The temporal pattern and rate of release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus) under nitrogen-enrichment and control conditions

J. Exp. Mar. Biol, Ecol., 153 (1991) 63-74

63

© 1991 Elsevier Science Publishers B.V. All rights reserved 0022.0981/91/$03.50

JEMBE 01669

The temporal pattern and rate of release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus) under nitrogen-enrichment and control conditions John Stimson and Robert A. Kinzie III Zoology Department and Hawaii Institute of Marine Biology, Univers{O,of Hawaii, Honoluht, Hawaii, USA (Received 14 March 1990; revision received 24 May 1991; accepted, 17 June 1991) I

Abstract: The rate of loss of zooxanthellae from intact Pocilloporadamicornis(Linnaeus) was determined for colonies growing in laboratory tanks supplied with either ambient seawater or seawater enriched with dissolved inorganic N. Algal release peaked during midday in both treatments. Corals in N-enriched water released 40% more algae. U - t surface area" day- ~than did control corals. However, algal densities in the N-enriched corals were three times higher than in controls, so specific release rate was lower for N-enriched corals. Lipid content of the N-enriched corals was also lower than in the controls. These results suggest that N enrichment results in: greater algal standing stock and a reduced rate of transfer of photosynthate to the host. N enrichment more than doubled algal densities in this coral indicating that zooxanthellae in situ may be nutrient limited and that algal densities are, to some extent, a function of nutrient levels in the external environment and not entirely regulated by the host. Key words: Coral; N-enrichment; Release; Zooxanthella

INTRODUCTION

Recent studies suggest that some corals can ahnost or completely meet their metabolic requirements with tronslocated material received from their zooxanthellae (Muscatine et al., 1984, 1989b). Others have suggested that under some conditions, corals may in fact have excess energy (Falkowski et al., 1984) which may be released by the coral as mucus and mucus-lipid (Crossland et al., 1980a,b; Crossland, 1987). Energy budgels have been constructed for a few corals (Davies, 1984; Edmunds & Davies, 1986) and indicate that there may be production, which does not appear as growth reproduction or respiration, a result which suggests fixed C is released by corals. In addition to the loss of particulate C as mucus or mucus lipid, corals are also known to lose or release zooxanthellae (Marshall, 1972; Hoegh-Guldberg et al., 1987), therefore a complete assessment of the C budget of corals should include quantification of C lost in the form of zooxanthellae.

Correspondence address: J. Stimson, Zoology Department and Hawaii Institute of Marine Biology, University of Hawaii, Honolulu, HI 96822, USA. Hawaii Institute of Marine Biology Contribution 865.

64

J. STIMSON AND R.A, KINZIE I11

Crossland et al. (1980a) estimated that 40~ of fixed C was lost as mucus and mucus lipid, but until recently no estimate was available of the fraction of fixed C lost as zooxanthellae. Hoegh-Guldberg et al. (1987), working with SO,Iophorapistilktta, found a daily release rate of zooxantheUae equivalent to 0.1% of the standing stock of algae. They concluded that algal release was not a significant pathway of fixed C loss in that association. Our study was initiated to determine whether the loss of zooxanthellae from Pocilloporadamicornis (Linnaeus) constitutes an important loss of fixed C from the coral, and to investigate how elevated inorganic N levels would influence algal standing stock and release rates. The effect of N enrichment was included because it is known that algal density, algal division rate, and therefore possibly release rates can be influenced by the ambient inorganic N concentrations experienced by their hosts (Wilkerson et al., 1983; Hoegh-Guldberg & Smith, 1989; Museatine et ai., 1989a). We realize that the mechanisms controlling zooxanthellae densities are not understood, but have chosen to use the phrase "algal release" even though it implies that algal densities are under the control of the host.

