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First published online February 29, 2008
Journal of Experimental Biology 211, 860-865 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012807
Uptake of dissolved free amino acids by the scleractinian coral Stylophora pistillata
1 Centre Scientifique de Monaco, Avenue Saint Martin, MC-98000, Monaco
2 Laboratoire de Chimie Marine, Institut Universitaire Européen de la
Mer, Place Nicolas Copernic, F-29280 Plouzane, France
* Author for correspondence (e-mail: rgrover{at}centrescientifique.mc)
Accepted 19 January 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: coral, isotopic enrichment, uptake, kinetics, dissolved free amino acid
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), whereas they thrive in oligotrophic environments containing
sub-micromolar nutrient concentrations
(Bythell, 1990;
Furnas, 1991;
Szmant, 2002). This high
productivity results from an efficient multiscale strategy, combining high
flow rates above the reef (Thomas and
Atkinson, 1997), high nutrient recycling by the reef community
(Szmant-Froelich, 1983;
Uthicke, 2001) and coral
mixotrophy (Titlyanov et al.,
2000). At the coral level, mixotrophy multiplies the nutrient
sources, by combining prey capture
(Ferrier-Pagès et al.,
2003), uptake of dissolved molecules
(Bythell, 1990) and utilization
of photosynthates produced by the symbiotic zooxanthellae living in the
endodermal cells of the animal (Muscatine
and Cernichiari, 1969). Previous studies have shown that corals
are opportunistic with respect to nitrogen and retain both inorganic sources,
such as ammonia and nitrate (Falkowski et
al., 1993; Grover et al.,
2002; Grover et al.,
2003; Hoegh-Guldberg and
Williamson, 1999; Marubini and
Davies, 1996; Wilkerson and
Trench, 1986), and organic sources such as urea
(Grover et al., 2006) and
dissolved free amino acids (DFAA)
(Al-Moghrabi et al., 1993;
Ferrier, 1991;
Hoegh-Guldberg and Williamson,
1999).
DFAA is part of the dissolved organic nitrogen (DON), which is a
heterogeneous mixture of urea, dissolved combined amino acids (DCAA), nucleic
acids and unidentified species (Bronk,
2002). As DFAA are excreted and consumed by a large variety of
marine organisms, they are subject to rapid turnover that leads to several
orders of magnitude variability in seawater concentrations. DFAA therefore
represent approximately 10% of the DON pool with concentrations ranging
between 0.001 and 0.7 µmol l–1
(Bronk, 2002).
DFAA uptake by corals has not been thoroughly investigated, and previous
works are all based on depletion measurements of a DFAA-enriched medium using,
de facto, elevated concentrations. HPLC techniques have been used to
monitor the specific amino acid uptake by different coral species
(Ferrier, 1991;
Hoegh-Guldberg and Williamson,
1999). The concentrations used, however, ranged from 4 to 5.6
µmol l–1, and were several times higher than those in
situ. Finally, Al-Moghrabi et al.
(Al-Moghrabi et al., 1993)
performed physiological approaches by using a unique radiolabelled DFAA
(valine) in order to figure out uptake regulation by light.
The present work investigates the uptake of a natural mixture of DFAA, at in situ concentrations, in the scleractinian coral Stylophora pistillata, using 15N-labeled products. DFAA uptake was measured with different incubation times, DFAA concentrations and light levels. To highlight a possible discrimination between various DFAA, depletion measurements were also performed, whereby corals were incubated with single amino acids.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Temperature was maintained at 26±0.2°C using heaters connected to
temperature controllers. Submersible pumps were used for water agitation and
metal halide lamps (Philips, HPIT, Guildford, Surrey, UK) provided light with
a 12 h:12 h light:dark photoperiod. Salinity and irradiance were measured
using a conductivity meter (Meter LF196, WTW, Weiheim, Germany), and a 4
quantum sensor (Li-Cor, LI-193SA, Lincoln, NE, USA), respectively. Corals were
fed twice a week with Artemia salina nauplii during the healing
period. They remained unfed 3 weeks before and during the experiments,
however, to avoid external nitrogen input that could interfere with DFAA
uptake (Grover et al., 2002;
Muller-Parker et al.,
1988).
