stripping method to remove symbiotic dinoflagellates [31] and then maintained in the dark for over 4 months with two monthly repeated stripping applications during that period. 13C Isotope Incubation The stable isotope 13C in the form of NaH13CO3 (Sigma- Aldrich) was used in an artificial seawater (ASW) mixture adapted from Harrison et al [32]. The final concentration of dissolved inorganic 13 carbon (DI13C) at 2 mM corresponded to that of normal seawater at pH 8.2, 33–34 ppt. Control ASW used non- isotopic NaH12CO3 in the same quantity. Anemones were removed from culture conditions and placed in separate polycar- bonate chambers containing 13C-enriched ASW, under normal culture light and temperature conditions (n = 5 symbiotic and n = 3 aposymbiotic for each extraction point). Different individual symbiotic anemones were placed in control environments for the same experimental duration (n = 5 for each extraction point). The different ASW treatments were replaced every 24 hr. The anemones were fed once four days prior to, but not, during experimental treatments. Sample anemones and any expelled dinoflagellate pellets were removed at 24 hr, 48 hr, 72 hr and 120 hr from ASW treatments, snap frozen in liquid nitrogen and then stored at 280uC prior to processing. The respective expelled dinoflagellate pellets from the 13C-enriched ASW treatment were collected at each time point, but due to low quantities were later combined to provide an integrated measurement over the total experiment duration from 0 to 120 hr prior to processing. Lipid and Fatty Acid Extraction Sampled anemones and dinoflagellates previously stored at 280uC were thawed and placed into an RVC2-18 (Christ) speed vac connected to a MZ2C (Vacuubrand) vacuum pump and desiccated to a dried pellet overnight. The dried pellets of each sample were then weighed. The total lipid fraction from each pellet was extracted using modified methods of Folch et al [33]. Each individual pellet was placed into a glass homogenizer together with 2 mL of fresh chloroform/methanol (2:1) and homogenized. The homogenate for each individual sample was then returned to a 15 mL falcon tube. The homogenizer was rinsed with 2 mL of chloroform/methanol (2:1) and added to the original homogenate in a 15 mL falcon tube, vortexed and stored for 2 hr in the dark at 4uC. The chilled suspension was removed and filtered using a 22 mm glass fibre filter and placed in a new 15 mL falcon tube. The filter was then rinsed with 1 mL of chloroform/methanol (2:1), which was added to the filtered homogenised sample. 1 mL of 0.1 M KCl in Milli-Q water was added to the combined 5 mL of homogenate filtered extract, vortexed and placed in the dark at 4uC for 1 hr or until the aqueous and organic phases had separated. The aqueous phase was removed and remaining organic phase washed with 5 mL of methanol/water (1:1), the phases were allowed to separate for 1 hr before the aqueous phase was removed and the process repeated (x3). Following the final rinse the organic phase was dried, weighed and the resulting pellet resuspended in 500 mL of 100% acetonitrile (B and J Brand HPLC grade, Lomb Scientific) with the aid of a 1.5 mL tube tissue homogenizer (Eppendorf). High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) of Fatty Acids The FAs from the total lipid extracts were analyzed using an Agilent 1200 Series HPLC instrument (Agilent Technologies, Inc., Santa Clara, USA) coupled to an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, USA). In Cnidarian-Dinoflagellate Symbiosis Lipidomics addition to the HPLC unit, the following components were installed: micro degasser (G1379B), binary pump SL (G1312B), high performance autosampler SL (G1367C) thermostat column compartment SL (G1316B) and a diode array detector SL (G1315C). Chromatography was performed using a Luna 3m C18 (2) 100 A˚ 7564.60 mm column (Phenomenex, Torrance, USA). Milli-Q water and 100% acetonitrile were used as the A1 and B1 solvents, respectively. For sample analysis, 10 mL of each lipid extract sample was injected using the autosampler. The components of each extraction were analyzed using a 40 min run with a HPLC flow rate of 0.8 mL/min. The gradient elution profile used during each analysis run is shown in Table S1. The LTQ XL mass spectrometer was equipped with an Ion Max electrospray ion source and run in negative ion mode with the following instrument settings: spray voltage 24.5 kV, capillary voltage 2130 V, tube voltage 100 V and capillary temperature 320uC. The nitrogen flow rates at the ESI source were set as follows: sheath gas 32 (arbitrary units) and auxiliary gas 8 (arbitrary units). No sweep gas was used. Nitrogen was sourced using a Domnick Hunter LCMS30-1-E nitrogen generator (Parker Hannifin Ltd, Industrial Division, England). The Luna C18 column was subjected to a cleaning regime every 10 sample runs with a 1:1 mixture of ethanol and hexane for approximately 20 min to remove non-specific highly-hydrophobic compounds within sample extracts, which do not elute from the column using 100% acetonitrile. A series of FA standards (Sapphire Bioscience, Waterloo, NSW, Australia) were used to identify retention times (tR) of specific m/z profiles associated with known FAs (Table S2). In previous trials, MS spectra were analyzed within a mass range of m/z 200–1000 (data not shown), however no FA signals and ion profiles of interest to this study were observed above m/z 500. Consequently, for data collection the MS spectra were recorded during the entire 40 min HPLC runs within a mass range of m/z 200–550. Spectra were recorded in profile mode with a maximum ion injection time of 50 ms and an automatic gain control setting of 3.00e+04. The number of microscans was set at 4 to minimize the file size of the MS data. The percentage of each isotopologue by number of ions was determined from the MS spectrum by converting the peak areas of each isotopologue into a relative percentage (normalized abundance). The peak span measurement was taken at 50% across FA peak heights on the ion chromatogram. The isotopologues containing the additional 13C stable isotope was then added to give a measurement of total 13C incorporation for a given time point. Isotopologues were verified as being associated with the respective FA by displaying the same elution profile and by the use of collision-induced dissociation for selected FA ion profiles (data not shown). In addition, the observed distributions of 13C-labeled isotopologues were clearly matched to the theoretical distributions predicted mathematically. Quantification of Extraction Efficiency in Negative Ion MS/MS In order to determine whether the described method resulted in the same extraction efficiency of FAs from the total lipids from different samples of anemones of different symbiotic states, the quantitation of arachidonic acid (AA) was used as a traceable marker. The sample analysis run time was reduced from 40 min to 15 min and the gradient elution was adapted accordingly (Table S3) using a flow rate of 0.8 mL/min. The MS/MS of the AA [M- H]2 ion at m/z 303.6 was performed with a normalized collision energy of 22% and an isolation width of 1 Th. The max injection time was changed to 30 ms and the automatic gain control was set to 10000. MS/MS spectra were acquired from m/z 80–320. The PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46801