Growth and Metabolic Responses of the Giant Clam- Zooxanthellae Symbiosis in a Reef-Fertilisation Experiment

MARINE ECOLOGY PROGRESS SERIES Vol. 170: 131-141, 1998 Published September 3 Mar Ecol Prog Ser

Growth and metabolic responses of the giant clam- zooxanthellae symbiosis in a reef-fertilisation experiment

'Department of Biochemistry and Molecular Biology and 'Department of Zoology, James Cook University of North Queensland,Townsville, Queensland 4811, Australia

ABSTRACT: To evaluate the impact of elevated nutrients on reef organisms symbiotic with zooxanthellae, giant clams Tridacna maxima were exposed daily to increased ammonia and phosphate (N, P, N+P) in their natural reef environment for 3 to 6 mo. The results strongly corroborate the major responses of the syn~bioticassociation to nutrient enr~chlnentpreviously observed (wlth T gigas) under controlled outdoor conditions.Exposure of the clams to elevated N (10 pM)increased zooxanthellae density, reduced zooxanthellae size, down-regulated N uptake by zooxanthellae freshly isolated from their hosts, and reduced glutamate in the clam haernolymph,with increased pools of some free amino acids (methionine,tyrosine) In the zooxanthellae These results confirm that the zooxanthellaein giant clams are N limited jn situand have free access to inorganic N from the sea water. There is also cor- roborating evidence thatthe zooxanthellae are P limited jn situ as well, possibly due to host interfer- ence While the N-Pratios of the animal host reflected ambient N and P concentrations in the sea water, those of the zooxanthellae d~dnot. Regardless of P exposure (2 FM P) of the clams, zooxanthellaeN:P ratios were cons~stent'lyhigh (>30:1) and phosphate concentrations in the clam haemolymph bathing the zooxanthellae tube system consistently low (<0.1 PM). These field findings, consistentwith previous laboratory observations,confirm the Limiting roles of both N and P in the giant clam-zooxanthellae symbiosis. That significant changes occurred earlier and at lower nutrient loading compared to some reef organisms investigatedwithin the same experimental framework further demonstratesorganism- level responses of a potential bio-indicatorof the early onset of eutrophication in reef waters.

KEYWORDS: Giant clam . Zooxanthellae . Symb~os~sNutrient limitat~on. Bio-indicators . Eutrophi- cation

INTRODUCTION tropical waters (Hawkins & Klumpp 1995). Adapted to such an impoverished environment, the giant clams' Like many marine invertebrates symbiotic with pho- growth and physiology can be strongly influenced by el- tosynthetic dinoflagellates (Symbiodinium sp.) called evated nutrient concentrations from natural or anthro- zooxanthellae, tndacnid clams are able to thrive in the pogenic events. For instance, it has previously been nutrient-poor tropical waters of the Indo-Pacific region. demonstrated that a major effect of chronically elevated The tridacnids' efficient utilisation of particulate organic nutrients (mainly N) on giant clams is enhancement of matter through filter-feeding (Klumpp et al. 1992), and soft-tissue production (Hastie et al. 1992, Belda et al. the algal symbionts' capability to recycle scarce nutrients 1993b, Fitt et al. 1993), with a simultaneous weakening are characteristics that are advantageous in oligotrophic of the shell structure (5 to 10 pM N, 2 to 10 pM P) (Belda et al. 1993a). While the former effect is appealing to on- P shore ventures in the commercial mariculture of these bi- 'Present address:Manne Science Institute. University of the Philippines, Diliman,Quezon City 1101, The Philippines valves, the effect on calcification has adverse implica- "Addressee for correspondence. tions for calcifying organisms in reef waters with E-mail: [email protected] elevated nutrient concentrations.