Measurements of dissolved free amino acids (DFAA) in seawater by spectrofluorimetry
The contribution of planktonic bacteria to DFAA uptake was checked by
measuring DFAA concentrations in 3 tanks filled with natural seawater, after
0, 12 and 24 h of incubation. Natural DFAA concentrations in the incubation
medium were also measured every day and before each experiment. They were
taken into account in the calculations of the total DFAA concentrations during
the experiments. This DFAA quantification was performed using a
spectrofluorometer (Xenius SAFAS, Monaco, Monaco) according to the method of
Parsons et al. (Parsons et al.,
1984). Briefly, o-phthalaldehyde reacts with DFAA in the presence
of β-mercaptoethanol to produce fluorescent compounds, the fluorescence
intensity being proportional to DFAA concentration. Seawater samples were
first filtered through a 0.22 µm syringe filter (Acrodisc, PALL, East
Hills, NY, USA) to remove any particle that could interfere with the
fluorescence measurements. 5 ml of each sample was added to 5 ml of reactive
solution (o-phthalaldehyde + β-mercaptoethanol) and mixed for 2 min.
Samples were then transferred into a 4 ml quartz SUPRASIL cell and excited at
a 342 nm wavelength. Emission wavelengths between 400 nm and 500 nm were
recorded in order to quantify the maximal fluorescence intensity. Standard
solutions of glycine from 0.2 to 1.0 µmol l–1 were
prepared for internal calibration and to set up the photomultiplier voltage.
DFAA concentration in the samples was obtained according to the following
formula:
 | (1) |
DFAA depletion experiments
In order to assess the relative uptake of the main DFAAs by S.
pistillata, eleven amino acids (glycine, valine, alanine, glutamate,
glutamine, aspartate, asparagine, histidine, leucine methionine, serine)
(Aldrich, St Quentin, Falavier, France) were tested separately. Each DFAA was
dissolved into 0.22 µm filtrated seawater at a final concentration of 3
µmol l–1, and transferred into three 250 ml beakers, each
containing a coral microcolony. Beakers were incubated during 6 h in the light
(300 µmol photons m–2 s–1), and at a
constant temperature of 26.5°C, using a water bath. Seawater was
continuously stirred using a magnetic stirrer. DFAA depletion was monitored in
each beaker by sampling 5 ml of the medium every hour. This depletion was
linear during the first 3 h, before decreasing asymptotically, due to the
lowering of the DFAA concentration in the medium. Only the linear decrease was
taken into account for the uptake rate calculations. Results were normalized
to skeletal surface area and expressed as nmol DFAA h–1
cm–2.
DFAA uptake kinetics
Experiments using 15N-enriched DFAA (thereafter called
15N-DFAA) were performed to measure DFAA uptake rates for different
incubation times, DFAA concentrations and light intensities.
15N-DFAA originated from an algal mix (98% 15N
enrichment, ISOTEC, Sigma-Aldrich, St Quentin, Falavier, France) whose
composition was close to the natural seawater DFAA composition.
|
To assess the effect of the incubation length on DFAA uptake rate, microcolonies were incubated either in 0.5 or 3 µmol l–1 15N-DFAA-enriched seawater during 2, 5, 7 and 21 h under a constant light intensity of 300 µmol photons m–2 s–1 (N=24 microcolonies). This experiment confirmed that the uptake rate was constant and linear during the whole incubation. The concentrations used (0.5 and 3 µmol l–1) were considered as `normal' and `high', compared to that in situ. To assess the effect of light on the uptake rates, microcolonies were incubated either in 0.5 or 3 µmol l–1 15N-DFAA-enriched seawater for 7 h and under three light intensities: 0 (dark), 160 and 300 µmol photons m–2 s–1. Finally, for the determination of DFAA uptake rates versus concentration, microcolonies were incubated in six different 15N-DFAA concentrations equal to 0.2, 0.5, 1, 3, 8 and 13 µmol l–1 for 7 h and under a constant light intensity of 300 µmol photons m–2 s–1 (N=18).
