Mixtures of similarly acting compounds in Daphnia magna: From gene to metabolite and beyond
Introduction
In order to understand how organisms respond to environmental challenges there is a clear need for model species that are ecologically well defined, widely distributed geographically and easy to maintain and manipulate experimentally in the laboratory (Daphnia Genomics Consortium, 2005). The traditional (invertebrate) model species in biology, such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, have substantial shortcomings in this respect since little is known of their natural ecology and the selective pressures which individuals and populations are routinely exposed to in the environment. In contrast, the cladoceran Daphnia magna is one of the most studied organisms in the field of freshwater ecology. This is due to its global distribution within the temperate zone and prominent role in a remarkably diverse spectrum of aquatic ecosystems (mainly freshwater lakes, ponds, streams and rivers) where they serve as primary grazers of algae, bacteria and protozoans as well as being a primary prey item for fish (Tessier et al., 2000). Besides their prominent role in freshwater ecology studies, Daphnia are also one of the most commonly used organisms in aquatic toxicity assays representing 8% of all available experimental data for aquatic animals within toxicological databases (Denslow et al., 2007). This is mainly due to their sensitivity to a broad range of chemicals, small size, short life-cycle and ease of culturing (generation time in the laboratory is only one to two weeks, which rivals that of most other model eukaryotes). Moreover, the reproductive cycle of Daphnia is well suited for experimental genetic studies. Under normal conditions they reproduce parthenogenetically, providing a highly effective means for ensuring a defined genetic background. Additionally, D. pulex was recently the subject of a concerted sequencing project (Colbourne et al., 2005) which will certainly enhance the development of additional genomic Daphnia tools in the near future.
Several ecotoxicogenomic based studies featuring genomics and transcriptomics using Daphnia have recently been published (a.o. Heckmann et al., 2006, Heckmann et al., 2008, Iguchi et al., 2006, Poynton et al., 2007, Poynton et al., 2008, Soetaert et al., 2006, Soetaert et al., 2007, Vandenbrouck et al., 2009). These studies — which are mostly microarray based — are indicative of the potential of genomics approaches to gain insights into unknown modes of action (MoA) of chemicals (Heckmann et al., 2008, Poynton et al., 2007, Soetaert et al., 2006, Soetaert et al., 2007) and possibly in providing an aid for the classification of chemicals (Poynton et al., 2008, Watanabe et al., 2007). However, in order to properly interpret these molecular responses there is also a clear need to establish the link between these early warning ‘omics’ responses and the more classical ecotoxicological endpoints such as survival, growth and reproduction at the organism or population level. Other ‘omics’ based techniques (besides microarrays) might provide essential mechanistic information to complete these relationships (Oberemm et al., 2005). However to our knowledge, only a limited amount of studies have been published describing large scale protein analyses (hence proteomics) with Daphnia spp. (Schwerin et al., 2008, Zeis et al., 2009, Fröhlich et al., 2009). Also studies describing metabolomic profiling are scarce. The first Daphnia spp. based metabolic study was published quite recently (Taylor et al., 2009). FT-ICR mass spectrometry was used in that particular study to gain insight into the copper mode of action in daphnids. Since metabolomic based research is very well suited for functional genomics analysis, we report the (first) metabolic profile of D. magna using a combined Nuclear Magnetic Resonance (NMR) spectroscopy and Gas Chromatography Mass spectrometry (GC-MS).
The two substances assessed in this study (fluoranthene and pyrene) are both polycyclic aromatic hydrocarbons (PAHs). These common pollutants mostly occur as mixtures and are associated with urbanization and suburbanization in freshwater systems. However little is known about their effects on the physiological properties of macroinvertebrates. Therefore in this study our aim was to gain insights in the effects caused by pyrene and fluoranthene exposure as single compounds and binary mixtures to D. magna. Therefore, effects were measured at different biological levels of organization to provide an integrated view of many possible responses. Acute exposure experiments were performed to measure endpoints of a lower biological level (gene expression, metabolomic profiling, and energetic content) while a chronic exposure experiment was performed to evaluate endpoints with a higher biological relevance (survival, growth and reproduction). The results of the study indicate the potential for forming direct connections between the genome and the phenotype of organisms in populations and communities, both in natural environments and in settings modified by human disturbance.
Section snippets
Maintenance of D. magna cultures
D. magna clones were cultured in 1 L glass recipients containing aerated and bio-filtered tap water, each holding 20–25 individuals. The medium was renewed three times weekly and simultaneously the water fleas were fed a mixture of Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii in a 3:1 ratio (4 × 105 cells/mL). A constant temperature of 20 ± 1 °C and a photoperiod of 14 h light/10 h dark were maintained throughout the experiment.
Toxicant exposures
The organisms were exposed to two PAHs, fluoranthene and
Gene information
Differential gene expression was determined according to the criteria described in the Materials and methods section. In total, 47 mRNA fragments were selected as differentially expressed in (at least) one of the single compound treatments. Exposure to fluoranthene caused significant responses in 34 genes, while 27 gene fragments responded to pyrene treatment. In general, there were more genes repressed than induced in both treatments (Fig. 2) and 14 genes were common among both treatments. The
Discriminating PAHs: pyrene and fluoranthene
The molecular responses of two compounds with presumed similar modes of action were assessed in this study. The physicochemical properties of the chemicals show several similarities. Both are 4-ring structures which share the same molecular weight (Table 1), but fluoranthene is characterized by a slightly higher water solubility and Log Kow value.
Chronic exposures (21 days) of daphnids to different concentrations of fluoranthene and pyrene resulted in severely impaired reproduction in both cases
Conclusion
The toxicity of two PAHs was evaluated on different levels of biological organization. It was shown that these two substances with presumed similar modes or action based on chemical characteristics also display similar biological modes of action. Transcriptomic fingerprints of pyrene and fluoranthene could not be separated using hierarchical clustering. Metabolomic profiles (of the single compounds) with similar levels of toxicity could be separated using PLD-DA but based on the identity of the
Acknowledgments
This research was financially supported by the European Union (European Commission, FP6 Contract No. 003956). JLG is sponsored by the Royal Society (UK). OAHJ is grateful to Miss Karolina Łada and Dr. Mahon Maguire for useful discussions on the manuscript.
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These authors contributed equally to the experimental design, experimental work, data analysis, interpretation, and to the preparation of this manuscript.