Spanier, Ine Swillen, Evelyne Vanvlasselaer, Dino Verreydt & Ellen Decaestecker

Daphnia as a model species in chemical stress ecology and ecotoxicogenomics: towards improved ecological risk assessment of chemical pollution

PROJECT DESCRIPTION: We use Daphnia as our premier model organism to investigate the response of freshwater biota to chemical pollution (primarily metals and insecticides). This response is studied at multiple levels of organization, such as gene expression, physiology, life-history up to the population level, including rapid micro-evolutionary responses.
Our primary aim is to contribute to the improvement of existing methods for ecological risk assessment and the setting of water quality criteria for chemicals. In general, this is achieved through research towards:

  1. Understanding the mode of action by which chemicals exert their toxic effects (ecotoxicogenomics)
  2. Quantifying the combined and interactive effects of chemicals with natural stressors and/or climate change (e.g. hypoxia, harmful algal blooms) (chemical stress ecology)
  3. The development of predictive models to extrapolate from the laboratory to the field, from lower to higher levels of organization, and from single-stressor to multi-stressor effects.
    Within this general context, we are currently working with Daphnia sp. on following research topics:
  4. The cost-of-adaptation and cross-tolerance hypotheses. Are populations of Daphnia that have genetically adapted to chemical stress (e.g., Cd) more or less tolerant to other stressors (e.g. cyanobacteria)? And if so, what is the genomic explanation? (Dieter De Coninck, in collaboration with Indiana University)
  5. DNA-methylation in Daphnia magna: How many and which loci are methylated? How is this modified by exposure to stressful conditions (chemicals, fish predation)? Which role can it play in plasticity and adaptive responses (Dr. Michiel Vandegehuchte, ESF Project STRESSFLEA).
  6. Linking synergistic effects of insecticides and cyanobacteria exposure in D. pulex at the level of gene expression to the individual level (changes in life-history traits) using whole-genome expression micro-arrays. (Jana Asselman, in collaboration with Indiana University)
  7. The genetic variation and (broad sense and narrow sense) heritability of fitness traits under metal stress in natural (Belgian) populations of D. magna. In a next step, we will explore the physiological and genetic basis of this variation. (Marlies Messiaen, in collaboration with KULeuven)

    PROJECT LEADERS: Karel De Schamphelaere & Colin Janssen
    INVESTIGATORS: Michiel Vandegehuchte, Marlies Messiaen, Dieter De Coninck & Jana Asselman

Neuroendocrinology and peptidergic systems in Daphnia

PROJECT DESCRIPTION: We are interested in the neuroendocrinology of Daphnia magna and Daphnia pulex. Neuropeptides and protein hormones play pivotal roles in most if not all regulatory systems of all crustaceans usually acting with diurnal or seasonal rhythms. These factors are certainly regulating growth by moulting, circadian behaviours such as dial vertical migration, and reproduction. Currently we are focussing on the unravelling of all neuropeptide and protein hormone genes and derived products of D. pulex, their localisation in the central and peripheral nervous system, and the study of elements of the Daphnia circadian clock. Neuropeptide and protein identifications by chromatographic, electrophoretic and mass spectrometric techniques and precursor cloning and synthesis of peptides for bioassaying are used to identify neuroendocrine and neuromodulatory key players, partly in collaboration with Leopold Ilag's group from the Dept. of Analytical Chemistry at Stockholm University.

PROJECT LEADER: Heinrich Dircksen
INVESTIGATORS: Heinrich Dircksen & Johannes Strauss

Ecological genomics of body size variation in Daphnia

PROJECT DESCRIPTION: Background: The insulin signaling pathway (ISP) has a key role in major physiological events like carbohydrate metabolism and growth regulation. The ISP has been well described in vertebrates and in a few invertebrate model organisms but remains largely unexplored in non-model invertebrates. This study is the first detailed genomic study of this pathway in a crustacean species, Daphnia pulex.
Results: The Daphnia pulex draft genome sequence assembly was scanned for major components of the ISP with a special attention to the insulin-like receptor. Twenty three putative genes are reported. The pathway appears to be generally well conserved as genes found in other invertebrates are present. Major findings include a lower number of insulin-like peptides in Daphnia as compared to other invertebrates and the presence of multiple insulin-like receptors (InR), with four genes as opposed to a single one in other invertebrates. Genes encoding for the Dappu_InR are likely the result of three duplication events and bear some unusual features. Dappu_InR-4 has undergone extensive evolutionary divergence and lacks the conserved site of the catalytic domain of the receptor tyrosine kinase. Dappu_InR-1 has a large insert and lacks the transmembranal domain in the beta-subunit. This domain is also absent in Dappu_InR-3. Dappu_InR-2 is characterized by the absence of the cystein-rich region. Real-time q-PCR confirmed the expression of all four receptors. EST analyses of cDNA libraries revealed that the four receptors were differently expressed under various conditions.
Conclusions: Duplications of the insulin receptor genes might represent an important evolutionary innovation in Daphnia as they are known to exhibit extensive phenotypic plasticity in body size and in the size of defensive structures in response to predation.

