Results Extraction Efficiency and Arachidonic Acid Concentration The outcomes of the comparative deuterated (D8) and non- deuterated AA concentration extraction efficiency method from total lipids, showed no difference between aposymbiotic and symbiotic anemones (Figure S1A and B). Stable Isotope Incorporation during Lipogenesis Controls. A naturally occurring 13C incorporation of ap- proximately 1–2% was detected within all FAs from different samples. There was no additional 13C incorporation in FAs from either DI13C ASW treated aposymbiotic anemones, or non- enriched DIC ASW treated symbiotic anemones as highlighted by the absence of additional isotopologues of docosahexaenoic acid (DHA C22:6: m/z 327) (Figure 1A and B) or FA lipogenesis profiles over time (Figure 2, 3, 4, and 5). Enriched DI13C media treated symbiotic A. pulchella and expelled dinoflagellates. Apart from very long chain fatty acids (LCFA), the same FAs were detected in all samples. Cnidarian-Dinoflagellate Symbiosis Lipidomics However, incorporation of enriched 13C was only detected into different FAs within both symbiotic anemones and expelled dinoflagellates collected from the enriched DI13C ASW, which also varied between FAs over time. During FA synthesis the 13C was incorporated in different ways according to the FA, and was either an immediate and rapid incorporation with a Vmax between 48 hr and 72 hr followed by a plateau profile, or a continuous linear incorporation over time, or little/no incorporation at all. Enriched 13C isotope incorporation was skewed towards an abundance of isotopologues with a high 13C content indicating a dependency upon the DI13C rather than an alternate source (12C) (Figure 2, 3, 4, and 5) with the exception of two very long-chain FA with a binomial skewed profile (Figure 5C-D). The FAs derived from enriched 13C ranged from tetradecanoic/ myristic acid (C14:0 ) to a long-chain FA, octacosaoctaenoic acid (OOA) (C28:8). Specifically, the isotopic incorporation into myristic acid (C14:0 ) within symbiotic anemones increased significantly from 24 hr (f = 18.33, P,0.01) until a Vmax 34.05% 6 Std Dev 8.2 at 48 hr. After 48 hr, the incorporation and synthesis rate was consistent over time (Figure 2A). At 120 hr, the 13C incorporation Figure 1. The spectrum profile of the fatty acid, DHA (C22:6, n–3) within (A) a aposymbiotic Aiptasia pulchella removed from the DI13C ASW at 72 hr and (B) within a symbiotic A. pulchella removed from the non-13C DIC ASW at 72 hr. (R.A.% = Relative Amount). doi:10.1371/journal.pone.0046801.g001 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46801