Cnidarian-Dinoflagellate Symbiosis Lipidomics Figure 3. See Figure 2 legend. doi:10.1371/journal.pone.0046801.g003 Std Dev 1.33; f = 29.36, P,0.05). The value for the CEDP C18:4 was 9.84% (Figure 3D). Dihomo-c-linoleic acid (C20:3) synthesis in symbiotic anemones showed a consistent linear rate of incorporation over 72 hr (Vmax 31.63% 6 Std Dev 5.57) with a significant increase between 24 hr to 72 hr (f = 49.1, P,0.01). The incorporation between 72 hr and 120 hr then plateaued, although the CEDP was higher at 42.12% (Figure 4A). The remaining C20 fatty acids detected, namely eicosatetraenoic/arachidonic acid (C20:4; AA) and eicosapentae- noic/timnodonic acid (C20:5; EPA) were similar to stearidonic acid (C18:4), with little incorporation over time (Figure 4B and C respectively). There was a slight, but significant increase in symbiotic anemone AA incorporation between 48 hr (3.09% 6 Std Dev 0.38) and 120 hr (5.42% 6 Std Dev 0.75)(f = 4.21, P,0.05). However, the negligible difference between other time points and 24 hr (,2% difference between all time points), suggests minimal incorporation into AA over the full time series, and that if host utilization of translocated 13C into FA lipogenesis occurs then it occurs around or after 120 hr. CEDP AA incorporation was lower than symbiotic anemones at 120 hr at 2.04% (Figure 4B). Incorporation into eicosapentaenoic/timno- donic acid, (C20:5; EPA) at 120 hr was higher than AA in symbiotic anemones at 7.87% (6 Std Dev 4.52) and in the CEDP at 13.36%. However, as with AA, although the increase was significant compared to earlier time points (f = 5.25, P,0.05), there was no difference between 24 hr (3.12% 6 Std Dev 0.76) to 72 hr (3.61% 6 Std Dev 0.33). There was no additional 13C incorporation across any time points in docosatetraenoic/adrenic acid (C22:4) synthesis in treated symbiotic anemones or CEDP (f = 0.44, P.0.05; Figure 4D). During synthesis of docosapentaenoic/clupanodonic acid (C22:5, DPA) incorporation remained low (,5%) with the exception of a significant spike above all other time points at 48 hr to 9.46% Std Dev 2.25 (f = 6.12, P,0.05) (Figure 5A). The isotopic incorpo- ration into the essential FA, docohexaenoic acid (C22:6, DHA) appeared to be linear until a Vmax at 72 hr (33.75% 6 Std Dev 4.57) (Figure 5B). The CEDP DHA incorporation was greater than the 120 hr symbiotic anemones at 31.67%. Finally, two very long-chain FAs were identified within symbiotic anemones and CEDP samples (Figure 5C and D). Unusually, one of the very long-chain FA identified was an n–6 pathway derived product, octacosaheptaenoic acid (C28:7; OHA) and not an n–3 concom- itant pathway product, displaying a linear increase after 48 hr (significant after 72 hrs; f = 178, P,0.001) reaching a maximum average incorporation of 24.95% at 120 hr and a CEDP incorporation of 27.94% (Figure 5C). The second very long-chain FA, octacosaoctaenoic acid (C28:8; OOA), had the highest incorporation of all FAs with a Vmax at 72 hr of 53.76% 6 Std Dev 7.42 and is a n–3 pathway derived product (Figure 5D). The CEDP isotopic incorporation into the C28:8 of 37.50% was higher than the C28:8 incorporation within symbiotic anemones at 120 hr, which was significantly reduced from the incorporation at 72 hr PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46801