PROJECT LEADER: France Dufresne

Functional genomics of endocrine disruption in Daphnia

PROJECT DESCRIPTION: Wastes generated from intensive agricultural practices pose significant risks to aquatic ecosystems by affecting the endocrine system of vertebrate and invertebrate animals. The overall goal of this project is to develop and test novel organismal biosensors for the screening of selected high risk target compounds with endocrine disrupting properties from agricultural effluent. Using Daphnia magna we combine novel functional genomics technologies to test the endocrine and neuroendocrine response of Daphnia to these compounds.

PROJECT LEADER: Andreas Heyland

Evolutionary genetics of reproduction

PROJECT DESCRIPTION: Research focuses on sexual and asexual reproduction, primarily using the planktonic crustacean, Daphnia pulex as a model organism. Daphnia pulex occurs as two forms, cyclical parthenogens with sexual reproduction and obligate parthenogens with no sexual reproduction. Both forms co-occur in southern Ontario but are rarely found in the same pond. We have been determining sex allocation variation in the cyclical parthenogens and the avoidance of the cost of males in the obligate parthenogens. Some obligate parthenogen clones can produce males that can mate with sexual females from the cyclical parthenonogens and generate new obligately parthenogenetic clones. Current experiments are using microsatellite genetic markers to examine the genetic structure of Daphnia pulex populations, competition between the two reproductive forms and the evolutionary dynamics of the formation of new obligately parthenogenetic clones.

COLLABORATORS: Melania Cristescu & Teri Crease

Motor proteins in insects and beyond

PROJECT DESCRIPTION: Motor proteins have extensively been studied in the past and consist of large superfamilies. They are involved in diverse processes like cell division, cellular transport, neuronal transport processes, or muscle contraction, to name a few. E.g. vertebrates contain up to 60 myosins and about the same number of kinesins that are spread over more than a dozen distinct classes.
In this project, we performed the comparative genomic analysis of the motor protein repertoire of 21 completely sequenced arthropod species using the owl limpet Lottia gigantea as outgroup. Arthropods contain up to 17 myosins grouped into 13 classes. The myosins are in almost all cases clear paralogs, and thus the evolution of the arthropod myosin inventory is mainly determined by gene losses. Arthropod species contain up to 29 kinesins spread over 13 classes. In contrast to the myosins, the evolution of the arthropod kinesin inventory is not only determined by gene losses but also by many subtaxon and species-specific gene duplications. All arthropods contain each of the subunits of the cytoplasmic dynein/dynactin complex. Except for the dynein light chains and the p150 dynactin subunit they contain single gene copies of the other subunits. Especially the roadblock light chain repertoire is very species-specific.
All 21 completely sequenced arthropods, including the twelve sequenced Drosophila species, contain species-specific sets of motor proteins. The phylogenetic analysis of all genes as well as the protein repertoire placed Daphnia pulex closest to the root of the Arthropoda. The louse Pediculus humanus corporis is the closest relative to Daphnia followed by the group of the honeybee Apis mellifera and the jewel wasp Nasonia vitripennis. After this group the rust-red flour beetle Tribolium castaneum and the silkworm Bombyx mori diverged very closely from the lineage leading to the Drosophila species.

