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2445 The Journal of Experimental Biology 201, 2445–2453 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JEB1546 NITROGEN RECYCLING OR NITROGEN CONSERVATION IN AN ALGA–INVERTEBRATE SYMBIOSIS? J.-T. WANG AND A. E. DOUGLAS* Department of Biology, University of York, PO Box 373, York, YO1 5YW, UK *Author for correspondence (e-mail: [email protected]) Accepted 1 June; published on WWW 27 July 1998 Summary When corals and allied animals are deprived of their and the concentration of protein amino acids in the free symbiotic algae, the ammonium content in their tissues amino acid pool of the animal, which were depressed by rises. This is commonly interpreted as evidence for darkness and algal depletion, were restored by exogenous nitrogen recycling (i.e. algal assimilation of animal waste carbon compounds. It is concluded that organic carbon, whether derived from algal photosynthate or exogenously, ammonium into amino acids that are released back to the promotes the animal’s capacity for ammonium assimilation animal), but it can also be explained as nitrogen conservation by the animal (i.e. reduced net ammonium and reduces ammonium production from amino acid production in response to the receipt of algal degradation. These processes contribute to nitrogen conservation in the animal, but they confound the photosynthetic carbon). This study discriminated between interpretation of various studies on nitrogen recycling by these interpretations in two ways. First, the increased symbiotic algae. ammonium concentration in the sea anemone Aiptasia pulchella, caused by darkness or depletion of the alga Symbiodinium, was partially or completely reversed by Key words: Aiptasia pulchella, Symbiodinium, zooxanthella, supplementing the medium with organic carbon symbiosis, nitrogen recycling, amino acid, glutamine synthetase, compounds (e.g. α-ketoglutarate). Second, the activity of ammonium. the ammonium-assimilating enzyme glutamine synthetase Introduction Many members of the phylum Cnidaria (corals, sea anemones, jellyfish, hydrozoans, etc.) bear symbiotic algae, usually dinoflagellates of the genus Symbiodinium (zooxanthellae) in marine groups (corals, sea anemones, etc.) and Chlorella in the freshwater hydras (Trench, 1993; Douglas, 1994). For the marine symbioses, Symbiodinium species have been implicated both in the provision of photosynthesisderived carbon and in nitrogen recycling, and these processes are widely considered to underpin the ecological success of Cnidaria–alga symbioses in shallow, low-nutrient waters (Muscatine and Porter, 1977; Falkowski et al. 1993). The biochemical basis of photosynthate release by Symbiodinium is well established. Many radiotracer studies have revealed high rates of photosynthate release from intact Symbiodinium cells, principally as glycerol and organic acids (e.g. Muscatine and Cernichiari, 1969; Trench, 1971b), and carbon budget analyses indicate that alga-derived carbon contributes much or all of the animal’s carbon requirements for basal respiration (Muscatine, 1990). The evidence for nitrogen recycling by Symbiodinium is, however, far from conclusive. Nitrogen recycling is biochemically complex, involving the bidirectional translocation of nutrients: first, the transfer of the animal’s waste nitrogen compounds (mostly ammonium in Cnidaria; see Shick, 199l) to the algal cells, which assimilate the nitrogen into compounds of nutritional value to the animal; and, second, the translocation of these latter compounds back to the animal (Lewis and Smith, 1971; Douglas, 1994) (Fig. 1A). To date, there is only partial evidence for nitrogen recycling. Cnidaria are known to generate ammonium at high rates, principally by deamination of amino acids used as respiratory substrates (Rees and Ellard, 1989; Shick, 1991), and Symbiodinium can assimilate ammonium (e.g. Domotor and D’Elia, 1984; Wilkerson and Muscatine, 1984; Trench, 1993; McAuley and Smith, 1995). There are, however, no quantitatively reliable estimates either of the flux of ammonium derived from animal catabolism to algal cells in the symbiosis or of the translocation of organic nitrogen compounds from the algae to the animal. The photosynthetic products released by the algae are predominantly non-nitrogenous compounds, but small amounts of amino acids, especially alanine, have been reported in some studies of photosynthate release by Symbiodium (e.g. Trench, 1971b; Lewis and Smith, 1971; Sutton and Hoegh-Guldberg, 1990), and glycoconjugates exuded from Symbiodinium could also contribute to the nitrogen nutrition of the animal (Markell and Trench, 1993). The basis of this study is the controversy that has arisen over 2446 J.