DFAA uptake kinetics by freshly isolated zooxanthellae
In order to obtain freshly isolated zooxanthellae (FIZ), tissue was removed
from the skeleton of three big colonies and zooxanthellae were isolated by
centrifugation as described below. They were then re-suspended in 200 ml
filtered seawater and divided into several beakers for a 5 h incubation with
three different 15N-DFAA concentrations equal to 0.5, 3 and 7
µmol l–1 (using triplicate samples for each
concentration). Experiments were performed in the light (160 µmol photons
m–2 s–1) and under a constant temperature of
26.5°C. At the end of the incubation, each sample was filtered through a
pre-combusted (450°C) GF/F filter and rinsed with a small volume of
filtered seawater. Filters were then dried at 60°C for 8 h and stored in a
desiccator until analysis. Results will be expressed as
%15Nenrichment and will not be converted into uptake
rate, because the exact number of zooxanthellae on the filter could not be
determined.
|
).
Briefly, coral tissue was removed from the skeleton with a flow of argon under
pressure in 5 ml filtered seawater and homogenized using a Potter tissue
grinder. Animal and zooxanthellae fractions were then separated by
centrifugation and zooxanthellae were rinsed three times in filtrated seawater
to avoid animal contamination. Samples were freeze-dried for conservation
until spectral analysis.
Isotopic enrichment quantification and determination of DFAA uptake rates
15N/14N isotopic ratios in animal tissue and
zooxanthellae were determined using an Isotope Ratio Mass Spectrometer (IRMS)
and compared to natural 15N/14N. The %15N
enrichment in the coral corresponds to the amount of nitrogen transferred from
seawater into the animal and vegetal constituents of the coral. This flow can
be converted into uptake rate (
) using the equation derived from Dugdale
and Wilkerson (Dugdale and Wilkerson,
1986) and presented in Grover et al.
(Grover et al., 2002), which
takes into account the sample biomass and the skeletal surface area. In the
present study, as in previous ones using the same technique
(Grover et al., 2002;
Grover et al., 2003;
Grover et al., 2006), even
after animal and algal separation, the results were normalized to skeletal
surface area, in order to be comparable with other studies on nitrogen fluxes.
Considering that animal and algal biomasses in a coral colony are not the
same, however, normalization to animal tissue or zooxanthellae dry mass
appeared to be more suitable for determining the contribution of each partner
of the symbiosis for DFAA accumulation. Uptake rates in this study will
therefore be expressed in ng N h–1 mg–1
animal tissue and mg–1 zooxanthellae, and in ng N
h–1 cm–2. Skeletal surface area of the
microcolonies was measured according to the wax technique
(Stimson and Kinzie,
1991).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Uptake rates of the 11 amino acids incubated separately with microcolonies of S. pistillata are presented in Fig. 1. Mean rates varied between 4.08 and 11.28 nmol h–1 cm–2 and were minimal and maximal for glutamate and histidine, respectively. There was no significant correlation between uptake rates and DFAA characteristics (hydrophobicity, acidity heteroatom or chemical functions). Comparable results were obtained when data were normalized to protein concentration. In this case, uptake rates varied between 11.36 and 31.42 nmol DFAA h–1 mg–1 protein.
Kinetics experiments showed that 15N-DFAA uptake was linear in animal tissue and in zooxanthellae for up to 21 h incubation at concentrations of both 0.5 µmol l–1 (Fig. 2A) and 3 µmol l–1 (Fig. 2B). At both concentrations, DFAA uptake rates, normalized to surface area, were twice as high in the animal tissue than in the zooxanthellae. Indeed, at 0.5 µmol l–1 DFAA, representing an in situ concentration, uptake rates were equal to 4.3 and 1.8 nmol N h–1 cm–2 for animal tissue and zooxanthellae, respectively. At 3 µmol l–1 DFAA, these rates were twice as high, with 9.8 and 5.3 nmol N h–1 cm–2 in animal tissue and zooxanthellae, respectively. Normalized to biomass, DFAA appeared to be 5–7 times more concentrated in the zooxanthellae than in the animal tissue. At 0.5 µmol l–1 DFAA, uptake rates ranged from 0.5 nmol N h–1 mg–1 tissue to 2.4 nmol N h–1 mg–1 zooxanthellae. At 3 µmol l–1 DFAA, uptake rates were equal to 1.0 nmol N h–1 mg–1 tissue and 7.0 nmol N h–1 mg–1 zooxanthellae.
|
 | (2) |
max is the maximal DFAA uptake rate, [DFAA] is the
concentration, and Km the solute concentration at which
DFAA uptake is half-maximal.
