Development of an NMR microprobe procedure for high‐throughput environmental metabolomics of Daphnia magna

Abstract

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.

Analysis of Sub-Lethal Toxicity of Perfluorooctane Sulfonate (PFOS) to Daphnia magna Using 1H Nuclear Magnetic Resonance-Based Metabolomics

“…Metabolite identification was performed using published 1 H NMR metabolite resonances as well as previous work in our laboratory with NMR-based metabolomics of D. magna [31,46,47]. Metabolites that were observed to make a significant contribution to the separation observed in the PCA scores plot included a number of amino acids, the sugar glucose, the nucleobase uracil, and the energy molecule adenosine triphosphate (ATP).…”

“…In addition, an unexposed control population (n = 10, 12 individuals each) was included as a reference comparison to the PFOS exposed populations. Based on previous experiments [31], each sample was comprised of 12 adult daphnids. This allows for the ~1 mg dry mass (7–8 daphnids) that is required for extraction to obtain a suitable 1 H NMR spectrum.…”

“…Previous research in our laboratory developed and tested different extraction methods for D. magna and also carried out detailed identification of polar metabolites using both one-dimensional and two-dimensional NMR spectroscopy [31]. A range of polar metabolites were extracted using a D 2 O-based buffer from D. magna based on the optimized method reported in Nagato et al [31].…”

“…As such, further information is required to determine the toxic mode of action (MOA) of PFOS in daphnids over a range of sub-lethal concentrations. This study focuses on the metabolic responses of adult D. magna with PFOS exposure using 1 H nuclear magnetic resonance (NMR) analysis of polar metabolites [31]. NMR-based metabolomics is widely used to study the toxicity of a range of environmental pollutants to various organisms that exist in different ecosystems [32,33,34,35,36,37,38,39].…”

“…Previous studies have used NMR-based metabolomics to characterize the sub-lethal toxicity of Daphnia species [18,22,23,27,42] to various anthropogenic stressors. Detailed NMR mapping of the D. magna metabolome has been performed recently and metabolite assignments have been verified using two-dimensional NMR experiments [31]. In this study, we build upon previous method development and toxicity studies to investigate how PFOS alters the metabolome of D. magna .…”