-T. WANG AND A. E. DOUGLAS A Fig. 1. Current hypotheses of nitrogen relationships in alga–invertebrate symbioses. (A) Algal recycling of animal-derived nitrogenous waste compounds, e.g. ammonium, to organic nitrogen compounds, e.g. essential amino acids, which are translocated to the animal cells. (B) Processes proposed to influence the ammonium content of the animal tissues and ammonium efflux from animals: (i) algal assimilation of animal-derived ammonium, as predicted by nitrogen recycling; (ii) promotion (+) of ammonium assimilation by the animal (in response to an undefined property of the algal cells) resulting in nitrogen limitation of algal proliferation, as predicted by the nitrogen limitation hypothesis; (iii) depression (−) of ammonium production by amino acid deamination in the animal through the preferential utilisation of alga-derived photosynthetic compounds as respiratory substrates, as predicted by the nitrogen conservation hypothesis; and (iv) promotion (+) of ammonium assimilation in the animal by alga-derived photosynthate (although not specified in the nitrogen conservation hypothesis of Rees, 1986, this process would tend to conserve the organic nitrogen pools of the animal). Waste nitrogenous compounds Animal Nitrogenous compounds of nutritional value to the animal Alga B (i) Waste ammonium in animal (ii) (−) Ammonium production in animal, e.g. by amino acid deamination experimental results commonly interpreted as indirect evidence for nitrogen recycling. When Cnidaria are incubated in darkness or treated to eliminate their symbiotic algae, they generally exhibit increased rates of ammonium efflux into the medium. This has been demonstrated for corals and sea anemones (e.g. Kawaguti, 1953; Cates and McLaughlin, 1976; Szmant-Froelich and Pilson, 1977; Wilkerson and Muscatine, 1984) and also for the freshwater hydra–Chlorella symbiosis (Rees, 1986). These data are commonly interpreted as evidence that the algae are a major sink for animal-derived ammonium (Fig. 1Bi), as predicted by the nitrogen recycling hypothesis (Fig. 1A). However, two further processes could contribute to, or even account for, these observations: alga-stimulated assimilation of ammonium by the animal (Fig. 1Bii,iv) and alga-induced reduction in ammonium production by the animal (Fig. 1Biii). These putative processes are predicted by the hypotheses of nitrogen conservation by the animal and nitrogen-limitation of the algal population (see legend to Fig. 1B). The nitrogen conservation hypothesis was first proposed for the freshwater hydra–Chlorella symbiosis by Rees (1986) and Rees and Ellard (1989), but it is equally applicable to algal symbioses in marine Cnidaria. These authors specifically proposed that the utilisation of amino acids as respiratory substrates by the animal is reduced by the receipt, from the algal cells, of photosynthetic carbon compounds which are used preferentially in animal respiration (Fig. 1Biii). The resultant conservation of nitrogenous compounds in the animal tissues would promote the persistence of these symbioses in low-nitrogen environments. (iii) Algal cells Alga-derived organic carbon compounds transferred to animal Algal assimilation of animal ammonium (+) (iv) (+) Animal assimilation of ammonium, e.g. via glutamine synthetase The chief evidence for nitrogen limitation of the symbiotic algae (Cook and D’Elia, 1987) is that the algal proliferation rates and population size are increased by supplementing the medium with ammonium (e.g. Hoegh-Guldberg and Smith, 1989; Falkowski et al. 1993; Muller-Parker et al. 1994). Rees (1986) has argued that the availability of ammonium to the algal cells is restricted by the activity of an ammoniumassimilating enzyme, glutamine synthetase, in the animal tissues. In the hydra experimentally deprived of their algae, the activity of animal glutamine synthetase is depressed, raising the possibility that this enzyme may be partly, or completely, responsible for the elevated ammonium efflux in these animals. The impact of eliminating the algal population on the glutamine synthetase activity in the animal tissues of marine Cnidaria has, however, not been studied. The hypotheses of nitrogen conservation and nitrogen limitation are broadly compatible with each other (see discussion of Rees, 1986), but neither is readily compatible with high rates of ammonium assimilation by the algae, as required by the nitrogen recycling hypothesis. An expectation of the nitrogen conservation hypothesis, but not of the nitrogen recycling hypothesis, is that exogenous organic carbon compounds and photosynthetic algae should have comparable effects on the nitrogen metabolism of symbiotic Cnidaria. Specifically, the nitrogen conservation hypothesis predicts that elimination of the algae or their photosynthetic activity should have two linked effects on the animal tissues: to depress concentrations of free amino acids (because of their utilisation as respiratory substrates) and to elevate the ammonium concentration (the product of amino Nitrogen metabolism in an alga–invertebrate symbiosis 2447 acid degradation). These effects would be reversed by exogenous organic carbon compounds. The nitrogen conservation hypothesis also predicts that treatments that result in decreased free amino acid concentrations and elevated ammonium concentration should also depress the activity of enzyme(s) that assimilate ammonium and/or increase the activity of ammonium-producing enzymes. In animals, glutamine synthetase is an important enzyme of ammonium assimilation, and transamination reactions are the major route for removing amino groups from amino acids (Lehninger, 1970; Shick, 1991). The study described here was designed to test these predictions of the nitrogen conservation hypothesis. Materials and methods Maintenance of Aiptasia pulchella The study was conducted on a clonal culture of Aiptasia pulchella bearing Symbiodinium sp. of clade-b, sensu Rowan and Powers (1991) (A. E. Douglas, unpublished results). The culture was derived from an animal collected from a