MATERIALS AND METHODS

Both control and nutrient-enriched (experimental) corals were maintained in a flowthrough seawater system. Control corals were supplied with Kaneohe Bay seawater ( ~ 0.6 ~M NH4+; Snidvongs, 1987). Experimental corals received the same seawater to which a solution of ammonium chloride was continuously added by a peristaltic pump, bringing the NH4+ level to 17 I~M. It has been established that NH4+ is the form of N most readily taken up by corals from seawater (D'Elia & Webb, 1977; Museatine & Porter, 1977; Muscatine & D'Elia, 1978; Wilkerson & Trench, 1986). Header tanks with preset overflow levels kept flow rates in the experimental tanks constant at ~ 4 I. rain - t. Vigorous aeration provided water movement. P. damicornLv(type Y; Richmond & Jokiel, 1984), < 10 cm in diameter, were collected on 22 November 1986 from the windward reef fiat of Coconut Island, Kaneohe Bay, Oahu, Hawaii, and immediately placed in one of two experimental or two control tanks. Tanks were 1.2 x 1.2 m and 50 cm deep; watcr was maintained at a depth of 30 cm. 1 day after collection the corals were stained with alizarin red-S dye (Barnes, 1970) to create a band in the skeleton from which to measure subsequent linear growth at the branch tips. Algal release rates were measured for l-h periods on 4 days in February and March 1987 to determine the time course of algal release. In preparation for the measurement of algal release rates, corals from each treatment were transferred from the holding tanks into individual 3-1, open, straight-sided, translucent plastic containers. The containers had an overflow opening 2 cm below the rim to which a collecting tube was attached. Each coral was transferred underwater into its container and was handled only by its

RATE OF RELEASE OF ZOOXANTHELLAE

65

dead basal portion to minimize stress to living tissues. Handling living corals in a less careful manner could have initiated mucus discharge and disrupted coral tissue, thus possibly increasing algal loss. During the day on which algal release rate was measured, containers holding the coral to be sampled were supplied with N-enriched or control seawater at a rate of 4 1.11-~, according to the treatment it came from. Seawater supplied to these containers was filtered through a 5-~um filter. Water in each container was aerated to further increase water movement. All containers were immersed in a running seawater bath to regulate water temperature. One layer of plastic window screen was used ~.~'reduce solar irradiance to the level normally experienced by the corals in their somewhat deeper holding tanks. Irradiance levels experienced by the corals on the days that algal release was measured, were recorded over l-h intervals using a submersible quantum sensor with a 4-pi collector (Biospherical Instruments, Model QSI 140). Polyps of all corals were extended, indicating that experimental manipulations caused negligible stress. Outflow from each container was collected in a carboy over the 1-11period of the runs. At the end of the hour of collection this outflow water was filtered through Millipore membrane filters ( < 5 ~tm pore size, 47 mm diameter) using a vacuum pump at a vacuum of < 38 mm Hg. On the following dates outflows were collected from two treatment and Iwo control corals at 0900, 1100, 1300 and 1500: 12, 19 and 26 February 1987. On 27 and 28 March 1987 outflow was collected over a 20-h period from two sets of four corals each. Each set contained two corals from each treatment and each set was sampled during alternate l-h periods. Algal release rate was measured by making 20-25 counts of the density of cells on each filter using a quadrat of 300 x 54/.tin at a magnification of 160 x. These counts were converted to cells released, coral- i. h - ~ by multiplying the average count, quadrat- ~times the ratio of the total filter surface area (1750 mm z) to quadrat area. These estimates of the total cells released were normalized to living tissue area of each coral. To obtain estimates of the surface area of the individual P. damicornis colonies the following procedure was used. Cubes of dry calcium carbonate of known surface areas were preweighed and briefly dipped in melted paraffin at a temperature of 59 °C. The blocks were at room temperature prior to being dipped. The increase in mass due to the surface coat of paraffin was determined and these increments of mass were plotted against the surface area of the blocks. The weight of wax added to the surface of the dried skeletons of the experimental P. damicornis was measured and these increments were converted to surface area using a graph of the mass increment vs. surface area developed from the blocks. This method was verified in the following way. Four colonies, ~ 6 cm in diameter, were fixed in formalin and decalcified in 4 ~ nitric acid. Flat disks of tissue 7 mm in diameter were cut from the decalcified tissue of the colony. The disks and the balance of the tissue from each colony were rinsed in tap water, dried and weighed. A proportionality was then constructed to estimate the area of the whole colony using the weight and area of the disks and the total dry weight of the soft tissues of the colony. The areas of colonies of known linear dimensions, which were estimated by this method, agreed

