Experimental material

Anemonia viridis (Forskal), green colour morph, were collected from a tide pool at Morfa Nefyn, North Wales, UK (Ordinance Survey grid reference 274, 410) and maintained in aerated artificial seawater (Instant Ocean Salts; Aquarium Systems, Sarrebourg, France) of salinity 35‰ and pH 8.2 at 16°C under a 16 h:8 h light:dark regime at 10–30 μmol photons m^–2 s^–1 photosynthetically active radiation (PAR). Experiments were conducted 3–5 days after the animals had received their weekly feed of chopped squid and were performed under the same light and temperature conditions as for anemone culture.

Replicate groups of three freshly excised tentacles of A. viridis (taken from three separate individuals) were incubated in 1 ml seawater buffer, comprising 420 mmol l^–1 NaCl, 26 mmol l^–1 MgSO₄, 23 mmol l^–1 MgCl₂, 9 mmol l^–1 KCl, 9 mmol l^–1 CaCl₂, 2 mmol l^–1 NaHCO₃ and 10 mmol l^–1 Hepes, pH 8.2 ( Wang and Douglas, 1997), for 30 min prior to an experiment, by which time the tentacles had relaxed. Where appropriate, algal photosynthesis was inhibited by 5 μmol l^–1 DCMU (dichlorophenyl dimethyl urea) added 15 min before the experiment. Experiments were initiated by adding radiolabelled substrate (details below) and, unless stated otherwise, comprised a 5-min time course, to minimize the extent of animal metabolism of the radioactive substrate whilst generating sufficient radioactivity for quantification. Samples without added radioactivity were included in all experiments as controls.

NaH¹⁴CO₃ incubation under photosynthesizing and non-photosynthesizing conditions

After the incubation with 0.37 MBq (10 μCi) NaH¹⁴CO₃ (ICN Pharmaceuticals, Costa Mesa, CA, USA), the tentacle samples were rinsed in ice-cold 0.5 mol l^–1 NaCl and homogenized in a hand-held glass homogenizer in 1 ml ice-cold 0.5 mol l^–1 NaCl. The protein content of each homogenate was quantified using the Bio-Rad protein micro-assay kit (Bio-Rad Laboratories GmbH, Munchen, Germany) according to the manufacturer's instructions, with bovine serum albumin as standard. The homogenate was centrifuged at 1000 g for 5 min at 4°C, and the pellet was washed twice by centrifugation and resuspension with 1 ml 0.5 mol l^–1 NaCl. The supernatants were combined to form the animal fraction, and the final suspension of algal cells was designated the algal fraction. The cell density in the algal fraction was quantified using an improved Neubauer haemocytometer (six replicate counts per sample). To quantify ¹⁴C incorporation, 25 μl of each fraction was acidified with 4 mol l^–1 acetic acid and shaken for 60 min to remove unfixed ¹⁴C. Scintillation cocktail (Ultima-Gold™ XR; Packard Bioscience B.V., Gröningen, The Netherlands) was then added, and the radioactivity quantified in a scintillation counter (Packard Tri-Carb Scintillation Analyser) using pre-set ¹⁴C windows. The counts in the samples incubated without added radioactivity were subtracted from values for the experimental samples.

Samples of the algal and animal fractions were incubated in 5% trichloroacetic acid (TCA) for 15 min at room temperature and then centrifuged at 10 000 g for 15 min. The supernatant (TCA-soluble fraction) was extracted three times with ether to remove the TCA, evaporated to dryness in a Speed-Vac (SC110; Savant Instruments, Holbrook, NY, USA) and then resuspended in 80 μl and 200 μl of deionised water for algal cell and animal samples, respectively. (The animal samples required a larger volume to dilute the salt; ethanol extraction was not used to remove the salt because this procedure substantially reduced the yield of radioactivity.) The lipid fraction was extracted from the TCA-insoluble fraction with 1 ml methanol:chloroform (2:1) for 15 min at room temperature followed by centrifugation at 10 000 g for 15 min. The chloroform layer containing the lipid was separated from the methanol by the addition of 0.4 ml 0.1 mol l^–1 KCl. The final pellet that contained protein and nucleic acids was solubilized by incubation in 0.2 ml 1 mol l^–1 NaOH at 100°C for 10 min and then neutralized with 0.2 ml 1 mol l^–1 HCl. Samples of each fraction were removed for the quantification of radioactivity. The recovery of radioactive compounds after fractionation was, on average, 90% and 65% for the algal and animal samples, respectively. It is unclear why recovery was lower for the animal samples but it seems unlikely to be due to the salt content as similar recovery figures were achieved after salt removal by Streamer et al. ( 1988).