PROJECT LEADER: Martin Kollmar
INVESTIGATORS: Florian Odronitz & Bjoern Hammesfahr

Quantitative proteomics

PROJECT DESCRIPTION: Proteins fulfill most of the functions that are crucial for cellular survival. In addition, it is becoming increasingly clear that proteins rarely act alone, but that they constitute intricate networks, both among themselves and with other biomolecules. This system is both robust and dynamic, allowing a cell to respond to external cues, and an organism to develop from an embryonic to a mature state. Our interest is to understand cellular behavior from this perspective, realizing that one needs to study proteins collectively rather than in isolation, and dynamically rather than under a static condition.
Our research is centered on quantitative proteomics, combining biochemistry, analytical chemistry, mass spectrometry and bioinformatics. Our lab is equipped with state-of-the-art mass spectrometric technology (Thermo Orbitrap Velos, Bruker Maxis Qq-Tof) that we use for the development of quantitative proteomic techniques using stable-isotope labeling (e.g. SILAC and chemical approaches), and their application to various (model) organisms, ranging from yeast, via Daphnia, to mammals.
Our biological interest falls apart in three main topics:
1. Investigating the changing proteome in haematopietic stem cells as they progress from a quiescent to an activated state.
2. Investigating protein turnover, defined by protein synthesis and degradation. Using chemical biology tools, we can selectively capture proteins that are newly synthesized upon cellular stimulation, isolating them from the background of 'old' (pre-existing) proteins. Profiling these newly synthesized proteins quantitatively over time provides a valuable link between genome regulation and protein output.
3. Identifying proteins that interact with DNA in a sequence-specific manner. This is complementary to the concept of chromatin IP, where we don't ask the question where a particular protein binds to the genome, but rather what proteins bind to a defined genomic region, such as enhancer elements.

PROJECT LEADER: Jeroen Krijgsveld

Inducible defences in Daphnia: Molecular basis and multiple stressors

PROJECT DESCRIPTION: Predation is a key factor in the evolution of prey species and the dynamics of prey communities. Phenotypic plasticity in defensive traits appears to be an appropriate mechanism to cope with the variable hazard of a frequently changing predator system.
Formations of protective devices in Daphnia, such as helmets and spines, are prominent examples of these chemically induced defenses in response to predator stress. These often dramatic phenotypic changes are ideal for exploring the adaptation to specific environmental conditions from their ecological to their genetic basis.
In the framework of the Daphnia Genomics Consortium we study the molecular basis of adaptation to predation stress, focusing on morphological plastic defensive traits.
Molecular mechanism involved in the adaptation to an environment cannot be exclusively deduced from genomic or transcriptional data. Comprehensive datasets addressing the protein level, therefore, are indispensable for a functional characterization of a specific trait. Hence, a proteome approach (in close collaboration with Georg Arnold and Thomas Fröhlich, LFUGA; LMU Munich) will be applied to show differences and similarities in protein expression in Daphnia faced to vertebrate and invertebrate predators to discover the proteins and pathways controlling different defensive strategies.
In addition our research addresses basic aspects of the effects of multiple stressors (micropollutants, parasites) on the evolution of predator-prey interactions. This might contribute to the understanding of how genotypes, phenotypes and the environment interact to effect individual fitness.

PROJECT LEADER: Christian Laforsch
INVESTIGATORS: Georg Arnold & Thomas Fröhlich (Proteomics), Wolfgang Engelbrecht, Quirin Herzog, Olivia Hesse, Hannes Imhof, Max Rabus, Kathrin Schoppmann, Robert Sigl & Linda Weiss (Group)

Evolutionary genomics

PROJECT DESCRIPTION: We are using the Daphnia pulex system to investigate four major issues in evolutionary genomics. (1) By comparing complete genome sequences from lineages with and without genetic recombination, we are obtaining the first direct insight into the molecular consequences of permanently abstaining from sexual reproduction. (2) We are also using this system to determine the mechanisms by which introns arise within protein-coding genes in natural populations. Daphnia pulex is the first organism to reveal introns in the process of establishing themselves. (3) We are performing mutation-accumulation experiments to estimate the rate and complete molecular spectrum of spontaneously arising mutations. (4) From comparative sequence analysis of the genomes of multiple D. pulex lineages as well as other Daphnia species, we are hoping to obtain insight into the mechanisms responsible for the preservation vs. demise of duplicate genes.


The evolution and function of Wnt signaling in protostomes

PROJECT DESCRIPTION: The Wnt genes encode secreted glycoprotein ligands that are important for many developmental processes in animals. The 13 Wnt gene subfamilies appeared early in animal evolution, but have since undergone numerous lineage specific loses and duplications. We employ the common house spider Achaearanea tepidariorum as a model to investigate the expression and function of Wnt genes and other components of Wnt signaling in chelicerates in comparison to other protostomes, including Daphnia, to understand the evolution of these genes and networks.