seawater dike at Tongkang (22 °N), Taiwan, and was maintained in aerated artificial sea water (Instant Ocean Salts) of salinity 35 ‰ and pH 8.2 at 25 °C under 12 h:12 h light:dark regime at 50 µmol m−2 s−1 photosynthetically active radiation (P.A.R.) The animals were fed twice a week with 1-day-old Artemia nauplii. The animals used for experiments had an oral disc 5–7 mm in diameter and their tentacles bore 6×106 to 7×106 algal cells mg−1 protein. They are termed the ‘control’ animals. The algal population in A. pulchella was depleted by cold shock, following the procedure of Steen and Muscatine (1987). Symbiotic animals were maintained at 4 °C for 4 h, and then returned to the culture at 25 °C in continuous darkness, apart from the limited periods required for feeding and maintenance. Over 1 month, the population of algal cells in the tentacles of the animals declined to 1000 cells mg−1 animal protein, but no further decrease was consistently obtained over 6 months. These animals are termed ‘alga-depleted’, and they were used for experiments between 1 and 3 months after the cold-shock treatment. Unless stated otherwise, the alga-depleted animals were maintained in continuous darkness during experiments. For studies on the impact of organic carbon compounds on A. pulchella, animals were maintained in 250 ml of artificial sea water, supplemented with 10 mmol l−1 (final concentration) fumarate, glucose, glycerol, α-ketoglutarate or succinate (Sigma Chemical Co.). The pH of media with organic acid supplements was adjusted to pH 8.2 with KOH. The cultures were maintained in darkness, and the medium was changed daily. Biochemical assays on the animal fraction Freshly excised tentacles of A. pulchella were used for biochemical analysis because the density of algae was high and uniform in these tissues of the control animals (J.-T. Wang and A. E. Douglas, unpublished results). For all assays, each sample consisted of 30 tentacles, cut from A. pulchella with sharp scissors and homogenised in 0.5 ml of ice-cold 3.5 % NaCl with a hand-held glass tissue grinder. Each homogenate was centrifuged at 20 000 g at 4 °C for 5 min, and the supernatant was decanted and retained. The supernatant had no algal contamination, as indicated by the absence of intact algal cells by light microscopy and undetectable chlorophyll content, and it was therefore used as the animal fraction. The ammonium content of the animal fraction was assayed spectrophotometrically, using the ammonia diagnostic kit (Technical Bulletin 171-UV) of Sigma Chemical Co., in which the amination of α-ketoglutarate is quantified by the oxidation of NADPH and associated reduction in absorbance at 340 nm. Prior to amino acid analysis, the protein in the animal fraction was precipitated by adding 230 µl of absolute ethanol to each 100 µl sample, followed by centrifugation at 20 000 g for 3 min at 4 °C. The supernatant was stored at −20 °C until analysis. The amino acids (100–300 µmol l−1) were derivatized with 1 mmol l−1 o-phthaldialdehyde for 1 min (Jones et al. 198l) and quantified by high-performance liquid chromatography (HPLC) using a Beckman System Gold delivery system with C18-ultrasphere column and Shimadzu RF-551 fluorescence detector. This procedure detects all protein amino acids, except cysteine and proline, and many non-protein amino acids. The reference amino acid mixture was AA-S-18 (Sigma), supplemented with γ-aminobutyric acid, asparagine, glutamine, ornithine, taurine and tryptophan. Glutamine synthetase activity was quantified by the transferase assay (Pahel et al. 1982), in which the synthesis of γ-glutamyl hydroxamate from glutamine is determined spectrophotometrically at 540 nm. Each sample of animal fraction was combined with an equal volume of imidazole buffer (pH 7.4) containing 40 mmol l−1 imidazole–HCl, 0.6 mmol l−1 MnCl2,, 0.5 mol l−1 sucrose, 20 mmol l−1 dithiothreitol and 200 µg ml−1 cetyltrimethylammonium bromide. Half of the combined sample was incubated at 100 °C for 15 min, for use as a sample blank, against which the activity of the other half of the sample was quantified. The specificity of the reaction was confirmed by the 90 % reduction in activity in samples pre-incubated with 5 mmol l−1 L-methionine sulphoximine, a specific inhibitor of glutamine synthetase. Enzyme activity is expressed as nmol product formed mg−1 animal protein min−1 at 30 °C. The activities of aspartate aminotransferase and alanine aminotransferase in the animal fraction were quantified using the transaminase kit (procedure 505) of Sigma Chemical Co., in which the keto-acid product is reacted with 2,4-dinitrophenylhydrazine and determined spectrophotometrically at 500 nm. The specificities of the reactions for aspartate aminotransferase and alanine aminotransferase were confirmed by reductions in activity by 96 % and 97 %, respectively, when the substrates (aspartate and alanine) were omitted from the reaction mixture. The enzyme activities are expressed as nmol glutamate formed mg−1 animal protein min−1 at 25 °C. The protein content of the animal fraction was determined using the protein assay kit of Bio-Rad Chemical Co., according 2448 J.