Download nitrogen recycling or nitrogen conservation in an alga–invertebrate

2445
The Journal of Experimental Biology 201, 2445–2453 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JEB1546
NITROGEN RECYCLING OR NITROGEN CONSERVATION IN AN
ALGA–INVERTEBRATE SYMBIOSIS?
J.-T. WANG AND A. E. DOUGLAS*
Department of Biology, University of York, PO Box 373, York, YO1 5YW, UK
*Author for correspondence (e-mail: [email protected])
Accepted 1 June; published on WWW 27 July 1998
Summary
When corals and allied animals are deprived of their
and the concentration of protein amino acids in the free
symbiotic algae, the ammonium content in their tissues
amino acid pool of the animal, which were depressed by
rises. This is commonly interpreted as evidence for
darkness and algal depletion, were restored by exogenous
nitrogen recycling (i.e. algal assimilation of animal waste
carbon compounds. It is concluded that organic carbon,
whether derived from algal photosynthate or exogenously,
ammonium into amino acids that are released back to the
promotes the animal’s capacity for ammonium assimilation
animal), but it can also be explained as nitrogen
conservation by the animal (i.e. reduced net ammonium
and reduces ammonium production from amino acid
production in response to the receipt of algal
degradation. These processes contribute to nitrogen
conservation in the animal, but they confound the
photosynthetic carbon). This study discriminated between
interpretation of various studies on nitrogen recycling by
these interpretations in two ways. First, the increased
symbiotic algae.
ammonium concentration in the sea anemone Aiptasia
pulchella, caused by darkness or depletion of the alga
Symbiodinium, was partially or completely reversed by
Key words: Aiptasia pulchella, Symbiodinium, zooxanthella,
supplementing the medium with organic carbon
symbiosis, nitrogen recycling, amino acid, glutamine synthetase,
compounds (e.g. α-ketoglutarate). Second, the activity of
ammonium.
the ammonium-assimilating enzyme glutamine synthetase
Introduction
Many members of the phylum Cnidaria (corals, sea
anemones, jellyfish, hydrozoans, etc.) bear symbiotic algae,
usually dinoflagellates of the genus Symbiodinium
(zooxanthellae) in marine groups (corals, sea anemones, etc.)
and Chlorella in the freshwater hydras (Trench, 1993; Douglas,
1994). For the marine symbioses, Symbiodinium species have
been implicated both in the provision of photosynthesisderived carbon and in nitrogen recycling, and these processes
are widely considered to underpin the ecological success of
Cnidaria–alga symbioses in shallow, low-nutrient waters
(Muscatine and Porter, 1977; Falkowski et al. 1993).
The biochemical basis of photosynthate release by
Symbiodinium is well established. Many radiotracer studies have
revealed high rates of photosynthate release from intact
Symbiodinium cells, principally as glycerol and organic acids
(e.g. Muscatine and Cernichiari, 1969; Trench, 1971b), and
carbon budget analyses indicate that alga-derived carbon
contributes much or all of the animal’s carbon requirements for
basal respiration (Muscatine, 1990). The evidence for nitrogen
recycling by Symbiodinium is, however, far from conclusive.
Nitrogen recycling is biochemically complex, involving the
bidirectional translocation of nutrients: first, the transfer of the
animal’s waste nitrogen compounds (mostly ammonium in
Cnidaria; see Shick, 199l) to the algal cells, which assimilate the
nitrogen into compounds of nutritional value to the animal; and,
second, the translocation of these latter compounds back to the
animal (Lewis and Smith, 1971; Douglas, 1994) (Fig. 1A).
To date, there is only partial evidence for nitrogen recycling.
