peared to have little incorporation. Such delayed incorporation may indicate either a rapid turnover and thus low accumulation, or that synthesis is retarded by a potential negative feedback loop via docosahexaenoic acid (DHA; C22:6) concentration, which is synthesized via the intermediate DPA; C22:5 (Figure 5A). The potential activity of a negative feedback loop control on EPA production correlates with the decrease in DHA at 120 hr. However, no control loop has previously been described and requires further research. The high incorporation into the symbiotic anemone’s C22:6 up until 72 hr followed by a significant decrease at 120 hr is possibly due to feedback control or resource limitation. The CEDP C22:6 incorporation remained within the range of the symbiotic anemones, again suggesting the dinoflagellates were responsible for DI13C utilization (Figure 5B). The evidence presented here of high incorporation and accumulation in end n–3 pathway essential FAs, such as DHA indicated a priority to produce large amounts of these products and correlates with similar production and accumulation in previous studies [8,9,14,43]. The importance of n–3 derived essential FAs such as DHA, to supplement animal diets is well known [3]. A large supply of essential FAs may also be important in symbiosis regulation and could correlate with a proportion of DHA conversion to very LCFA, such as octacosaoctaenoic acid (C28:8, n–3). The multinomial peak profile of isotopologues indicated 13C incorporation was skewed towards both high and low content isotopologues. The latter indicated use of 13C in elongation of a pool of pre-treatment non- enriched, normally proportioned 12C/13C DHA isotopologues (m/z 327) to OOA (C28:8, n–3; m/z 407) to form a series of isotopologues from m/z 328 to m/z 413 (6613C added during elongation of DHA) (Figure 5D). The presence of OOA in dinoflagellates is already known, although the synthesis and novel adaptation to the n–3 pathway was unknown [47]. The 13C incorporation of OOA was the highest of all fatty acids in the symbiotic anemones and although the function remains unknown, OOA could lead to downstream metabolites or storage whereby later catabolism leads to additional DHA. The description of the interaction between DHA and OOA (C28:8, n–3) is further complicated by the incorporation into octacosaheptaenoic acid (OHA; C28:7, n–6) (Figure 5C) which displays the similar multinomial peak of the C28–8,n–3. The less pronounced peak distribution of this n–6 LCFA, indicated that it is either an intermediate between the n–3 DHA and the LCFA, OOA (C28:8, n–3), or a direct derivative of one of the two and therefore indicating a direct and close interaction between n–3 and n–6 enzyme activity, which is independent of upstream pathway integration (Figure 7). The functions of these particular highly produced essential FAs, OOA and OHA within this symbiosis and dinoflagellates are unknown and are currently the focus of further research. The absence of incorporation in enriched DI13C treated aposymbiotic control anemones and non-13C ASW treated symbiotic anemones (Figure 1A-B), combined with finding that the 13C-fatty acid profiles of CEDPs and symbiotic anemones do not differ greatly, suggests symbiotic dinoflagellates are essential for DI13C incorporation into the FAs of symbiotic anemones. In support of this conclusion is the finding that all FAs found to incorporate 13C can be assembled as a schematic pathway that aligns with n–3 and n–6 algal lipogenesis (Figure 7). Although late n–6 pathway FA were detected within both aposymbotic and symbiotic anemone hosts, there was no direct evidence of DI13C derived FA synthesis or processing of endosymbiont derived FAs within the cnidarian host. An alternative explanation for the findings is that symbiont derived FAs were translocated to the host, but not used in host FA synthesis and rapidly catabolized into Cnidarian-Dinoflagellate Symbiosis Lipidomics undetectable non-FA metabolites, such as ATP. Whilst this is a possibility and cannot be dismissed, it is also unlikely as there was no evidence of the host free FAs, including downstream longer chain FAs, containing enriched isotopic incorporation. If symbiont derived FA or derived catabolized carbon were made available to the host it is unlikely that they would be discriminated against for incorporation for host FA elongation and lipogenesis due to the important nature of these metabolites. The difference in rates of carbon fixation as a result of the symbiosis that have been reported in 14C FA incorporation studies and this 13C study are quite considerable, and raises questions as to where the radioisotope FA signals that were detected were actually located and if there was considerable symbiont contam- ination involved? In respect to this, attempts were made in independent trials (data not shown) to obtain algal contaminate- free samples using this highly sensitive technique and it was concluded that obtaining symbiotic host fractions that were free of symbiont FA contamination (as indicated by the presence of LCFAs) was not consistently possible. The consensus of evidence from this study suggests that DI13C derived FAs via the symbiotic dinoflagellate were not used directly or utilized via potential symbiont derived triacylglycerol stores in host FA lipogenesis during the treatment period. Further, if metabolites, including lipids and FAs produced through the symbiosis help meet 90–99% of host energy requirements [15,19] and the respective lipid energy stores are derived from dinoflagellate photosynthates [8] then the results of this study imply either that the utilization of DIC in host FA lipogenesis is subjected to a 120 hr (5 day+) lag period or that the required carbon is derived from an alternative source. DI13C incorporation profiles were mainly below 50% and clearly showed 12C still being incorporated by the dinoflagellate, suggesting additional sources of carbon are important such as host respiration and/or heterotrophy are important [12,28]. Host heterotrophy influences partner lipid content and can provide 35% of the daily metabolic requirements of healthy symbiotic corals, and 100% in bleached individuals [12,29]. The fact that anemones were not fed during the experimental treatment could explain why some FA synthesis declined between 72 hr and 120 hr, and is potentially in response to reduced supplies of a limiting resource, such as nitrogen for the symbiont in the form of ammonia via host catabolism [19]. The transferable enriched isotopic incorporation method presented here clearly demonstrat- ed how one form of carbon is differentially utilized and distributed during FA lipogenesis in cnidarian-dinoflagellate symbiosis. This has important connotations for understanding both the health and integrity of the individual dinoflagellate and the symbiotic relationship with the cnidarian host. Supporting Information Figure S1 (A). The total quantity of AA (arachidonic acid; C20:4, n–6) within symbiotic and aposymbiotic A. pulchella used to calculate the extraction efficiency from a mixture of both non-enriched and enriched DI13C media at 72 hr was not significantly different between symbiotic (1.029 mg/mg of tissue ± Std Dev 0.138) and aposymbiotic anemones (0.944 mg/mg of tissue ± Std Dev 0.279) (t(6) = 0.56, P = 0.6). (B) The fatty acid extraction efficiency from total lipid extractions from symbiotic and aposymbiotic Aiptasia pulchella. There was no significant difference in extraction efficiency (t(6) = 0.96, P = 0.37) between aposymbiotic (43.01% 6 Std Dev 12.19) and symbiotic A. pulchella (48.97% 6 Std Dev 10.46) (n = 6, Error bars = Std Dev). (TIF) PLOS ONE | www.plosone.org 12 October 2012 | Volume 7 | Issue 10 | e46801