MARINE ECOLOGY PROGRESS SERIES Published October 5 Mar. Ecol. Prog. Ser. I

Influence of the population density of zooxanthellae and supply of ammonium on the biomass and metabolic characteristics of the reef hystrix and pis tilla ta

Ove Hoegh-Guldberg*,G. Jason Smith**

Department of Biology, University of California Los Angeles, 405 Hilgard Ave, Los Angeles, California 90024-1606, USA

ABSTRACT: In May 1987, the population density of zooxanthellae in the reef around Lizard Island (Great Barrier Reef) varied within and between colonies. This naturally occurring variabhty made it possible to examine the effect of the population density of zooxanthellae on the physiological characteristics of S. hystnx and its zooxanthellae. As the population density of zooxanthel- lae increased, the chlorophylla content and maximum rate of photosynthesis of the zooxanthellae decreased. Phytoplankton studies suggest that cellular chlorophyll content will increase if cells are self- shaded or will decrease if cells are nitrogen-limited. To test the hypothesis that zooxanthellae in S. hystrix and Stylophora pistillata are nitrogen-limited at their highest population densities, colonies of S. hystrix and S. pistillata with high densities of zooxanthellae were incubated in aquaria to which ammonium (ca 10 to 40pfi4) was added at regular intervals. After 3wk, the population density, chlorophylla content and maximum rate of photosynthesis of the zooxanthellae had significantly increased, indicating that the biomass of zooxanthellae in reef corals can be lirmted by the availability of inorganic nitrogen to the association.

INTRODUCTION that maintain the ratio of symbiont to host biomass (Muscatine & Pool 1979). Little is known about these Zooxanthellae are endosymbionts control mechanisn~s. fcund in all reef-building corals and a wide range cf If the populatio:: density of zooxanthellae is con- other tropical invertebrates (Trench 1979). In these trolled by the host, then there are several possible symbioses, there is an intimate association of host and points at which control could occur. Muscatine & Pool symbiont metabolism, and a bilateral exchange of inor- (1979) proposed 3 main mechanisms: (1) the expulsion ganic and organic substrates (Cook 1985). One of the and/or (2) digestion of excess zooxanthellae; and/or (3) more remarkable features of symbiotic associations the inhibition of the growth of zooxanthellae via either between invertebrates and zooxanthellae is that they the restricted access to essential nutrients or via the are long-lived and that neither host nor symbiont out- production of growth-inhibiting factor(s), which grows the other (Drew 1972). This balance between includes the putative host factor that causes the selec- host and symbiont has prompted several authors to tive release of metabolites from zooxanthellae (Mus- propose that there are regulatory control mechanisms catine 1967). The first 2 mechanisms act on excess zooxanthellae after their production via cell division, Present addresses: and in the few associations that have been investi- Department of Biological Sciences, University of Southern gated, do not appear to be important in regulating the California, Los Angeles, California 90089-0371, USA Department of Molecular Genetics and Cell Biology, Uni- population size of zooxanthellae in 'normal' situations versity of Chicago, Chicago, Illinois 60637, USA, and Hop- (Fitt & Trench 1983, Hoegh-Guldberg et al. 1987, luns Marine Station, Pacific Grove, California 93950, USA Hoegh-Guldberg & Smith 1989).

O Inter-Research/Printed in F. R. Germany Mar. Ecol. Prog. Ser 57: 173-186, 1989

There is indirect evidence that the inhibition of the no evidence that the host produces substances that growth of zooxanthellae (the third category proposed inhibit the growth or division of the zooxanthellae, The by Muscatine & Pool 1979) is active as a regulatory observation that the growth rates of zooxanthellae are mechanism in symbioses between zooxanthellae and density-dependent (Hoegh-Guldberq & Hinde 1986, invertebrates. Demonstration of a putative 'host factor' Smith 1986),with the highest rates of growth occurring that induces zooxanthellae to release organic carbon at the lowest population densities, suggests that there (Muscatine 1967, Trench 1971) suggests that, by this may be some feature(s) of the host cell environment mechanism, the host may control the size of the carbon that inhibits the division of the zooxanthellae at high pool of the zooxanthellae. Inorganic nutrient supply population densities. Whether or not these features of has also been considered as a point of control although the host cell environment are part of active host control there has been no evidence that unequivocally demon- or merely features of the host cell environment pro- strates that the host manipulates the supply of inor- duced by high population densities of zooxanthellae is ganic nutrients to the zooxanthellae. Similarly, there 1'; unclear. Cook & D'Elia (1987) argue that the population

Fig 1,(a1 Ftow-through seawater aquana used to maintain daughter colonies (d) after during cultivation from parent colonies of Semlopota hystrix and Stylophora pstillata. Colonies of S- hystnx with (b) many and (c) few zooxanthellae in their tissues. Colony m (d) is 4 cm from base to tip Hoegh-Guldberg & Smith: Nltrogen limitation and zooxanthellae ~n corals 175