Cnidaria are known to generate ammonium at high rates,
principally by deamination of amino acids used as respiratory
substrates (Rees and Ellard, 1989; Shick, 1991), and
Symbiodinium can assimilate ammonium (e.g. Domotor and
D’Elia, 1984; Wilkerson and Muscatine, 1984; Trench, 1993;
McAuley and Smith, 1995). There are, however, no
quantitatively reliable estimates either of the flux of ammonium
derived from animal catabolism to algal cells in the symbiosis
or of the translocation of organic nitrogen compounds from the
algae to the animal. The photosynthetic products released by
the algae are predominantly non-nitrogenous compounds, but
small amounts of amino acids, especially alanine, have been
reported in some studies of photosynthate release by
Symbiodium (e.g. Trench, 1971b; Lewis and Smith, 1971;
Sutton and Hoegh-Guldberg, 1990), and glycoconjugates
exuded from Symbiodinium could also contribute to the
nitrogen nutrition of the animal (Markell and Trench, 1993).
The basis of this study is the controversy that has arisen over
2446 J.-T. WANG AND A. E. DOUGLAS
A
Fig. 1. Current hypotheses of nitrogen relationships in
alga–invertebrate symbioses. (A) Algal recycling of
animal-derived nitrogenous waste compounds, e.g.
ammonium, to organic nitrogen compounds, e.g.
essential amino acids, which are translocated to the
animal cells. (B) Processes proposed to influence the
ammonium content of the animal tissues and
ammonium efflux from animals: (i) algal assimilation
of animal-derived ammonium, as predicted by
nitrogen recycling; (ii) promotion (+) of ammonium
assimilation by the animal (in response to an
undefined property of the algal cells) resulting in
nitrogen limitation of algal proliferation, as predicted
by the nitrogen limitation hypothesis; (iii) depression
(−) of ammonium production by amino acid
deamination in the animal through the preferential
utilisation of alga-derived photosynthetic compounds
as respiratory substrates, as predicted by the nitrogen
conservation hypothesis; and (iv) promotion (+) of
ammonium assimilation in the animal by alga-derived
photosynthate (although not specified in the nitrogen
conservation hypothesis of Rees, 1986, this process
would tend to conserve the organic nitrogen pools of
the animal).
Waste nitrogenous
compounds
Animal
Nitrogenous compounds
of nutritional value to
the animal
Alga
B
(i)
Waste ammonium in
animal
(ii)
(−)
Ammonium
production in
animal, e.g. by
amino acid
deamination
experimental results commonly interpreted as indirect
evidence for nitrogen recycling. When Cnidaria are incubated
in darkness or treated to eliminate their symbiotic algae, they
generally exhibit increased rates of ammonium efflux into the
medium. This has been demonstrated for corals and sea
anemones (e.g. Kawaguti, 1953; Cates and McLaughlin, 1976;
Szmant-Froelich and Pilson, 1977; Wilkerson and Muscatine,
1984) and also for the freshwater hydra–Chlorella symbiosis
(Rees, 1986). These data are commonly interpreted as evidence
that the algae are a major sink for animal-derived ammonium
(Fig. 1Bi), as predicted by the nitrogen recycling hypothesis
(Fig. 1A). However, two further processes could contribute to,
or even account for, these observations: alga-stimulated
assimilation of ammonium by the animal (Fig. 1Bii,iv) and
alga-induced reduction in ammonium production by the animal
(Fig. 1Biii). These putative processes are predicted by the
hypotheses of nitrogen conservation by the animal and
nitrogen-limitation of the algal population (see legend to
Fig. 1B).
The nitrogen conservation hypothesis was first proposed for
the freshwater hydra–Chlorella symbiosis by Rees (1986) and
Rees and Ellard (1989), but it is equally applicable to algal
symbioses in marine Cnidaria. These authors specifically
proposed that the utilisation of amino acids as respiratory
substrates by the animal is reduced by the receipt, from the
algal cells, of photosynthetic carbon compounds which are
used preferentially in animal respiration (Fig. 1Biii). The
resultant conservation of nitrogenous compounds in the animal
tissues would promote the persistence of these symbioses in
low-nitrogen environments.
