# Alkaline phosphatase activity of reef-building corals

Claire Godinot, C. Ferrier-Pagès, S. Sikorski, R. Grover

► To cite this version:

Claire Godinot, C. Ferrier-Pagès, S. Sikorski, R. Grover. Alkaline phosphatase activity of reef-building corals. Limnology and Oceanography, 2013, 58 (1), pp.227-234. 10.4319/lo.2013.58.1.0227. hal-04148998

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Alkaline phosphatase activity of reef-building corals

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3

C. Godinot,ᵃ,ᵇ,\* C. Ferrier-Pagès,ᵃ,ᵇ S. Sikorski,ᵃ and R. Groverᵃ,ᵇ

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**Running head:** Reef corals organic phosphate use

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<footnote>ᵃ Centre Scientifique de Monaco (CSM), Principality of Monaco</footnote>

<footnote>ᵇ Laboratoire Européen Associé (LEA) CSM-Centre National de Recherche Scientifique
(CNRS), Monaco, Principality of Monaco</footnote>

<footnote>\* Corresponding author: cgodinot@centrescientifique.mc</footnote>
## 8 Abstract

Extracellular alkaline phosphatase activity (APA) was measured in four tropical (Stylophora pistillata, Pocillopora damicornis, Pavona cactus, Galaxea fascicularis) and two Mediterranean (Oculina patagonica, Cladocora caespitosa) symbiotic coral species, both in the coral host and associated symbionts, as well as in one tropical non-symbiotic coral (Tubastraea sp.). The effects of light, feeding, and bleaching (loss of symbionts) were also tested in *Stylophora pistillata*. Host APA increased both with long-term starvation, irradiance, and bleaching suggesting that APA is linked to the metabolic activity of the host and symbionts, and to their phosphate limitation or repletion status. The comparison of APA between coral species containing different symbiont clades suggests that clade C is less efficient than clades A and B, but this result remains to be confirmed. At environmental phosphate concentrations, if the total amount of phosphate generated by APA is taken up by the coral colony, it can supply 0.3 - 1.6 mmol P m⁻² d⁻¹. In comparison, dissolved inorganic phosphorus (DIP) and particulate organic phosphorus (POP) have been shown supply 0.2 - 1.6 and 0.9 mmol P m⁻² d⁻¹, respectively, highlighting the importance of APA activity in symbiotic corals.

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## 25 Acknowledgements

We thank Cécile Rottier for assistance in coral maintenance and physiological measurements, Didier Zoccola and Anthony Bertucci for assistance in zooxanthellae clade determination, and Michael Holcomb for use of his bleached *S. pistillata*. We also thank two anonymous reviewers for their helpful comments on this manuscript. This research was supported by the government of the Principality of Monaco. Financial support to C.G. was provided by the École Normale Supérieure and the Centre Scientifique de Monaco.

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33 **Introduction**

Phosphorus is essential to living organisms because it enters into the composition of many biological molecules as phosphate (e.g., deoxyribonucleic acids, ribonucleic acids, phospholipids), and has a role in signaling pathways and metabolic reactions (mostly via the role of adenosine triphosphate, ATP). Corals can fulfill their phosphate needs in several ways. The first way is through the degradation of particulate organic phosphorus (POP), after the capture of plankton caught by the coral host with its stinging tentacles (Johannes et al. 1970; Wijgerde et al. 2011). POP in reef waters corresponds to a mean concentration of ca. 0.10 µmol P L⁻¹ (Furnas et al. 1988; Charpy 2001). Coral acquisition of phosphate from POP has been seldom examined. A recent study estimated that extracoelenteric zooplankton feeding in the species *Galaxea fascicularis* may provide up to 0.9 ± 0.3 mmol P m⁻² d⁻¹ (Wijgerde et al. 2011), which was however obtained with a relatively high prey density (4100 nauplii L⁻¹). Phosphate is also acquired from dissolved inorganic phosphorus (DIP), directly through an active uptake mediated by the dinoflagellate symbionts (D'Elia 1977; Jackson and Yellowlees 1990; Godinot et al. 2009). This DIP presents concentrations between 0.05 and 0.50 µmol P L⁻¹ in reef waters (Atkinson 1987a; Charpy 2001), and contribute 0.2 to 1.1 mmol P m⁻² d⁻¹ to reef benthos (Steven and Atkinson 2003; Cuet et al. 2011), and 1.6 mmol P m⁻² d⁻¹ to corals (Godinot et al. 2009) at these concentrations. The last source of phosphate is represented by dissolved organic phosphorus (DOP), which can be hydrolyzed into phosphate by metalloenzymes called phosphatases. DOP concentrations in reef waters are in the same range as DIP concentrations, i.e., generally lower than 0.5 µmol P L⁻¹ (Crossland and Barnes 1983; Larned 1998). While the coral acquisition of phosphate from DIP has been frequently studied, little research has been done on DOP degradation, and only isolated symbionts from scleractinian corals were considered so far (Jackson et al. 1989; Annis and Cook 2002). However, DOP may provide a significant alternative source of phosphate for corals, in
replacement or in addition to DIP and POP, because it has been shown that phosphate uptake
from DOP hydrolysis by reef benthos phosphatases could be as fast as DIP uptake (Atkinson
1987b).

