The high metabolic productivity of dinoflagellates residing within the coral (Anthozoa, Cnidaria) host is believed to contribute to the formation of coral reefs through the translocation of carbon rich products from the symbiont to the host. Lipids are the main energy store in cnidarians and the primary products derived from photosynthetically-fixed carbon translocated from the dinoflagel- late symbionts to the host [8]. Total lipids from the symbiosis (triacylglycerols, wax esters, phospholipids and free FAs), account for 10–46% of the cnidarian tissue dry weight [9,10]. Previous studies reported that metabolites, including lipids vary in composition and abundance according to the carbon source and species [11,12]. Furthermore, changes in environmental stimuli such as light and temperature, elicit a marked change in lipid production and subsequently the potential storage and availability of energy for coral-dinoflagellate symbiosis, which has indirect implications for reef health [9,13,14]. Photoautotrophs rely upon CO2 diffusion for organic carbon fixation using the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to produce metabolites such as sugars, amino acids, proteins and lipids. In the marine environment, photoautotrophs have a limited availability of dissolved CO2 for photosynthesis due to ambient seawater pH and associated gaseous saturation states [15,16]. The dominant form of dissolved inorganic carbon (DIC) in the marine environment is bicarbonate (HCO32; ,95% of the total DIC in seawater at ,pH 8.2) and in order for marine photoautotrophs, such as Chlorophyta and Dinophyta, to access and utilize the more abundant form of DIC, photoautotrophs use carbon- concentrating mechanisms (CCMs) such as the enzyme, carbonic anhydrase (CA). CA catalyzes the reversible conversion between HCO3- and CO2, to allow diffusion of DIC across bipolar membranes and supplement limited ambient CO2 for photosyn- thesis and enable RUBISCO to function efficiently [16]. A combination of respiration and CA activity in cnidarian hosts of symbiotic dinoflagellates [17] are important CO2 supplies for symbiont metabolite production [18], which in part, meet the energy requirements of both the symbiont and host [15,19]. Understanding the complex metabolic contribution of each partner within a symbiotic interaction requires the translocation and rates of specific metabolite production to be determined. Previous 14C radioisotope studies have indicated direction of transfer into either, or both the host and symbiont, as well as the rate of carbon fixation from DIC source to total products [11,20]. In other studies, 14C incubations from 5–60 min to several hours have shown that photosynthetic fixation occurs rapidly, resulting in, almost immediate incorporation into both host and symbiont products, including total lipids [20,21,22,23]. However, the use of 14C is problematic because of radioisotope regulations, safe work practise and difficulties associated with using radioisotopes within protected environments. In addition, although detection of radioisotope incorporation into metabolites may suggest a carbon source to a product, there may be potential resolution restrictions for identification and measurement of metabolite synthesis rates when compared to the application of alternative techniques. In previous studies the use of 14C may also underestimate translo- cation by up to 50% [24]. For example, short-term isotopic incubation may clearly show rapid synthesis and translocation of one form of metabolite over another that may take longer to accumulate. The resulting and subsequent extrapolation may infer importance of that metabolite over another form, which may actually not be the case. For example, Whitehead and Douglas [23], clearly showed with short-term 14C incubations, rapid synthesis and translocation of trichloroacetic acid soluble com- pounds such as the sugar, glucose and citric acid cycle compound, succinate between symbiont and host. These translocated metab- Cnidarian-Dinoflagellate Symbiosis Lipidomics olites were in relatively large quantities compared to ‘‘barely detectable’’ levels of lipid incorporation, which were dependent on algal photosynthesis. Whitehead and Douglas [23] as a result, proposed the results of the study did not represent complete translocation, which may be subject to temporal variability in synthesis rates and subsequent potential delayed translocation of other metabolites, such as lipids. Tracking a source of carbon to specific metabolite synthesis may benefit from the application of an alternative technique. Seques- tration of carbon from different sources can be characterized by the isotopic profile of mixed 12C and 13C proportions. For example, the isotopic ratio of carbon fixation via a phototrophic dinoflagellate is subtly different to that of the animal host via heterotrophy and by measuring the ratio of 13C:12C can indicate the trophic source of a compound through a carbon ratio signature [25,26,27,28,29]. In naturally occurring organic compounds, the isotopic fraction ratio of 13C to 12C is approximately 1–2% irrespective of source [13,30], and naturally-occurring multiple isotopologues containing 13C (.1–2%) are of low abundance. By increasing the isotopic fraction available for fixation, through an enrichment of a source such as DI13C, the likelihood of 13C incorporation thus increases beyond the naturally-occurring 1–2% and subsequently can act as a biomarker for metabolite synthesis. Here, we introduce an alternative isotopic method to the use of radioisotopes to track a carbon source into sequential incorpora- tion of metabolites. Specifically, this technique is favoured over using GC-MS (and its inherent greater level of molecular fractionation) for the following three reasons. Firstly, as there is no inherent fragmentation for each measured FA, an array of isotopologues that illustrate a distribution of different isotopic carbon within molecular structures, that can also be used to measure rates of synthesis. Secondly, if so required, a specific target ion can be selected for molecular structural determination and verification. Thirdly, samples do not require derivatization and can be directly analysed from the extraction. This study focused upon the lipogenesis of free FAs from a known source and concentration of DIC to determine which pathways are active within each organism that is using the DIC source in FA synthesis, the rate of synthesis, and the potential of translocation between organisms and subsequent further use in this symbiosis. The symbiotic anemone, Aiptasia pulchella (Anthozoa, Cnidaria) was chosen as a model symbiotic cnidarian-dinoflagellate consortium for this study, for two main reasons: Firstly, large cultures can be maintained easily and individuals of a uniform size can be specifically selected for experiments to minimize potentially variability in metabolism relating to different size. Secondly, A. pulchella can be maintained in a healthy aposymbiotic state and therefore offers an ideal control for experiments focused upon the symbiosis with the dinoflagellate. However, to assess the method transferability, a separate scleractinian hermatypic coral model and an algal culture were also used to demonstrate the broader application of the technique described here. Materials and Methods Culture Conditions Aiptasia pulchella were initially supplied through a local aquarium supplier. The cultures of the symbiotic sea anemone, A pulchella were maintained at Moreton Bay Research Station (MBRS), North Stradbroke Island, QLD under a 12 hr light (350–400 mmol m22 s21 from a metal halide unit)/12 hr dark regime for over a year. The culture temperature was maintained at 25–26uC. Anemones were fed once a week with frozen brine shrimp nauplii. Aposymbiotic anemones were produced using an adapted cold PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46801