(iii)
Algal cells
Alga-derived
organic carbon
compounds
transferred to
animal
Algal assimilation of
animal ammonium
(+)
(iv)
(+)
Animal assimilation
of ammonium, e.g.
via glutamine
synthetase
The chief evidence for nitrogen limitation of the symbiotic
algae (Cook and D’Elia, 1987) is that the algal proliferation
rates and population size are increased by supplementing the
medium with ammonium (e.g. Hoegh-Guldberg and Smith,
1989; Falkowski et al. 1993; Muller-Parker et al. 1994). Rees
(1986) has argued that the availability of ammonium to the
algal cells is restricted by the activity of an ammoniumassimilating enzyme, glutamine synthetase, in the animal
tissues. In the hydra experimentally deprived of their algae, the
activity of animal glutamine synthetase is depressed, raising
the possibility that this enzyme may be partly, or completely,
responsible for the elevated ammonium efflux in these animals.
The impact of eliminating the algal population on the
glutamine synthetase activity in the animal tissues of marine
Cnidaria has, however, not been studied.
The hypotheses of nitrogen conservation and nitrogen
limitation are broadly compatible with each other (see
discussion of Rees, 1986), but neither is readily compatible
with high rates of ammonium assimilation by the algae, as
required by the nitrogen recycling hypothesis.
An expectation of the nitrogen conservation hypothesis, but
not of the nitrogen recycling hypothesis, is that exogenous
organic carbon compounds and photosynthetic algae should
have comparable effects on the nitrogen metabolism of
symbiotic Cnidaria. Specifically, the nitrogen conservation
hypothesis predicts that elimination of the algae or their
photosynthetic activity should have two linked effects on the
animal tissues: to depress concentrations of free amino acids
(because of their utilisation as respiratory substrates) and to
elevate the ammonium concentration (the product of amino
Nitrogen metabolism in an alga–invertebrate symbiosis 2447
acid degradation). These effects would be reversed by
exogenous organic carbon compounds. The nitrogen
conservation hypothesis also predicts that treatments that result
in decreased free amino acid concentrations and elevated
ammonium concentration should also depress the activity of
enzyme(s) that assimilate ammonium and/or increase the
activity of ammonium-producing enzymes. In animals,
glutamine synthetase is an important enzyme of ammonium
assimilation, and transamination reactions are the major route
for removing amino groups from amino acids (Lehninger,
1970; Shick, 1991). The study described here was designed to
test these predictions of the nitrogen conservation hypothesis.
Materials and methods
Maintenance of Aiptasia pulchella
The study was conducted on a clonal culture of Aiptasia
pulchella bearing Symbiodinium sp. of clade-b, sensu Rowan
and Powers (1991) (A. E. Douglas, unpublished results). The
culture was derived from an animal collected from a seawater
dike at Tongkang (22 °N), Taiwan, and was maintained in
aerated artificial sea water (Instant Ocean Salts) of salinity
35 ‰ and pH 8.2 at 25 °C under 12 h:12 h light:dark regime at
50 µmol m−2 s−1 photosynthetically active radiation (P.A.R.)
The animals were fed twice a week with 1-day-old Artemia
nauplii. The animals used for experiments had an oral disc
5–7 mm in diameter and their tentacles bore 6×106 to
7×106 algal cells mg−1 protein. They are termed the ‘control’
animals.
The algal population in A. pulchella was depleted by cold
shock, following the procedure of Steen and Muscatine (1987).
Symbiotic animals were maintained at 4 °C for 4 h, and then
returned to the culture at 25 °C in continuous darkness, apart
from the limited periods required for feeding and maintenance.
Over 1 month, the population of algal cells in the tentacles of
the animals declined to 1000 cells mg−1 animal protein, but no
further decrease was consistently obtained over 6 months.
These animals are termed ‘alga-depleted’, and they were used
for experiments between 1 and 3 months after the cold-shock
treatment. Unless stated otherwise, the alga-depleted animals
were maintained in continuous darkness during experiments.