In reef waters, DOP is present as phosphomonoesters, phosphodiesters, and
phosphonates (molecules that contain a C-P bond). In this first study, we concentrated on the
use of phosphomonoesters. Phosphatases hydrolyzing phosphomonesters are classified as acid
and alkaline depending on their pH optimum; and each type has different substrate
specificities, catalytic mechanisms, and inhibition pathways (Hollander 1971). Phosphatase
activity has been shown to be a good indicator of phosphate deficiency and low
environmental DIP in a large number of marine organisms, such as cyanobacteria (Yentsch et
al. 1972), dinoflagellates (Dyhrman and Palenik 1999), and symbiotic dinoflagellates (Annis
and Cook 2002). Coral hosts and their symbionts (commonly called zooxanthellae), either
freshly isolated (FIZ) or cultured (CZ), exhibit phosphatase activity. Only two studies have
investigated phosphatases in FIZ and CZ, showing both acid and alkaline phosphatases
activities (AcPA and APA, respectively) with intracellular (Jackson et al. 1989) and
extracellular (Annis and Cook 2002) activities reported. Some other studies only mentioned
the presence of intracellular (Goreau 1956) and extracellular (Hayes and Goreau 1977)
phosphatase activity in coral hosts.

This study therefore first aimed at examining the alkaline phosphatase activity in the
scleractinian coral *Stylophora pistillata* under different environmental conditions. We
therefore tested the effect of light, feeding, and bleaching (loss of symbionts) of the coral
host. The following hypotheses were tested: APA should increase with light as previously
observed with DIP (D'Elia 1977; Godinot et al. 2009), and APA should vary with the
availability of other phosphate sources. It should therefore increase with a loss of symbionts,
which leads to a decreased DIP uptake and a release of DIP (D'Elia 1977; Muller-Parker et al.
1990); and it should decrease following feeding of the host, which corresponds to a POP
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supply. It was indeed previously reported that zooxanthellae (zoox) APA significantly
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increased after 5 weeks of host starvation in FIZ or after cultivation in P-limited medium in
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CZ (Jackson et al. 1989; Annis and Cook 2002). The second aim of this study was to compare
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APA in coral species containing different symbiont clades. All these questions have never
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been addressed in corals, but are essential and timely because phosphate uptake is related to
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net carbon production (Atkinson 1992), and should therefore be one of the key factors
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determining primary productivity of corals.
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## 92 Methods

93 Organisms

94 APA was assessed in four tropical symbiotic species (*Stylophora pistillata*,
95 *Pocillopora damicornis*, *Pavona cactus*, and *Galaxea fascicularis*), two Mediterranean
96 symbiotic species (*Oculina patagonica* and *Cladocora caespitosa*), and one tropical non-
97 symbiotic species (*Tubastraea sp.). APA was also assessed in bleached *S. pistillata*, prepared
98 by culturing nubbins for 6 weeks in the dark. Microscopy observations confirmed that
99 symbiont density was 3 orders of magnitude lower in bleached nubbins ($6.7 \times 10^3$ zoox cm$^{-2}$)
100 than in symbiotic ones ($2.5 \times 10^6$ zoox cm$^{-2}$).

101 All colonies were maintained in aquaria supplied with natural seawater (exchange rate
102 2% h⁻¹) containing low nutrient concentrations (DOP < 0.1 µmol P L⁻¹, DIP < 0.05 µmol P L⁻¹,
103 nitrate < 0.4 µmol N L⁻¹ and ammonium < 0.5 µmol N L⁻¹). Seawater salinity was constant
104 at 38.2, pH was 8.1 ± 0.1, and temperature was maintained at 25.0 ± 0.5°C for tropical corals
105 and 18.0 ± 1.0°C for Mediterranean ones. Irradiance was 150 µmol photons m⁻² s⁻¹ for
106 symbiotic corals, with a light:dark cycle of 12 h:12 h. Since corals were cultured at this light
107 intensity and adapted to it, it corresponded to a maximal rate of photosynthesis. The non-
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symbiotic *Tubastraea* sp. and bleached *S. pistillata* were cultured in the dark. All corals were fed twice a week with *Artemia salina* nauplii, but were used for the experiments three days after the feeding (also for the experiments involving FIZ).

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## Isolation of symbionts

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For each experiment with FIZ, symbionts were extracted from 5 coral nubbins using a commercial-type Water-Pick. They were further purified by three centrifugations (2000 rotations per minutes for 5 min at 25°C) followed by re-suspension in seawater (Johannes and Weibe 1970). Aliquots were frozen for subsequent cell counts, chlorophyll and protein determinations. Zooxanthellae phylogeny was determined to the clade level according to Santos et al. (2002), using chloroplastic ribosomal deoxyribonucleic acid sequences.