PROJECT LEADER: Alistair P. McGregor
INVESTIGATORS: Our research on Wnt signaling is carried out in collaboration with Ralf Janssen, Martine Le Gouar, Matthias Pechmann, Francis Poulin, Renata Bolognesi, Evelyn E. Schwager, Corinna Hopfen, John K. Colbourne , Graham E. Budd, Susan J. Brown, Nikola-Michael Prpic, Carolin Kosiol, Michel Vervoort, Wim G. M. Damen, & Guillaume Balavoine

Ecological developmental biology on arthropod polyphenisms

PROJECT DESCRIPTION: Phenotypes of organisms are not determined completely genetically, but vary according to environmental factors (phenotypic plasticity). Some organisms express several discrete adaptive phenotypes (polyphenism). Polyphenism can be explained as the modification of postembryonic development to produce alternative phenotypes. In our laboratory, we are studying on the polyphenism in daphniids, ants, termites and aphids, in terms of the developmental mechanisms of phynotype-specific characters. We are working on these topics, in terms of the alteration of body plan in response to environmental signals, and trying to understand the evolutionary process of the interaction between ontogeny and environment.
Daphnia provide us a model system studying the evolution of polyphenic development. We are currently working on the molecular basis underlying Daphnia polyphensim, mainly focusing on a representative phenomenon, inducible defense. In D. pulex, juveniles form neckteeth in response to kairomones, released by predatory Chaoborus larvae. To reveal the developmental mechanism of defensive morph, we observed detailed embryogenesis and postembryonic development. The kairomone-exposed embryos possessed the thickened epidermis at the back of head, where the neckteeth would be formed. The direct exposure on embryos and neonates showed that the reception and developmental mechanisms are still working even at the postembryonic stages. Investigations on growth rate and reproduction suggested that the there are several developmental regulations in response to kairomone. Based on these observations, we screened genes responsible for this developmental process. Results of candidate gene approach and differential display implied that several morphogenetic factors and endocrine pathways including juvenile hormone pathway are involved in the defense morph formation in D. pulex. We are currently trying to compare these developmental mechanisms between intra- and inter-species in order to elucidate evolutionary processes of phenotypic variation among Daphnia species.

INVESTIGATORS: Hitoshi Miyakawa & Naoki Sugimoto

Adaptive control systems of Daphnia in response to environmental changes

PROJECT DESCRIPTION: The planktonic microcrustaceans Daphnia play a central role in the ecology of standing freshwater and in their habitats, they are exposed to severe spatial and temporal changes in environmental conditions including water temperature and oxygen content or the quantity and quality of food (algae). Consequently, these zooplanktonic organisms have evolved quite a number of complex control systems to compensate for environmental change including behavioral, physiological, biochemical, and genetic mechanisms. In addition to analyses of these mechanisms on the organismal level, we are mainly interested in the cellular responses to environmental change including the principles of sensing and processing of environmental information and the downstream processes on the level of gene expressions and enzyme activations. One of the major regulatory elements under changing oxygen and temperature conditions is the respiratory protein hemoglobin, which has been studied in some detail by our working group. Other "single candidate" approaches deal with functional adjustments of metabolic enzymes (lactate dehydrogenase, mitochondrial enzymes) upon environmental change. These studies are complemented by proteomic approaches (and transcriptomic approaches in the near future) to identify further genes and proteins essential for environmental acclimation and adaptation. Recent proteomic studies revealed, for instance, an essential role of carbohydrate metabolism during hypoxia acclimation and protein metabolism during temperature acclimation. In the last years, main research focused on the cellular processes in Daphnia in response to environmental extremes, which included the mechanisms of stress sensing, the role of antioxidative defense systems, and the processes responsible for the functional integrity of macromolecules under stress.

PROJECT LEADER: Ruediger J. Paul
INVESTIGATORS: Bettina Zeis, Susanne Schwerin, Doerthe Becker & Yann Reydelet


PROJECT DESCRIPTION: Chemoreception, regarded as vitally important for all animals, encompasses olfaction, pheromone detection, hormonal signaling and various other essential processes. Similar responses to chemical cues are shared in a phylogenetically broad array of animals, implying that there is an ancient solution to the problem of detecting and discriminating odorants, and hence the potential to extrapolate from one system to others. However, to date our knowledge of chemoreception is based largely on the study of terrestrial chemoreception, and while lobsters and crabs behaviors have been studied, neither of their genomes is available for comparison. The Daphnia genome therefore is the first to offer us the opportunity to investigate aquatic chemoreception from a molecular and ecological perspective. We have identified a set of 58 gustatory receptor genes (Grs) of which 49 Grs are members of a Daphnia-specific clade (when compared to insects), but some of which resemble known arthropod receptors (e.g. 2-3 sugar receptors). This relatively small set of receptor genes is presumably the basis for much of Daphnia's complex behavioral repertoire. Our project currently focuses on identifying Grs which are differentially expressed in the sexes and embryonic stages, investigates gene sequence divergence and selection across sexual and asexual lineages, with a particular interest in sex-biased genes, and aims to characterize the evolution of the Gr gene family across the Daphnia genus. Our main goal is to establish a foundation for the molecular and evolutionary understanding of aquatic chemoreception in a model crustacean, using bioinformatics, gene evolution theory, and molecular techniques.

