Metabolomics of Microliter Hemolymph Samples Enables an Improved Understanding of the Combined Metabolic and Transcriptional Responses of Daphnia magna to Cadmium

Omic technologies offer unprecedented opportunities to better understand mode (s)-of-toxicity and downstream secondary effects by providing a holistic view of the molecular changes underlying physiological disruption. Crustacean hemolymph represents a largely untapped biochemical resource for such toxicity studies. We sought to characterize changes in the hemolymph metabolome and whole-body transcriptome to reveal early processes leading to chronic toxicity in the indicator species, Daphnia magna, after 24-h sublethal cadmium exposure (18 mu g/L, corresponding to 1/10 LC50). We first confirmed that metabolites can be detected and identified in small volumes (similar to 3-6 mu L) of D. magna hemolymph using Fourier transform ion cyclotron resonance mass spectrometry and NMR spectroscopy. Subsequently, mass spectrometry based metabolomics of hemolymph identified disruption to two major classes of metabolites: amino acids and fatty acids. These findings were compared to differentially expressed genes identified by a D. magna 44k oligonucleotide microarray, which included decreased levels of digestive enzymes and increased expression of cuticle proteins and oxidative stress response genes. The combination of metabolic and transcriptional changes revealed through KEGG pathway analysis and gene ontology, respectively, enabled a more complete understanding of how cadmium disrupts nutrient uptake and metabolism, ultimately resulting in decreased energy reserves and chronic toxicity.