-T. WANG AND A. E. DOUGLAS to manufacturer’s instructions, with bovine serum albumin as standard. Glutamine synthetase activity (nmol mg−1 min−1) 12 h:12 h L:D A 75 50 25 0 40 35 30 25 20 15 150 Alanine aminotransferase activity (nmol mg−1 min−1) Results The animal fraction of Aiptasia pulchella The first experiments explored the ammonium and free amino acid content of the animal fraction in control animals, i.e. A. pulchella containing the ‘normal’ population of symbiotic algae, and in alga-depleted animals, whose algal population had been experimentally depressed 6000- to 7000fold (see Materials and methods). The concentration of ammonium was significantly elevated in the alga-depleted animals, but the total free amino acid content did not differ significantly between the two treatments (Table 1A). The dominant amino acid in all the samples was taurine, a nonprotein amino acid which accounted for 57±2 % and 79±1.4 % (mean ± S.E.M., N=12) of the amino acids in the control and alga-depleted animals, respectively. All the protein amino acids were detected in the samples (apart from cysteine and proline, which cannot be quantified by the method used), and the animals also contained low concentrations of the nonprotein amino acids ornithine and γ-aminobutyric acid. The total concentration of protein amino acids was significantly higher in the free amino acid pool of control animals than in that of the alga-depleted animals (Table 1A). The activity of three enzymes in the animal fraction is shown in Table 1B. The animals with depleted algal population displayed depressed glutamine synthetase activity, relative to the control animals, an increase in alanine aminotransferase activity and no significant change in aspartate aminotransferase activity. Separate experiments had demonstrated that, when control animals are transferred from the 12 h:12 h L:D light regime to continuous darkness for 6–7 days, the algal population in their tentacles is reduced by 30 % ((J.-T. Wang and A. E. Douglas, unpublished results), a value more than 1000 times greater than the population in the alga-depleted animals. This provided the basis to explore the contribution of darkness per se and of the very low algal density to the low glutamine synthetase activity of the animal fraction in alga-depleted animals. Fig. 2 shows how the enzyme activity in control animals is influenced by incubation in darkness for 6 days, followed by a 12 h:12 h L:D regime (the routine culture conditions for control animals). The activity of glutamine synthetase and alanine aminotransferase, 100 Aspartate aminotransferase activity (nmol mg−1 min−1) Statistical procedures All variables were checked to conform to the assumptions of normality and homogeneity of variance, as indicated by the Kolmogorov–Smirnoff one-sample test and Bartlett’s test, respectively. For percentage variables, the data were arcsinsquare-root-transformed prior to analysis. The parametric tests used were t-test, least-squares regression and analysis of variance (ANOVA), with Fisher’s least significant difference (LSD) method for pairwise comparisons that contributed to significant ANOVA differences. 24 h D 0 B 3 6 9 12 15 C 125 100 75 50 25 0 2 4 6 8 10 12 14 16 Time (days) Fig. 2. Impact of light regime on the activity of enzymes in amino acid metabolism in the animal fraction of control animals of Aiptasia pulchella incubated in continuous darkness for 6 days and then (arrow) transferred to a 12 h:12 h L:D regime. Enzyme activities are expressed as nmol product mg−1 animal protein min−1, and mean values ± S.D. (six replicates) are shown. (A) Glutamine synthetase (ANOVA: F10,55=26.60, P<0.0001), (B) aspartate aminotransferase (F10,55=1.93, P>0.05), (C) alanine aminotransferase (F10,55=20.04, P<0.001). but not of aspartate aminotransferase, varied significantly over the experiment (details of ANOVAs are given in the in legend to Fig. 2). Subsequent application of Fisher’s LSD test confirmed that glutamine synthetase activity was significantly depressed, below the value on day 0, between days 2 and 13 of the experiment, and that alanine aminotransferase activity was significantly elevated, relative to day 0, on day 2 and between days 4 and 13 (P<0.05). The impact of organic carbon compounds on the animal fraction of Aiptasia pulchella When A. pulchella was incubated in darkness for 7 days in medium supplemented with an organic carbon compound, both Nitrogen metabolism in an alga–invertebrate symbiosis 2449 A a 125 Control (N =9) 75 b,c b,c d 0 125 B a Alga-depleted (N =3) a 100 b 75 b,c c c 50 25 a,b 60 Ammonium concentration (nmol mg−1 protein) c Control (N =3) a 80 b 100 Glutamine synthetase activity (nmol mg−1 protein min−1) C a 40 20 ND ND 0 125 D a Alga-depleted ( N =3) b 100 b b 75 50 50 c 25 ND 25 ND ND N N Carbon supplement (12 h:12 h Light condition L:D) S F Gly Glu ND ND ND 0 0 K N 24 h D N S (12 h:12 h L:D) F Gly Glu K 24 h D Fig. 3. Impact of organic carbon supplements on the animal fraction of Aiptasia pulchella. Glutamine synthetase activity in (A) control animals (ANOVA: F6,56=22.2, P<0.0001) and (B) alga-depleted animals (F3,8=116.94, P<0.0001), and ammonium concentration in (C) control animals (F4,10=5.50, 0.01<P<0.05) and (D) alga-depleted animals (F3,8=5.64, 0.01<P<0.05). Mean values + S.E.M. are shown (N=number of replicates; ND, not determined), and values that are not significantly different from each other (Fisher’s least significant difference test, P>0.05) are given the same superscript. Carbon supplements: N, none; S, succinate; F, fumarate; Gly, glycerol; Glu, glucose; K, α-ketoglutarate. Table 1. Nitrogenous compounds