For concentrations above 3 µmol l–1, the carriers of
the active transport were saturated, and a passive DFAA diffusion process
through animal membranes became apparent, according to the equation:
 | (3) |
Therefore, over the entire range of concentrations tested, DFAA uptake
through the animal tissue followed a combination of Michaelis–Menten and
linear kinetics, which can be pooled in the following unique equation:
 | (4) |
).
Note that the active transport is dominant up to approximately 16 µmol
l–1, the diffusive transport becoming dominant above this
concentration (when the linear curve crosses the Michaelis–Menten
curve). max and Km were determined using
the fitting software pro Fit 6.0.6 (Quantum Soft). The calculated values are
max=7.52 nmol N h–1 cm–2 and
Km=1.23 µmol l–1 DFAA. When normalized
to animal tissue dry mass, these values are max=0.62 nmol N
h–1 mg–1 tissue and
Km=0.63 µmol l–1 DFAA.
|
%15N enrichment in freshly isolated zooxanthellae (FIZ) versus DFAA concentration in the incubation medium is presented in Fig. 6. %15N in FIZ linearly increased with increasing DFAA concentration. Fig. 6 can be compared with the %15N enrichment of zooxanthellae in hospite (Fig. 7), where it apparently reached a maximum at 3 µmol l–1 DFAA. Fig. 7 is derived from the data in Fig. 3 (same trend), but represents the raw data of %15N enrichment in zooxanthellae in hospite.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hoegh-Guldberg and Williamson,
1999), the 15N technique exhibits some advantages, such
as: (i) it allows the amount of DFAA taken up by the animal to be
distinguished from that going into the zooxanthellae; (ii) kinetics can be
performed at low DFAA concentrations; and (iii) more specific details can be
obtained concerning both DFAA transport mechanisms through membranes and
nitrogen allocation.
When data were normalized to skeletal surface area, animal tissue presented
the highest uptake rate, as already observed for urea, which is another DON
source for corals (Grover et al.,
2006), in contrast to ammonium and nitrate, which were mainly
taken up by the zooxanthellae. If biomass is taken into consideration, all
nitrogen sources, including inorganic and organic, accumulated in the
zooxanthellae, suggesting that they are the main nitrogen users.
15N enrichment rapidly occurred in the zooxanthellae, since they
were labeled in less than 2 h. If we consider that external DFAA has to
migrate through the animal epithelial layers to reach the zooxanthellae, we
can then suppose that DFAA uptake occurred, partly or entirely, from the
seawater that fills the cœlenteric cavity. Considering that symbiotic
zooxanthellae live within the endodermal cells that constitute the
cœlenteron, this could explain the fast 15N enrichment in the
symbionts.
Usually, animals show preferences and a higher affinity for the eight
essential DFAAs (valine, leucine, isoleucine, tyrosine, phenylalanine,
histidine, methionine, lysine), since they are not capable of synthesizing
these amino acids. However, corals are not pure animals, due to the presence
of zooxanthellae in their tissue, and their capacity for DFAA synthesis
remains controversial (Fitzgerald and
Szmant, 1997; Wang and
Douglas, 1999). In our depletion experiments, all amino acids were
taken up, but with a significantly higher uptake rate for histidine compared
to the other amino acids (except asparagine). Such preferential uptake for
some amino acids was also evident in the work of Schlichter
(Schlichter, 1978) and Ferrier
(Ferrier, 1991) for the coral
species Montastrea annularis. However, it was not observed for three
other coral species [Madracis Mirabilis, Agaricia fragilis and
Favia fragum (Ferrier,
1991; Hoegh-Guldberg and
Williamson, 1999)]. These differences could either be due to
reciprocal inhibitory effects between DFAAs, or to species-specific
differences.
|
) for several scleractinian species (3–19 nmol N
cm–2 h–1), and for Pocillopora
damicornis (4.9–9.8 nmol N cm–2
h–1) (Hoegh-Guldberg and
Williamson, 1999). These two studies used equivalent DFAA
concentrations in the external medium (4–5.6 µmol
l–1). Schlichter and colleagues
(Schlichter, 1980;
Schlichter et al., 1986) were
the first to suggest that the absorption of amino acids through the membranes
of the epidermal cells of anthozoans takes place against gradients of up to
1:106 and requires a carrier-mediated trans-epithelial transport.