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## APA assays

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In this study, all experiments were conducted in seawater (pH = 8.1) and should therefore be considered as assays for APA. APA was measured using a colorimetric assay, which is based on the non-biological substrate p-nitrophenyl phosphate (pNPP) to yield a colored product, *p*-nitrophenolate (pNP), upon hydrolysis under alkaline conditions. Most measurements were performed with the symbiotic coral *S. pistillata* and its FIZ, but APA was also compared between different coral species cited above (*see* description below). In each case, 5 nubbins or 5 FIZ samples were considered. All APA experiments were performed using six beakers filled with 200-mL of filtered seawater (0.2 µm membrane filters, pH of 8.1), maintained in a water bath at 26°C or 18°C (for tropical and Mediterranean species, respectively). Light was supplied by 400 W HQI lights and measured using a spherical quantum sensor (LiCor LI-193).

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In experiments involving coral nubbins, individual nubbins suspended to a nylon wire were introduced in five beakers. A beaker without coral served as a control for APA activity in the filtered seawater and for any non-enzymatic hydrolysis of the pNPP substrate. Thirty

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133 minutes after nubbins were placed in the beakers (acclimatization period), the water was
134 enriched with 250 µmol P L⁻¹ of pNPP. Magnetic stirring bars ensured proper homogenization
135 inside the beakers. A 10-mL water sample was taken from each beaker immediately after
136 enrichment and then every 30 min for 120 min. Samples were immediately alkalinized with
137 2 mL of 1 mol L⁻¹ NaOH to stop the enzymatic reaction and yield the yellow colored p-
138 nitrophenolate (pNP). Absorbance was read at 400 nm after 30 minutes against a blank of
139 filtered seawater treated with NaOH. Concentrations of pNP were calculated from a standard
140 curve of pNP absorbance vs. concentration established during each experiment using known
141 concentrations of ACS reagent grade pNP in filtered seawater treated with NaOH. At the end
142 of the experiments, corals were frozen for subsequent normalization of the data.

143 For experiments involving FIZ, a concentrated zooxanthellae solution, obtained after
144 isolation of FIZ, was used to inoculate the five pNPP-enriched beakers, yielding
145 concentrations comprised between 2 × 10⁴ and 10⁵ FIZ mL⁻¹. Sampling protocol and sample
146 measurements were the same as above, except that samples were filtered (0.45-µm membrane
147 filters) prior to alkalinization to remove FIZ.

148 The working parameters for the APA assays were developed in preliminary tests. A
149 first preliminary test was performed to determine the optimal incubation length, using the
150 coral *S. pistillata* and its FIZ at various pNPP concentrations. During each assay, pNP
151 concentration increased linearly over time (independently of the pNPP concentration), during
152 ca. 180 min both for whole coral nubbins and FIZ. Thus, APA was considered linear over
153 these 180 min, and an incubation length of 120 min was chosen for the subsequent
154 experiments. We also examined the effect of pH on phosphatase activity in FIZ, since
155 previous studies suggested that pH optima is different for internal and external phosphatases,
156 and is likely related to the pH experienced by the zooxanthellae (Jackson et al. 1989; Annis
157 and Cook 2002). In the present study, external phosphatase activity of intact FIZ from *S*.
pistillata was predominantly alkaline, as phosphatase activity was 2.5 times higher at the pH of seawater (8.1) than at an acidic pH (5.8; Fig. 1). We therefore worked at a pH of 8.1 (in natural seawater, no buffer added) for the FIZ in the following experiments. We also examined the effect of initial pNPP concentration on APA to determine the kinetics characteristics of coral and zooxanthellae phosphatases. We measured host APA using pNPP concentrations of 0.05, 0.5, 1.0, 5.0, 25, 50, 250, 500, 1000, 2500, and 5000 µmol P L⁻¹, and FIZ APA using pNPP concentrations of 0.05, 5.0, 25, 50, 250, 1000, and 2500 µmol P L⁻¹. APA in the coral host was detectable at pNPP concentrations as low as 0.05, 0.5, and 1.0 µmol P L⁻¹ and was equal to 0.8 ± 0.1, 4.1 ± 0.4, and 5.5 ± 0.7 nmol P mg prot⁻¹ h⁻¹, respectively, and was 1.2 ± 0.2 in FIZ at a pNPP concentration of 0.05 µmol P L⁻¹. FIZ and host extracellular APA were strongly dependent upon DOP concentration, as already reported for the intracellular phosphatases of FIZ (Jackson et al. 1989), and curves fitted with the Michaelis–Menten equation (Fig. 2; Eq. 1):

$$171 \qquad APA = \frac{APA_{\max} [\text{pNPP}]}{[\text{pNPP}] + K_m} \qquad APA = \frac{APA_{\max} [\text{pNPP}]}{[\text{pNPP}] + K}$$