and enzyme activities in Aiptasia pulchella A Nitrogenous compounds Free amino acids Animals Control Alga-depleted Ammonium (nmol mg−1 protein) Total amino acids (nmol mg−1 protein) Protein amino acids (nmol mg−1 protein) 58±7 (14) 149±10 (9) 923±51 (12) 843±48 (12) 392±35 (12) 168±17 (12) t21=7.53, P<0.0001 t22=1.16, P>0.05 t22=4.96, P<0.001 B Activity of enzymes involved in amino acid metabolism Animals Control Alga-depleted Glutamine synthetase (nmol mg−1 min−1) Alanine aminotransferase (nmol mg−1 min−1) Aspartate aminotransferase (nmol mg−1 min−1) 126±6 (18) 27±2 (14) 44±5 (6) 85±2 (6) 32±2 (6) 31±1 (6) t30=14.08, P<0.0001 t10=7.95, P<0.0001 t10=0.52, P>0.05 Enzyme activities are measured as nmol product mg−1 animal protein min−1. The control animals (containing symbiotic algae) were maintained under 12 h:12 h L:D photoperiod and the alga-depleted animals in continuous darkness. Values are means ± S.E.M., with the number of replicates in parentheses. the control and alga-depleted animals exhibited a significant increase in glutamine synthetase activity (Fig. 3A,B) and a significant reduction in ammonium concentration (Fig. 3C,D). The effect varied between the compounds tested. Fumarate, for example, consistently increased glutamine synthetase to levels intermediate between those of animals incubated without a 2450 J.-T. WANG AND A. E. DOUGLAS N=12), respectively, of the total free amino acids; these values were significantly different (t22=13.02, P<0.0001, for arcsinsquare-root-transformed data). There was a commensurate change in the taurine content, which accounted for 48 % of the amino acids in the animals with the carbon supplement and for 78 % in those without the supplement. [Ammonium] (nmol mg−1 protein) 120 100 80 60 40 20 0 0 30 60 90 120 150 Glutamine synthetase activity (nmol product mg −1 protein min −1) Fig. 4. Regression analysis of ammonium concentration on glutamine synthetase activity in the animal fraction of control animals (䊏) and alga-depleted animals (䊐) incubated in darkness for 7 days. The regression equation for the control animals is: y=−0.22x+78.7 (r=0.58, P<0.05), and that for the alga-depleted animals is: y =−0.33x+113 (r=0.63, P<0.05), where y is the ammonium concentration and x is the glutamine synthetase activity. carbon supplement in continuous darkness and control animals in the 12 h:12 h L:D regime; and α-ketoglutarate stimulated glutamine synthetase activity to values characteristic of control animals in the 12 h:12 h L:D light regime. In these experiments, the ammonium content of the algadepleted animals was generally higher than that of darkincubated control animals under the same treatment. To explore this further, the ammonium content of each sample was regressed on the corresponding value of glutamine synthetase activity (Fig. 4). The regression equations (see legend to Fig. 4) for the control and alga-depleted animals had negative slopes that were not significantly different from each other (t23=0.53, P>0.05), but the intercept of the equation for alga-depleted animals was significantly greater than that for control animals (t23=12.69, P<0.0001). This result suggests that the control animals have an appreciable sink for ammonium, additional to glutamine synthetase, that is absent from alga-depleted animals. As considered in the Discussion, the additional sink may be the algal population. The impact of one organic compound, α-ketoglutarate, on the free amino acid content of the alga-depleted animals was examined. After 7 days of incubation with and without the carbon supplement, the protein amino acid contents of the animal fraction was 50±1.8 % and 20±1.3 % (mean ± S.E.M., The animal fraction of alga-depleted Aiptasia pulchella incubated under illuminated conditions When alga-depleted animals were transferred to the 12 h:12 h L:D regime, the residual population of symbiotic algae increased. Twelve weeks later, it was noted that the algal population in the tentacles varied between animals over three orders of magnitude: from animals with 2×103 algal cells mg−1 protein, just double the mean algal population in the dark-maintained alga-depleted animals, to animals with 2×106 cells mg−1 protein, nearly one-third of the algal population in control animals. Although the cellular basis of this wide variation has yet to be established, it did provide the opportunity to investigate the impact of algal density on the glutamine synthetase activity and ammonium content of the animal fraction. Consistent with the differences observed between control and alga-depleted animals (Table 1), the activity of glutamine synthetase in the animal fraction increased, and the ammonium concentration decreased, with density of algae. The data were log-transformed prior to analysis (this increased the correlation coefficients of both glutamine synthetase activity and ammonium concentration with algal density). As shown in Fig. 5, with regression equations in the legend, the slopes of the regressions of log(glutamine synthetase activity) and log(ammonium concentration) on log(algal density) were similar in magnitude, but opposite in sign, at +0.114 (S.D. 0.0177) and −0.120 (S.D. 0.0323), respectively. In other words, for a 10-fold increase in algal density, the animal is predicted to exhibit a 24–30 % increase in glutamine synthetase activity and an equivalent decrease in ammonium content. Discussion