density of zooxanthellae per unit tissue volume in syrn- these tubs for 4 wk. The tips from each colony were biotic invertebrates is so high normally (in excess of 106 kept together and separate from the t~psoriginating cell ml-' tissue) that the availability of nutrients, such from the other colonies. as NH4+ originating from host catabolism, would limit The tips prepared from the partly-bleached colonies the growth of zooxanthellae. of Seriatopora hystrix ranged from brown to white, due Interestingly, the natural stability characterizing to the varying abundance of zooxanthellae (Fig. lb,c; populations of endosymbiotic algae (and hence limited Hoegh-Culdberg & Smith 1989). After 4 wk, coral tis- variation in the population dens~tyof symbionts) has sue had grown over the portions of skeleton exposed hampered investigation of putative regulatory mechan- when tips were broken off the parent colonies. Only isms operating in the maintenance of steady state popu- those tips (referred to hereafter as colonies) with a lation densities. The reef corals Seriatopora hystrix and complete tissue cover were used in the experiments Stylophora pistillata are widespread in the Indo-Pacific described below. region and have been observed to undergo periodic loss Experimental design. Effectof population density of of zooxanthellae (Hoegh-Guldberg & Smith 1989) zooxanthellae on the biomass and oxygen flux of which has been termed 'bleaching'. During the recovery Seriatopora hystrix: The effect of variation in popula- of colonies following a bleaching event, coral colonies tion density of zooxanthellae on the biomass and oxy- are found to have widely varying population densities of gen flux of S. hystrix was investigated by closed vol- zooxanthellae. Partially bleached colonies provide a ume oxygen flux measurements (see 'Measurement natural system with which to explore the influence of techniques', below). This procedure was followed by symbiont population density on coral biomass and measurements of the population density, mitotic index metabolic characteristics. These data along with nu- and chlorophyll a content of the zooxanthellae; and by trient enrichment experiment with 'unbleached' corals the measurement of the soluble protein content and are used to address the question as to whether the surface area of 14 colonies originating from the first population density of zooxanthellae in reef corals is parent colony and 13 colonies from the second parent limited by the availability of inorganic nitrogen. colony. Effect of external nutrient supply: The effect of increasing the ambient ammonium concentration was MATERIALS AND METHODS investigated. Ten colonies of Seriatopora hystrix each with about 1.0 X 106 zooxanthellae cm-' and 10 col- Collection and maintenance of corals. During Janu- onies of Stylophora pistillata with about 0.5 X 106 zoo- ary-February 1987, colonies of Seriatopora hystrix xanthellae cm-2 were randomly assigned to either of 2 Dana 1846 growing at Lizard Island, Great Barrier 481 aquaria that received a continual supply of fresh Reef, began to lose zooxanthellae (Hoegh-Guldberg & seawater (27 "C; flow rate = 193 + 15 m1 min-l, n = 4). Smith 1989). The cause of this phenomenon is The aquaria received 25 % incident solar irradiance by unknown although there is evidence that suggests that shading the tanks with black plastic mesh. A solution of elevated sea temperatures may be responsible (Glynn 10.0 mMNH,Cl (220 ml) was added to one of the tanks 1984). Three months later (May 1987). 2 recovering ('+ NH4+') every 6 h for the first 4 d and every 12 h for (partly bleached) and 2 normally pigmented colonies of the next 15 d. Each addition provided an NH,+ concen- S. hystrix (ca 30 cm in diameter), and 2 normally pig- tration of 45.8 uM which decreased to ca 10 p.M after mented colonies of Stylophora pistillata Esper 1797, 6 h, and to ca 2 pM after 12 h (calculated assuming were removed from the substrate, using a mallet and dilution by the continual inflow and outflow of sea- chisel, and transported to the Lizard Island Research water at the flow rate described above). After 19 d the Station (L.I.R.S.). The colonies had similar growth mor- incubation was terminated and the colonies removed phologies and were selected randomly from popula- for the measurement of oxygen flux and biomass tions growing at 5 m along the edge of the fringing reef (including the cell diameter of zooxanthellae). opposite Research Beach. These colonies were origi- Measurement techniques. Closed volume measure- nally separated from each other by more than 5 m. In ment of oxygen flux: The photosynthetic and respira- the laboratory, ca 20 branch tips (3 to 5 cm length; Fig. tory rates of colonies were measured using Clark-type Id) were broken off from each colony and were oxygen electrodes inserted into small plastic chambers attached with monofilament netting to microscope (ca 35ml) containing single colonies. Each chamber slides fitted into small racks and immersed in tubs contained a stirbar powered by a submersible magnetic receiving a continuous supply of seawater (Fig. la; stirrer ('Variomag' magnetic stirrer, Telesystems Inc.). Hoegh-Guldberg & Smith 1989). Incident irradiance hght was provided by a dual optic fiber light source (ca 1800 pm01 rnp2 S-') was reduced to 25 % in tanks (Reichert Scientific Instruments). The colonies were shaded by black plastic mesh. The tips were left in exposed to a series of irradiances and the hyperbolic Mar. Ecol. Prog. Ser. 57: 173-186, 1989

