Computational protocol: NMR Profiling of Metabolites in Larval and Juvenile Blue Mussels (Mytilus edulis) under Ambient and Low Salinity Conditions

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[…] The 1D NMR spectra were processed using ACD/NMR Processor Academic Edition Software. Data were Fourier transformed with a backward linear prediction for the first 2 points and then baseline corrected, phased, and calibrated to the TSP peak prior to analysis. For relative quantification, we manually selected and integrated the peaks for TSP, alanine, β-alanine, taurine, glycine, betaine, homarine, and maleic acid (). For each compound of interest, we calculated the concentration following the method of Bharti and Roy []: (1)Mi =(IiHiIsHs)*Ms where M is the relative concentration, I is the integral of the peak(s), and H is the number of protons contributing to the signal for the peak of interest (i) relative to the maleic acid standard (s). The concentrations for the metabolites of interest were then standardized to the mean TSP concentration and adjusted for dry weight, to yield the relative concentration in µmol g−1 dry tissue weight. To make direct comparisons of the metabolites quantified from the larval and juvenile samples, we adjusted the dry weights for the larval samples to account for the mass contributed by the larval shell. For this analysis, we prepared Whatman GF/C™ Glass Microfiber filters (25 mm diameter) by soaking in reverse osmosis (RO) water for 1 h and drying them overnight at 65 °C. The filters were then ashed at 350 °C overnight in a Thermo Isotemp® (Waltham, MA, USA) Muffle Furnace and weighed. We placed a subset of larvae from each larval sample onto a filter and rinsed using 10 mL of 0.5 M ammonium formate to remove any residual salts from the seawater. The prepared samples were dried overnight at 65 °C and weighed (total dry weight), prior to ashing at 350 °C overnight to determine the ash-free dry weight (AFDW). The ratio of AFDW relative to dry weight was used to determine the proportion of the dry weight accounted for by the tissue (and not shell). The amino acid concentrations for each of the larval samples were then scaled by this proportion.The 2D NMR spectra were processed using SpinWorks 3.1 NMR data processor. We Fourier transformed the data using complex forward linear prediction, predicting from 2048 to 4096 points in the F2 dimension and from 512 to 2048 points in the F1 dimension. Spectra were baseline corrected, phased, and calibrated to the TSP peak at 0 ppm. The coupling partners were recorded for each peak from the representative veliger, pediveliger, gill, mantle, and adductor samples and put into an Excel spreadsheet. The identification of metabolites was completed manually through reference to the primary literature and the Human Metabolome Database []. Tissue-specific or stage-specific variation in the relative concentration of the key metabolites alanine, taurine, glycine, betaine, β-alanine and homarine were determined by analyzing the solute concentrations for larval and juvenile mussels held under ambient, control conditions using 1H NMR spectroscopy. To test for tissue- and stage-specific differences in the relative quantities of these metabolites we ran a one-way MANOVA in SPSS Statistics 22.0 (IBM Corporation, Armonk, NY, USA), with sample type (juvenile tissue or larval stage) as a fixed-factor, main effect using a Type III Sum of Squares model with an experiment-wide α = 0.05. We examined the univariate effects of sample type on the concentration of individual amino acids using separate one-way ANOVAs with sample type as a main effect against a Type III Sum of Squares model and adjusting the critical value (p = 0.05/6 = 0.008) to account for multiple comparisons.We investigated temporal variation for the five most abundant metabolites contributing to the FAA pool in juveniles under hypoosmotic conditions, glycine, alanine, taurine, betaine, and homarine. To test for the effects of low-salinity exposure on the concentrations of alanine, betaine, glycine, homarine, and taurine in juveniles, we used three one-way MANOVAs, one for each tissue type, with salinity treatment (control versus 24, 48, or 72 h exposure to 20 ppt) as a fixed-factor main effect using a Type III Sum of Squares model with an experiment-wide α = 0.05. We examined the univariate effects of salinity treatment on the concentration of individual amino acids using separate one-way ANOVAs with treatment as a main effect against a Type III Sum of Squares model and adjusting the critical value (p = 0.05/5 = 0.01) to account for multiple comparisons within each tissue type. Similarly, we investigated the temporal variation in six amino acids (those listed above plus β-alanine) in veliger and pediveliger larvae using two one-way MANOVAs. We examined the univariate effects of salinity treatment on the concentration of individual amino acids in larval stages using separate one-way ANOVAs with treatment as a main effect against a Type III Sum of Squares model and adjusting the critical value (p = 0.05/6 = 0.008) to account for multiple comparisons within each stage. Four cases were excluded from analysis because of sample loss during preparation. […]

Pipeline specifications

Software tools ACD/NMR Processor Academic Edition, SPSS
Databases HMDB
Applications Miscellaneous, NMR-based metabolomics
Organisms Mytilus edulis