66

J. STIMSON AND R.A. K1NZIE Ill

closely with the areas of skeletons of colonies of similar linear dimensions estimated by dipping the skeleton in wax. Release rates of algae were analyzed using a two-factor ANOVA: treatments (N enrichment and control), days (the four dates on which algal release was measured) and hour (0900, 1100, 1300 and 1500). Algae were collected from individual corals over each of the four 1-h periods in a day, so these four measures on each coral are not independent; different corals were used on each day of analysis. The statistical design used for the analysis of these data took into account the fact that each coral was sampled four times in a day, i.e., the values obtained from each coral on a day were considered as repeated measures. Daily release rates were computed by fitting a polynomial equation to the data on cells released per hour vs. hour for the hours between 0700 and 1800. The polynomial was used because it provides an expression from which estimated hourly release rates for the two treatments can be obtained. Lipid content and algal densities were obtained from tissues of branches removed from experimental and control corals at 2- to 7-day intervals. Sample branches 3 cm in length were taken from two control and two experimental corals each sampling day and were immediately fixed in 10~o seawater-formalin. After 24 h of fixation, each branch was decalcified in 4~o nitric acid; the decalcified tissue was rinsed in freshwater and air-dried. Lipid content was estimated by placing the dry tissue of the branches in individual vials and adding 6 cc of chloroform-methanol (2 : 1) to extract the lipid. The solvent-lipid mixture was decanted into premassed Ai weighing dishes, along with two chloroform-methanol rinses of each tissue sample. The mixture was evaporated at 55 °C for 24 h. The mass of both the extracted lipid and the remaining dried tissue were then determined and the tissue-lipid content expressed as a percentage of the summed masses, The density of zooxanthellae (n' era- 2 of coral tissue) was estimated from 7 mm diameter disks of tissue cut from the tissue of decalcified branches. These disks were approximately the largest flat areas of tissue which could be obtained in between verrucosities and branch bifurcations. The glove-like tissue of the decalcified branch was placed in water in a plastic Petri dish, a fiat area of the tissue > 1.5 cm from the tip was located and a disk was cut from this flat area with a cork borer. The disk was ground with 1 ml water in a glass Ten Broeck tissue grinder and the algal cells in the resulting suspension were counted on a hemacytometer. Tissue mass (animal plus plant)" U - ~surface area of coral was estimated from similar 38.5-mm z disks. Each disk was separated from the organic skeletal matrix, dried at 55 °C and weighed to the nearest 0.1 mg. RESULTS

Both experimental and control corals showed a diel pattern of algal release (Fig. 1). Release rates in the morning were ~ 100 cells" c m - 2. h - ~. Control corals reached a

RATE OF RELEASE OF ZOOXANTHELLAE 0

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Fig. 1. Algal release rate for control and N-enriched corals on following days: 12, 19 and 26 February and 27 and 28 March 1987. Points ( + st.) at 0900, 1100, 1300 and 1500 represent mean release rates for sets of eight corals; all other points represent release rate of a single coral. Curves represent second degree polynomials fitted to data ofeach treatment between 0700 and 1800. Solid curve is fitted to N enrichment data, dashed curve to control data.

peak release rate of ~ 1000 cells.cm - 2 " h-1 by 1100. N-enriched corals reached a maximum of > I000 cells.cm-2"h -I at ,~,1300. These peak release rates were estimated from predicted values obtained using two second degree polynomial equations fitted to data from the hours between 0700 and 1800 (Table I). By 1900 release rates fell to ~ 100 cells, cm- 2. h - ~ and remained at these low levels throughout the night. Mean release rates were compared at four times during the day when the most replication was available with the use of a repeated measures design ANOVA. This analysis showed that the two treatments were significantly different, that the hours were

TA BI.I" I Results of polynomial regression analysis of algal release data for hours from 0700 to 1800. Source

dr

MS

F

P

I 1

70.72 57.58 73. I 1 2.1 I

33.54 27.31 34.67

< 0.00 I < 0.001 < 0.00 I

1 I

55.51 51.84 54.28 1.32

20.92 39.08 40.91

< 0.00 I <0.001 <0.001

Control corals

Model Linear Quadratic Error

2

45

N-enriched corals

Model Linear Quadratic Error

2

45

68

J. STIMSON AND R.A. KINZIE II1

significantly different and that tile treatment x (Table I1). Examination of means indicates algal were higher tha~ the controls at 1100, 1300 and two treatments at 0900 were quite similar (Fig.

hours interaction was not significant release rates for the N-enriched corals 1500, while lower release rates o f the 1, Table ll).