function was used to model the variation of oxygen (Deluxe model, Teledyne). The volume of the resulting flux with irradiance. Data were fitted using repeated homogenate was measured, mixed thoroughly and iterations and the Marqhardt algorithm (Marqhardt three 2ml samples stored at -20°C for protein meas- 1963). urement. A S % buffered formalin solution (2 ml) was An Acorn BBC microcomputer was used to collect added to another 10ml subsample of the homogenate, and analyze data during the experiments. hght was which was set aside for measurement of the concen- measured using a light-dependent resistor (Cat. tration and mitotic index (ratio of dividing cells to total # LDR1, Dick Smith, USA) calibrated with a LI-COR cells) of the zooxanthellae in the homogenate. Three quantum sensor (L1 1935A). Temperature was meas- 16 m1 subsamples were deposited on GF/C (Whatman) ured using a small waterproof copper/constantan ther- filters. The filters were covered with aluminum foil to mocouple (and electronics employing an AD595 tem- exclude light and were frozen (-20°C) until analyzed perature chlp, Analogue Devices) mounted on the for chlorophyll a. The total protein concentration of the inside of the chamber. The Clark-type polarographic homogenate was determined using the Hartree (1972) electrodes were constructed according to the method of modification of the Lowry protein assay (Lowry et al. Mickel et al. (1983) and, together with the light and 1951). Chlorophyll fluorescence was determined by temperature sensors, connected via a multi-gain and grinding the frozen filters in 3.2ml of acetone and multi-channel amplifier (Hoegh-Guldberg 1989) to the centrifuging the resulting solution until clear. The Acorn BBC computer. Light, temperature and oxygen amount of fluorescence due to chlorophyll was meas- were measured every 30s as described, by Hoegh- ured using a Turner Fluorometer (Model 111; excita- Guldberg & Smith (1989). Each colony was allowed to tion filters: Corning C/S2A and C/S5-60; emission fil- adjust to chamber conditions for at least 20 min before ter: Corning C/S 2-64). Specific fluorescence measure- each experiment. Rates of respiration in the dark were ments were determined from the concentration of measured in chambers covered with several sheets of chlorophyll a (r2= 0.90) measured in 30 samples using black plastic. The oxygen flux of each colony at each the methodology of Jeffrey & Humphrey (1975). irradiance was measured at least twice. Temperature The concentration of zooxanthellae and the homoge- during each experiment was kept between 27 and nate was measured using a hemacytometer (Bright- 28 "C. If temperature increased above 28 "C or the con- Lne, American Optical Corp.) and 8 replicate cell centration of oxygen in the chamber decreased below counts. The mitotic index was determined by counting 2 ppm, the experiment was terminated and the the number of dividing cells in 3 samples of 500 zoo- chamber flushed with aerated seawater. Colonies were xanthellae. In a separate study, the variation of mitotic allowed to adjust to new chamber conditions for 20 min index over a die1 cycle was examined by sampling 2 before the experiment was restarted. Total changes in colonies (collected and maintained as described above) oxygen were calculated by multiplying the changes in every 3 h. In addition, 2 sites within each colony of oxygen concentration by the total volume of the cham- Senatopora hystrix were examined. The first site was bers corrected for the volume of the colonies. restricted to within 0.5cm of the tip ('tip'), while the Immediately following each experiment, the colonies second was restricted to an area greater than 3 cm from were placed aside for biomass measurements. Rates of the tip ('base'). change in oxygen concentration were standardized (1) The specific growth rates and division times of zoo- to the surface area, (2) to the protein content of each xanthellae in Seriatopora hystni and Stylophora pistil- colony, (3) .to the chlorophyll a content and (4)to the lata were calculated using the formulae described by number of zooxanthellae found within a colony. The Wilkerson et al. (1983). The diameters of zooxanthellae respiratory rate of each colony (the rate of consumption isolated from S. hystrix and S. pistillata were deter- of oxygen by the colony in darkness, r,) and the net mined by photographing zooxanthellae [n = 500 cells), photosynthetic rate (the net rate production of oxygen using a Leitz photomicroscope and by measuring their of each colony at saturating irradiances, p, and diameters from projected images of known magnifica- the ratio of the gross photosynthetic rate (r, +p, ,,et ,,,) tion. The diameters of the projected images were meas- and the respiratory rate were calculated. The conven- ured using a linear potentiometer connected to an tions for these quantities are as described by Muscatine Acorn BBC computer The potentiometer was cali- (1980), with p, ,,,,, ,,, abbreviated to p,, ,,,. brated to a precision of -t 0.1 pm. Measurement of biomass parameters: Following The surface area of the bare skeletons was measured oxygen flux measurements, the colonies were placed using the Varathane method of Hoegh-Guldberg back in the holding tubs until 09:OO h. At this time, the (1988) and the aluminum foil method of Marsh (1970). colonies were removed and blotted on moist tissue Total protein, chlorophyll a content and zooxanthell.ae paper (to remove excess water) and the tissue on the number were determined by multiplying each sample colonies was removed using a dental Water-Pik parameter by the total volume of the homogenate. Hoegh-Guldberg & Smith: Nitrogen limitation and zooxanthellae in corals 177

RESULTS between 03:OO and 07:OO h, and were lowest around 14:OOh. Mitotic indices also varied as a function of Effect of population density of zooxanthellae on location within the colonies of S. hystrix. Populations of biomass, respiration and photosynthesis of zooxanthellae located within 0.5 cm of the colony branch Seriatopora hystrix tips had higher mean mitotic indices (although not significantly so, p > 0.05) than populations located more Biomass and oxygen flux parameters followed simi- basally (Table 1; Fig. 2). Specific growth rates of zooxan- lar trends in colonies derived from separate parent thellae within S. hystrix varied between 0.0407 and colonies of Seriatopora hystrix (p > 0.05). Conse- 0.0817 d-'. Because the division of populations of zoo- quently, data for the 2 groups of colonies were pooled. xanthellae in S, hystrix was phased, the duration of the Values reported in the following text represent means doublet stage (td) could be estimated (Wilkerson et al. k standard errors of the mean (SEM). 1983, Smith & Hoegh-Guldberg 1987; Tablel). Estimates of td for zooxanthellae in S. hystrivwere 0.55 f Mitotic index 0.061 d and 0.57 f 0.050 d for the first and second day respectively. The mitoticindices (MI)measured between The mitotic index of the zooxanthellae within Sena- 09:OO and 12:OO h varied as a function of the population topora hystnjr varied over the die1 cycle (Fig. 2). Mitotic density of zooxanthellae (D) in S. hystrix (p 10.05, n = indices were highest in the pre-dawn to dawn period 24; Fig.3).