For studies on the impact of organic carbon compounds on
A. pulchella, animals were maintained in 250 ml of artificial
sea water, supplemented with 10 mmol l−1 (final concentration)
fumarate, glucose, glycerol, α-ketoglutarate or succinate
(Sigma Chemical Co.). The pH of media with organic acid
supplements was adjusted to pH 8.2 with KOH. The cultures
were maintained in darkness, and the medium was changed
daily.
Biochemical assays on the animal fraction
Freshly excised tentacles of A. pulchella were used for
biochemical analysis because the density of algae was high and
uniform in these tissues of the control animals (J.-T. Wang and
A. E. Douglas, unpublished results). For all assays, each
sample consisted of 30 tentacles, cut from A. pulchella with
sharp scissors and homogenised in 0.5 ml of ice-cold 3.5 %
NaCl with a hand-held glass tissue grinder. Each homogenate
was centrifuged at 20 000 g at 4 °C for 5 min, and the
supernatant was decanted and retained. The supernatant had no
algal contamination, as indicated by the absence of intact algal
cells by light microscopy and undetectable chlorophyll content,
and it was therefore used as the animal fraction.
The ammonium content of the animal fraction was assayed
spectrophotometrically, using the ammonia diagnostic kit
(Technical Bulletin 171-UV) of Sigma Chemical Co., in
which the amination of α-ketoglutarate is quantified by the
oxidation of NADPH and associated reduction in absorbance
at 340 nm.
Prior to amino acid analysis, the protein in the animal
fraction was precipitated by adding 230 µl of absolute ethanol
to each 100 µl sample, followed by centrifugation at 20 000 g
for 3 min at 4 °C. The supernatant was stored at −20 °C until
analysis. The amino acids (100–300 µmol l−1) were derivatized
with 1 mmol l−1 o-phthaldialdehyde for 1 min (Jones et al.
198l) and quantified by high-performance liquid
chromatography (HPLC) using a Beckman System Gold
delivery system with C18-ultrasphere column and Shimadzu
RF-551 fluorescence detector. This procedure detects all
protein amino acids, except cysteine and proline, and many
non-protein amino acids. The reference amino acid mixture
was AA-S-18 (Sigma), supplemented with γ-aminobutyric
acid, asparagine, glutamine, ornithine, taurine and tryptophan.
Glutamine synthetase activity was quantified by the
transferase assay (Pahel et al. 1982), in which the synthesis of
γ-glutamyl hydroxamate from glutamine is determined
spectrophotometrically at 540 nm. Each sample of animal
fraction was combined with an equal volume of imidazole
buffer (pH 7.4) containing 40 mmol l−1 imidazole–HCl,
0.6 mmol l−1 MnCl2,, 0.5 mol l−1 sucrose, 20 mmol l−1
dithiothreitol and 200 µg ml−1 cetyltrimethylammonium
bromide. Half of the combined sample was incubated at 100 °C
for 15 min, for use as a sample blank, against which the activity
of the other half of the sample was quantified. The specificity
of the reaction was confirmed by the 90 % reduction in activity
in samples pre-incubated with 5 mmol l−1 L-methionine
sulphoximine, a specific inhibitor of glutamine synthetase.
Enzyme activity is expressed as nmol product formed mg−1
animal protein min−1 at 30 °C.
The activities of aspartate aminotransferase and alanine
aminotransferase in the animal fraction were quantified using
the transaminase kit (procedure 505) of Sigma Chemical Co.,
in which the keto-acid product is reacted with 2,4-dinitrophenylhydrazine and determined spectrophotometrically at
500 nm. The specificities of the reactions for aspartate
aminotransferase and alanine aminotransferase were confirmed
by reductions in activity by 96 % and 97 %, respectively, when
the substrates (aspartate and alanine) were omitted from the
reaction mixture. The enzyme activities are expressed as
nmol glutamate formed mg−1 animal protein min−1 at 25 °C.
The protein content of the animal fraction was determined
using the protein assay kit of Bio-Rad Chemical Co., according
2448 J.-T. WANG AND A. E. DOUGLAS
to manufacturer’s instructions, with bovine serum albumin as
standard.