TABLI, II ANOVA of release data (cells released' cm - 2. h - t). ANOVA model used was a repeated-measures design, which took into account that within 1 day an individual coral contributed four values, one at each of four sampling hours. Analysis was performed on transformed data (In Y). Source Between treatments (control vs. N-enriched) Among Hours Treatment x Hours Among corals (treatments) Hour x coral (treatments) Total Class means Treatment Control N-enriched Days 12 19 26 27 Treatment Control Control Control Control N-enriched N-enriched N-enriched N-enriched

Hour 9 I1 13 15 9 ii 13 15

df

MS

F

P

1

4.80

6.51

0.023

3 3 14 42 63

15.87 2.81 0.74 !.09

14.43 2.56

<0.001 0.068

Sample size

Mean of transformed values

Mean of backtransformed values

32 32

5.82 6.37

337 584

16 16 16 16

6.32 5.84 6.25 5.97

556 344 518 391

8 8 8 8 8 8 8 8

5.69 7.10 6.25 4.23 5.34 7.27 7.04 5.82

296 1212 518 69 208 1436 1141 337

An estimate of daily algal release rate for corals in each treatment was obtained by summing predicted hourly release rates estimated from the polynomial regressions between 0700 and 1800. F r o m t h i s , ' w e estimate that control corals lose ~ 4 3 0 0 cells" c m - 2. d a y - i (Table III). A control coral o f 400 cm 2 (,~8.5 cm in diameter) would lose 2.24 x 106 cells, d a y - ~ . Corals in N-enriched water released cells at a significantly higher rate, 6000 cells" c m - 2. d a y - ~ (Table III), = 1.4 times the release rate o f the controls.

RATE O F R E L E A S E O F Z O O X A N T H E L L A E

69

Specific release rates (number of algae released" day- ~relative to the standing stock of algae) can be estimated for the corals in both treatments. Algal density in the N-enriched corals in March was 1.2 x l0 t' zooxanthellae, cm-2 (Table III), 3.1 times the density in controls (Table III). Using these algal densities and the absolute release rates in Table llI, the specific release rate of algae (cells.cell - j .day - t ) can be estimated for both treatments (Table 1I); control corals had a specific release rate of 5.0 × 10 - 3 cells" cell- ~. day-t, almost two times that of the N-enriched corals. TA III.1-~ III Algal densities and estimates ofrelease rates ofalgal cells by control and N-enriched P. damiconziscolonies. Algal release rates were obtained by adding predicted hourly means derived from curves in Fig. 1. Control Algal density (cells. cm - 2 coral tissue) SO Number o f corals Algal release rate (. cm - 2. day - J) Specific release rate d a y - 1

N-enriched

0.4 x l0 t' 0.12 x 106 3 4397 11.1 x I0 -'a

1.2 x 10r' 0.28 x 10r' 5 6000 5.0 x I0 -a

Lipid content of the N-enriched corals was indistinguishable from that of the controls for the first 2 wk of treatment, but after this time lipid content of the N-enriched corals dropped to ~ 2/3 that of the controls (Fig. 2). 40

• Nitrogen

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22 Nov

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Fig. 2. Percent lipid content in branches o f control and N-enriched corals. Split symbols represent two readings o f same value.

DISCUSSION

This study of release rates of zooxanthellae and the study of Hoegh-Guldberg et ai. (1987) both indicate that release rates are low, but the studies show different patterns of release through the day.