PROJECT LEADER: Carolina Peñalva-Arana

Epigenetic inheritance and the control of offspring development in Daphnia

PROJECT DESCRIPTION: Maternal effects have typically been studied from the perspective of maximising offspring fitness. However, recent work suggests that maternal effects evolve to optimize maternal fitness, irrespective of whether this increases or decreases the fitness of individual offspring. Consequently, mothers may have considerable control over how their offspring develop. In most cases, maternal effects are manifest as variation in the provisioning of offspring. However, the maternal environment may also induce epigenetic effects that alter offspring phenotypes and fitness. For example, in humans, 'fetal programming' has been linked with obesity, heart disease, and metabolic disorders. In this study we tested the hypothesis that epigenetic inheritance influences offspring phenotypes by comparing gene expression profiles of Daphnia pulex offspring that were exposed to a similar or very different nutritional environment to that experienced by their mother. Our findings demonstrate that offspring development is strongly influenced by the immediate nutritional environment but is not influenced by the maternal environment. These findings suggest that resource availability does not induce epigenetic inheritance in this system.

PROJECT LEADER: Stewart Plaistow
INVESTIGATORS: Steve Paterson & John Colbourne

Transposable element annotation in Daphnia

PROJECT DESCRIPTION: We identify transposable elements from the completed draft genome sequences of Daphnia, a cyclically parthenogenetic, aquatic microcrustacean of the class Branchiopoda.

COLLABORATORS: Ellen Pritham, Mina Rho, Haixu Tang & Michael Lynch

Causes and phenotypic consequences of gene copy number variation

PROJECT DESCRIPTION: Recent studies indicate copy number variation (CNV) represents a large source of the genetic variation observed in human populations and have uncovered strong associations between CNV and disease, including complex phenotypes. However, the environmental contributions to CNV remain unknown, in part because there are few animal models available for environmental genomics studies, which seek to understand how genome structure and function evolve in response to environmental change. Our group makes use of the NIH model, Daphnia, to test the central hypothesis that exposure to environmental contaminants increase the rate of mutations giving rise to CNV, and that this variation has functional consequences on gene expression, phenotype, fitness, and population structure. To accomplish this goal we are developing mutation accumulation (MA) lines derived in the absence and presence of cadmium in order to define the spectra of CNV and measure the per generation rate at which they spontaneously arise in individuals. We are studying past populations that have been captured and preserved in lake sediments. Here we scan genomes from populations that have adapted to over a century of mining pollution in order to characterize the magnitude, distribution, functional consequences, and evolutionary path of CNV in relation to the metal adapted phenotype. Finally, we are conducting quantitative trait loci experiments to determine the functional significance of CNV by establishing cause and effect relationships between copy number variants and metal tolerance. Collectively, these studies quantitatively assess whether environmental exposure affects the risk for spontaneous CNV, and do so in context of their contributions to individual health parameters that influence tolerance (i.e., adaptation and susceptibility) and disease. Answers to these questions have profound implications for the long-term health of human populations that are living longer and doing so in the presence of a greater diversity of chemicals that can modify DNA.

INVESTIGATORS: Joe Shaw, John Colbourne, Mike Lynch & Mike Pfrender


Physiological responses to climate change

PROJECT DESCRIPTION: We are interested in understanding how organisms respond to changes in abiotic conditions that are expected to occur with further climate change (e.g., warmer temperatures, ocean acidification) and shifts in human land and water use practices (e.g., reduced flow into estuarine and riparian habitats). Our studies use organismal physiology (e.g., respiration rates, tolerance thresholds), biochemical (e.g., enzymology), and genomic level (e.g., transcriptomic and proteomic) analyses, and include a broad range of organisms including adult and larval crabs, algae and Daphnia. Daphnia projects we are presently involved with include the potential for adaptive shifts in responses to thermal and salinity variability, and the evolutionary diversity of crustacean lactate dehydrogenases.