(1) where [pNPP] is the initial concentration of p-nitrophenyl phosphate introduced in the beaker; K_m is the pNPP concentration at which activity is half-optimal, and corresponds to the inverse of the affinity of the alkaline phosphatase towards the pNPP substrate; and APA_max is the activity when the saturation concentration of pNPP has been reached. In both cases, APA saturated for pNPP concentrations between 250 and 500 µmol P L⁻¹, and in subsequent experiments, APA was measured at a pNPP concentration of 250 µmol P L⁻¹ (i.e., near the saturating concentration for both corals and FIZ). Other preliminary tests revealed that APA was strongly affected by light level and coral nutrition. These parameters were therefore tightly controlled in each of the subsequent experiments, to test their individual effect. Therefore, unless otherwise specified, light level was maintained at 150 ± 10 µmol photons m⁻² s⁻¹, and corals were fed three days before the experiments. The set of experimental
conditions tested is summarized in Table 1. Moreover, for experiment 1 (Table 1), the dark incubation was conducted at the end of the 12-hour dark period. For experiment 3 (Table 1), APA measurements were also performed in the dark, at the end of the night, to prevent metabolic changes associated with photosynthesis in the symbiotic nubbins.

## Data normalization and statistical analyses

APA was expressed as units of pNPP hydrolyzed per hour, which corresponds to one molecule of phosphate liberated by the enzyme per molecule of pNPP hydrolyzed. It was then normalized for the coral host either to the chlorophyll (Chl) and zooxanthellae (zoox) content (using the same methods as in Godinot et al. 2009), to the surface area of the nubbins (using the wax-dipping method described in Stimson and Kinzie 1991), or to the protein (prot) content (after extraction in 1 mol L⁻¹ NaOH for 30 min at 90°C, measured using the commercially available BC Assay Interchim kit). Protein content is a good estimator of the biomass, since there is a linear relationship between protein content and dry weight in *S. pistillata* (unpublished results). Data for the FIZ were normalized to protein content, and to cell density and chlorophyll content. All data are expressed as the mean ± standard error of 5 replicates. The software GraphPad Prism (version 5.0) was used to perform linear regressions to examine the effect of light, and to study concentration-dependent APA using non-linear regressions with Michaelis-Menten fittings. For the other experiments, StatView (version 5.0) was used to perform *t*-tests, and one-way and two-ways analysis of variance (ANOVA) on the data, followed by Fisher's protected least significant difference (PLSD) tests.

## Results

Since we used nubbins entirely covered with tissue and intact symbiont cells, phosphatase activities should have been from external phosphatases only. In the following results, we will refer to 'FIZ APA' for the activity related to the symbionts only, and to 'host
208 APA' for the activity related to the entire symbiotic association, but mainly coming from the
209 host because only the host tissue was exposed to the medium. Moreover, normalization of
210 APA per protein and skeletal surface area led to the same results, and only the normalization
211 per protein is presented. It is considered as the most appropriate to compensate for the
difference in biomass per skeletal surface area between the various coral species used, and
212 between coral hosts and FIZ. In the clade experiment, results are also presented with the
213 normalization to chlorophyll content.
214

215 **Effect of light and feeding on the APA of S. pistillata**

216 APA increased linearly with light in both host ($r^2$ = 0.61, $F_{1,18}$ = 29, *p* < 0.0001) and
217 FIZ ($r^2$ = 0.87, $F_{1,18}$ = 120, *p* < 0.0001; Fig. 3), being c.a. 3.0 times higher at 350 µmol
218 photons m⁻² s⁻¹ than in the dark. APA also significantly increased over time in both host and
219 FIZ when corals were starved (1-way ANOVA, both *p* < 0.0001, $F_{3,16}$ = 16.9 for host, and
220 $F_{3,16}$ = 57.4 for FIZ; Fig. 4). Host APA was the highest after 6 weeks of starvation (165.5 ±
221 20.3 nmol P mg prot⁻¹ h⁻¹). At this moment, activity was 4 times higher than in fed corals
222 (40.0 ± 2.4 nmol P mg prot⁻¹ h⁻¹), and remained 2.8 times higher until 9 weeks of starvation.
223 FIZ APA was the highest after 3 weeks of starvation (154.6 ± 4.3 nmol P mg prot⁻¹ h⁻¹), being
224 1.5 times higher than in FIZ from fed corals (100.0 ± 7.0 nmol P mg prot⁻¹ h⁻¹). After this
225 point, APA decreased back to the level of FIZ from fed corals.

226 **Effect of the symbiotic status**

227 In the non-symbiotic *Tubastraea* sp., light had no effect on APA, as APA was not
228 significantly different in the dark (18.5 ± 2.9 nmol P mg prot⁻¹ h⁻¹) and in the light (19.3 ± 2.2
229 nmol P mg prot⁻¹ h⁻¹; *t*-test, *t* = 0.27, *df* = 9, *p* = 0.76,). Host APA in the dark was not
230 significantly different between *Tubastraea* sp. and symbiotic *S. pistillata* (mean of 18.3 ± 1.6
231 nmol P mg prot⁻¹ h⁻¹; 1-way ANOVA, *p* = 0.89, $F_{2,12}$ = 0.21; Fig. 5), but increased by 6.4 fold
232 in bleached *S. pistillata* (115.1 ± 20.4 nmol P mg prot⁻¹ h⁻¹; 1-way ANOVA, *p* = 0.0001, *F*<sub>2,12</sub> = 21.85).