Chlorophyll a concentration l0 The amount of chlorophyll a per area increased with the population density of zooxanthellae (r2= 0.77, n = 0 28, p < 0.001; Fig. 4a). The amount of chlorophyll a per

0020 0620 1220 1820 0020 0620

Time of Day 0 1 0.5 1.0 1.5 2.0 2.5 3.0 Fig. 2. Senatopora hystru. Mitotic index of populations of - Population Density of Zooxanthellae zooxanthellae as a function of time of day. Data from 2 col- (X 1o6 cell cm-') onies area shown (m)areas within 0.5- cm of the tip of colony branches (= 'tips'; Table l); (A) areas located > 3 cm away Fig. 3. Seriatopora hystrix. Mitotic index (MI)as a function of from branch tips (= 'bases'; Table 1). Points represent mean the population density of zooxanthellae (D). Points represent number of divibng zooxdnthellae in 1500 cells. Bars indicate number of dividing zooxanthellae in l500 zooxanthellae; he night-time is MI= 3.523 - 0.326(D),(r2= 0.28, p 50.05, n = 21)

Table 1. Seriatopora hystrix. Average (MI) and maximal mitotic lndices (MI,,), and calculated specific growth rates (p) and duration of cytokinesis (td) for zooxanthellae growing in 2 regions of 2 colonies ('tip' refers to areas within 0.5 cm of branch tip, 'base' refers to areas 3 cm from branch tip, numbers distinguish the 2 colonies sampled). Shown are means + standard error of the mean (SEM)

'parameter Day Base 1 Tip 1 Base 2 Tip 2

M1 (%) Pooled MIrnaX Day 1 Day 2

,P (d-l) Day 1 Day 2 Td (d) Day 1 Day 2 178 Mar. Ecol. Prog. Ser. 57: 173-186, 1989

zooxanthella varied inversely with the population density of zooxanthellae (r2 = 0.45, n = 28, p < 0.01; Fig. 4b). Zooxanthellae at the lowest population densities had the highest chlorophyll a contents.

Photosynthesis and respiration

The net and gross photosynthetic rate per area of colonies of Seriatopora hyslrix at Light saturation (p, ,,,) ,,,) increased as a function of the population density of zooxanthellae (r2= 0.80, n = 27; Figs. 5 and 6a). The respiratory rate per area (r,) tended to increase (though the slope of the line was not significantly greater than 0; Fig. 5, p > 0.05) with increasing population densities of zooxanthellae. The mean rate of respiration was 31.1 + 4.45 pg O2 cm-' h-' (N = 28). The ratio of the gross

POPULATION DENSrrY OF ZOOXANTHEUAE (106 cell cm2) Fig. 5. Seriatopora hystrix. Maximum net rate of photosyn- thesis (p, .,, ,,,) and colony resp~ratoryrates (r,) as a function of the population density of zooxanthellae (D). Regression lines are: p,.. ,,,, = 32.14(D),r2 = 0.80 and r, = 31.06 + 4.448 ~igO2 h-' (trend not significant, p > 0.05)

photosynthetic rate (p,, ma,, per area) and r, (per area) was linearly related to the population density of zoo- xanthellae in S. hystrix (r2 - 0.78; Fig. 7). The value of p,, ,,, per zooxanthellae cell declined with increasing population density of zooxanthellae (Fig. 6b; r2 = 0.72). Thls trend paralleled the decline in the chlorophyll a content with population density. Zooxanthellae at the lowest population densities had gross photosynthetic rates that were almost double those of zooxanthellae at the highest population densities. When standardized to chlorophyll a, p,, ,,, did not exhibit any significant variation (p > 0.10) with the population density of zooxanthellae and averaged 33.6 + 3.16 pg O2 (ug ch1a)-' h-' (n = 28).

Effect of elevated external NH4+on biomass, respira- POPULATION DENSrPl OF ZOOXAKMELLAE (106 cell cm2) tion and photosynthesis of S. hystrix and S. pistillata Fig. 4. Seriatopora hys6ix. Amount of chlorophylls (a) per Biomass parameters area (Chl aA) and (b) per zooxanthella (Chl aZ) as a function of the population density of zooxanthella. Regression lines are: After 19 d of elevated ammonium, several changes in (a) Chl aA = 2.639(~)'740 (rZ= 0.78. p < 0.001 where p is the probability that exponents are equal to 0) and (b) ChlaZ = the biomass and metabolism of Seriatopora hystrix and 2.340(~)-".~~~(rZ= 0.45, p < 0.01) Stylophora pistillata had occurred. In both , col- Hoegh-Guldberg & Smith: Nitrogen limitation and zooxanthellae in corals 179

0 0.5 1.0 15 20 25 3.0 POPULATION DENSIT OF ZOOXAhrmELLAE (l 0scell an? Fig. 7. Senatopora hystrix. Ratio of maximum rate of gross photosynthesis (p, ,, and colony respiration rate (r,) as a function of population density of zooxanthellae. Regression line is p,, ,,,/r, = 1.171(D) + 0.843, r2 = 0.65

tent and the population density of zooxanthellae per unit protein were greater after ammonium enrichment for S. pistillata than for S. hystriw(Tab1e 2).The average mitotic index, measured at the end of the experiment (Table 2), did not differ between treatments.