When Aiptasia pulchella is incubated in darkness or its population of Symbiodinium is subtantially depleted, it exhibits an increased ammonium concentration in its tissues (this study) and an increased ammonium efflux into the medium (Wilkerson and Muscatine, 1984). These differences in ammonium content and efflux rates would, traditionally, be attributed to algal assimilation of animal-derived ammonium and cited as evidence for nitrogen recycling by the algae (see Introduction and Fig. 1Bi). This explanation is confounded by the demonstration here that the effects of darkness and of depletion of the algal population on the ammonium concentration in the animal tissues are partially or completely reversed by exogenous carbon compounds (Fig. 3). The most parsimonious explanation for the results of this study is that organic carbon, whether derived from algal photosynthate or exogenous sources, promotes the assimilation of ammonium 2.2 2.2 2.1 2.1 2.0 2.0 1.9 1.9 1.8 1.8 1.7 1.7 1.6 1.6 1.5 1.5 1.4 3.03.0 3.53.5 4.0 4.0 4.5 4.5 5.0 5.0 5.5 6.0 log10(ammonium concentration) log10(glutamine synthetase activity) Nitrogen metabolism in an alga–invertebrate symbiosis 2451 1.4 6.5 6.5 log10(algal density) Fig. 5. Regression analysis of glutamine synthetase activity (䊏) and ammonium concentration (䊐) of the animal fraction on algal density (cells mg−1 animal protein), for alga-depleted Aiptasia pulchella incubated in a 12 h:12 h L:D regime for 12 weeks. The regression equations are: log(glutamine synthetase activity)=0.114log(algal density)+1.09 (r=0.90, P<0.05) and log(ammonium concentration)= −0.120log(algal density)+2.43 (r=0.76, P<0.05). (Fig. 1Biv) and/or depresses the production of ammonium in the animal tissues (Fig. 1Biii). This study has obtained strong evidence for the promotion of ammonium assimilation in the animal by organic carbon compounds, principally via glutamine synthetase, an enzyme that catalyses the ATP-dependent amination of glutamate to produce glutamine. The treatments that resulted in an elevated ammonium concentration (darkness, depletion of the algal population) also depressed glutamine synthetase activity (Table 1B), and the organic carbon supplements enhanced glutamine synthetase activity (Fig. 3). Further, the finding that the regressions of glutamine synthetase activity and ammonium concentration on algal density had closely similar slopes of opposite sign (Fig. 5) suggests that glutamine synthetase may play an important role in mediating the impact of algal density on the ammonium content of the animal. It is unlikely, however, that algaderived photosynthate contributes to the substrate pool of glutamate for glutamine synthetase in the animal tissues, because the principal fates of algal photosynthate translocated to the animal are respiration and incorporation into the lipid fraction, not the amino acid or protein fractions (e.g. Trench, 1971a; Battey and Patton, 1987; Wang, 1998). Perhaps the algal photosynthate and exogenous carbon compounds enhance the respiration rates of the animal, and the resultant increase in ATP content and energetic status of the animal tissues may result in increased glutamine synthetase activity. Consistent with this proposal, both feeding and the receipt of algal photosynthate are known to enhance rates of respiratory oxygen consumption in Cnidaria (Muller-Parker, 1984: Tytler and Davies, 1984; Harland and Davies, 1995), and a favourable energetic status promotes glutamine synthetase activity in some algae (e.g. GarciaFernandez et al. 1995). Further research is required to test this interpretation because, at present, nothing is known about the regulation of glutamine synthetase activity in Cnidaria beyond the expectation that, as with all other eukaryotes, it is not controlled by the complex adenylylation/deadenylylation system found in most bacteria (White, 1995). The reduction in glutamine synthetase activity in Cnidaria deprived of their algae has been reported previously only in the freshwater hydra–Chlorella symbiosis (Rees, 1986), and our data serve to generalise those findings from freshwater to marine systems and across two classes (Hydrozoa to Anthozoa). The impact of algae on the glutamine synthetase activity in hydra was considered exclusively in terms of its proposed function to reduce the availability of ammonium to the algal cells (Fig. 1Bii). As described above, data from this study on A. pulchella indicate that glutamine synthetase is modulated by alga-derived photosynthate (Fig. 1Biv) and not by the presence of algae per se. [The reduced glutamine synthetase activity of hydra bearing Chlorella, which release relatively little photosynthate (Rees, 1986), suggests that this interpretation may apply to the hydra association.] The implication of our data is that the translocation of algal photosynthate would tend, via increased glutamine synthetase activity, to depress the availability of ammonium to the algal cells. If ammonium were the principal, or sole, nitrogen source utilised by the algae, then nitrogen limitation could be a direct consequence of the translocation of photosynthate to the animal tissues. However, the relative importance of ammonium and organic nitrogen compounds to the nitrogen nutrition of the algae in symbiosis is uncertain; Symbiodinium can certainly assimilate both ammonium (see Introduction) and amino acids (e.g. Carroll and Blanquet, 