PROJECT LEADER: Jonathon Stillman

C2H2 Zinc Finger genes in Daphnia

PROJECT DESCRIPTION: A recent comparative genomic analysis tentatively identified roughly 40 orthologous groups of C2H2 Zinc-finger proteins that are well conserved in "bilaterians" (i.e. worms, flies, and humans). Here we extend that analysis to include a second arthropod genome from the crustacean, Daphnia pulex. Most of the 40 orthologous groups of C2H2 zinc-finger proteins are represented by just one or two proteins within each of the previously surveyed species. Likewise, Daphnia were found to possess a similar number of orthologs for all of these small orthology groups. In contrast, the number of Sp/KLF homologs tends to be greater and to vary between species. Like the corresponding mammalian Sp/KLF proteins, most of the Drosophila and Daphnia homologs can be placed into one of three sub-groups: Class I-III. Daphnia were found to have three Class I proteins that roughly correspond to their Drosophila counterparts, dSP1, btd, CG5669, and three Class II proteins that roughly correspond to Luna, CG12029, CG9895. However, Daphnia have four additional KLF-Class II proteins that are most similar to the vertebrate KLF1/2/4 proteins, a subset not found in Drosophila. Two of these four proteins are encoded by genes linked in tandem. Daphnia also have three KLF-Class III members, one more than Drosophila. One of these is a likely Bteb2 homolog, while the other two correspond to Cabot and KLF13, a vertebrate homolog of Cabot. Consistent with their likely roles as fundamental determinants of bilaterian form and function, most of the 40 groups of C2H2 zinc-finger proteins are conserved in kind and number in Daphnia. However, the KLF family includes several additional genes that are most similar to genes present in vertebrates but missing in Drosophila.

INVESTIGATORS: Arun Seetharam, Yang Bai & Gary W. Stuart

ABC proteins in the Daphnia pulex genome

PROJECT DESCRIPTION: The large gene superfamily of ABC proteins encodes membrane proteins involved in transmembrane transport, and proteins with other cell biological functions. In the present study, we identified ABC proteins in the Daphnia pulex genome, providing for the first time a survey of ABC transporters in a crustacean species. Daphnia possesses at least 64 ABC proteins, comprising members of all eight metazoan ABC subfamilies A to H. ABC proteins involved in fundamental cell biological functions were highly conserved between Daphnia and other animals. Similar to insects, Daphnia lacks homologues to the TAP protein, which is involved in antigene processing, and the chloride channel cystic fibrosis transmembrance conductance regulator (CFTR). A number of ABC proteins function as drug efflux transporters, and have high (eco-)toxicological relevance. Within this group, Daphnia possesses two proteins homologous to multidrug resistance (MDR) P-glycoprotein and six proteins homologous to Multidrug resistance-associated proteins (MRPs). Daphnia ABC transporters showed a high number of gene duplications in the subfamilies G and H. In conclusion, the survey of ABC proteins in the Daphnia pulex genome showed that this gene superfamily is as complex in crustaceans as it is in other metazoans.

INVESTIGATORS: Armin Sturm, Phil Cunningham & Michael Dean

Mechanism of gene regulation in response to environmental stimuli

PROJECT DESCRIPTION: Daphnia undergo characteristic changes in morphology and physiology in response to various environmental cues. These changes include the increasing the quantity of hemoglobin (Hb) in hemolymph in response to hypoxia that results in a change in body color from colorless to red, the development of defensive structures such as elongated helmet and neck teeth in response to chemical substances released by predators, and the switch from parthenogenetic to sexual reproduction under unfavorable environmental conditions such as food shortage, high population density, or exposing certain insecticides. We are interested in the molecular mechanisms of Hb gene regulation in response to hypoxia and switching the mode of reproduction. To elucidate these mechanisms, the following researches are currently being undertaken.
1. Regulation of Hb gene expression in response to hypoxia – To elucidate the regulation of hemoglobin genes, we search new regulatory elements related to expression of hemoglobin. We have found the non-protein coding RNA (ncRNA) gene in the hemoglobin gene cluster. We now focus on the function of this non-protein coding RNA. We investigate the expression pattern of this ncRNA during embryogenesis and in the adult.
2. Molecular mechanism of gametogenesis in Daphnia – To understand molecular mechanism of switching the reproduction mode, we have focused on mechanisms of germ cell proliferation and differentiation in the ovary and testis. During gametogenesis, meiotic maturation of gonad and early embryogenesis, the translation of maternal mRNAs is spatially and temporally controlled by several RNA binding proteins. We have isolated several cDNAs encoding RNA binding protein and analyzed expression pattern of them in the gonad.

