Mixtures of similarly acting compounds in Daphnia magna: From gene to metabolite and beyond

Daphnia are an important and widely studied model species in ecological and toxicological studies throughout the world and an official (OECD) recommended test organism. Their small size, wide distribution and easy growth conditions make this organism ideal for functional genomics based studies, including metabolic profiling and transcriptomics. In this study we used an integrated systems approach in which transcriptomic, metabolomic and energetic responses of juvenile (4 days old) daphnids were evaluated in response to exposure to two poly aromatic hydrocarbons (pyrene and fluoranthene) and binary mixtures thereof. In addition, these responses were linked to responses measured during chronic experiments (21 days) assessing survival, growth and reproductive traits. Custom Daphnia magna microarrays were used to assess transcriptomic changes. Hierarchical cluster analysis did not result in a clear distinction between the single compounds suggesting similar molecular modes of action. Cluster analysis with both the single compounds and the binary mixture treatments resulted in a separation of treatments based on differences in toxic ratios rather than component differences. Changes in the metabolic profiles of the organisms were investigated using Nuclear Magnetic Resonance Spectroscopy and Gas and Liquid Chromatography Mass Spectrometry. These multivariate metabolomic datasets were analyzed with Principal Components Analysis and Partial Least Squares Discriminant Analysis. The major metabolite changes responsible for the differences observed indicated a disturbance in aminosugar metabolism in all cases. The study demonstrates the potential of ‘omics’ to provide screening tools for monitoring of the freshwater environment — in invertebrate species — which is reasonably rapid, cost — effective and has the potential to greatly increase the amount of information obtained from aquatic toxicology testing.