234 **Comparison of APA in different coral hosts and zooxanthellae clades**

235 When results obtained for the FIZ were normalized to the protein content, there was no
236 significant difference in APA between the clades (1-way ANOVA, *p* = 0.18, *F*<sub>2,27</sub> = 4.81; data
237 not shown). Conversely, a significant difference in host APA was observed between species
238 (1-way ANOVA, *p* < 0.0001, *F*<sub>5,24</sub> = 21.7; Fig. 6A). Differences were only related to
239 differences in the chlorophyll content normalized per protein content between coral species,
240 and were unrelated to the tropical or temperate status of the host, to the symbiont density, to
241 the chlorophyll content per surface area, or to the zooxanthellae clade (1-way ANOVAs, all
242 *p* > 0.4). For FIZ, results were also normalized to the chlorophyll content. With this
243 normalization, there was a significant difference in APA between species (1-way ANOVA,
244 *p* < 0.0001, 192.6; Fig. 6B), which was related to the zooxanthellae clade (1-way ANOVA,
245 *p* < 0.0001, 232.7, respectively).

246

247 **Discussion**

248 This study reports on the alkaline phosphatase activity in coral hosts and their isolated
249 symbionts under various environmental conditions. It is the first to examine APA in coral
250 hosts, and to highlight the importance of host metabolism, and phosphate limitation or
251 repletion, on this activity. It also emphasizes for the first time the effects of light and
252 zooxanthellae clade on APA, and confirms that host feeding has a negative effect on this
253 activity.

254 **Effects of metabolic activity on APA**

255 In symbiotic corals, APA was dependent upon symbiont activity, and likely upon
256 zooxanthellae photosynthetic activity, since APA was 3.0 times lower in the dark than at 350
μmol photons m⁻² s⁻¹ in a symbiotic coral (Fig. 3) while it did not increase with light in a non-symbiotic one. APA was also 6.4 times higher in bleached than in symbiotic colonies (Fig. 5), suggesting a phosphate limitation in bleached corals, as previously demonstrated (Annis and Cook 2002). In symbiotic corals, most of the phosphate requirements are supplied by the activity of the symbionts, which continuously take up DIP from the surrounding seawater (D'Elia 1977; Godinot et al. 2009). The loss of symbionts in bleached colonies corresponds to a reduction of DIP uptake, and even to a release of it (Muller-Parker et al. 1990), which may both lead to a deficit of phosphate in the symbiotic association. Therefore, the host has to rely on organic phosphate (and increased APA) to fulfill its high phosphate metabolic demand (Godinot et al. 2011). Conversely, in the non-symbiotic *Tubastraea* sp., APA was 6.2 times lower than in bleached *S. pistillata* (Fig. 5), suggesting that despite the fact that *Tubastraea* also loses DIP (D'Elia 1977), its total phosphate demand may be lower. Alternatively, although non-symbiotic, bleached, and symbiotic corals, were supplied the same zooplankton regime before the experiments, their feeding rates remain to be examined in order to determine if their phosphate inputs from POP were different.

In symbiotic *S. pistillata*, host APA was also affected by heterotrophy and starvation. The observed increase in APA with long-term starvation of the host (Fig. 4) can be related to a lack of phosphate availability in the symbiosis. Indeed, feeding provides the host with a source of POP (Johannes et al. 1970; Wijgerde et al. 2011), and this source disappears with starvation, which therefore might trigger the increase of host APA. The same feeding effect was observed in FIZ (Fig. 4) and was also reported previously in FIZ (Jackson et al. 1989; Annis and Cook 2002). After a prolonged starvation (more than 3 weeks in FIZ and more than 6 weeks in the host), APA decreased again, probably due to a reorganization and/or reduction of the coral metabolic activity, including a possible shut-down of metabolic machinery and the recycling of macromolecules. It is in fact well established that starvation lowers protein
and chlorophyll concentrations, symbionts density (Titlyanov et al. 2001; Ferrier-Pagès et al. 2003; Houlbrèque et al. 2003), and rates of respiration and photosynthesis (Szmant-Froelich and Pilson 1984; Davy et al. 2006). Such reduction of the metabolism may either be due to the catabolism of alkaline phosphatases or may have led to a lower phosphate demand explaining the APA decrease.

Results obtained finally showed that light increased host and FIZ APA (Fig. 3), maybe due to the higher metabolic activity of corals in terms of respiration rates in the light (Porter et al. 1984). Another explanation can be a faster depletion of phosphate stocks of the host by symbiont photosynthesis, as phosphate is a component of ATP and enters into the composition of other compounds of the Calvin cycle (Hall and Rao 1987), and as DIP demand has been shown to increase with light (Jackson and Yellowlees 1990; Godinot et al. 2009).