PROJECT LEADER: Shinichi Tokishita

Molecular mechanisms of inducible defenses in Daphnia sp.

PROJECT DESCRIPTION: The model freshwater crustacean Daphnia shows a remarkable ability to respond to changing environmental conditions with phenotypic plasticity.
Different Daphnia species are capable to form morphological, behavioral and life-history adaptation in response to an increased risk of predation. This immense physiological competence of Daphnia to sense and interact with its environment can only be explained by a well-developed nervous system capable to compute environmental signals, which are subsequently translated into changes in gene expression as a response. The ability to discriminate between different environmental hazards such as a diversity of predator species is commonly regarded as one component in the structuring of natural ecosystems and therefore predator- prey interactions are considered as a primary force driving evolution. We are investigating the molecular mechanisms of inducible defenses from predator detection to differential gene expression. In order to unravel the underlying mechanisms we apply a variety of genomic and optical techniques to measure environmentally controlled differential gene expression with the aim to understand the pathways involved in phenotypic plasticity.

PROJECT LEADER: Ralph Tollrian
INVESTIGATORS: Florian Leese, Linda Weiss & Christoph Mayer

Using metabolomics approaches to assess toxicity in Daphnia magna

PROJECT DESCRIPTION: Currently there is widespread interest in exploiting 'omics approaches to screen for chemical toxicity in the context of ecological risk assessment and environmental monitoring. Direct infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) based metabolomics provides a sensitive and non-targeted analysis of potentially a thousand endogenous metabolites. Our previous work has shown that mass spectra can be recorded from whole-organism homogenate or haemolymph of single adult Daphnia magna. More recently we have developed robust multivariate classification models, with minimal classification error rates, and discovered perturbations to specific metabolic pathways that can discriminate between the acute toxicities of four chemicals to D. magna using FT-ICR MS metabolomics3. This work focused on model toxicants (cadmium, fenvalerate, dinitrophenol and propranolol) with different modes-of-action. Based upon the results, we concluded that whole-organism homogenates are the more information-rich sample type for use in predictive toxicology. The ability to measure metabolic fingerprints from individual daphniids raises the potential of correlating molecular toxicity to whole-organism chronic endpoints that are of ecological significance, in particular reproductive output. In our most recent work we have discovered metabolic biomarker signatures in individual D. magna exposed to model toxicants that are strongly predictive of reproductive output (in preparation). Furthermore, we has determined that biomarkers of reduced reproductive output can be elucidated that are either toxicant specific or more generic.

  1. NS Taylor et al. (2009) A new approach to toxicity testing in Daphnia magna: application of high throughput FT-ICR mass spectrometry metabolomics. Metabolomics 5(1):44-58.
  2. HC Poynton et al. (submitted) Integration of metabolomics and transcriptomic signatures offers a coordinated model of cadmium toxicity in Daphnia magna.
  3. NS Taylor et al. (2010) Discriminating between Different Acute Chemical Toxicities via Changes in the Daphniid Metabolome. Toxicological Sciences 118(1):307-317.

    PROJECT LEADER: Mark R. Viant
    INVESTIGATORS: Mark Viant, Nadine Taylor, Tom White & Alex Gavin

Genes involved in responding to predator odors and in adaptation to cyanobacterial toxins

PROJECT DESCRIPTION: Daphnia is well known for its high plasticity in many ecologically relevant traits. Plasticity in physiology, morphology, behavior and life-history helps to optimize fitness in fluctuating environmental conditions. In the Daphnia world, highly fluctuating parameters are the type of available food and the risk of being eaten by a predator. Infochemicals from predators induce adaptive diel vertical migration behavior and life-history changes in Daphnia. Using bioassay-guided fractionation of the infochemicals by HPLC crossed with gene-expression analyses and proteomics, we aim at identifying the infochemicals and deciphering the molecular basis of the response in Daphnia. In many lakes, Daphnia have to coexist with cyanobacteria, which are a very low-quality food for Daphnia. One reason, among others, for this low quality of cyanobacteria is the production of inhibitors and toxins, among which the production of protease inhibitors is particularly widespread in cyanobacteria. We have identified the targets of these inhibitors in Daphnia and look for micro-evolutionary adaptation of Daphnia populations to cyanobacterial toxins at the level of sequences and expression of target genes.