The TCA-soluble fraction was further separated into neutral, acidic and basic fractions using cation (Dowex 50 × 8 H⁺; Sigma-Aldrich, Dorset, UK) and anion (Dowex 1 × 8; Sigma-Aldrich) exchange columns, following the procedure of Quick et al. ( 1989). To identify individual radiolabelled compounds, samples of the TCA-soluble fraction were separated by thin layer chromatography/thin layer electrophoresis, as in Wang and Douglas ( 1997), for organic acids, phosphate esters, sugars and sugar alcohols and as in Wang and Douglas ( 1999) for amino acids. The chromatography/electrophoresis plates were exposed to Kodak X-ray film for 4–6 weeks, and radioactive spots were identified by comparison with authentic compounds. The identification of the sugars, sugar alcohols and amino acids was verified by reverse-phase HPLC using the procedures of Ashford et al. ( 2000) and Karley et al. ( 2002).

¹⁴C organic substrate incubations

The analyses were performed as for NaH¹⁴CO₂ incubations with the following modifications. All experiments were performed under non-photosynthesizing conditions (in the presence of 5 μmol l^–1 DCMU) in autoclaved seawater buffer supplemented with the antibiotics ampicillin (1 mg ml^–1) and streptomycin (50 μg ml^–1) to inhibit the metabolism of any contaminating bacteria ( Wang and Douglas, 1998). Organic substrates {[U-¹⁴C]glucose, [U-¹⁴C]glycerol, [2,3-¹⁴C]succinate, [2,3-¹⁴C]fumarate (Sigma-Aldrich), [U-¹⁴C]malate and [1,5-¹⁴C]citrate (Amersham Pharmacia Biotech UK Ltd, Amersham)} were supplied at a concentration of 1 mmol l^–1 and 0.11 MBq ml^–1 for single substrate incubations and 0.5 mmol l^–1 and 0.06 MBq ml^–1 for double substrate incubations (i.e. giving a total concentration of 1 mmol l^–1 and 0.11 MBq ml^–1). The chemical purity of radiochemical compounds was> 99%, as determined by thin layer chromatography. The experimental design was developed following preliminary experiments on the uptake of organic substrates by tentacle samples over time from 5 s to 5 min, which revealed minimal nonspecific binding of ¹⁴C with >98% recovery of radioactivity from the anemone fraction, i.e. <2% in the algal cells. Algal cells and animal tissue were therefore not separated in the definitive experiments. The experimental material was homogenized in ice-cold deionised water, and the TCA-soluble fractions, which were essentially salt-free, were resuspended in 80 μl volumes after evaporation.

NaH¹⁴CO₃ fixation and assimilation

The rate of NaH¹⁴CO₃ fixation by the excised tentacles of A. viridis in the light was 3.70.29×10⁵ d.p.m. mg^–1 total protein h^–1 (mean ± s.e.m., N=19), equivalent to 0.09±0.008 d.p.m. algal cell^–1 h^–1, and fixation was linear in incubations of 5–60 min duration with 30±1.5% of ¹⁴C recovered from the animal tissue. Fixation in the presence of 5 μmol l^–1 DCMU was, on average, 6% of that in samples without DCMU.

Incorporation of radioactivity into the principal chemical fractions of the algal cells and animal tissue after 5-min incubations is shown in Fig. 2. The distribution of radioactivity (expressed as d.p.m. mg^–1 protein) across the chemical fractions varied significantly between the algal cells and animal tissues and was significantly altered by the DCMU treatment (statistical analysis shown in legend to Fig. 2). The two principal factors contributing to this variation were: (1) the greater incorporation of radioactivity into the TCA-soluble fraction of animal tissues (94% of the total) compared with algal cells (59%) under photosynthesizing conditions and (2) the disproportionate reduction in ¹⁴C incorporation into the lipid fraction in the DCMU-treated samples relative to those under photosynthesizing conditions. The radioactivity in the neutral, acidic and basic fractions of the TCA-soluble fraction is shown in Fig. 3, with statistical analysis in the legend. For the samples under photosynthesizing conditions, the distribution of radioactivity differed significantly between the algal cells and animal tissues, with recovery from the neutral, acid and basic fractions in approximate ratios of 2:1:2 for algal cells and 1:2:1 for the animal tissues. The distribution of radioactivity was also significantly altered by DCMU treatment.