70

J. STIMSON AND R.A. KINZIE III

The diei pattern in the algal release rate observed here (Fig. 1) could result from phased cell division (Wiikerson et al., 1983). The cell cycle of algae from P. damicornis is not known, but Wilkerson et al. (1983)did not find evidence of phased division in zooxanthellae from S. pistillata or most other coelenterates, and Muller-Parker (1984) did not find evidence of synchronous or phased division of algal cells in Aiptasia pulchella. Fitt & Trench (1983), however, have reported phased cell division in cultures of algae from two anemones including Aiptasia tagetes and a jellyfish. Phased cell division was observed by Wilkerson et al, (1983) in the scyphozoan Mastigias sp. These animals migrate down into nutrient-rich water (~ 15/~m NH4+ ) where they stay for 2 h each night. The phased cell division in Mastigias was attributed by Wilkerson et al. (1983) to the brief nightly exposure to waters with high N levels. Unlike Mastigias, the P. damicornis colonies were not exposed to diurnal change in N levels (ambient or elevated), yet still showed a strong did pattern ofrelease, suggesting the pattern may be related to changes in light level. The midday peak in cell release from P. damicornis could be a response to the buildup of photosynthetic products such as fixed C or 0 2 in the coral tissue. Crossland et al. (1980a) show a rapid increase in C 14 incorporation into tissue lipid and photosynthate production during the morning hours, and Crossland (1987) shows that the release of mucus and mucus lipid by corals follow diurnal changes in light level, suggesting that these materials or their precursors could be showing a diel pattern in the coral tissues. Barnes (1970) showed a diurnal cycle in calcification which is also thought to be enhanced by algal activities (Pearse & Muscatine, 1971; Fang et al., 1989). Such a diurnal pattern of photosynthate concentration could provide a changed chemical environment resulting in a diurnal pattern of algal release. A second possible explanation of the diel peak in algal release in both treatments is based on the dependency of NH4+ upt.ake on light level. NH 3 uptake by symbiotic corals has been demonstrated to depend on the presence of the zooxanthellae (Muscatine & D'Elia, 1978; Wilkerson & Trench, 1986). NH 2 uptake by zooxanthellae has in turn been demonstrated to be light-dependent (Muscatine & D'Elia, 1978; Wilkerson & Trench, 1986). The pat'~ern ofalgal release observed here could result from an increase in NH4+ level in the host tissue in morning hours, which could stimulate algal division (Wilkerson et al., 1983) arid subsequent release. N-enriched corals showed higher absolute rates of algal release (cells released.cm - 2 . d a y - I ) , while the control corals had higher specific release rates (Table III). These release rates were measured when the corals had been in the system for from 2-4 monlhs and we assume their algal population had reached an equilibrium with the algal densities in the N-enriched corals at 3.1 x the density of the controls (Table III). We do not believe that the higher specific rate of release for controls is due to a higher rate of cell division in controls because Snidvongs (1987) found that C. cell - t for zooxanthellae of P. damicornis was not significantly altered by increases in ambient NH2- levels, indicating that mean cell size did not decrease due to higher division rates in nutrient-enriched conditions. A second alternative is that algal cells

RATE OF RELEASE OF ZOOXANTHELLAE

71

were retained longer in N-enriched corals; a longer period of retention of algal cells might be achieved if the N-enriched corals were growing faster producing more animal tissue and retaining more algal cells which colonized this new space. Meyer & Schultz (1985) observed higher growth rates in corals when schooling fish lived close to the corals, significantly increasing ambient NH4+ levels (Meyer et al., 1983). More rapid skeletal growth and concomitant growth of coral soft tissue could result in increased retention of zooxanthellae and thus a lower specific rate of release of algae. However, no significant difference in the linear growth rate of branch tips of control and experimental corals was detected in this experiment, and no difference was apparent between the two treatments in the amount of coral tissue.cm -2 of surface area (control: = 28.1 mg.cm -2, SD = 9.27, n = 12; N-enriched: E = 24.2mg'cm -2, SD = 8.5, n = 11). In a later experiment of similar design, performed during summer months the linear growth of branch tips of N-enriched corals (7 and 15 # M N H4+ ) was significantly less than that of controls (Stimson, unpubl, data). The higher specific release rate for controls may be the result of greater competition for nutrients among algal cells (less capability of animal cells to support algal cells) under a low N regime. Addition of N resulted in: increased algal cell density and increased absolute release rate of algae, lowering of lipid levels in the coral-algal samples and perhaps a decrease in rate of skeletal extension. Since Snidvongs (!987) did not find a difference in C cell between algal cells from P. damiconlis grown in high and low NH4+ the differences in lipid levels probably are attributable to differences in the lipid level in coral tissues. The difference suggests that the larger number of cells released.day-~ and possibly produced' d a y - ~ by N-enriched corals may have resulted in a reduction in the rate of translocation of photosynthate, including lipid and its precursors, to the coral tissues. This is consistent with the reduced skeletal growth found in the subsequent trial. This study was originally undertaken to assess the rate of loss from corals of fixed C in the form of algal cells. It seems that this loss is minor relative to C loss by other paths (Crossland et al., 1980a; Crossland, 1987). Zooxanthellae of P. damicornis in ambient conditions contain ~312 pg C" cell-~ (Snidvongs, 1987), thus control corals would have released 1.3 Fg C' cm - z ' day-~ in the form of algal cells: 1.29 x 1 0 - 6 g C ' c m - 2 " d a y - t = 0.3 x 1 0 - g g C'cell -I × cells released'cm -z" day-5 N-enriched corals would have released ~. 1.8 Fg C. c m - 2. d a y - ~. This loss of C can be compared with the net fixation rate of C in corals. Net fixation'day-~ for P. damiconlis colonies can be estimated from the data ofJokiel & Morrissey (1986). For corals 4-8 cm in diameter, the values are 74-224 Fg C" cm - 2. day- 5. Corals in this study were 8 cm in diameter. Given these rates and coral sizes, daily C loss as algal cells from the symbiotic system represents ~ (1.3/224) × 100 = 0.6% of the net C fixation by the symbionts. Net fixation, day- 5 for P. damicornis is similar to that estimated for S. pistillata by McCloskey & Muscatine (1984), Muscatine et al. (1984) and Porter et al. (1984). They present figures for daily net production ranging from 122.55 to 267.6 Fg C' cm- z. day- 5. Hoegh-Guldberg et al. (1987) estimate that the proportion of fixed C lost through the release of zooxanthellae is < 1% in S. pistillata.