POPULATION DENSrrY OF ZOOXANTHELlAE (106 cell an? Photosynthesis and respiration Fig. 6. Seriatopora hystrix. Maxlmuln gross rate of photosyn- thesis (pcg ,,,) as a function of population density of zooxan- Although the respiratory rates per unit area of both thellae. (a) Results standardized to coral surface area, p,, ,,, Seriatopora hystrix and Stylophora pistillata were unaf- (cm-') = 35.004(D) + 28.088 (r2= 0.72);and (b) results stan- fected by the addition of ammonium (with NH,+; Fig. dardized to zooxanthella number, pCg,,, (zooxanthella-l) = 9a and 10a; Table 3), the photosynthetic response to 63.416(~)-'"' (r2= 0.38, p < 0.05 where p is probability that exponent is equal to 0) irradiance of S. hystrix and S. pistillata was affected. On a per area basis, p,, ,,, of NH,+-treated colonies was 131 % (S. hystrix, p c0.05; Fig. 9a; Table 3) and onies incubated in seawater with elevated ammonium 195 '10 (S. pist~llata, p .< C.05; Fig. 10a; Table3) that of concentrations were darker. This color change was untreated controls. associated with an increased presence of zooxanthellae The differences between NH4+-treated and control as indicated by increased chlorophyll a and number of corals were reversed when photosynthetic rates were zooxanthellae per surface area (Fig. 8a, b). The standardized to chlorophyll a (Figs. 9b and lob; Table chlorophyll acontent of zooxanthellaeincreased with the 3). Maximum rates of photosynthesis (photosynthetic addition of ammonium ions (31 % increase in S. pistillata, capacity) were still significantly different. In both and 90% increase in S. hystrix), although the mean species, however, the gross photosynthetic capacity of diameter of zooxanthellae was unaffected by elevated NH,+-treated corals was lower than that measured for ammonium (Fig. 8c, d).The size range of zooxanthellae in untreated controls (p < 0.01; Table 3). Photosynthetic colonies receiving ammonium, however, did include efficiencies (a)were reduced by the addition of NH4+ larger values than the size ranges of zooxanthellae from (p < 0.05; Table 3). Trends were also modified when untreated corals. Although the amount of protein per results were expressed per zooxanthella. In S. pistillata, coral surface area tended to be higher in corals that photosynthetic capacity per cell was reduced when received ammonium, the amount of protein per coral colonies were treated with ammonium. In S. hystriv surface area was not significantly influenced by the however, photosynthetic capacity per cell was unaf- addition of ammonium. Increases in chlorophyll a con- fected by the addition of ammonium (Table3). Mar. Ecol. Prog. Ser. 57: 173-186, 1989

DISCUSSION Table 2. Seriatopora hystriw and Stylophorapistillata. Average mitotic indices of zooxanthellae. protein concentration, and Several Lines of evidence suggest that the partially chlorophyll a and the number of zooxanthellae per protein for colonies incubated in seawater containing elevated ammonia bleached corals used ln this study were healthy, Firstly1 (10 to 40 ~~h/n.Control corals were incubated in normal sea- new tissue grew equally as rapidly on broken ends of water shown, are means I SEM. *.(With NH;) treatments fragments that had low population densities of zooxan- significantly different from controls (p 50.05) thellae as on the broken ends of fragments that had high population densities of zooxanthellae (pers. obs.). Parameter Control With NH: Secondly, although elevated colony respiratory rates have been associated with exposure to high water S. hystrix temperatures (Coles & Joluel 1977, Hoegh-Guldberg & Mitotic index 3.2 -t 0.80 3.1 f 0.27 Smith 1989), respiration rates in this study did not vary ("/.l Protein per area 0.59 -t 0.323 0.66 * 0.600 as a function of the population density of zooxanthellae (mg (hence extent of bleaching) and were within the range Chlorophyll a 8.75 f 4.04 13.5 f 4.49 of values reported for Seriatopora hystrix previously (mg g protein -') (Bunis et al. 1983, Hoegh-Guldberg & Smith 1989). Zooxanthellae 2.11 f 1.025 2.78 ? 1.55 (106 cell mg protein-') Thirdly, mitotic indices in corals with low population densities of zooxanthellae are higher than the mitotic S. pistillata Mitotic index 1.6 f 0.26 1 1 2 0.56 indices of zooxanthellae in corals with higher popula- ("/U tion densities, suggesting a healthy rate of division by Protein per area 0.57 +. 0.281 0.82 * 0.357 low density populations of zooxanthellae. (mg cmm2) Chlorophyll a 5.6 f 3.14 19.4 k8.97 (mg g protein-') Effect of population density of zooxanthellae on Zooxanthellae 0.55 f 0.124 1.49 k0.248 (106 cell mg protein-') biomass, respiration and photosynthesis of Seriatopora hystrix Mitotic index kerson et al. 1983).The division of zooxanthellae in the corals Stylophora pistillata, Fungia repanda and Pocil- The proportion of dividing zooxanthellae (mitotic lopora damicornis from Lizard Island is also phased index) in Seriatopora hystriv vanes over the die1 cycle (Smith & Hoegh-Guldberg 1987). These observations (Fig. 2). This is indicative of phased cell division (Wil- are in direct contrast to the observations of Wilkerson et

Fig. 8. Stylopora pistilla- ta and Seriatopora hys- triw. (a) Chlorophyll a, (b) number of zooxanthellae per coral surface area, (c) zooxanthella chlorophyll a content, and (d) cell diameter for colonies in- cubated in elther normal seawater (control) or searvater containing ele- vated levels of am- monium ions (ca 20 PM). Control NH~+ Bars indicate standard d! error of the mean Hoegh-Guldberg 8: Smith: N~trogenIln~itat~on and zooxanthellae in corals 181