Our observations confirm this hypothesis, since DFAA uptake in S.
pistillata followed a bimodal process with a major contribution of the
carrier-mediated transport at DFAA concentrations lower than16 µmol
l–1. Such bimodal transport has been emphasized
(Al-Moghrabi et al., 1993) for
valine uptake by Galaxea fascicularis, in the light, which also
showed an active transport at concentrations lower than 20 µmol
l–1. The value of the carrier affinity
(Km=1.23 µmol l–1) found in this work
for the DFAA mixture highlights how well corals are adapted to the low levels
usually found in seawater. Uptake of amino acids by freshly isolated
zooxanthellae (Fig. 6) followed
a passive diffusion process, since no saturation in the uptake occurred even
when amino acids concentrations were as high as 13 µmol
l–1. This result suggests that the saturation curve obtained
with in hospite zooxanthellae
(Fig. 3) is due to a
limitation, by the animal tissue, of amino acid transport (carrier-mediated
transport observed in Fig.
4).
The effect of light on DFAA uptake is complex. Indeed, light enhanced DFFA
uptake, but only at concentrations above 0.5 µmol l–1,
suggesting that a minimal concentration is required to activate this process.
Moreover, this enhancement was not proportional to light intensity since it
was saturated at 160 µmol photons m–2
s–1. Finally, the same phenomenon was observed both in the
zooxanthellae and the animal tissue (Fig.
5). These observations strongly suggest that zooxanthellae are
actively involved in DFAA uptake, but their contribution should necessarily be
indirect and linked to their photosynthetic activity. It is indeed known that
the transfer of photosynthates from the zooxanthellae to the animal is a
source of energy, enhancing the animal metabolism and the incorporation of
nitrogen into proteins. This mechanism has been called the `light-enhanced
amino acid assimilation' (Al-Moghrabi et
al., 1993). In contrast to our observations and those of
Al-Moghrabi et al. (Al-Moghrabi et al.,
1993), a faster DFAA uptake in the dark was observed for the
species Pocillopora damicornis
(Hoegh-Guldberg and Williamson,
1999). This inconsistency could be due to species specificity, but
remains to be further investigated.
Taking into account results obtained in previous studies concerning the
uptake of dissolved inorganic and organic nitrogen by S. pistillata
(Grover et al., 2002;
Grover et al., 2003;
Grover et al., 2006), a simple
model can be designed to evaluate the importance of DFAA compared to the other
nitrogen sources for animal tissue growth. For this purpose, we considered the
daily uptake rates of each nitrogen source measured for the whole coral colony
after 12 h incubation in the light (Fig.
8). These daily rates were compared to the daily nitrogen
requirements for animal tissue growth. This latter value is based on the daily
coral tissue growth and the mass of nitrogen per mg of coral tissue measured
in our samples (55±9 ng N mg–1 tissue). Since no
measurement of tissue growth was available for S. pistillata, we
calculated it by multiplying the tissue mass by the specific growth rate
ua expressed as day–1. This specific
growth rate ua was calculated according to the empirical
formula given by Muscatine et al.
(Muscatine et al., 1985),
depending on the surface area of each sample. Finally, the % contribution of
each nitrogen source to tissue growth was calculated by dividing the daily
nitrogen uptake rate by the daily nitrogen requirement for tissue growth
(Fig. 8). These calculations do
not take into account nitrogen excretion by the coral, which is negligible for
Stylophora pistillata (Rahav et
al., 1989). The model suggests that total dissolved nitrogen
provides 99% of the daily nitrogen necessary for tissue growth. Organic
sources (urea+DFAA) only provide 24% of the tissue requirements, with a major
contribution (21%) of DFAA. Inorganic nitrogen sources
(NH4++NO3–) therefore
account for 75% of the tissue needs. Since these inorganic sources are mainly
taken up by the zooxanthellae (Grover et
al., 2002; Grover et al.,
2003), most of the dissolved nitrogen uptake is due to
zooxanthellae activity.
In summary, our results show that DFAA can represent an important source of
nitrogen for corals at in situ concentrations (200–500 nmol
l–1), with uptake rates as high as those measured for DIN at
the same concentrations. DFAA uptake by Stylophora pistillata shows
no discrimination, allowing the uptake of any available amino acid through the
animal membranes, depending on the DFAA concentration in the surrounding
water. A `light-enhanced amino acid assimilation' process
(Al-Moghrabi et al., 1993) has
been confirmed, suggesting DFAA uptake is a diurnal event.
|
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