72

j. STIMSON AND R.A. KINZIE III

Up to 40'Z, of net C fixation can be lost. day- J as dissolved organic C (Crossland et al., 1980a,b; Muscatine etal., 1984; Davies, 1984; Edmunds & Davies, 1986). Evidently, the loss of C through algal release is small relative to other patbs of C loss. While it is often stated that algal density in corals is relatively constant (Drew, 1972; Porter et al., 1984), this parameter has in fact been shown to vary in response to growth conditions (Kinzie et al., 1984; Kinzie & Hunter, 1987). The increased algal density observed under nutrient enrichment in this study and studies by Dubinsky et al. (1990) and Meyer & Sehultz (1985) provides some insight into the question of regulation of symbiont density. In this study and in that of Dubinsky et al. (1990), the density of zooxanthellae within host tissues was essentially tripled by nutrient addition. Muscatine & Pool (1979) pointed out that for a number of symbiotic associations, the ratio of algal cell volume to host cell volume is relatively constant, and that algal division rate exceeds that of host cells. The results of NH4+ addition presented here suggest that endosymbiont density in host cells need not be regulated at a constant value, but instead can vary in response to altered nutrient availability, even though there is no apparent increase in the amount of coral tissue on an areal basis. That is, what sets algal density in host cells is, in part, nutrient avaihtbility or competition among algal cells for nutrients. The nutrient availability in host cells, in turn, evidently responds to changes in nutrient concentration in the external environment. Thus, algal densities may not be simply regulated by the host, but may also reflect nutrient availability in the external environment. ACKNOWLEDGEMENTS We gratefully acknowle"ge the use of the facilities of the Hawaii Institute of Marine Biology and the support from NIH/MBRS Grant GM08125-16 to R.A. Kinzie. Dr. Satoru Taguchi's comments improved the manuscript.

REFERENCES Barnes, D.J., 1970. Coral skeletons: an explanation of their growth and structure. Science, Vol. 170, pp. 1305-1308. Crossland, C.J., 1987. In situ release of nmcus and DOC-lipid from the corals Acropora variabilis and SO,Iophora pisti#ata in different light regimes. Coral Reefs, Vol.6, pp. 35-42. Crossland, C.J., D.J. Barnes & M.A. Borowitzka, 1980a. Diurnal lipid and mucus production in the staghorn coral Acropora acumblata. Mar. Biol., Vol.60, pp. 81-90. Crossland, C.J., D.J. Barnes, T. Cox & M. Devereaux, 1980b.Compartmentation and turnover of organic carbon in the staghorn coral Acroporafomwsa. Mar. Biol., Vol. 59, pp. 181-187. Davies, P.S., 1984.The role ofzooxanthellaein the nutritional energyrequirements ofPocillopora eydouxi. Coral Reefs, Vol.2, pp. 181-186. D'Elia, C. F. & K. L.Webb, 1977.The dissolvednitrogen fluxofreefcorals. Proc. Thirdlnt. Coral Reef Syrup. Miami, Vol. 1, pp. 325-330. Drew, E.A., 1972.The biologyand physiologyof alga-invertebrate symbiosis II. The density of symbiotic

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