Nuclear magnetic resonance (NMR) is the primary platform used in high-throughput environmental metabolomics studies because its non-selectivity is well suited for non-targeted approaches. However, standard NMR probes may limit the use of NMR-based metabolomics for tiny organisms because of the sample volumes required for routine metabolic profiling. Because of this, keystone ecological species, such as the water flea Daphnia magna, are not commonly studied because of the analytical challenges associated with NMR-based approaches. Here, the use of a 1.7-mm NMR microprobe in analyzing tissue extracts from D. magna is tested. Three different extraction procedures (D2O-based buffer, Bligh and Dyer, and acetonitrile : methanol : water) were compared in terms of the yields and breadth of polar metabolites. The D2O buffer extraction yielded the most metabolites and resulted in the best reproducibility. Varying amounts of D. magna dry mass were extracted to optimize metabolite isolation from D. magna tissues. A ratio of 1-1.5-mg dry mass to 40 µl of extraction solvent provided excellent signal-to-noise and spectral resolution using (1)H NMR. The metabolite profile of a single daphnid was also investigated (approximately 0.2 mg). However, the signal-to-noise of the (1)H NMR was considerably lower, and while feasible for select applications would likely not be appropriate for high-throughput NMR-based metabolomics. Two-dimensional NMR experiments on D. magna extracts were also performed using the 1.7-mm NMR probe to confirm (1)H NMR metabolite assignments. This study provides an NMR-based analytical framework for future metabolomics studies that use D. magna in ecological and ecotoxicity studies.
Poor diet quality frequently constrains the growth and reproduction of primary consumers, altering their population dynamics, interactions in food webs, and contributions to ecosystem services such as nutrient cycling. The identification and measurement of an animal's nutritional state are thus central to studying the connections between diet and animal ecology. Here we show how the nutritional state of a freshwater invertebrate, Daphnia magna, can be determined by analyzing its endogenous metabolites using hydrogen nuclear magnetic resonance-based metabolomics. With a multivariate analysis, we observed the differentiation of the metabolite composition of animals grown under control conditions (good food and no environmental stress), raised on different diets (low quantity, nitrogen limited, and phosphorus limited), and exposed to two common environmental stressors (bacterial infection and salt stress). We identified 18 metabolites that were significantly different between control animals and at least one limiting food type or environmental stressor. The unique metabolite responses of animals caused by inadequate nutrition and environmental stress are reflective of dramatic and distinctive effects that each stressor has on animal metabolism. Our results suggest that dietary-specific induced changes in metabolite composition of animal consumers hold considerable promise as indicators of nutritional stress and will be invaluable to future studies of animal nutrition.
Environmental contextPerfluoroalkyl acids are persistent environmental contaminants that are also found in soils. We use a metabolomics approach based on nuclear magnetic resonance analyses to investigate the responses of earthworms to exposure to sub-lethal levels of two perfluoroalkyl acids. The results indicate that this metabolomics approach is able to delineate the toxic mode of action of contaminants present at sub-lethal levels. AbstractMetabolomics entails the analysis of endogenous metabolites within organisms exposed to an external stressor such as an environmental contaminant. We utilised 1H NMR-based metabolomics to elucidate sub-lethal toxic mechanisms of Eisenia fetida earthworms after exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Earthworms were exposed to a range of concentrations of PFOA (6.25 to 50 μg cm–2) and PFOS (3.125 to 25 μg cm–2) by contact tests for 2 days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, and the polar fraction was analysed by 1H NMR spectroscopy. NMR-based metabolomic analysis revealed heightened E. fetida toxic responses with higher PFOA and PFOS exposure concentrations. Principal component analysis (PCA) exhibited significant separation between control and exposed earthworms along PC1 for all PFOA and PFOS exposure concentrations. Leucine, arginine, glutamate, maltose and adenosine triphosphate (ATP) are potential indicators of PFOA and PFOS exposure as these metabolite concentrations fluctuated with exposure. Our data also indicate that PFOA and PFOS exposure may increase fatty acid oxidation and interrupt ATP synthesis due to a disruption in the inner mitochondrial membrane structure. NMR-based metabolomics has promise as an insightful tool for elucidating the environmental toxicology of sub-lethal contaminant exposure.
1H nuclear magnetic resonance (NMR)-based metabolomics was used to characterize the response of Daphnia magna after sub-lethal exposure to perfluorooctane sulfonate (PFOS), a commonly found environmental pollutant in freshwater ecosystems. Principal component analysis (PCA) scores plots showed significant separation in the exposed samples relative to the controls. Partial least squares (PLS) regression analysis revealed a strong linear correlation between the overall metabolic response and PFOS exposure concentration. More detailed analysis showed that the toxic mode of action is metabolite-specific with some metabolites exhibiting a non-monotonic response with higher PFOS exposure concentrations. Our study indicates that PFOS exposure disrupts various energy metabolism pathways and also enhances protein degradation. Overall, we identified several metabolites that are sensitive to PFOS exposure and may be used as bioindicators of D. magna health. In addition, this study also highlights the important utility of environmental metabolomic methods when attempting to elucidate acute and sub-lethal pollutant stressors on keystone organisms such as D. magna.
Environmental context. Phenanthrene is a persistent soil contaminant, whose toxic mode of action in earthworms has not been fully examined. We adopt a metabolomics approach, using 1 H nuclear magnetic resonance (NMR) spectroscopy, to measure the response of earthworms to sub-lethal phenanthrene exposure. The results indicate that NMR-based metabolomics may be used to monitor responses to sub-lethal levels of contaminants and to delineate their toxic mode of action.
Abstract.1 H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida to sublethal phenanthrene exposure. E. fetida were exposed via contact tests to six sub-lethal (below the measured LC 50 of 1.6 mg cm
À2) concentrations of phenanthrene (0.8-0.025 mg cm À2 ) for 48 h. Multivariate statistical analysis of the 1 H NMR spectra of earthworm tissue extracts revealed a two-phased mode of action (MOA). At exposures below 1/16th of the LC 50 , the MOA was characterised by a linear correlation between the metabolic response and exposure concentration. At exposures !1/16th of the LC 50 , the metabolic response to phenanthrene appeared to plateau, indicating a distinct change in the MOA. Further data analysis suggested that alanine, lysine, arginine, isoleucine, maltose, ATP and betaine may be potential indicators for sub-lethal phenanthrene exposure. Metabolite variation was also found to be proportional to the exposure concentration suggesting that NMR-based earthworm metabolomics is capable of elucidating concentrationdependent relationships in addition to elucidating the MOA of sub-lethal contaminant-exposure.
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