Glutamine synthetase
activity (nmol mg−1 min−1)
12 h:12 h L:D
A
75
50
25
0
40
35
30
25
20
15
150
Alanine aminotransferase
activity (nmol mg−1 min−1)
Results
The animal fraction of Aiptasia pulchella
The first experiments explored the ammonium and free
amino acid content of the animal fraction in control animals,
i.e. A. pulchella containing the ‘normal’ population of
symbiotic algae, and in alga-depleted animals, whose algal
population had been experimentally depressed 6000- to 7000fold (see Materials and methods). The concentration of
ammonium was significantly elevated in the alga-depleted
animals, but the total free amino acid content did not differ
significantly between the two treatments (Table 1A). The
dominant amino acid in all the samples was taurine, a nonprotein amino acid which accounted for 57±2 % and 79±1.4 %
(mean ± S.E.M., N=12) of the amino acids in the control and
alga-depleted animals, respectively. All the protein amino
acids were detected in the samples (apart from cysteine and
proline, which cannot be quantified by the method used), and
the animals also contained low concentrations of the nonprotein amino acids ornithine and γ-aminobutyric acid. The
total concentration of protein amino acids was significantly
higher in the free amino acid pool of control animals than in
that of the alga-depleted animals (Table 1A).
The activity of three enzymes in the animal fraction is shown
in Table 1B. The animals with depleted algal population
displayed depressed glutamine synthetase activity, relative to
the control animals, an increase in alanine aminotransferase
activity and no significant change in aspartate aminotransferase
activity.
Separate experiments had demonstrated that, when control
animals are transferred from the 12 h:12 h L:D light regime to
continuous darkness for 6–7 days, the algal population in their
tentacles is reduced by 30 % ((J.-T. Wang and A. E. Douglas,
unpublished results), a value more than 1000 times greater than
the population in the alga-depleted animals. This provided the
basis to explore the contribution of darkness per se and of the
very low algal density to the low glutamine synthetase activity
of the animal fraction in alga-depleted animals. Fig. 2 shows
how the enzyme activity in control animals is influenced by
incubation in darkness for 6 days, followed by a 12 h:12 h L:D
regime (the routine culture conditions for control animals). The
activity of glutamine synthetase and alanine aminotransferase,
100
Aspartate
aminotransferase
activity
(nmol mg−1 min−1)
Statistical procedures
All variables were checked to conform to the assumptions
of normality and homogeneity of variance, as indicated by the
Kolmogorov–Smirnoff one-sample test and Bartlett’s test,
respectively. For percentage variables, the data were arcsinsquare-root-transformed prior to analysis. The parametric tests
used were t-test, least-squares regression and analysis of
variance (ANOVA), with Fisher’s least significant difference
(LSD) method for pairwise comparisons that contributed to
significant ANOVA differences.
24 h D
0
B
3
6
9
12
15
C
125
100
75
50
25
0
2
4
6
8
10
12
14
16
Time (days)
Fig. 2. Impact of light regime on the activity of enzymes in amino
acid metabolism in the animal fraction of control animals of Aiptasia
pulchella incubated in continuous darkness for 6 days and then
(arrow) transferred to a 12 h:12 h L:D regime. Enzyme activities are
expressed as nmol product mg−1 animal protein min−1, and mean
values ± S.D. (six replicates) are shown. (A) Glutamine synthetase
(ANOVA: F10,55=26.60, P<0.0001), (B) aspartate aminotransferase
(F10,55=1.93, P>0.05), (C) alanine aminotransferase (F10,55=20.04,
P<0.001).
but not of aspartate aminotransferase, varied significantly over
the experiment (details of ANOVAs are given in the in legend
to Fig. 2). Subsequent application of Fisher’s LSD test
confirmed that glutamine synthetase activity was significantly
depressed, below the value on day 0, between days 2 and 13
of the experiment, and that alanine aminotransferase activity
was significantly elevated, relative to day 0, on day 2 and
between days 4 and 13 (P<0.05).