Various compounds were radioactively labelled in both the algal cells and animal tissue of samples incubated with NaH¹⁴CO₃ under photosynthesizing conditions ( Table 1). Differences between the algal cells and the animal tissue included the detection of ¹⁴C-labelled glycerol in the algal cells but not in the animal tissue, and intense labelling of malate in the animal tissue as opposed to citrate in the algal cells. The ¹⁴C content of DCMU-incubated samples was so low that identification of individual radiolabelled compounds was not feasible.

Uptake and assimilation of ¹⁴C-labelled organic substrates into the animal tissue

Tentacles of A. viridis incubated under non-photosynthesizing conditions with each of the six organic substrates (glucose, glycerol, malate, citrate, succinate and fumarate) accumulated radioactivity equivalent to between 2.74 nmol substrate mg^–1 total protein and 4.20 nmol substrate mg^–1 total protein over the 5-min incubation period ( Table 2). The pattern of incorporation of radioactivity into the principal chemical fractions was very similar for all the organic substrates tested. Radioactivity was recovered predominantly from the TCA-soluble fraction (99%), with a small amount in the protein/nucleic acid fraction (approximately 0.8%) and barely detectable amounts (0.2%) in the lipid fraction. For all the organic substrates, the distribution of radioactivity between the neutral, acidic and basic fractions of the TCA-soluble fraction was different to that recorded from the control experiments where the symbiosis was supplied with NaH¹⁴CO₃ under photosynthesizing conditions ( Fig. 4). For every substrate, radioactivity was detected almost exclusively in just two of the three TCA-soluble fractions: little radioactivity was recovered from the neutral fraction (≤0.7% of total ¹⁴C in the TCA-soluble fraction) when an organic acid was supplied or from the basic fraction when glucose (4% of the total) or glycerol (5% of the total) was supplied. For most organic substrates tested, the most strongly radiolabelled fraction corresponded to that of the substrate supplied. Exceptionally, 58% of the total radioactivity derived from exogenous ¹⁴C-labelled malate was detected in the basic fraction.

Individual radioactively labelled compounds recovered from tentacles that had been incubated in the various organic substrates are shown in Table 3, which includes the control data obtained for tentacles incubated with NaH¹⁴CO₃ ( Table 1) for comparison. For all substrates tested, four ¹⁴C-labelled compounds – malate and three sugar phosphates– were detected in common with the control. The most strongly labelled compounds were those corresponding to the substrate supplied and to glucose 6-phosphate. In general, when an organic acid was supplied, a greater number of ¹⁴C-labelled compounds were detected in common with the control compared with when glucose and glycerol were supplied. However, certain ¹⁴C-labelled compounds detected in the control samples (notably proline) were only detected in the experimental samples supplied with ¹⁴C-labelled glucose.

When the tentacle samples were incubated with two ¹⁴C-labelled substrates simultaneously, the total uptake varied between 4.25 nmol mg^–1 total protein and 5.21 nmol mg^–1 total protein ( Table 2). Incorporation of radioactivity into the principal fractions was very similar to single substrate incubations, with 99% of the radioactivity recovered from the TCA-soluble fraction. The pattern of incorporation that most closely resembled the control incubations with NaH¹⁴CO₃ was obtained with glucose and either succinate or fumarate as substrates ( Fig. 5).

The profile of labelled compounds in double substrate incubations ( Table 4) was identical to the combined profile of compounds labelled in previous experiments using the same substrates applied singly ( Table 3). For the incubations with glucose and succinate or glucose and fumarate, the radiolabelled compounds were very similar to those labelled in the control experiments incubated with NaH¹⁴CO₃. The products of double substrate incubations that included glycerol were also similar to the results from the control experiments, but two of the most strongly labelled compounds, glycerol and an unknown amino acid, in these double substrate incubations were not detected in the control incubations.

In summary, the profile of metabolites in the animal tissues derived from exogenous glucose and succinate/fumarate, but no other exogenous compounds tested, was closely similar to the equivalent profiles derived from photosynthetically fixed compounds translocated from the algal cells. This comparison of metabolite profiles (see Fig. 1) suggests that glucose and a dicarboxylic acid (probably succinate and/or fumarate), or metabolically allied compounds, are important mobile photosynthetic compounds in the Anemonia viridis–Symbiodinium symbiosis.