Colonies of the scleractinian coral Acropora millepora were collected from Nelly Bay (Magnetic Island), Orpheus Island and Davies Reef in the central Great Barrier Reef in October 2007 at depths of approximately 5 m. Colonies used for acrylate isolation were frozen at -20°C until analysed. The colonies used for acrylate quantification were placed in liquid nitrogen within 10 minutes of collection and later stored at -80°C until analysed. Acrylate was isolated by extracting the frozen coral three times with methanol. The dried methanol extract was partitioned between methanol and hexane. The methanol soluble fraction was separated by reverse phase-C18 silica gel flash column chromatography using increasing proportions of methanol in water, to give nine fractions. The methanol extract and all fractions from the chromatography were analyzed by 1H NMR spectroscopy.The concentration of acrylate was measured from coral nubbins (10 g wet mass), which were extracted three times with methanol and then extracted with dichloromethane (x3) followed by hexane (x5), to extract the remaining lipids, and give a combined dichloromethane/hexane extract. The total organic extract was calculated from the sum of the combined extracts. After extraction, the nubbins were air-dried and weighed to determine the skeleton mass plus nonextractable cellular material. The nubbins were then bleached with 50% bleach, rinsed with MilliQ water three times, air-dried and weighed to determine the skeleton mass. From this, the non-skeletal fresh mass and the non-skeletal dry mass for each sample were determined.Duplicate weighed portions of the methanol extracts were dissolved in 1 ml of the deuterated methanol and 1H NMR spectra were acquired. The amounts of acrylate in the methanol extracts were quantified by measuring the integrals of the three acrylate proton signals and the benzene protons signal and converting to an absolute value by reference to the standard curve. The amount of acrylate was reported as µmol/g non-skeletal fresh mass and µmol/g/ non-skeletal dry mass. The total carbon present in the total organic extract (which also contained NaCl salt) was determined, and the % of carbon present as acrylate carbon in the total organic extract was calculated. Duplicate methanol extracts were dissolved in methanol/water (1:1) and samples containing 2 mg of extract were transferred to tin sample cups and the solvent was allowed to evaporate. The dichloromethane/hexane extracts were dissolved in dichloromethane and duplicate samples prepared in a similar manner. Total carbon analysis was carried out using a LECO TruSpec CN analyser.