1984; Bester et al. 1997) efficiently from low external concentrations. The data in Fig. 4 provide circumstantial evidence that the algal cells in symbiosis utilise animal-derived ammonium. The significantly lower intercept for the regression of ammonium concentration on glutamine synthetase activity in control animals than in algadepleted animals suggests that the former assimilate ammonium by a route, additional to animal glutamine synthetase, that is absent from the latter. This may be the algal cells. Symbiodinium is known to be capable of sustained ammonium assimilation, even in darkness (McAuley and Smith, 1995, and references cited therein). In developing the nitrogen conservation hypothesis, Rees and Ellard (1989) emphasized the importance of algal photosynthate as an alternative respiratory substrate to the 2452 J.-T. WANG AND A. E. DOUGLAS amino acid pool of the animal (Fig. 1Biii). This study provides only qualified evidence for this interpretation. The expectation that darkness and depletion of the algal population would enhance animal transaminase activity (see Introduction) was borne out for one of the two transaminases examined (Table 1B; Fig. 2B,C), and the expectation that algal depletion would depress the free amino acid concentrations was confirmed for the protein amino acids, but not for total amino acids, in the free amino acid pool of the animal tissues (Table 1A). This latter result may reflect the role of free amino acids in the regulation of cell volume in sea anemones (Shick, 1991), such that the total free amino acid content of the animal tissues is constrained to vary within very narrow limits, in medium of a given salinity. As with some other sea anemone species (e.g. Male and Storey, 1983; Deaton and Hoffmann, 1988; Herrera et al. 1989), the principal osmolyte in A. pulchella may be taurine, an amino acid that cannot, generally, be utilised as a respiratory substrate by animals (Huxtable, 1992). The concentration of protein amino acids (but not total amino acids) in the animal’s free amino acid pool is, therefore, the most appropriate test of the prediction (above) that algal photosynthate may enhance the free amino acid concentration in the animal tissues. Consistent with this prediction of the nitrogen conservation hypothesis, the protein amino acids contribute 42 % and 50 %, respectively, of the total free amino acid pool in control animals and in alga-depleted animals incubated with an exogenous carbon compound, but just 20 % of the total in alga-depleted animals in routine culture. In conclusion, this study has revealed the crucial importance of organic carbon to the nitrogen metabolism of the animal tissues in the Aiptasia pulchella–Symbiodinium symbiosis. Whether derived from algal photosynthate or exogenous sources, this organic carbon may both promote animal assimilation of ammonium via glutamine synthetase (Fig. 1Biv) and reduce the rates of ammonium production by providing an alternative respiratory substrate to amino acids (Fig. 1Biii). The impact of alga-derived photosynthate on ammonium production and consumption by the animal confounds the interpretation of experimental designs traditionally used to explore the role of the algae in nitrogen recycling. As a result, the relative contributions of nitrogen conservation and nitrogen recycling to the overall nitrogen relations of alga–invertebrate symbioses remain unresolved. The key issue to establish is the relative strength of the ammonium assimilatory capabilities of algal and animal cells. This may vary with environmental circumstance and between different associations, with substantial implications for both the regulation of algal population increase and the nitrogen nutrition of the animal. We thank Dr K. Soong (National Sun Yat-sen University, Taiwan) for providing us with Aiptaisia pulchella and Dr T. L. Wilkinson for his constructive comments on the manuscript. This research was funded by a studentship to J.T.W. provided by the Ministry of Education, Taiwan. References BATTEY, J. F. AND PATTON, J. S. (1987). Glycerol translocation in Condylactis gigantea. Mar. Biol. 95, 37–46. BESTER, C., LIPSCHULTZ, F., REAM, R., TOMASINO, T. AND TRAPIDOROSENTHAL, H. (1997). Effects of exposure to exogenous amino acids on the cellular physiology of cultured zooxanthellae. Proc. 8th int. Coral Reef Symp. 2, 1287–1290. CARROLL, S. AND BLANQUET, R. S. (1984). Alanine uptake by isolated zooxanthellae of the mangrove jellyfish, Cassiopea xamachana. I. Transport mechanisms and utilization. Biol. Bull. mar. biol. Lab., Woods Hole 166, 409–418. CATES, N. AND MCLAUGHLIN, J. J. A. (1976). Differences of ammonia metabolism in symbiotic and aposymbiotic Condylactis and Cassiopea spp. J. exp. mar. Biol. Ecol. 21, 1–5. COOK, C. B. AND D’ELIA, C. F. (1987). Are natural populations of zooxanthellae ever nutrient-limited? Symbiosis 4, 199–212. DEATON, L. E. AND HOFFMANN, R. J. (1988). Hypoosmotic volume regulation in the sea anemone Metridium senile. Comp. Biochem. Physiol. 91C, 187–191. DOMOTOR, S. L. AND D’ELIA, C. F. (1984). Nutrient uptake kinetics and growth of zooxanthellae maintained in laboratory culture. Mar. Biol. 80, 93–101. DOUGLAS, A. E. (1994). Symbiotic Interactions. Oxford: Oxford University Press. FALKOWSKI, P. G., DUBINSKY, Z., MUSCATINE, L., AND MCCLOSKEY, L. (1993). Population control in symbiotic corals. BioSci. 