PROJECT LEADER: Eric von Elert
INVESTIGATORS: Anke Schwarzenberger, Patrick Fink, Christian Küster & Christopf Effertz

Genetic engineering of Daphnia

PROJECT DESCRIPTION: The waterflea Daphnia magna have been used as the subject of the ecology, evolution and environmental sciences for decades. While Daphnia genome project accelerated our understanding of daphniid biology at molecular levels, understanding of gene functions was still a great problem. In order to clarify gene functions of Daphnia, we are developing genetic engineering technology on Daphnia magna. Combined with DNA microarray technique, which we previously developed, this technology will help us to understand daphniid biology and contribute to evolutional, ecological, environmental and molecular biology.

PROJECT LEADER: Hajime Watanabe
INVESTIGATORS: Hajime Watanabe & Yasuhiko Kato

Resurrection ecology and environmental genomics: eutrophication on microevolutionary dynamics in Daphnia

PROJECT DESCRIPTION: How do anthropogenic environmental alterations affect the evolution of biotic entities and at what levels are these effects manifested? Traditional organismal and ecosystem approaches to this question indicate marked effects that are a result of changes in multiple pathways (e.g., metabolic, trophic). Given the important role organisms play in ecosystem processes, it is surprising that shifts in microevolutionary trajectories of biota have rarely been considered in the light of ecosystem-level changes (and vice versa). Here, the PIs will study the microevolutionary trajectories of physiologically relevant genes, pathways and systems that are involved in the handling of phosphorus (P), whose availability in the environment (especially freshwater systems) has increased drastically due to cultural eutrophication (e.g. high use of P-rich fertilizers).
The freshwater microcrustacean Daphnia pulex, which plays a major role in the freshwater
P-cycle (major sinks, remineralization) will serve as the model organism. Daphniid responses
to P-limitation at the organismal level are well-documented. Daphnia are ideal for such studies because they are cyclical parthenogens that produce resting eggs that can lay
dormant in lake sediments. Diapause can be broken experimentally in decades-old eggs and
viable DNA can be extracted from eggs that are centuries old. Moreover, the D. pulex
genome has been sequenced and microarray chips are available. Preliminary gene
expression have been obtained under contrasting P-supply environments (Jeyasingh et al. 2011, in press), and will be used to identify candidate loci. Therefore, the D. pulex - P-environment system is well-suited to analyze the reciprocity between organismal (i.e., genetic and physiological) change and ecosystem change in a set of well-studied Minnesota lakes along a eutrophication gradient. As such, this work is at the interface between evolutionary biology and ecosystem science - two disciplines rarely integrated.

PROJECT LEADERS: Lawrence J. Weider & Punidan D. Jeyasingh
INVESTIGATORS: Priyanka Chowdhury, John K. Colbourne, Billy Culver, Dagmar Frisch & Philip Morton

Comparative analysis of nervous system development in arthropods

PROJECT DESCRIPTION: Within euarthropods (insects, crustaceans, myriapods and chelicerates) the morphological and molecular mechanisms of early nervous system development have been analyzed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. We have confirmed that neuroblasts are present in the branchiopod Daphnia magna; however, in contrast to insects, malacostracan and branchiopod neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. Although conserved neural genes are involved in the formation of neural stem cells, asymmetric divisions and generation of neural precursors in D. magna, their expression patterns suggest that they perform different/additional functions. This raises the question how the molecular mechanisms of neuroblast selection have been modified during evolution to allow for the segregation of neuroblasts in insects on the one hand and the maintenance of neuroblasts in the neuroepithelium of crustaceans on the other hand. We are analyzing the function and interactions of genes that are known to be required for epithelial-mesenchymal transitions and the morphological consequences of the expression of these genes in the crustacean Daphnia magna. We are also analysing genes that are involved in the maintenance of neural stem cells in vertebrates (e.g. Notch signalling, EGF receptor signalling, Sox genes) and are comparing their functions in insects and crustaceans.

PROJECT LEADER: Angelika Stollewerk
COLLABORATORS: Dieter Ebert (University of Basel), Gerhard Scholtz (Humboldt-University of Berlin)
INVESTIGATORS: Petra Ungerer, Joakim Eriksson, Beate Mittmann, Marleen Klann

Figure: Expression of the neural genes snail (red) and prospero (blue) in neuroblasts of a Daphnia magna embryo

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