0 Inter-Research 1998 Resale of fullarticle not permitted 132 Mar Ecol Prog Ser

It is notable that such gross changes in clam tissue sponses in situor confound the effects of nutrients. In production with N-enrichment are accompanied by the case of sewage outfall in Kaneohe Bay (Kinsey more subtle effects on a range of metabolic parameters 1988), there was a pronounced shift in community including: C:N:P ratios of the animal tissue; ammo- structure. The resulting enhancement of phytoplank- nia/ammonium concentration in the clarn haernolymph ton growth favoured the dominance of particle feeders, (Fitt et al. 1995); and densrty. chlorophyll a content, loss of available substratum, reduction in light penetra- and ammonium uptake rates of the zooxanthellae tion, and decline in benthic primary production. In an (Belda et al. 1993b). While these responses to elevated early experiment of Kinsey & Domm (1974), however, nutrients (along with a lack of change in other proper- where nitrogen (mainly urea) and phosphorus were ties) provide useful information on the metabolic rela- added daily to a patch reef over 8 mo, a striking 50% tionship between the clam and its algal symbionts, reduction in calcification of the reef system was evi- they are also indicative of the nutrient regimen In dent (Kinsey & Davies 1979), along with a 25% en- which the symbiotic partners are growing. As such, hancement in primary productivity due to increased these parameters may represent powerful environ- benthic algae. Clearly, experimental manipulations in mental tools for evaluating current and perhaps recent the field are necessary to better understand the impact nutrient exposure of reef organisms. Identification of of eutrophication on reef organisms. bio-indicators of the early onset of eutrophication on The Enrichment of Nutrients on a Coral Reef Experi- coral reefs should prove extremely useful. ment or ENCORE is the latest initiative and the first Studies elucidating the effects of inorganic nutrients fully replicated and controlled field experiment on the on coral reef organisms have been few until recently. long-term effect of elevated nutrients on reef commu- One of the first collaborative programs to address the nities. This large-scale manipulative reef-fertilisation issue of nutrient effects on reef-building corals was the experiment was carried out on Australia's Great Bar- 1991 US-Israel workshop at the Hawaii Institute of rier Reef (GBR) by the Great Barrier Reef Marine Park Marine Biology in Kaneohe Bay, Hawaii, USA (Jokiel Authority from 1993 to 1996 in response to a global et al. 1994). Exposure of 2 species of Hawaiian corals, concern about the effect of eutrophication on coral Pocillopora darnicornis and Montipora verrucosa, in reefs. This interdisciplinary undertaking, consisting of tanks containing elevated N (20 and 50 pM N as acclimation, nutrient enrichment, and recovery (NH,),SO,) significantly affected a broad spectrum of phases, was designed to: (1) provide quantitative evi- growth and metabolic parameters of the symbiotic dence on the relative impacts of elevated N and P on partners. N enrichment increased area1 concentrations various reef organisms, and the processes that regulate of zooxanthellae and chlorophyll, host protein and these responses; and (2) test the usefulness of parame- zooxanthellae N, but did not affect host C:N:P ratio ters proposed as sub-lethal indicators of eutrophication (Muller-Parker et al. 1994a, b); increased zooxanthel- (Larkum & Steven 1994). The present study on giant lae mitotic index and growth rate, and disrupted the clams was part of this initiative. cell division phase (Hoegh-Guldberg 1994); decreased In this paper, the results of an in sifuexperiment on a glutamine synthetase (GS) activity of the host as well natural population of Tridacna maxima are presented. as the GS activity and ammonium uptake capacity of The major aim of this study was to test if clams (T max- freshly isolated zooxanthellae (Yellowlees et al. 1994); ima) periodically exposed to increased nutrients in and increased g1n:glu (g1utamine:glutamic acid) and their natural environment would behave in a manner pool sizes of most free amino acids in the zooxanthellae similar to clams (7.gigas) similarly investigated in an (McAuley 1994). essentially artificial environment (Belda et al. 1993a, b; It should be noted that the above studies were con- Belda & Yellowlees 1995). Indeed, the present field ducted under carefully controlled conditions in an arti- findings are, in most cases, consistent with previous ficial environment and in the absence of other major laboratory observations, and together constitute valu- organisms. They do not guarantee that parallel studies able information that 1s useful in reaching definitive in situwould yield similar results. Although such labo- conclusions regarding the impact of nutrlent enrich- ratory studies are quite useful and necessary in isolat- ment on reef organisms such as giant clams and their ing the effects of a complex range of environmental zooxanthellae. factors on reef organisms, they are necessarily limited by the handling sensitivity of the experimental organisms and the inadequate simulation of conditions in the MATERIALS AND METHODS field, which is usually an extremely complex and heterogeneous environment. The presence of other or- Experimental design and maintenance. Thls study ganisms competing for available nutrients, for in- constituted a part of ENCORE, a large-scale, manipu- stance, could conceivably decrease the clam's relative reef-fertilisation experiment undertaken on the Belda-Ba~ll~eel al..In situ nutrienteffects on reef clams 133