al. (1988), who reported that zooxanthellae in the tis- lae from Seriatopora hystrix. In corals from Jamaica, sues of all symbiotic cnldarians studied to date (except smaller zooxanthellae have higher growth rates (Wil- zooxanthellae in Mastigias sp.; Wilkerson et al. 1983) kerson et al. 1988). Zooxanthellae from S. pistillata lack phased cell division. Wilkerson et al. (1988) prop- (larger diameter, slower growth) and S. hystrix (smaller osed that the lack of phased cell division in symbiotic diameter, higher growth) show a similar trend. Zooxan- populations of zooxanthellae, normally a characteristic thellae in S. hystrix also showed higher rates of division of growth-restricted phytoplankton populations, is due in the tips of the coral colony, as described by Wilker- to 'host control and the need to regulate zooxanthellae son et al. (1988) for Acropora cervicornis and Agaricia population density'. Given the prevalence of phased ayaricites. Maximal growth rates of tissue have division in zooxanthellae populations from corals col- also been reported from regions at the tip of coral lected at Lizard Island, this statement requires recon- branches (Gladfelter 1983). The mitotic index of zoo- sideration. xanthellae in S. hystrix was also lower at the highest Specific growth rates calculated from the mitotic population densities of zooxanthellae (Fig. 3). The indices measured in this study ranged between 0.040 trend with population density, however, was not as and 0.082d-' for Seriatopora hystrix and 0.028 and dramatic as that demonstrated for zooxanthellae grow- 0.032 d-' for Stylophora pistillata (Table 1).These val- ing in the nudibranch, Pteraeolidia ianthina (Hoegh- ues compare well with those reported for S. pistillata Guldberg & Hinde 1986, Hoegh-Guldberg et al. 1986). growing in the Red Sea (range 0.013 to 0.094 d-'; Wilkerson et al. 1983, Muscatine et al. 1985) and are lower than the growth rate of zooxanthellae reported Chlorophyll a previously for S. pistjllata from the Great Bamer Reef (0.182 d-l; Patton & Burns 1983). As pointed out by The amount of chlorophyll a per area increased as the Wilkerson et al. (1988), the majority of symbiotic zoo- population density of zooxanthellae increased (Fig. 4a). xanthellae fall into the lower growth range, with zoo- Opposing this trend was the decreasing amount of xanthellae from the hydrocoral Millepora dichotoma chlorophyll a per algal cell as population density (Hoegh-Guldberg et al. 1987) and S. pist~Uata from increased (Fig. 4b). This trend has not been reported Patton & Burns (1983) being exceptionally high. before for zooxanthellae, and may be inhcative of The mean cell volume of zooxanthellae from reduced nitrogen availability to zooxanthellae at high Stylophora pistillata is ca 60 % larger than zooxanthel- population densities. Chlorophyll per cell in algae has

Table 3. Seriatopora hystrixand Stylophora pistillata. Maximum rates of gross photosynthesis (P,, ,,,), colony respiratory rates (I,) and photosynthetic efficiency (a) standardized to surface area, chlorophyll a and the number of zooxanthellae for colonies incubated in seawater contaimng elevated concentrations of ammonium Ions (10 to 40 pA4. Control corals were incubated in normal seawater Shown are means +_ SEM. . (With NH:) treatments significantly different from control treatments (p5 0.05)

Denom~nator PC9 mnx TC a 02h-') (W 02h-'] (lig o2m* S pn~ol-' h-')

S. hystrix Surface area W~thNH: (cm2) Control

Chlorophyll a With NH: (W) Control

Zooxanthellae With NH: (106cell) Control S, pistillata Surface area With NH: (cm2) Control

Chlorophyll a With NH: (W) Control

Zooxanthellae With NH; (106 cell) Control Mar Ecol. Prog. Ser. 57: 173-186, 1989

been reported to vary with 2 factors. The chlorophyll a would be expected to be limiting as discussed by Cook & content of zooxanthellae has been reported to increase D'ELia (1987). Smith (1988) and Cook et al. (1988) found with decreasing ambient light in analogy to sun-shade that the chlorophyll content of zooxanthellae in the sea differences in terrestrial plant communities (Wethey & anemone Aptasia pallida decreased as the host was Porter 1976, Falkowski & Dubinsky 1981, Chang et al. starved, presumably because of reduced intracellular 1983, Porter et al. 1984). The reduced availability of inorganic nutrient concentrations. Smith & Muscatine inorganic nitrogen has also been demonstrated to (198613) and Cook et al. (1988) also reported decreases in reduce the chlorophyll content of marine algae (Fal- mitotic index and population density, and increases in kowski 1980, Dawes et al. 1984, Graneli & Sundback zooxanthella C : N ratios, signs that are also indicative of 1985). Given that the lowest chlorophyll contents are in nitrogen limitation. zooxanthellae Living at the highest population densities, the second mechanism provides the best explanation of the variation of chlorophyll per algal cell with popula- Photosynthesis and respiration tion density. As population densities of zooxanthellae increase, light available to resident zooxanthellae The maximum net photosynthetic rate (p, ,,, ,,, per would tend to decrease as self-shading by members of cm2) was strongly dependent on the population density the population increases. On the other hand, the nitro- (D) of zooxanthellae in Seriatopora hystrix (Fig. 5; gen available to zooxanthellae at the highest densities pcnetmRx = 32.14(D) + 0.43, r2 = 0.80). The population