The impact of organic carbon compounds on the animal
fraction of Aiptasia pulchella
When A. pulchella was incubated in darkness for 7 days in
medium supplemented with an organic carbon compound, both
Nitrogen metabolism in an alga–invertebrate symbiosis 2449
A
a
125
Control (N =9)
75
b,c
b,c
d
0
125
B
a
Alga-depleted (N =3)
a
100
b
75
b,c
c
c
50
25
a,b
60
Ammonium concentration
(nmol mg−1 protein)
c
Control (N =3)
a
80
b
100
Glutamine synthetase activity
(nmol mg−1 protein min−1)
C
a
40
20
ND ND
0
125
D
a
Alga-depleted ( N =3)
b
100
b
b
75
50
50
c
25
ND
25
ND
ND
N
N
Carbon supplement
(12 h:12 h
Light condition
L:D)
S
F
Gly Glu
ND
ND
ND
0
0
K
N
24 h D
N
S
(12 h:12 h
L:D)
F
Gly Glu
K
24 h D
Fig. 3. Impact of organic carbon supplements on the animal fraction of Aiptasia pulchella. Glutamine synthetase activity in (A) control animals
(ANOVA: F6,56=22.2, P<0.0001) and (B) alga-depleted animals (F3,8=116.94, P<0.0001), and ammonium concentration in (C) control animals
(F4,10=5.50, 0.01<P<0.05) and (D) alga-depleted animals (F3,8=5.64, 0.01<P<0.05). Mean values + S.E.M. are shown (N=number of replicates;
ND, not determined), and values that are not significantly different from each other (Fisher’s least significant difference test, P>0.05) are given
the same superscript. Carbon supplements: N, none; S, succinate; F, fumarate; Gly, glycerol; Glu, glucose; K, α-ketoglutarate.
Table 1. Nitrogenous compounds and enzyme activities in Aiptasia pulchella
A Nitrogenous compounds
Free amino acids
Animals
Control
Alga-depleted
Ammonium
(nmol mg−1 protein)
Total amino acids
(nmol mg−1 protein)
Protein amino acids
(nmol mg−1 protein)
58±7 (14)
149±10 (9)
923±51 (12)
843±48 (12)
392±35 (12)
168±17 (12)
t21=7.53,
P<0.0001
t22=1.16,
P>0.05
t22=4.96,
P<0.001
B Activity of enzymes involved in amino acid metabolism
Animals
Control
Alga-depleted
Glutamine synthetase
(nmol mg−1 min−1)
Alanine aminotransferase
(nmol mg−1 min−1)
Aspartate aminotransferase
(nmol mg−1 min−1)
126±6 (18)
27±2 (14)
44±5 (6)
85±2 (6)
32±2 (6)
31±1 (6)
t30=14.08,
P<0.0001
t10=7.95,
P<0.0001
t10=0.52,
P>0.05
Enzyme activities are measured as nmol product mg−1 animal protein min−1.
The control animals (containing symbiotic algae) were maintained under 12 h:12 h L:D photoperiod and the alga-depleted animals in
continuous darkness.
Values are means ± S.E.M., with the number of replicates in parentheses.
the control and alga-depleted animals exhibited a significant
increase in glutamine synthetase activity (Fig. 3A,B) and a
significant reduction in ammonium concentration (Fig. 3C,D).
The effect varied between the compounds tested. Fumarate, for
example, consistently increased glutamine synthetase to levels
intermediate between those of animals incubated without a
2450 J.-T. WANG AND A. E. DOUGLAS
N=12), respectively, of the total free amino acids; these values
were significantly different (t22=13.02, P<0.0001, for arcsinsquare-root-transformed data). There was a commensurate
change in the taurine content, which accounted for 48 % of the
amino acids in the animals with the carbon supplement and for
78 % in those without the supplement.