GBR to document the responses of coral reefs to nutri- shell, the mantle was excised from the rest of the soft ent enrichment. This multi-agency project was con- tissues. Resldual sea water and haemolymph were ducted at the relatively pristine One Tree Island Reef blotted from the soft tissues with paper towels. The tis- (23" 30' S, 152" 06' E) in the Mackay/Capricorn Section sues were weighed separately to the nearest 0.01 g. of the GBR Marine Park. The experimental design Whole-clam, viscera, and mantle tissues were freeze used here has already been described in detail by dried and analysed for C and N composition using a Larkun~& Steven (1994). CHN analyser (LECO CHN-600, Australian Institute of In total, 96 indigenous Tridacna maxima, ranging Marine Science, Townsville; CARLO ERBA EA-1108- from 15 to 30 cm in shell length, were collected from the CHN, James Cook University), and for N and P compo- eastern crest of One Tree Island reef and randomly al- sition using the hydrogen peroxide-sulfuric acid diges- located to 12 patch reefs or micro-atolls of similar size tion technique of Allen (1974). (16 to 25 m across), volume (46 to 152 m3),and benthic Measurement of nutrient levels in clam haemo- composition. These micro-atolls were, in turn, ran- lymph. During extraction of the soft tissues from the domly allocated to 1 of 4 nutrient treatments, namely, shell, the haemolymph was collected in a beaker and nitrogen (N),phosphorus (P),N+P, or no nutrient (con- the volume recorded. The haemolymph was centri- trol), such that there were 3 micro-atolls per treatment. fuged at 2000 X g for 10 min to remove cells and The clams were acclilnated on the micro-atolls for debris. Haemolymph (30 ml) was immediately about 7 mo during the pre-nutrient enrichment phase of analysed for ammonia using a modification (Wilkerson the ENCORE experiment (February to August 1993). & Trench 1986) of the method of Liddicoat et al. (1975). Using computer-controlled nutrient-dispersal units, After adding the appropriate reagents, the samples NH,C1 and/or KH2P0, were simultaneously added to were centrifuged at 1000 X g (10 min) to remove insol- 9 micro-atolls to achieve, at the time of addition, con- uble material before measuring the absorbance in a centrations of 10 FM N and/or 2 pM P, respectively. Fer- spectrophotometer. The rest of the haemolymph was tilisation occurred once at each low tide, when the wa- stored frozen (-20°C) prior to phosphate and phospho- ter contained within the micro-atolls was isolated from rus determinations. The frozen haemolymph was later the surrounding lagoon for about 4 to 5 h. Despite thawed and mixed with 10% (w/v) trichloroacetic acid ponding of the micro-atolls, nutrient concentrations to precipitate protein (see Deane & O'Brien 1980). This were detectable above background levels (0.8 to 1.8 was removed by centrifugation at 2000 X g (10 min) PM N, 0.18 to 0.30 pM P) for only 2 to 3 h after addition. and the supernatant analysed for phosphate (Strick- The micro-atolls were therefore exposed to a pulse of land & Parsons 1972) and total phosphorus (Koroleff nutrients rather than a fixed concentration for the dura- 1983). All assays were done in triplicate for each clam. tion of ponding. This initial nutrient enrichment phase Measurement of zooxanthellae biomass. The man- ran from September 1993 to December 1994. tle was cut into small pieces and homogenised in A second nutrient enrichment phase consisting of 0.45-pm-filtered sea water (FSW), the resulting more frequent (thrice every low tide) and higher nutri- homogenate centrifuged at 1000 X gfor 2.5 min, before ent loading (20 pM N, 4 pM P) was carried out from decanting the animal tissue. The algal pellet was then January to December 1995. Nevertheless, the present resuspended in FSW and the animal tissue removed by study on giant clams bras confined to the second phase filtration through a double layer of clinical gauze cloth. of ENCORE, when nutrients were added only once Most of the remaining zooxanthellae from the animal every low tide. tissue were washed through the filter with FSW. The Measurement of clam biomass. Two clams were col- total zooxanthellae filtrate was then centrifuged at lected from each micro-atoll after 1 and then 3 mo of 2000 X g for 2.5 min, the supernatant discarded, and fertilisation. Before removal of the clams from the the resulting algal pellet resuspended in FSW. The micro-atolls, a wooden wedge was inserted between zooxanthellae were washed twice more or until the the gaping valves in order to keep the valves open for supernatant was clear, before finally resuspending the later extraction of the soft tissues from the shell. Once algal pellet in 20 m1 of FSW. in the laboratory, each clam was immediately inverted A known volume (0.25 ml) of the final zooxanthellae onto a beaker to drain any sea water trapped inside its suspension was preserved in buffered 10% formalin mantle cavities. Using a scalpel, the soft-tissue mass solution. Zooxanthellae density and size were deter- was extracted from the shell by cutting the mantle and mined under a compound microscope for 6 replicate adductor muscles away from the inner surface of both subsamples from each algal suspension using a valves, being careful not to rupture any visceral organs haemocytometer. A known volume (10 ml) of the final in the process. zooxanthellae suspension was also centrifuged at Shell length (longest axis) was measured to the near- 1000 X g for 2.5 min and the algal pellet stored at est mm using calipers. After removing the clam from its -20°C. The frozen pellets were later lyophilised over 134 Mar Ecol Prog Ser 170: 131-141, 1998

