HPLC-MS instrument settings were reset to a source voltage of 3.2 kV, sheath gas flow rate 40 (arbitrary units), auxiliary gas flow rate 20 (arbitrary units), capillary voltage 228 V, capillary temperature 320uC and tube lens voltage 275 V. The ion chromatogram for the m/z 259.3 fragment ion (carbon dioxide neutral loss) was displayed using a range of m/z 258.8–259.8. The area of the chromatographic peak corresponding to AA (tR = 5.6 min) was integrated using the genesis algorithm without the use of a prior smoothing function within the Xcalibur TMsoftware (Vers. 2.0.7, Thermo Scientific). For quantification, a standard curve of AA concentration was produced for comparison with the quantity of AA in experimental samples. A dilution gradient of AA standard (Sapphire Bioscience, Waterloo, NSW, Australia) was produced in that 5 mL injection volumes resulted in quantities of 156.25, 312.5, 625 and 1250 pg, respectively. A series of A. pulchella samples from the 72 hr incubation were diluted 1:200 and repeatedly run alternately to standard dilutions such that any drift in analysis response could be both observed and corrected for. Two standard curves were constructed by averaging the peak areas for run 1 & 2 and 2 & 3 for the AA dilution series. The amount of AA within the samples and standards was verified from a linear trendline formulae and the amount of AA in 5 mL (the injection volume) of the 1:200 diluted A. pulchella samples. The amount of AA was then converted to a concentration and adjusted to determine the AA quantity in undiluted samples. Octadeuterated arachidonic acid (D8-AA) (Sapphire Bioscience, Waterloo, NSW, Australia) was used as a traceable marker throughout the extraction process to measure the extraction efficiency of the FAs from the total lipids. The D8-AA standard was added to tissue homogenate at a final concentration of 0.8 ng/ mL prior to extraction and then compared to the known concentration of AA (see above) within each sample following the extraction. The D8-AA, which has eight deuterium atoms within its molecular structure, has a distinct isotopologue mass profile compared to the endogenous AA. D8-AA has an m/z of 311, however, ions of m/z 306–310 ions were also observed in the ESI-MS spectrum of the deuterium-labeled standard. The m/z 310 ion was the most abundant and likely represents the D7-AA isotopologue. It is assumed that the ionization efficiency of AA and the D8-AA standard are the same, since the isotopologues are chemically identical (although a very small difference in retention times is observed between different deuterated isotopologues). For a direct comparison of endogenous AA to the D8-AA standard, the m/z 303 ion of AA and the m/z 310 ion of the D8-AA standard were converted to mole fractions. Within an AA standard, the m/z 303 ion was calculated to have a mole fraction of 0.80. For the D8- AA standard, the mole fraction of the m/z 310 ion was determined to be 0.31. The chromatograms for the m/z 303 and 310 from a LC-MS spectrum of a sample containing both endogenous AA and D8-AA standard were integrated with the genesis function without prior smoothing. The peak area for the m/z 303 ion was then divided by the mole fraction 0.80 to give a peak area representative of all the isotopologues of AA (AA peak area). This process was repeated for the m/z 310 ion of the D8-AA standard using the mole fraction of 0.31 to give a peak area representative of all the isotopologues of D8-AA (D8-AA peak area). The previously known concentration of AA within an experimental sample was converted from ng/mL to molarity and used to calculate the molarity of the D8-AA by multiplying the molarity of AA by D8- AA peak area/AA peak area. The molarity calculated for D8-AA was then converted to ng/mL using the molecular mass of D8-AA. The measurement of extraction efficiency was then calculated by Cnidarian-Dinoflagellate Symbiosis Lipidomics dividing the measured D8-AA concentration by the pre-extraction known concentration (0.8 ng/mL) and expressed as a percentage. The FAs with increased 13C profiles were compared to known lipogenesis pathways from the online databases (Lipid library (www.lipidlibrary.aocs.org), lipidmaps (http://www.lipidmaps.org) and KEGG 1.3 lipid pathways (www.genome.jp/kegg/pathway). Transferability of Methods: Coral and Dinoflagellate Culture Model Systems To assess transferability of the DI13C incorporation method for other model systems that utilize dissolved HCO3- as a carbon source for metabolite synthesis and specifically lipogenesis the hermatypic coral Acropora millepora (Anthozoa, Cnidaria) complete with integral clade C3 symbionts and a separate clade B1 dinoflagellate culture were incubated in the same 13C enriched ASW as the A. pulchella model for 48 hr. Clade B1 culture was chosen as a comparable model culture as this clade is known to form a symbiosis with A. pulchella [34]. The incubation method for A. pulchella was adapted to accommodate the differences in the model systems as described below. The hermatypic coral, acropora millepora model. For the A. millepora model, an area of approximately 3610 cm Ø at the tips of the corymbose branches (8–10 main branches) from three colonies approximately 50 cm across was removed and immedi- ately placed in polycarbonate chambers containing the DI13C enriched ASW, and partly immersed in flow-through outdoor aquaria. Portions of coral colonies were removed from Heron Island Reef flat, Great Barrier Reef, Australia (see acknowledge- ments for permit details). The enriched 13C ASW was exchanged every 12 hr for a period of 48 hr and maintained in ambient light and temperature conditions (1100–2000 mmol m22 s21; 26– 27uC). The cluster of branches was then removed and frozen in liquid N2 for later processing. The processing method was adapted from the A. pulchella model to account for removal of tissue from the skeleton whereby a frozen 2 cm length of one branch of each individual colony was incubated in the chloroform/methanol extraction mixture in the dark overnight at 4uC. The remainder of the extraction protocol was as previously described for A. pulchella. Dinoflagellate clade B1 culture. Clade B1 dinoflagellate cultures were maintained in accordance with Rosic and Hoegh- Guldberg [35]. The method of 13C incorporation was adapted for use with the culture model by mixing the F2 media (Guillard’s Marine Water Enrichment – Solution, Sigma) with the 13C enriched ASW as used with the A. pulchella model. After 48 hr the enriched treated culture were removed from the media by centrifugation at 2775 RCF for 5 min. Following removal of the supernatant, the pellet was resuspended in ASW and re- centrifuged to rinse excess enriched media. Following an additional rinse the culture was centrifuged at 92006g for 10 min and the pellet frozen in liquid N2 for later processing. The culture pellets were then treated in the same way as for the A. pulchella extractions. Statistics. All statistical test comparisons were undertaken with MinitabTM v16 software. Two-tail paired t-tests were performed on total arachidonic acid concentration and total lipid extraction efficiencies. Following Anderson-Darling tests for normality and box-plot tests for heteroscedasticity, one-way ANOVA tests were performed with multiple tukey pairwise post- hoc comparisons for the isotopic incorporation (n = 4) over time. Tukey post-hoc comparisons were tested to set significance values of P,0.05 and P,0.01 as part of the program. A two-tail two- sample t-test was used to test between treatments where an observed difference between controls was noted. PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46801