Application of ¹H-NMR Metabolomic Profiling for Reef-Building Corals

<div><p>In light of global reef decline new methods to accurately, cheaply, and quickly evaluate coral metabolic states are needed to assess reef health. Metabolomic profiling can describe the response of individuals to disturbance (i.e., shifts in environmental conditions) across biological models and is a powerful approach for characterizing and comparing coral metabolism. For the first time, we assess the utility of a proton-nuclear magnetic resonance spectroscopy (¹H-NMR)-based metabolomics approach in characterizing coral metabolite profiles by 1) investigating technical, intra-, and inter-sample variation, 2) evaluating the ability to recover targeted metabolite spikes, and 3) assessing the potential for this method to differentiate among coral species. Our results indicate ¹H-NMR profiling of <i>Porites compressa</i> corals is highly reproducible and exhibits low levels of variability within and among colonies. The spiking experiments validate the sensitivity of our methods and showcase the capacity of orthogonal partial least squares discriminate analysis (OPLS-DA) to distinguish between profiles spiked with varying metabolite concentrations (0 mM, 0.1 mM, and 10 mM). Finally, ¹H-NMR metabolomics coupled with OPLS-DA, revealed species-specific patterns in metabolite profiles among four reef-building corals (<i>Pocillopora damicornis, Porites lobata, Montipora aequituberculata,</i> and <i>Seriatopora hystrix</i>). Collectively, these data indicate that ¹H-NMR metabolomic techniques can profile reef-building coral metabolomes and have the potential to provide an integrated picture of the coral phenotype in response to environmental change.</p></div

Flow-through tank conditions prior to sampling of reef-corals at the National Museum for Marine Biology and Aquarium.

<p><b>*</b>Measurements span the 2-week acclimation period in July 2011.</p><p>Flow-through tank conditions prior to sampling of reef-corals at the National Museum for Marine Biology and Aquarium.</p

OPLS-DA Model Results.

<p>*R²X and R²Y represent the goodness of fit between the X (metabolite data) and Y (predictor values) matrices. Q² assesses the accuracy and predictability of the model. A Q² value close to 1.0 represents a more predictive model.</p><p>OPLS-DA Model Results.</p

Reef-building corals have species-specific ¹H-NMR profiles.

<p>OPLS-DA models comparing ¹H-NMR profiles from (A) <i>Montipora aequituberculata, Pocillopora damicornis</i>, <i>Porites lobata</i> and <i>Seriatopora hystrix</i> and (B) between <i>M. aequituberculata</i> and <i>P. damicornis</i> only. Separation within and between species is represented by the t-orthogonal- and t-axis, respectively. Model statistics are reported (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111274#pone-0111274-t002" target="_blank">Table 2</a>). (C and D) Corresponding loading plots showing ¹H-NMR-bin coefficients. Variables driving separation in the 4-species OPLS-DA model (A) are identified with numbers corresponding to unknowns (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111274#pone-0111274-t004" target="_blank">Table 4</a>). Only significant variables are indicated for each model. Ala = Alanine, Trig = Trigonelline, Thre/Lac = Threonine/Lactate.</p

¹H-NMR profiles of <i>Porites compressa</i> are reproducible within and between coral colonies.

<p>(A) PCA comparing <i>Porites compressa</i>¹H-NMR metabolite profiles between technical, intra-colony and inter-colony samples. Profiles from inter-colony <i>P. compressa</i> samples were obtained using two extraction methods: method 1 and method 2 (B) Boxplots of percent relative standard deviation (% RSD) scores across ¹H-NMR variables comparing technical, intra- and inter-colony variability. The median is indicated (black bar) along with the quartile ranges and outlying values (open circles). Letters denote Kruskal-Wallis test results (p<0.001). Groups connected by the same letter are not significantly different.</p

Separation in metabolite profiles after experimental addition of alanine, glucose, and glycolic acid.

<p>(A) OPLS-DA model comparing the control, 0.1 mM, and 10 mM metabolite spiking treatments. Separation within and between treatments is represented by the t-orthogonal- and t-axis, respectively. Model statistics are reported (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111274#pone-0111274-t002" target="_blank">Table 2</a>). (B) Corresponding loading plot showing ¹H-NMR bin coefficients. Bins arising from each spiking compound are indicated. Ala = alanine, Glu = glucose, Gly = glycolic acid.</p

Kruskal-Wallis test results comparing spiking treatments.

<p>*ND = Not Detected.</p><p>Kruskal-Wallis test results comparing spiking treatments.</p