24 h and weighed to the nearest 0.1 mg. The samples 0.06 M KH2P04/0.06M K2HP04 (pH 6.65), so that the were then stored with silica gel at 20°C until analysed final total amino-acid concentration was between 15 for C and N composition. and 45 PM. From each sample, 10 p1 of sample was Measurement of N uptake by freshly isolated separated on a Waters NOVA-PAK"' CLscolumn with a zooxanthellae. Prior to nutrient-uptake experiments, mobile phase described by the manufacturers (Waters) freshly isolated zooxanthellae (FIZ) were maintained as gradient 2. Amino-acid derivatives were detected in the dark by wrapping them in aluminium foil. The with a fluorescence detector (Waters model 420 AC). density of the FIZ suspension was estimated and, if The software Maxima 820 (Millipore) was used to necessary, an appropriate dilution was made to obtain determine individual peak areas and concentrations. It a density of ca 108 cells ml-l. Solutions (20 ml) of 20 PM was not possible to separate serine and histidine so ammonium (N) in FSW were prepared in triplicate in data from these amino acids were pooled for analysis. small glass scintillation vials. To obtain a final density Zooxanthellae samples were thawed and resus- of ca 106 cells ml-' in the incubation vials, 0.2 m1 of the pended in FSW. Cell counts were made using a FIZ suspension was added to each of the triplicate vials haemocytometer. The zooxanthellae were lysed by at 15 s intervals. Triplicate vials without FIZ were kept resuspension in 80% ethanol, centrifuged at 15000 X g as controls for the determination of bacterial N uptake for 5 min, and the supernatant analysed for amino in FSW. The FIZ were then incubated in ammonium acids by high-performance liquid chromatography solution for 30 to 60 rnin in the light (50 to 200 pE m-' (McAuley 1994). S-'; 26 to 28°C). After incubation, the FIZ were succes- Analysis of data. To test the hypothesis that elevated sively filtered on GF/C glass-fibre filters at 15 s inter- levels of N and P had no effect on the various clam and vals and the filtrates were immediately analysed for zooxanthellae parameters at a = 0.05, data were ammonia. analysed using analysis of variance (ANOVA) for 2 or- Measurement of amino-acid contents of the clam thogonally arranged fixed factors (N and P), with a third haemolymph and zooxanthellae. For amino-acid random factor (micro-atoll) nested within each treat- analysis, 3 other clams were removed from each of the ment combination (Underwood 1981). AI1 statistical micro-atolls after approximately 6 mo in the different analyses were performed using the analytical software nutrient regimens. These clams were killed and the package, Statistix 3.0 (Analytical Softwareo 1985). haemolymph collected and frozen as previously Cochran's test for the assumption of homoscedasticity described. Zooxanthellae were extracted from the of variances underlying the analytical procedure was mantle, pelleted at 1200 X g for 2 min, and frozen. made prior to performing each analysis (Underwood The total amino-acid concentration (excluding pro- 1981). Except for the FIZ N uptake and some amino- line) in the haemolymph was determ~nedfor each clam acid data (Cochran'stest, p < 0.05),no serious violations using a modification of the method described by of the assumptions were evident in the different data Magne & Larher (1992). Haemolymph samples were sets (Cochran's test, p > 0.05). Data for N uptake by FIZ thawed and centrifuged at 1600 X g for 10 min. To and some amino acids in zooxanthellae were log-trans- 100 p1 of haemolymph, 1.4 m1 of sodium citrate (pH 4.5) formed to conform to the assumption of homogeneity of and 1 m1 of a ninhydrin/ascorbic acid solution (1% nin- variances (Cochran's test, p > 0.05). Where observed F hydrin, 0.03 % ascorbic acid dissolved in ethylene gly- ratios were less than unity, significance tests at a = 0.05 col monomethyl ether) were added. The solution was based on the reciprocal of the observed F ratios were shaken and placed in a boiling water bath for 15 min performed (Underwood 1981). In all cases, F ratios before being cooled to room temperature. After addi- were not improbably small (p > 0.05), which implies tion of 3 m1 of 60% ethanol, the absorbance was mea- that the assumptions of the analysis were not seriously sured at 570 nm and the total amino-acid concentration violated (Underwood 1981).Where the ANOVA results was calculated using glycine as a standard. were significant, Tukey's test was used to compare Haemolymph samples from control and N-supple- treatment means (Zar 1984). mented clams were also analysed for individual amino acids. The haemolymph was thawed, centrifuged for 5 rnin at 800 X g and diluted between 100- and 300-fold RESULTS using dH20. The amino acids were derivatised by addition of 10 p1 o-pthaldialdehyde (Pierce) (5 mg ml-' Significant differences between control and N- in 0.5 M potassium borate pH 10 and 4 p1 ml-' 2-mer- enriched clams were obtained within 3 mo of fertilisa- captoethanol) to 100 p1 of each sample immediately tion of the clams. Observed trends in the syrnblotic prior to analysis. Samples were then diluted with a partners' N:P ratios and the host haemolymph's nutri- solution of 18.2% acetonitrile, 18.2% 2-mercap- ent levels after a month of fertilisation were consistent toethanol, 18.2% isopropyl alcohol, and 45.4 % of with trends after 3 mo. Belda-Baillie et al.: In situ nutrient effects on reef clams 135

N:P ratios in clam viscera and zooxanthellae Table 1. Mean (ASE)N:P atomic ratios of zooxanthellae isolated from Tridacna maxima In controls or supplemented with After 3 mo of fertilisation, addition of P alone signifi- ammonium (N) and phosphate (P) on micro-atolls on One Tree Island Reef cantly decreased the N:P ratio in the soft tissues of Tri- dacna maxima (ANOVA: F = 6.57, df = 1,8, p < 0.05), indicating increased phosphorus content (Tukey, p < Treatment I mo (n) 3 mo (n) 0.05) (Fig. 1). Addition of N alone, on the other hand, Control 48.7 i 7.8 (6) 33.1 t 2.0 (6) significantly increased the N:P ratio (ANOVA: F= 8.13, N 45.9 i 3.0 (6) 38.0 + 3.8 (4) df = 1,8, p c 0.05), indicating increased nitrogen con- P 48.0 i 3.4 (6) 33.0 i 0.6 (6) tent (Tukey, p < 0.05). Addition of N+P did not appear N+P 44.3 i 3.0 (6) 34.0 & 2.2 (6) to affect the N:P ratio of T. maxima, with no significant Mean 46.7 + 2.2 (24) 34.2 + 0.9 (22) interaction between N and P (ANOVA: F = 1.00, df = 1,8, p 0.05). It is assumed that increased N and P simultaneously increased nitrogen and phosphorus N uptake by FIZ content, resulting in a constant N:P ratio. There was no significant variation in N:P ratios due to the different After 3 mo of fertilisation, FIZ from N-treated clams micro-atolls (ANOVA: F = 0.85, df = 8,12, p > 0.05). took up significantly less ammonium (ANOVA: F = Most of the viscera samples taken after only 1 mo of 10.46, df = 1,8, p < 0.05), and FIZ from P-treated clams fertilisation were lost due to a malfunction of storage took up significantly more ammonium (ANOVA: F = equipment. Only 4 samples were saved and analysed 6.99, df = 1,8, p 4 0.05), than those from control clams giving the following N:P ratios: (1) Control = 28:1, (2)N (Tukey, p < 0.05) (Fig. 2). FIZ from N+P- treated clams = 36 * 2:1, and (3) P = 24:l. The observed trend, though had N-uptake rates similar to those from control clams based on single readings, was similar to the trend after (Tukey: p > 0.05). There was no significant interaction 3 mo of fertilisation. between Nand P (ANOVA: F= 0.01, df = 1,8, p > 0.05), In contrast to the animal tissue, the N:P ratios of the nor was there variation due to the different micro- zooxanthellae after 3 mo of fertilisation were similar in atolls (ANOVA: F= 1.39, df = 8,ll,p > 0.05). A similar all treatments (ANOVA: for N effect, F= 0.01, df = 1,8, trend was evident after only 1 mo but was not signifi- p>0.05; forpeffect, F=0.49, df = 1,8, p>0.05), witha cant (ANOVA, p > 0.05). mean (*SE) value of 34.2 * 0.9 across all treatments (Table 1).There was no significant interaction between N and P (ANOVA: F =1.36, df = 1,8, p > 0.05) and no Zooxanthellae density and size variation due to the different micro-atolls (ANOVA: F= 2.19, df = 8,12, p > 0.05) A similar trend was observed After 3 mo of fertilisation, zooxanthellae densities in a month after fertilisation (Table 1). the N- and N+P-treated clams were higher than those

