Cnidarian-Dinoflagellate Symbiosis Lipidomics Figure 5. See Figure 2 legend. doi:10.1371/journal.pone.0046801.g005 symbiont cells, with respect to the metabolites being produced, where they are utilized and if they are actually available for potential translocation. The main model system used was the anemone, Aiptasia pulchella together with its clade B1 Symbiodinium sp. dinoflagellate symbiont. The method’s transferability was demonstrated using other model systems, however as cross-model optimization was not the focus of this study, we suggest targeted method adaptation associated with specific model use, to enhance specific isotopic incorporation depending upon the required application. The efficiency of FA extraction method was not significantly different between aposymbiotic and symbiotic anemones samples. This method, unlike previous symbiotic cnidarian lipid studies [9,13,14,40] enables a measure of extraction efficiency to be calculated and, as such, could be applied in future studies for inter-study comparison. The recovered FAs from sample total lipids were consistent between symbiotic and aposymbiotic samples, but potentially amounted to less than half of the available FAs. In future, the efficiency of the extraction method could be potentially improved by the use of additional steps, such as sonication or the heating of solvents. The naturally occurring abundance of isotopologues with 1–2% 13C [13,30] represented by 1 or 2 Da above the monoisotopic FA ion (pure 12C) in the MS spectrum were present in all samples (Figure 1, 2, 3, 4, 5, and 6). Differential amounts and rates of increased 13C incorporation in FAs from enrichment could indicate that either, priority is given to carbon allocation for certain FAs over others, and/or the rates of production are affected by synthesized or catabolized downstream products. The influence of these processes, alternative sources of carbon, potential turnover rates or storage capacity should be taken into account when examining individual FA profiles and potential functions as was done here, and noted in other isotope ratio studies [41]. Throughout the incubation period of enriched 13C stable isotope used in this study, the incorporation of 13C during FA lipogenesis was only detected in symbiotic anemones and within the combined expelled dinoflagellate pellets (CEDP’s) from different anemones. 13C incorporation varied in both sample types with different FAs, and the different profiles of isotopic incorporation over time proceeding down the cascading lipogen- esis pathways is likely to be a result of both transfer from shorter to longer chain synthesis and catabolism of each metabolite. For example, the isotopic incorporation profile of myristic acid (C14:0) was characterised by a rapid linear increase followed by a plateau (Figure 2A). The plateau may either indicate storage or utilization in secondary metabolite synthesis. The latter and potentially more significant within the symbiosis is the potential use of myristate in covalent attachment to the N-terminal of proteins known as N- myristoylation. The N-myristoylated proteolipids have important roles in membrane and cellular signal transduction cascades, and protein-protein interactions, including apoptosis and pathogenesis response, such as endothelial nitric-oxide synthase (NOS) and GTPase regulation [1]. These processes have previously been PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46801