Moreover, light levels used in this study are not as high as those encountered naturally in reefs, and APA might therefore be even higher under natural conditions, with a much faster hydrolysis of organic phosphate into inorganic phosphate.

## Inter-species comparison and effect of zooxanthellae clade

Results obtained in this experiment suggest for the first time a possible dependency of APA upon zooxanthellae clade, with the cosmopolitan clade C (Stat et al. 2008) being apparently less efficient than clades A and B (Fig. 6). Specific metabolic characteristics of the different clades may influence APA, possibly through differences in the phosphate demand and/or uptake. This theory remains however to be further investigated, because only one study has examined up to date the relationship between eutrophication and zooxanthellae clades (Lampert-Karako et al. 2008), and has found no differences. Since only one clade B coral was available for the present study, the preliminary results from this experiment need to be completed by expanding the number of samples within the same clade, as well as the number of clades investigated.
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Contribution of DOP to the phosphate demand of corals

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APA measured at DOP concentrations of 0.05 and 0.5 µmol P L⁻¹, which corresponds to the range of those occurring in situ (Crossland and Barnes 1983; Larned 1998), was equal to 0.8 ± 0.1 and 4.1 ± 0.4 nmol P mg prot⁻¹ h⁻¹ in the light, respectively, and 0.4 ± 0.1 and 1.9 ± 0.3 nmol P mg prot⁻¹ h⁻¹ in the dark, respectively (2.2 times lower compared to the light value). These values correspond to 0.3 to 1.6 mmol P m⁻² d⁻¹ (value × 2.2 mg prot cm⁻² × 12 h of day and night ×10,000). If all phosphate provided by DOP hydrolysis through alkaline phosphatase activity is entirely taken up by corals, DOP appears to be a significant source of phosphate for corals, even at low environmental concentrations. Indeed, it has been shown that DIP contributes 0.2 to 1.6 mmol P m⁻² d⁻¹ (Steven and Atkinson 2003; Godinot et al. 2009; Cuet et al. 2011) and POP brings of 0.9 ± 0.3 mmol P m⁻² d⁻¹ (Wijgerde et al. 2011). DOP may have an even more significant contribution if sources like phosphodiesters, polyphosphates and phosphonates are taken into consideration. Coral species can benefit from these sources using other phosphatases and phosphonatases. The question that remains to be examined is how much of the phosphate recycled from DOP can effectively be absorbed by coral colonies, since phosphate might be rapidly flushed out of the reef by water currents (Atkinson 1987b). Some additional studies, using a ³³P-labeled DOP source, may help answer this question, by tracing ³³P inside coral tissues.

In conclusion, this study showed that host and symbionts alkaline phosphatase activities are dependent upon light and host feeding, and upon the metabolic activity of the host and the symbionts (with a possible clade effect). We showed that DOP may represent a significant source of phosphate for corals in oligotrophic reefs, since at similarly low environmental concentrations (less than 0.5 µmol P L⁻¹), this source may bring as much phosphate as DIP (1.6 mmol P m⁻² d⁻¹). The present study thus needs to be followed by attempts at determining the amount of phosphate effectively recycled from various sources of
332 DOP and POP by corals, in order to complete our first calculation of the balance of phosphate

333 inputs from DIP, DOP, and POP in corals under oligotrophic conditions.
334 **References**

335 Annis, E. R., and C. B. Cook. 2002. Alkaline phosphatase activity in symbiotic dinoflagellates (zooxanthellae) as a biological indicator of environmental phosphate exposure. Mar. Ecol. Prog. Ser. **245**: 11-20.

338 Atkinson, M. J. 1987a. Rates of phosphate uptake by coral reef flat communities. Limnol. Oceanogr. **32**: 426-435.

340 Atkinson, M. J. 1987b. Alkaline phosphatase activity of coral reef benthos. Coral Reefs **6**: 59-62.

342 Atkinson, M. J. 1992. Productivity of Enewetak Atoll reef flats predicted from mass transfer relationships. Continental Shelf Research **12**: 799-807.

344 Charpy, L. 2001. Phosphorus supply for atoll biological productivity. Coral Reefs **20**: 357-360.

346 Crossland, C. J., and D. J. Barnes. 1983. Dissolved nutrients and organic particulates in water flowing over coral reefs at Lizard Island. Aust. J. Mar. Freshw. Res. **34**: 835-844.

348 Cuet, P., C. Pierret, E. Cordier, and M. J. Atkinson. 2011. Water velocity dependence of phosphate uptake on a coral-dominated fringing reef flat, La Réunion Island, Indian Ocean. Coral Reefs **30**: 37-43.

351 D'Elia, C. F. 1977. The uptake and release of dissolved phosphorus by reef corals. Limnol. Oceanogr. **22**: 301-315.

353 Davy, S. K., K. J. T. Withers, and R. Hinde. 2006. Effects of host nutritional status and seasonality on the nitrogen status of zooxanthellae in the temperate coral *Plesiastrea versipora* (Lamarck). J. Exp. Mar. Biol. Ecol. **335**: 256-265.