Control N P N+P Control N P N+P Nutrient regimens Nutrient regimens

Fig. 1. Tndacna maxima. Mean N:P atomic ratios of the vis- Fig. 2. Symbiodin~um sp. Mean ammonium-uptake rates of ceral tissues of giant clams in controls or supplemented for 3 freshly isolated zooxanthellae (FIZ) from Tndacna maxima in mo with ammonium (10 N) and/or phosphate (2 pM P) on controls or supplemented for 3 mo with ammonium (10 PM N) micro-atolls on One Tree Island Reef, Great Barrier Reef, Aus- and phosphate (2 PM P) on micro-atolls on One Tree Island tralia (error bars: +SE,n = 6) Reef (error bars. +SE, n = 6) Mar Ecol Prog Ser 170: 131-141, 1998

pooling of the micro-atoll and residual mean squares for testing of N effect (Underwood 1981). The micro- atoll factor had been safely assumed to be zero since it was not detected by an F ratio at p = 0.25 (instead of the usual p = 0.05), a test powerful enough to guard against a Type I1 error. There was no significant interaction between N and P (ANOVA: F = 3.41, df = 1,20, p > 0.05). No significant differences in zooxanthellae size were discernible after a month of fertilisation (ANOVA, p > 0.05).

Control N P N+P Ammonia, phosphate, and total phosphorus levels in haemolymph

After 3 mo of exposure to nutrients, there were no significant differences in the levels of phosphate, total phosphorus, and ammonia in the haemolymph (ANOVA: p > 0.05) in all treatments (Table 2). For ammonia in the haemolymph, there was a significant variation due to the different micro-atolls (ANOVA: F= 3.34, df = 8,12,p < 0.05).Ammonia levels were surpris- ingly high, while phosphate concentrations were very low (~0.1PM), similar to trends observed only a month Conlrol N P N+P after fertilisation (Table 2). After 3 mo of fertilisation, Nutrient regimens ammonia levels were reduced to about 50% of the Fig. 3. Symbiodjnium sp. (a) Mean density and (b) mean levels observed after a month of fertilisation (Table 2). diameter of zooxanthellae from Mdacna maxima in controls or supplemented with ammonium (10 pM N) and/or phosphate (2 pM P) for 3 mo on micro-atolls on One Tree Island Amino-acid contents of the clam haemolymph and Reef (error bars: *SE, n = 6) zooxanthellae in the control and P-treated clams (Fig. 3a). These dif- After 6 mo of fertilisation, increased N significantly ferences, however, were not statistically significant reduced glutamate (glu) in the clam h.aemolymph (ANOVA: for N effect, F= 2.77, df = l.,8, p > 0.05;for P (ANOVA:F= 11.33, df = 1,4,p < 0.05) (Fig.4a), with no effect, F = 0.02, df = 1,8,p > 0.05).The statistical sig- significant variation due to the different micro-atolls nificance of the effects due to the treatments was (ANOVA: F = 0.12, df = 4,12, p > 0.05). Gln:Glu, how- potentially obscured by the highly significant variation ever, and the rest of the amino acids [total, gln (gluta- found due to the different micro-atolls (ANOVA: F = mine), asp (asparatic acid), ser (senne),his (histidine), 7.91, df = 8,12, p i 0.001). There was no significant arg (arginine), gly (glycine), ala (alanine), tau (tau- interaction between N and P (ANOVA: F = 0.02, df = 1,8,p > 0.05). Zooxanthellae densities after a month of fertilisation showed no Table 2. Tridacna maxima. Mean (+SE)concentrations (pM)of inorganic phosphate (P,),total phosphorus (P,),and ammonia in the haemolymph trend and were not different of giant clams in controls or supplemented with ammonium (N) and between treatments (ANOVA: p > 0.05). phosphate (P) on micro-atolls on One Tree Island Reef (n= 6; nd: no data Similarly, addition of N and N+P, but not P, due to technical problem) significantly decreased the mean diameter of the zooxanthellae (ANOVA: for N effect, F = Treatment I mo 3 mo 4.28, df = 1,20,p i 0.05; for P effect, F= 0.59, p, P, Ammonla P, P, Ammonia df = 1,20, p > 0.05) (Fig. 3b). This significant effect of N on zooxanthellae size was Control <0.1 35.8i 2.3 43.5+ 4.2 l nd 22.4+ 3.2 N <0.1 37.3i 4.7 35.7 i 2.7 <0.1 nd 23.0 i 2.4 detected after removing the micro-atoll factor P <0.1 34.9k3.1 40.9i3.8 <0.1 nd 19.4i1.1 as a source of variation from the analysis in N+P <0.1 32.8* 2.6 41.8t 2.9 l nd 22.8i 2.0 view of its non-significant effect (ANOVA: Mean <0.1 35.2i 1.6 40.5 i 1.7 <0.1 nd 21.9k1.1 F = 0.99, df = 8,12, p = 0.49), and subsequent Belda-Baillie et al.. In situ nutrient effects on reef clams 137

