Effect of nitrogen-deprivation on the cell proliferation and lipid accumulation in <i>Symbiodinium</i>.

<p>(A) Growth of <i>Symbiodinium</i> cells cultivated in control versus nitrogen-deprivation media. The data represents mean ± SD (n = 3). (B) The visualization of neutral lipid accumulation using BODIPY 493/503 in control vs. nitrogen-deprived cultures. Scale bar, 10 µm.</p

Identifying corals displaying aberrant behavior in Fiji’s Lau Archipelago

<div><p>Abstract</p><p>Given the numerous threats against Earth’s coral reefs, there is an urgent need to develop means of assessing reef coral health on a proactive timescale. Molecular biomarkers may prove useful in this endeavor because their expression should theoretically undergo changes prior to visible signs of health decline, such as the breakdown of the coral-dinoflagellate (genus <i>Symbiodinium</i>) endosymbiosis. Herein 13 molecular- and physiological-scale biomarkers spanning both eukaryotic compartments of the anthozoan-<i>Symbiodinium</i> mutualism were assessed across 70 pocilloporid coral colonies sampled from reefs of Fiji’s easternmost province, Lau. Eleven colonies were identified as outliers upon employment of a detection method based partially on the Mahalanobis distance; these corals were hypothesized to have been displaying aberrant sub-cellular behavior with respect to their gene expression signatures, as they were characterized not only by lower <i>Symbiodinium</i> densities, but also by higher levels of expression of several stress-targeted genes. Although these findings could suggest that the sampled colonies were physiologically compromised at the time of sampling, further studies are warranted to state conclusively whether these 11 scleractinian coral colonies are more stress-prone than nearby conspecifics that demonstrated statistically normal phenotypes.</p></div

The ultrastructural examination of morphological changes and LD formation in <i>Symbiodinium</i> after nitrogen deprivation.

<p>Transmission electron micrographs of <i>Symbiodinium</i> in control (A, B) and nitrogen-deprivation media (five days: C–D; seven days: E–F). Insets in A, C and D were magnified as B, D and F, respectively. Arrows in A, C, and E indicated cell walls, while arrowheads in F indicated the OsO₄-negative “inclusion bodies”. Abbreviations: LD, lipid droplet; Ch, chloroplast; S, starch granule; P, pyrenoids; N, nucleolus.</p

Light microscopy of the LDs purified from <i>Symbiodinium</i> cells after different treatments.

<p>The LDs were suspended in the (A) pH 7.5 grinding buffer, (B) pH 6.5 grinding buffer or (C) treated by the trypsin digestion. (D) Phospholipid analyses by TLC showing the presence of phospholipids (PLs) in purified LDs (the top layer during the centrifugation) but not lower layer fractions after detergent (0.1% Triton X-100) washing.</p

LDs purification and protein analyses.

<p>(A) SDS-PAGE analyses of isolated LDs fraction and the LD purity assessment by RuBisCO western blotting. <i>Symbiodinium</i> spp. cells harvested after five days of nitrogen deprivation were homogenized and fractionated to purify LDs as shown in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087416#s2" target="_blank">Materials and methods</a>” section. The purity of LDs was examined based on the absence of RuBisCO contamination by western blotting. Proteins bands 1 to 5 were excised for mass spectrometric analysis. (B) BODIPY 493/503 staining of isolated LDs displayed the abundance of neutral lipid.</p

The change of fatty acid compositions in TAGs of <i>Symbiodinium</i> after five days of nitrogen deprivation.

<p>Relative amounts (%) of fatty acid compositions in purified TAGs from total <i>Symbiodinium</i> were determined (see the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087416#s2" target="_blank">Materials and methods</a>” section). The data represents mean ± SD (n = 3).</p

The TLC analysis of lipids extracted from <i>Symbiodinium</i> spp. cells.

<p>The comparison between control and nitrogen-deprivation treated cells (A). The lipid content of the purified LDs from <i>Symbiodinium</i> after five days of nitrogen deprivation is shown in (B).</p

Identification of lipid droplet proteins in <i>Symbiodinium</i> spp.

<p>a)MS/mps(p): Mowse score/number of total matched peptides (numbers of different matched peptides).</p

Temporal effects.

<p>Sampling time (three categorical groupings: <10:00, 10:00–14:00, and >14:00) was found to affect the multivariate molecular physiological response (a), though this was mainly due to temporal variation within the <i>Pocillopora acuta</i> dataset only (b). Several notable outliers have been labeled in the first two panels, and only a few biplot rays have been labeled in each due to spatial constraints. The blue, red, and green centroids represent 95% confidence for the <10:00, 10:00–14:00, and >14:00 sampling times, respectively. For the loading scores, please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185857#pone.0185857.s002" target="_blank">S1 Data</a>, and the legend for (a-b) is found in (a). The main driver of this multivariate temporal effect for <i>P</i>. <i>acuta</i> was the <i>Symbiodinium</i> zinc-induced facilitator-like 1-like (<i>zifl1l</i>) mRNA (see canonical scores in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185857#pone.0185857.s002" target="_blank">S1 Data</a>.), and its expression has been plotted for each species at the three sampling time intervals (c). In this panel, the letters above the normal quantile plots reflect Tukey’s honestly significant difference groups (<i>p</i><0.05) for the interaction of species (<i>P</i>. <i>acuta</i> vs. <i>P</i>. <i>damicornis</i>) and time (2-way ANOVA interaction effect, <i>p</i><0.001). One-way ANOVA <i>p</i>-values (time effects) for each species analyzed in isolation have been provided in an inset.</p

Tonga pics for Dryad

Zip file containing images of the majority of the coral reef sites surveyed, as well as whole-colony and "macro" images of the sampled colonies and their tissues/polyps, respectively