...... Control

IRRADIANCE (pm01@m) m2 S-') Fig. 9. Seriatopora hystriu. Net rate of photosynthesis as a Fig. 10 Stylophora pistillata. Net rate of photosynthesis as a function of irradiance for colonies incubated in either normal function of irradiance of colonies incubated in either normal seawater (control) or seawater containing elevated levels of seawater (control) or seawater containing elevated levels of ammonium ions. Top panel shows results standardized to ammonium ions. Top panel shows results standardized to coral surface area and bottom panel shows results standar- coral surface area and bottom panel shows results standar- dized to chlorophylls. Regression hes are hyperbolic func- &zed to chlorophyll a. Regression lines are hyperbolic func- tions, p, .., ,., = p,, ,,(l -e-""~? + r,, the parameters of tions (see legend to Fig. g),the parameters of which appear in which appear In Table 3 Table 3 Hoegh-Guldberg & Smith: Nitrogen limitat~onand zooxanthellae in corals 183

density of zooxanthellae did not significantly influence population densities of zooxanthellae in Seriatopora coral respiration and had a mean value of 31.06 f 4.448 hystrix colonies suggests that density-dependent fac- pg O2 cm-2 h-' (Fig. 5). This is in contrast to the increase tors may set an upper limit on zooxanthellae biomass in in the respiratory rate of symbiotic associations associ- S. hystrix. Experiments with the long-term enrichment ated with increasing population densities of zooxanthel- of seawater with ammonium were designed to eluci- lae in anemone (Smith 1984), nudibranch (Hoegh-Guld- date the role of the supply of inorganic nitrogen in berg et al. 1986) and coral (Smith & Muscatine 1986a) determining the carrylng capacity of reef corals for associations. In the present study, this trend may have zooxanthellae. been offset by higher metabolic rates of zooxanthellae at The repeated addition of ammonium to the flowing the lowest densities (Fig. 6, a,b).The ratio of p,, ,,,,, to r, water supply of Seriatopora hystrix and Stylophora increased linearly with the population density of zoox- pistillata colonies over 19d resulted in an increase in anthellae (Fig. 7). From these data, the density of zoox- several biomass parameters, and in colony photo- anthellae necessary for S. hystrix to be phototrophic synthetic rates. This is interpreted as direct evidence with respect to carbon can be calculated. Assuming a that the biomass of zooxanthellae in these 2 corals is photosynthetic quotient of 1.0 and that photosynthetic dependent upon ambient nitrogen supply for a given rates are saturated for 10 h d-l, then the population Light regime. These results are in agreement with those density of zooxanthellae necessary to produce enough of Muscatine et al. (1989) for S. pistillata in the Red Sea, carbon for 24 h for the respiration of both coral host and and with Meyers & Schultz (1985) for Acropora palmata zooxanthellae is calculated by equating: and Porites furcata in the Caribbean.

As PC g ,,, is equal to the sum of pc .., ,,, and r,, this Biomass parameters equation reduces to: Elevated levels of ammonium increased the average PC net lnax = 1,4(rc), (2) populabon density and chlorophyll a content of zooxan- and in terms of the population density (D) of zooxan- thellae in both coral species. The large variation of thellae in S. hystrix, becomes: values around each mean, however, indicates that the response was not uniform within each experimental population (Fig. 8). The response to nitrogen enrich- The population density at which S. hystrix becomes ment varied between species as well. Although zoo- phototrophlc with respect to carbon is then, 1.34 X 106 anthellae from Senatopora hystrix and Stylophora pis- zooxanthellae cm-2. S. hystrix norn~allyhas in excess of tillata accumulated similar amounts of chlorophyll a per 1.6 X 106 zooxanthellae cmF2 (this study) and may have cell during enrichment (ca 2.5 pg cell-'), the zooxan- up to 4.78 X 106 zooxanthellae cm-2 (Burris et al. 1983). thellae from S. hystrix accumulated a greater amount The fact that population densities of zooxanthellae in relative to the starting chlorophyll content. Conversely, 'unbleached' S, hystrix are in excess of that necessary the population density of zooxanthellae in ammonium- for phototrophic maintenance of respiration suggests enriched S. pistillata was double that of untreated con- that the photosynthetic contribution of zooxanthellae trols and the relative increase was an order of mag- can potentially support more than the respiration of the nitude greater than that observed in S. hystrix exposed association. to the same enrichment conditions (Fig. 8). These The maximum gross photosynthetic rate per algal differences resulted in S. pistillata colonies experien- cell (p,, ,,,) in Senatopora hystnx decreased as the cing a 3-fold greater increase in chlorophyll a per area population density of zooxanthellae increased (Fig. 6). than S. hystrix during enrichment. The protein content The decrease in pcg ,,, was a direct effect of the of S. pistillata colonies also increased to a greater decreasing chlorophyll content of zooxanthellae as extent than did the protein content of S. hystrix when their population density increased, and is supported by enriched with ammonium (Table 2). the independence of the chlorophyll specific p,, ,,, Despite the dramatic increase in the chlorophyll a from population density effects. On a per chlorophyll a content and population density of zooxanthellae in basis, p,, ,,, was 33.64 k 3.158 pg O2 (pg chl a)-' hp'. Stylophora pistillata (and to a lesser extent in Seriato- pora hystrix), the mitotic index of zooxanthellae was not affected by enrichment with ammonium (Table 2). Effect of elevated external NH4+ on biomass, photo- A similar result was observed for S. pistillata from the synthesis and respiration of S. hystrix and S. pistillata Red Sea (Muscatine et al. 1989). Recent work by Fitt The reduction in the chlorophyll content, metabolic (1988) and Cook et al. (1988) has shown that both rate and growth rate of zooxanthellae with increasing feeding and the addition of ammonium transiently Mar. Ecol. Prog. Ser. 57: 173-186, 1989