different micro-atolls (ANOVA, p < 0.05), potentially masking any effects of the nutrients on the amino-acid concentrations. Histidine (log-transformed) was nei- ther affected by N nor P (ANOVA: p > 0.05), with no significant interaction between N and P, nor a significant variation due to the different micro-atolls (ANOVA, p > 0.05). The rest of the amino-acid data [total, gln:glu, ala, arg, ile (isoleucine), leu (leucine),ser, val (valine)]did not meet the assumptions of paramet- ric analysis even after transformation (Cochran's test, p > 0.05) and were not analysed further.

DISCUSSION

This study is one of the few reported field studies on the impact of nutrients on reef organisms which involves adequate replication and controls (Ambari- yanto & Hoegh- Guldberg 1996, Bishop & Duckworth 1997, Hoegh-Guldberg et al. 1997, Kiene 1997, Larkum & Koop 1997, Steven & Broadbent 1997, Ten- tori et al. 1997, Ward & Harrison 1997). Our in situ observations on Tridacna maxima strongly corroborate major responses previously observed for T giyas under controlled outdoor conditions (Belda et al. 1993b, Belda & Yellowlees 1995). Changes in ambient nutrient concentrations In sea water are generally NuLrientregimens reflected in a range of growth and metabolic parame- Fig. 4. Tridacnamaxima and Symbiodinium sp (a) Mean ters in both the animal host and the algal symbionts, (*SE) concentration of glutamate (glu)in the haernolymph (n with the exception that inorganic P generally has no = 9) of glant clams in controls or supplemented with ammo- effect on the resident zooxanthellae population. It is nium (10 pM N) for 6 mo; (b) mean concentrations of methio- significant that the responses from T. maxima occurred nine (met) and tyrosine (tyr) (n = 9) in the zooxanthellae of as a result of a nutrient exposure that is not only peri- giant clams in controls or supplemented with ammonium (10 pMN) and/or phosphate (2 pM P) for 6 mo on the micro- odic, but also at N and P concentrations significantly atolls on One Tree Island Reef. lower than those continuously supplied to T. gigas onshore over an equivalent period (Belda et al. 1993b). These results confirm our previous conclusions that rine)] were unaffected by N (ANOVA: p > 0.05), with zooxanthellae in giant clams are nutrient limited, and no significant variation due to the different micro- demonstrate the potential of the giant clam-zooxan- atolls (ANOVA: p > 0.05), except for glycine (ANOVA: thellae symbiosis as a bio-indicator of the early onset of F=3.99, df = 4,12, p < 0.05). eutrophication in coral reefs. Addition of N, but not P, significantly increased methionine in the zooxanthellae (ANOVA: for N effect, F = 6.24, df = 1,8,p < 0.05; for P effect, F = 1.96, df = Nutrient limitation in clam zooxanthellae 1,8,p > 0.05) (Fig. 5b). Similarly, addition of N, but not P, significantly increased tyrosine (tyr) in the zooxan- The important limiting roles of both N and P in the thellae (ANOVA: for N effect, F = 16.97, df = 1,8, p < growth and physiology of the symbiotic partners are 0.05; for P effect, F= 1.82, df = 1,8,p > 0.05) (Fig. 4b). evident. Increases in either N or P were directly For both met and tyr, there was no significant interac- reflected in the N:P ratios of the animal host, but not tion between N and P, nor was there a significant vari- those of the zooxanthellae (Flg. 1, Table 1). The zoox- ation due to the different micro-atolls (ANOVA, p > anthellae's N:P ratios of >30:1 strongly suggests that 0.05). For most of the amino acids analysed from zoo- they are P limited in situ (see Atkinson & Smith 1983, xanthellae [gln, glu, asp, asn (asparagine), gly, lys (ly- Belda & Yellowlees 1995). Such P limitation is consis- sine), orn (ornithine), phe (phenylanine), thr (threo- tent with previous findings on freshly isolated zooxan- nine)], there was a significant variation due to the thellae from P-enriched Tridacna gigas, which exhib- Mar Ecol Prog Ser 170: 131-141, 1998

