Cnidarian-Dinoflagellate Symbiosis Lipidomics Figure 2. Isotopic incorporation lipid profiles of fatty acids in symbiotic A. pulchella. Subplots: Relative Amount (R.A.) of 13C incorporation (13C I) into the FA structures (%) over time. Key: R.A.% = Relative Abundance of isotopologues, m/z = Mass-to-Charge ratio of isotopologues, 13CI % = Isotopic incorporation in isotopologues, X = DI13C ASW treated symbiotic anemones (n = 5), Square symbol = DI13C ASW treated aposymbiotic anemones (n = 3), X = Non-isotopic DIC ASW treated symbiotic anemones (n = 5), Circle symbol = DI13C ASW Dinoflagellates expelled from treated symbiotic anemones (combined from treatment over time). Error Bars = Standard Deviation. Note: Where data not shown for time points = Signal was below detection. Spectrum data shown is from DI13C ASW symbiotic anemones at 72 hr. doi:10.1371/journal.pone.0046801.g002 in C14:0 from the cumulative combined expelled dinoflagellate pellets (CEDPs) was 29.58% and similar to symbiotic anemones (34.3% 6 Std Dev 3.33)(Figure 2A). The similar isotopic incorporation in FAs measured from the CEDP and symbiotic anemones continued across other FAs. The isotopic incorporation and synthesis rates of palmitic acid (C16:0) and palmiteolic acid (C16:1) were more linear over the total incubation period compared to C14:0 (Figure 2B and 2C). There was a significant increase from 24 hr - 48 hr for C16:0 (f = 59.06, P,0.01) and (C16:1) (f = 49.68, P,0.01). Incorporation steadily increased over time, although the amount at 72 hr was not significantly different to that of 48 hr for both FAs. There was a further significant increase (P,0.05) between 72 hr and 120 hr for both FAs (23.73% 6 Std Dev 1.92 for C16:0; Figure 2B and 26.22% 6 Std Dev 2.84 for C16:1; Figure 2C). The CEDP incorporation for both C16:0 and C16:1 was marginally greater than symbiotic anemones at 28.75% and 30.02% respectively. Stearic acid (C18:0) displayed a similar profile to C14:0, albeit with a lower incorporation. Vmax occurred at 48 hr with a highly significant increase from 24 hr (3.27% 6 Std Dev 2.8; f = 31.55, P,0.01) to 48 hr (12.19% 6 Std Dev 1.34) followed by no further significant changes in isotopic incorporation (Figure 2D). The same profiles were displayed in the monounsaturated octadece- noic/oleic acid (C18:1), polyunsaturated octadecadienoic/linoleic acid (C18:2) and the a/c linolenic acid (C18:3) (Figure 3A-C). For the purpose of this study, isotopic profiles of n–3 and n–6 derived C18:3 FAs were not analyzed separately. At 48 hr, incorporation significantly increased in C18:1 from 24 hr - 48 hr (2.35% 6 Std Dev 0.96 to 47.38% 6 Std Dev 4.16; f = 222.63, P,0.01). A reduction in incorporation at 72 hr into C18:1 did not become significant until 120 hr (26.21% 6 Std Dev 3.15; P,0.01) and was similar to CEDP at 31.6%. The linoleic acid (C18:2) isotopic incorporation also significantly increased from 24 hr to a Vmax at 48 hr (43.01% 6 Std Dev 5.73; f = 17.63, P,0.01), and like C18:1 was followed by reduced incorporation, which was significant at 120 hr (30.74% 6 Std Dev 0.65; P,0.05). At 120 hr, incorpo- ration within C18:2 from CEDP was higher than the symbiotic anemones at 37.12% (Figure 3B). The Vmax of linolenic acid (C18:3) occurred at 72 hr (40.87% 6 Std Dev 6.87). There was a significant increase between 24 hr and 48 hr (f = 47.77, P,0.01) and a decrease between 72 hr and 120 hr. The incorporation in CEDP was, however, similar to the symbiotic anemones at 72 hr (43.44%) (Figure 3C). In contrast to C18:3, the profile for octadecatetraenoic/stearidonic acid (C18:4) displayed very little incorporation in symbiotic anemones over time, until a significant increase from 72 hr (4.53% 6 Std Dev 1.27) to 120 hr, (10.05% 6 PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46801