356 Dyhrman, S. T., and B. Palenik. 1999. Phosphate stress in cultures and field populations of the dinoflagellate *Prorocentrum minimum* detected by a single-cell alkaline phosphatase assay. Appl. Environ. Microbiol. **65**: 3205-3212.
359

Ferrier-Pagès, C., J. Witting, E. Tambutté, and K. P. Sebens. 2003. Effect of natural

360

zooplankton feeding on the tissue and skeletal growth of the scleractinian coral

361

*Stylophora pistillata*. Coral Reefs **22**: 229-240.

362

Furnas, M., A. W. Mitchell, J. Wellington, and B. Brady. 1988. Dissolved and particulate

363

nutrients in waters of the Whitsunday Island Group. Research Publication no. 16,

364

Australian Institute of Marine Science.

365

Godinot, C., C. Ferrier-Pagès, and R. Grover. 2009. Control of phosphate uptake by

366

zooxanthellae and host cells in the scleractinian coral *Stylophora pistillata*. Limnol.

367

Oceanogr. **54**: 1627-1633.

368

Godinot, C., R. Grover, D. Allemand, and C. Ferrier-Pagès. 2011. High phosphate uptake

369

requirements of the scleractinian coral *Stylophora pistillata*. The Journal of

370

Experimental Biology **214**: 2749-2754.

371

Goreau, T. 1956. Histochemistry of mucopolysaccharide-like substances and alkaline

372

phosphatase in *Madreporaria*. Nature **177**: 1029-1030.

373

Hall, D. O., and K. K. Rao. 1987. Photosynthesis. Cambridge University Press.

374

Hayes, R. L., and N. I. Goreau. 1977. Cytodynamics of coral calcification, p. 433-438. *In* D.

375

L. Taylor [ed.], Proceedings, Third Int. Coral reef Symp. Rosenthal School of Marine

376

and atmospheric Science.

377

Hollander, V. P. 1971. Acid phosphatases, p. 449-498. *In* P. D. Boyer [ed.], The enzymes.

378

Academic Press.

379

Houlbrèque, F., E. Tambutté, and C. Ferrier-Pagès. 2003. Effect of zooplankton availability

380

on the rates of photosynthesis, tissue and skeletal growth of the scleractinian coral

381

*Stylophora pistillata*. J. Exp. Mar. Biol. Ecol. **296**: 145-166.

382

Jackson, A. E., and D. Yellowlees. 1990. Phosphate uptake by zooxanthellae isolated from

383

corals. Proc. R. Soc. Lond. B Biol. Sci. **242**: 201-204.
384 Jackson, A. E., D. J. Miller, and D. Yellowlees. 1989. Phosphorus metabolism in the coral-
385 zooxanthellae symbiosis: characterization and possible roles of two acid phosphatases
386 in the algal symbiont *Symbiodinium* sp. Proceedings of the Royal Society of London.
387 Series B **238**: 193-202.

388 Johannes, R. E., and W. J. Weibe. 1970. A method for determination of coral tissue biomass
389 and composition. Limnol. Oceanogr. **15**: 822-824.

390 Johannes, R.E., S.L. Coles, and N.T. Kuenzel. 1970. The role of zooplankton in the nutrition
391 of some scleractinian corals. Limnol. Oceanogr. **15**: 579-586.

392 Lampert-Karako, S., N. Stambler, D. J. Katcoff, Y. Achituv, Z. Dubinsky, and N. Simon-
393 Blecher. 2008. Effects of depth and eutrophication on the zooxanthella clades of
394 *Stylophora pistillata* from the Gulf of Eilat (Red Sea). Aquatic Conservation: Marine
395 and Freshwater Ecosystems **18**: 1039-1045.

396 Larned, S.T. 1998. Nitrogen- versus phosphorus-limited growth and sources of nutrients for
coral reef macroalgae. Mar. Biol. **132**: 409-421.

398 Muller-Parker, G., C. B. Cook, and C. F. D'Elia. 1990. Feeding affects phosphate fluxes in the
399 symbiotic sea anemone *Aiptasia pallida*. Mar. Ecol. Prog. Ser. **60**: 283-290.

400 Porter, J. W., L. Muscatine, Z. Dubinsky, and P. G. Falkowski. 1984. Primary production and
401 photoadaptation in light- and shade-adapted colonies of the symbiotic coral,
402 *Stylophora pistillata*. Proc. R. Soc. Lond. **222**: 161-180.

403 Santos, S. R., D. J. Taylor, R. A. Kinzie, M. Hidaka, K. Sakai, and M. A. Coffroth. 2002.
404 Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast
405 large subunit (23S)-rDNA sequences. Mol. Phylogenet. Evol. **23**: 97-111.

406 Stat, M., E. Morris, and R. D. Gates. 2008. Functional diversity in coral– dinoflagellate
407 symbiosis. Proc. Natl. Acad. Sci. **105**: 9256-9261.
408

Steven, A. D. L., and M. J. Atkinson. 2003. Nutrient uptake by coral-reef microatolls. Coral

409

Reefs **22**: 197-204.