ited properties similar to those of zooxanthellae cul- One surprising trend observed in this study was the tured in the absence of phosphate (Belda & Yellowlees much higher ammonium levels in the haemolymph of 1995). In the field, the density of symbiotic zooxanthel- Tridacna maxima (Table 2) than those which have lae increased with N- or N+P-enrichment, but not with been previously observed in T. gigas (Fitt et al. 1995). P alone (Fig. 3a) (see also Belda et al. 1993b, Belda & These high levels were in apparent conflict with the Yellowlees 1995, but cf. Steven & Broadbent 1997). high ammonium uptake rates in control clams (Fig. 2), When cultured outside their clam host (T. gigas), how- and suggest that ammonium concentration in the ever, zooxanthellae readily responded to P increases in haemolymph is not the sole determinant of ammonium sea water (Belda & Yellowlees 1995). It is interesting availability to the zooxanthellae, at least in T. maxima. that the inorganic phosphate concentration in clam Furthermore, any differences between treatments may haemolymph was found to be consistently low in T. have been potentially obscured by the significant vari- maxima and T gigas (i.e. <0.1 PM) (Table 2) (Deane & ation due to the different micro-atolls. It is interesting O'Brien 1980, Belda et al. 1993b). Such low concentra- that ammonia levels in the haemolymph after 3 mo of tions of inorganic phosphate in the haemolymph sur- fertilisation were reduced by about 50% from that rounding the zooxanthellal tube system only further found after a month of nutrient exposure (Table 2). illustrates the poor P environment of zooxanthellae in This is consistent with the decrease in the zooxanthel- situ, regardless of ambient P concentrations in the sea lae's N:P ratios over time (Table 1). water (Belda et al. 1993b) The observed amino-acid composition of the haemo- In addition to P limitation, our data likewise suggest lymph was unexpected since the g1n:glu ratio was N limitation in the symbiotic zooxanthellae. For unaffected by an increase in ammonium levels in sea instance, zooxanthellae from the N- or N+P-treated water and there was a drop in glu with N exposure clams had higher densities and were significantly (Fig. 4a). This is in direct contrast to the response smaller than zooxanthellae from the control or P- observed in the haemolymph of Tridacna gigas, in treated clams (Fig. 3a, b). This is consistent with the which a dramatic and immediate increase in gln con- reduced cross-sectional area observed for zooxanthel- centration was observed when clams were exposed to lae isolated from a young batch of Tridacna maxima 20 pM ammonium in sea water (Shepherd et al. 1998). exposed to N or N+P in a related ENCORE study G1n:glu is a biologically relevant indicator of N status (Ambariyanto & Hoegh-Guldberg 1996). That fast- relative to carbon availability for many microalgae dividing symbiotic zooxanthellae are usually smaller (reviewed in Cook et al. 1997), and if ammonium was than s1o.c~-dividing ones can be attributed to the assimilated by the zooxanthellae, one would expect a greater proportion of newly divided cells in the former rise in gln in the haemolymph, assuming that it is than in the latter (Hoegh-Guldberg et al. 1986, Wilker- released by the algae. It may be that the intrinsically son et al. 1988, Ambariyanto & Hoegh-Guldberg 1996), high ammonium concentrations in the haemolymph of and the greater allocation of energy to reproduction T. maxima are unaffected by exposure to N enrich- than to growth (i.e. increase in size). It is also interest- ment, indicating that there is no need for metabolic ing that zooxanthellae from clams exposed to N had adjustment following an increase in availability of significantly lower N-depletion rates than zooxanthel- ammonium to the clam. Alternatively, any increase in lae from control clams (Fig. 2) (see also Belda et al. gln concentration could have been immediate and 1993b), similar to laboratory observations of zooxan- short lived, and therefore missed during sampling. thellae from corals (Yellowlees et al. 1994). This sug- Indeed, gln:glu is sensitive to changes in either N or C gests that zooxanthellae from N-treated clams were availability over very short time periods (min or h), and exposed to increased ammonium in situ with increased might be most applicable for monitoring short-term N N concentrations in sea water. In contrast, zooxanthel- sufficiency (see Cook et al. 1997). lae from clams exposed to P had significantly higher N- Some pools of free amino acids in the zooxanthellae depletion rates than zooxanthellae from control clams (met, tyr) increased with N exposure of the clams (Fig. 2).This indicates that the resident zooxanthellae (Fig. 4b). However, these amino acids are non-basic had access to some internal sources of P that increased and may not be reliable indicators of N sufficiency in their demand for N, albeit an insufficient demand to zooxanthellae. Basic internal amino acid (BIAA. his, significantly enhance their growth or influence their lys, orn, arg) pools function as nitrogen-rich storage N:P ratio. Indeed, it has been shown that plant cells compounds in zooxanthellae, free-living dinoflagel- require small amounts of inorganic P in order to use lates, and other microalgae (reviewed in Cook et al. inorganic N (Ryther & Dunstan 1971). All these trends 1997). It is possible that any potential effects of N treat- suggest greater access of zooxanthellae in situ to ment on most free-amino-acid pools in the zooxanthel- increased N concentrations in sea water, than to P lae were obscured by the significant variation due to (Belda et al. 199313). the different micro-atolls. Alternatively, the BIAP Belda-Baillie et al.: In s~tu' nutrient effects on rer,f clams 139