43, 606–611. GARCIA-FERNANDEZ, J. M., LOPEZ-RUIZ, A., ALHAMA, J. AND DIEZ, J. (1995). Light regulation of glutamine synthetase in the green alga Monoraphidium braunii. Plant Physiol. 146, 577–583. HARLAND, A. D. AND DAVIES, P. S. (1995). Symbiont photosynthesis increases both respiration and photosynthesis in the symbiotic sea anemone Anemonia viridis. Mar. Biol. 123, 715–722. HERRERA, F. C., LOPEZ, I., EGEA, R. AND ZANDERS, I. P. (1989). Shortterm osmotic responses of cells and tissues of the sea anemone, Condylactis gigantea. Comp. Biochem. Physiol. 92A, 377–384. HOEGH-GULDBERG, O. AND SMITH, G. J. (1989). Influence of the population density of zooxanthellae and supply of ammonium ions on the biomass and metabolic characteristics of the reef corals Seriatopora hystrix and Stylophora pistillata. Mar. Ecol. Prog. Ser. 57, 173–186. HUXTABLE, R. J. (1992). Physiological actions of taurine. Physiol. Rev. 72, 101–163. JONES, B. N., PAABO, S. AND STEIN, S. (1981). Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labelling procedure. J. Liquid Chromatogr. 4, 565–586. KAWAGUTI, S. (1953). Ammonium metabolism of the reef corals. Biol. J. Okayama Univ. 1, 171–176. LEHNINGER, A. L. (1970). Biochemistry, 2nd edition. New York: Worth Publishers Inc. LEWIS, D. H. AND SMITH, D. C. (1971). The autotrophic nutrition of symbiotic marine coelenterates with special reference to hermatypic corals. I. Movement of photosynthetic products between the symbionts. Proc. R. Soc. Lond. B 178, 111–129. MALE, K. B. AND STOREY, K. B. (1983). Kinetic characterization of NADP-specific glutamate dehydrogenase from the sea anemone Anthopleura xanthogrammica: control of amino acid biosynthesis during osmotic stress. Comp. Biochem. Physiol. 76B, 823–829. MARKELL, D. A. AND TRENCH, R. K. (1993). Macromolecules exuded by symbiotic dinoflagellates in culture: amino acid and sugar composition. J. Phycol. 29, 64–68. Nitrogen metabolism in an alga–invertebrate symbiosis 2453 MCAULEY, P. J. AND SMITH, V. J. (1995). Effect of diel photoperiod on nitrogen metabolism of cultured and symbiotic zooxanthellae. Mar. Biol. 123, 145–152. MULLER-PARKER, G. (1984). Photosynthesis-irradiance responses and the photosynthetic periodicity in the sea anemone Aiptasia pulchella and its zooxanthellae. Mar. Biol. 82, 225–232. MULLER-PARKER, G., MCCLOSKEY, L. R., HOEGH-GULDBERG, O. AND MCAULEY, P. J. (1994). Effect of ammonium enrichment on animal biomass of the coral Pocillopora damicornis. Pac. Sci. 48, 273–283. MUSCATINE, L. (1990). The role of symbiotic algae in carbon and energy flux in reef corals. In Coral Reefs (ed. Z. Dubinsky), pp. 7587. Amsterdam: Elsevier. MUSCATINE, L. AND CERNICHIARI, E. (1969). Assimilation of photosynthetic products of zooxanthellae by a reef coral. Biol. Bull. mar. biol. Lab., Woods Hole 137, 506–523. MUSCATINE, L. AND PORTER, J. W. (1977). Reef corals: mutualistic symbiosis adapted to nutrient poor environments. BioSci. 27, 154–157. PAHEL, G., ROTHSTEIN, D. M. AND MAGASANIK, B. (1982). Complex glnA-glnL-glnG operon of Escherichia coli. J. Bacteriol. 150, 202–213. REES, T. A. V. (1986). The green hydra symbiosis and ammonium. I. The role of the host in ammonium assimilation and its possible regulatory significance. Proc. R. Soc. Lond. B 229, 299–314. REES, T. A. V. AND ELLARD, F. M. (1989). Nitrogen conservation and the green hydra symbiosis. Proc. R. Soc. Lond. B 236, 203–212. ROWAN, R. AND POWERS, D. A. (1991). A molecular genetic classification of zooxanthellae and the evolution of animal–algal symbioses. Science 251, 1348–1351. SHICK, J. M. (1991). A Functional Biology of Sea Anemones. London: Chapman & Hall. STEEN, R. G. AND MUSCATINE, L. (1987). Low temperature evokes rapid exocytosis of symbiotic algae by a sea anemone. Biol. Bull. mar. biol. Lab., Woods Hole 172, 246–263. SUTTON, D. C. AND HOEGH-GULDBERG, O. (1990). Host–zooxanthellae interactions in four temperate marine invertebrate symbioses: assessment of effect of host extracts on symbionts. Biol. Bull. mar. biol. Lab., Woods Hole 178, 175–186. SZMANT-FROELICH, A. AND PILSON, M. E. Q. (1977). Nitrogen excretion by colonies of the temperate coral Astrangia danae with and without zooxanthellae. Proc. 3rd int. Coral Reef Symp. 1, 417–423. TRENCH, R. K. (1971a). The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. I. The assimilation of photosynthetic products of zooxanthellae by two marine coelenterates. Proc. R. Soc. Lond. B 177, 225–235. TRENCH, R. K. (1971b). The physiology and biochemistry of zooxanthellae symbiotic with marine coelenterates. III. The effect of homogenates of host tissues on the excretion of photosynthetic products in vitro by zooxanthellae from two marine coelenterates. Proc. R. Soc. Lond. B 177, 251–264. TRENCH, R. K. (1993). Microalgal–invertebrate symbioses: a review. Endocytobiosis Cell Res. 9, 135–175. TYTLER, E. M. AND DAVIES, P. S. (1984). Photosynthetic production and respiratory energy expenditure in the anemone Anemonia sulcata (Pennant). J. exp. mar. Biol. Ecol. 81, 73–86. WANG, J.-T. (1998). Nutritional interactions between the alga Symbiodium and sea anemone Aiptasia pulchella. PhD thesis, University of York, UK. WHITE, D. (1995). The Physiology and Biochemistry of Prokaryotes. New York: Oxford University Press. WILKERSON, F. P AND MUSCATINE, L. (1984). Uptake and assimilation of dissolved inorganic nitrogen by a symbiotic sea anemone. Proc. R. Soc. Lond. B 221, 71–86.