410

Stimson, J., and R. A. Kinzie. 1991. The temporal pattern and rate of release of zooxanthellae

411

from the reef coral *Pocillopora damicornis* (Linnaeus) under nitrogen-enrichment and

412

control conditions. *J. Exp. Mar. Biol. Ecol.* **153**: 63-74.

413

Szmant-Froelich, A., and M. E. Pilson. 1984. Effect of feeding frequency and symbiosis with

414

zooxanthellae on nitrogen metabolism and respiration of the coral *Astrangia danae*.

415

*Mar. Biol.* **81**: 153-162.

416

Titlyanov, E. A., T. V. Titlyanova, K. Yamazato, and R. van Woesik. 2001. Photo-

417

acclimation of the hermatypic coral *Stylophora pistillata* while subjected to either

418

starvation or food provisioning. *J. Exp. Mar. Biol. Ecol.* **257**: 163-181.

419

Wijgerde, T., R. Diantari, M. W. Lewaru, J. A. J. Verreth, and R. Osinga. 2011.

420

Extracoelenteric zooplankton feeding is a key mechanism of nutrient acquisition for

421

the scleractinian coral *Galaxea fascicularis*. *The Journal of Experimental Biology* **214**:

422

3351-3357.

423

Yentsch, C. M., C. S. Yentsch, and J. P. Perras. 1972. Alkaline phosphatase activity in the

424

tropical marine blue-green alga, *Oscillatoria erythraea* ("Trichodesmium"). *Limnol.*

425

*Oceanogr.* **17**: 772-774.

426

427

428
429 **Table**

430

431 **Table 1**

432 Working parameters of the four experiments performed to measure APA in various

433 scleractinian corals.
<table><thead><tr><th>Experiment</th><th>Species (clade)</th><th>Symbiotic status</th><th>Coral nubbin or FIZ</th><th>Light (µmol photons m<sup>-2</sup> s<sup>-1</sup>)</th><th>Host feeding (measurements 3 days after feeding)</th><th>[pNPP] (µmol P L<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="4">1 - Effect of light</td><td>Stylophora pistillata (A)</td><td>symbiotic</td><td>coral<br>FIZ</td><td>0<br>150<br>250<br>350</td><td>control</td><td>250</td></tr><tr><td>Tubastraea sp.</td><td>asymbiotic</td><td>coral</td><td>0<br>150</td><td>control</td><td>250</td></tr><tr><td rowspan="2">S. pistillata (A)</td><td>symbiotic</td><td>coral<br>FIZ</td><td>150</td><td>control<br>starved 3 wks<br>starved 6 wks<br>starved 9 wks</td><td>250</td></tr><tr><td>bleached</td><td>coral</td><td></td><td></td><td></td></tr><tr><td rowspan="2">3 - Effect of symbiotic status</td><td>S. pistillata (A)</td><td>symbiotic</td><td>coral</td><td>0</td><td>control</td><td>250</td></tr><tr><td>S. pistillata (A)<br>Tubastraea sp.</td><td>bleached<br>asymbiotic</td><td>coral</td><td></td><td></td><td></td></tr><tr><td rowspan="7">4 - Effect of symbiont clade</td><td>S. pistillata (A)</td><td>symbiotic</td><td>coral<br>FIZ</td><td>150</td><td>control</td><td>250</td></tr><tr><td>Pocillopora damicornis (C)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pavona cactus (C)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Galaxea fascicularis (C)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cladocora caespitosa (A)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Oculina patagonica (B)</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
434 **Figure legends**

435
436 **Figure 1:** Effect of pH on APA of FIZ from *Stylophora pistillata* incubated with 250 µmol L⁻¹ pNPP. Data are presented as the mean ± standard error of 5 measurements.

438
439 **Figure 2:** Concentration-dependent APA in host and FIZ from *S. pistillata*. Data are mean ± standard error of 5 replicates.

441
442 **Figure 3:** Light-dependent APA in *S. pistillata* nubbins and FIZ incubated with 250 µmol L⁻¹ pNPP. Data are mean ± standard error of 5 replicates. Light was measured using a LiCor instrument and is expressed as µmol m⁻² s⁻¹ PAR.

445
446 **Figure 4:** Effect of starvation on APA in host and FIZ from the coral *S. pistillata* incubated with 250 µmol L⁻¹ pNPP. Data are mean ± standard error of 5 replicates.

448
449 **Figure 5:** Dark APA in the non-symbiotic tropical coral *Tubastraea* sp., and in symbiotic and in bleached *S. pistillata* incubated with 250 µmol L⁻¹ pNPP. Data are presented as the mean ± standard error of 5 measurements.

452
453 **Figure 6:** Comparison of APA in different (a) coral hosts (b) harboring symbionts from different clades. Data are presented as the mean ± standard error of 5 measurements, (a) normalized per protein content or (b) per chlorophyll content.
<figure><img src="image_2.png" /><figcaption>Fig. 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig. 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 4</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 5</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 6</figcaption></figure>

Coral species