pools may not be responsive indicators of nitrogen suf- water compared to giant clams and their algal sym- ficiency in clam zooxanthellae over the duration of the bionts. Although some in situ effects on giant clams study. were more subtle than those previously observed in the laboratory, they are still important given the short exposure time to the minimal nutrient loading in the Bio-indicators of early eutrophication in reef waters field, where biological competition and local tidal conditions affect nutrient availability. The general agreement of the results of this field The present findings indicate that giant clams and investigation and previous onshore studies (Belda et al. their algal syrnbionts are able to compete well with 1993b, Belda & Yellowlees 1995), despite the fact that other zooxanthellate reef organisms for available nutri- different species of clams were investigated in each ents, and that they have a potential use as sub-lethal case, is quite lucid. This has direct implications for tri- indicators of eutrophication in reef waters. The clam dacnids and, almost certainly, other symbiotic associa- zooxanthellae's nutrient status is particularly relevant tions in their natural environment. Reef organisms as an indicator of reef eutrophication, specifically with symbiotic with zooxanthellae are adapted to the poor respect to N enrichment. Compared to direct measure- nutrient conditions of reef waters. Consequently, any ment of temporally and spatially variable nutrient lev- chronic increase in nutrient levels substantially affects els in the water column, the zooxanthellae's nutrient the symbiotic partners. status integrates short-term signals of nutrient loading, One month of fertilisation of the micro-atolls did not and allows assessment of long-term changes in nutri- produce significant changes in the host and symbiont ent supply with repeated sampling (Cook et al. 1997). parameters examined, although the symbiotic part- Protocols for the non- destructive sampling of the man- ners' N:P ratios and haemolymph nutrient levels al- tle and adductor muscle (Benzie et al. 1993), growing ready showed trends consistent with those observed shell edges (see Belda et al. 1993a), and haemolymph later. Nevertheless, discernible effects of elevated nu- (Rees et al. 1993) are available Although tridacnid trient concentrations on clams and their zooxanthellae populations are declining and large species are listed were clearly evident after 3 mo and might have even as endangered (Lucas 19941, the small species like Tri- been significant at an earlier stage in the fertilisation. dacna crocea and T maxima still occur in reasonable In contrast, coral colonies of Pocillopora damicornis abundance within their natural geographic range and Stylophora pistillata incubated during the first half (Juinio et al. 1989, Lucas 1994). Furthermore, there is of the ENCORE nutrient-enrichment phase (10 1-IM N, scope for use of cultured clams as test organisms for 2 pM P) did not show significant changes, results monitoring purposes in reef waters. which the investigators attributed to the potential It is interesting to note that there was a lack of re- masking of treatment effects by season and coral sponse of some reef communities to nutrient enrichment parentage (Hoegh-Guldberg et al. 1997). Only during in the ENCORE experiment. For instance, primary pro- the latter intensified half of the nutrient-enrichment duction of the epilithic algal community on dead coral phase were these corals observed to have reduced cal- blocks (Larkum & Koop 1997), rates of surface erosion cification rates with P exposure and increased skeletal due to grazing or endolithic colonisation of micro- and extension rates with N exposure, even though the dif- macroborers of exposed experimental substrates ferences were insignificant (cf. Belda et al. 1993a). (Kiene 1997), and arnrnomum affinities of microphytes in Similarly, it took 6 mo of exposure of Acropora palifera sediments (Bishop & Duckworth 1997) were all unaf- colonies to 20 pM N and/or 4 pM P before significant fected by fertilisation. It has been suggested that nutrient increases could be discrened in zooxanthellae pigment conditions at the surface of the One Tree Island micro- concentrations and densities with N and N+P, respec- atolls are already sufficient to support these communities tively, and an increase or decrease in skeletal growth (Kiene 1997, Larkum & Koop 1997). These studies rates with P or N, respectively (Steven & Broadbent demonstrate that not all communities in oligotrophic reef 1997). Other ENCORE studies showed that nutrient waters, particularly non-symbiotic associations or non- enrichment did not affect the C:N:P ratios of the zoox- calcifying organisms, are necessarily nutrient-limited, anthellate soft coral Sarcophyton sp. (Tentori et al. In conclusion, this study underscores the limiting 1997), but reduced the larval settlement rates of the roles of both N and P In the giant clam-zooxanthellae reef coral A. longicyathus during the 5 d larval settle- symbiosis. It further provides valuable insights into ment period (Ward & Harrison 1997). organism-level responses of a symbiotic association to That some zooxanthellate corals did not manifest sig- nutrient enrichment in reef waters, demonstrating the niflcant results until much later and at higher nutrient potential use of giant clams and their zooxanthellae as concentrations demonstrates that they may have less bio-indicators of the early onset of eutrophication in sensitivity to changes in ambient nutrient levels in sea reef waters. 140 Mar Ecol Prog Ser 170 132-141, 1998

Acknou~l~~dgements.Financial support came from the sue growth and mctabohsm J Exp Mar B101 Ecol 190 .ACIAR-AIL);\B fellowship grant to C: A B -B and from a 263-290 C>t:RMPA grant to D.Y GBRMPA gave permission (ENCORE Hoegh-Guldberg 0 (1994) The population dynamlcs of sym- Permlt No G92/382) to use part of a natural population of Trl- biotic zooxanthellae In the coral Poclllopora darmconls dacnd nldxima on One Tree Island Reef, and Ovr Hoegh- exposed to elevated ammonla Pac Scl 4813) 263-272 Guldberg collected and placed the clams In the expermental Hoegh-Guldbcrq 0, Hlnde R, Muscatlne L (1986) Studies on micro-atolls. 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Editorial responsibility: David Klumpp (Contributing Editor), Submitted: August 16, 1997; Accepted: April 16, 1998 Townsville, Queensland, Australia Proofs received from author(s): August 3, 1998