stimulate the division frequency of zooxanthellae in Although coral photosynthetic capacities increased, the hydrozoan Ivlyrionema amboinense and the sea chlorophyll a specific photosynthetic rates and photo- anemone Aptasia pallida respectively. This may ex- synthetic efficiencles declined with ammonium enrich- plain the similarity of the mitotic indices from the 2 ment (Table3). Photosynthetic rates per cell decreased experimental treatments in this study. If the growth for zooxanthellae in Stylophora pistillata but were response of zooxanthellae to a sudden elevation in unaffected in Senatopora hystrix. This trend contrasts available nitrogen is short-lived, then sampling the that observed with the population density of zooxan- populations after 19d may have been too late to thellae in S. hystrix (see the first section of this discus- observe the response of the mitotic index to ammonium sion), where higher cellular chlorophyll was associated enrichment. An alternative to this hypothesis is that with increased photosynthetic capacities. The chloro- some other factor influencing the population density of phyll a accumulated during ammonium enrichment is zooxanthellae in S. hystrix and S. pistillata varied with utilized with reduced efficiency in the photosynthetic the addition of ammonium (e.g. the digestion or expul- performance of the zooxanthellae. This observation sion of zooxanthellae; Hoegh-Guldberg et al. 1987, may reflect increased shelf-shading at the high Hoegh-Guldberg & Smith 1989). The specific expulsion densities and high chlorophylla biomass per area rate of zooxanthellae from S. hystrix and S, pistillata obtained during ennchrnent, and is analogous to the under control conditions only ranges up to 0.002 d-' reduction in photosynthetic efficiency and absorptive (Hoegh-Guldberg et al. 1987, Hoegh-Guldberg & cross-sectional area observed for zooxanthellae in Smith 1989) and is an order of magnitude less than the cave-dwelling S. pistillata (Dubinsky et al. 1984, Porter estimated growth rate of zooxanthellae in both S. hys- et al. 1984). In the case of cave-dwelling S. pistillata, trix and S. pistdlata (Table 1). It is unlikely, therefore, environmental irradiance levels set the upper limit on that the increase in the population density of zooxan- zooxanthellae density. Clearly, habitat-specific pat- thellae during enrichment is due to a reduction in the terns in the population density of zooxanthellae will rate at which zooxanthellae are expelled from S. hystrix reflect the long-term balance between the influences of and S. pistillata. In the only study of the expulsion of irradance and nitrogen availability. zooxanthellae during enrichment with inorganic nitro- gen, Stimson (1988) reported that the expulsion of zooxanthellae from the coral PociUopora damicornis Availability of cytosolic inorganic nitrogen and limits increases rather than decreases when coral colonies to biomass of zooxanthellae in S. hystrix and are incubated in seawater enriched with ammonium. S. pistillata

Our study provides 2 pieces of evidence in support of Photosynthesis and respiration the conjecture that the population density of zooxan- thellae is limited by the nutrient supply to the corals. Ammonium enrichment induced an increase in the First, the decline in chlorophyll a content per zooxan- photosynthetic rate per surface area of both coral thella with increasing population densities of zooxan- species. The increase in photosynthetic capacity thellae in Seriatopora hystrix is similar to the chlorosis reflects the increase in the chlorophyll a per area, experienced by nutrient-limited phytoplankton popu- analogous to the trend seen with changes in the popu- lation~(Falkowski 1980, Dawes et al. 1984, Graneli & lation density of zooxanthellae in Seriatopora hystrix Sundback 1985, Cook et al. 1988). Second, the addition (Fig. 6b). Colony respiration rates were unaffected by of ammonium to the immediate environment of these the addition of ammonia. The ratio of pCg ,,, to r, corals resulted in a rapid increase in chlorophyll a con- increased by 45 % and 8296 in S. hystrix and tent and the population density of zooxanthellae in Stylophora pistillata, respecti.vely. Presumably, the S, hystrix and Stylophora pistillata. The reduction in increase in p,, ,,,/r, and the reduction in compensa- the photosynthetic performance of the zooxanthellae tion irradiance (Fig. 10) would eventually lead to (associated with the increase in chlorophyll a content increased growth of the colonies under these condi- per algal cell) does suggest, however, that while the tions. In the present study, only S. pistillata exhibited externally supplied nitrogen is available for assimila- an increase in protein biomass during the 19d expo- tion, the supply of nitrogen alone is not sufficient to sure period. Long-term studies involving the fertiliza- sustain the balanced growth by the zooxanthellae. The tion of patch reefs found a 50 % increase in community question as to whether corals act~vely regulate the photosynthesis (Kmsey & Domm 1974). Meyers & availability of nutrients to zooxanthellae remains Schultz (1985) reported an increase m tissue biomass unanswered. However, recent evidence supporting the and colony growth rates in corals exposed to the excre- existence of an ammonium-concentrating mechanism ment of schooling fish. in clams and corals (Summons et al. 1986) and the Hoegh-Guldberg & Smith: Nitrogen limitation and zooxanthellae in corals 185

identification of a cytosolic NADPH-dependent gluta- dinoflagellate Synlbiodinium microadriaticum Frueden- mate dehydrogenase unique to symbiotic corals thal by endodermal cells of the scyphistomae of Cassiopeia xamachana and resistance of the algae to host digestion. J. (Dudler et al. 1987) does suggest that some corals may Cell SCI.64: 195-212 have the facility to regulate the concentration of Gladfelter, E. H. (1983). Spatial and temporal patterns of ammonium in the immediate extracellular environment mitosis in the cells of the axial of the reef coral of the zooxanthellae. Acropora cervicornis. Biol. Bull mar biol Lab., Woods Hole 165. 811-815 Glynn, P. W. (1984). Widespread coral mortality and the 1982/ Acknowledgements. Support for this study was provided by a 1983 El Nifio warming event. Environ. Conserv. 11: Lizard Island/Australian Museum Bicentenary Fellowship to 133-146 O.H.G. and G.J.S. We would like to thank Drs Barbara Kojis, Graneli, E., Sundback, K. 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This article was submitted to the editor Manuscript first received: March 9, 1989 Revised version accepted: June 22, 1989