[Ammonium] (nmol mg−1 protein)
120
100
80
60
40
20
0
0
30
60
90
120
150
Glutamine synthetase activity
(nmol product mg −1 protein min −1)
Fig. 4. Regression analysis of ammonium concentration on
glutamine synthetase activity in the animal fraction of control
animals (䊏) and alga-depleted animals (䊐) incubated in darkness
for 7 days. The regression equation for the control animals is:
y=−0.22x+78.7 (r=0.58, P<0.05), and that for the alga-depleted
animals is: y =−0.33x+113 (r=0.63, P<0.05), where y is the
ammonium concentration and x is the glutamine synthetase activity.
carbon supplement in continuous darkness and control animals
in the 12 h:12 h L:D regime; and α-ketoglutarate stimulated
glutamine synthetase activity to values characteristic of control
animals in the 12 h:12 h L:D light regime.
In these experiments, the ammonium content of the algadepleted animals was generally higher than that of darkincubated control animals under the same treatment. To
explore this further, the ammonium content of each sample
was regressed on the corresponding value of glutamine
synthetase activity (Fig. 4). The regression equations (see
legend to Fig. 4) for the control and alga-depleted animals
had negative slopes that were not significantly different from
each other (t23=0.53, P>0.05), but the intercept of the
equation for alga-depleted animals was significantly greater
than that for control animals (t23=12.69, P<0.0001). This
result suggests that the control animals have an appreciable
sink for ammonium, additional to glutamine synthetase,
that is absent from alga-depleted animals. As considered in
the Discussion, the additional sink may be the algal
population.
The impact of one organic compound, α-ketoglutarate, on
the free amino acid content of the alga-depleted animals was
examined. After 7 days of incubation with and without the
carbon supplement, the protein amino acid contents of the
animal fraction was 50±1.8 % and 20±1.3 % (mean ± S.E.M.,
The animal fraction of alga-depleted Aiptasia pulchella
incubated under illuminated conditions
When alga-depleted animals were transferred to the
12 h:12 h L:D regime, the residual population of symbiotic
algae increased. Twelve weeks later, it was noted that the
algal population in the tentacles varied between animals over
three orders of magnitude: from animals with
2×103 algal cells mg−1 protein, just double the mean algal
population in the dark-maintained alga-depleted animals, to
animals with 2×106 cells mg−1 protein, nearly one-third of the
algal population in control animals. Although the cellular
basis of this wide variation has yet to be established, it did
provide the opportunity to investigate the impact of algal
density on the glutamine synthetase activity and ammonium
content of the animal fraction.
Consistent with the differences observed between control
and alga-depleted animals (Table 1), the activity of glutamine
synthetase in the animal fraction increased, and the ammonium
concentration decreased, with density of algae. The data were
log-transformed prior to analysis (this increased the correlation
coefficients of both glutamine synthetase activity and
ammonium concentration with algal density). As shown in Fig.
5, with regression equations in the legend, the slopes of the
regressions of log(glutamine synthetase activity) and
log(ammonium concentration) on log(algal density) were
similar in magnitude, but opposite in sign, at +0.114 (S.D.
0.0177) and −0.120 (S.D. 0.0323), respectively. In other words,
for a 10-fold increase in algal density, the animal is predicted
to exhibit a 24–30 % increase in glutamine synthetase activity
and an equivalent decrease in ammonium content.
Discussion
When Aiptasia pulchella is incubated in darkness or its
population of Symbiodinium is subtantially depleted, it exhibits
an increased ammonium concentration in its tissues (this study)
and an increased ammonium efflux into the medium
(Wilkerson and Muscatine, 1984). These differences in
ammonium content and efflux rates would, traditionally, be
attributed to algal assimilation of animal-derived ammonium
and cited as evidence for nitrogen recycling by the algae (see
Introduction and Fig. 1Bi). This explanation is confounded by
the demonstration here that the effects of darkness and of
depletion of the algal population on the ammonium
concentration in the animal tissues are partially or completely
reversed by exogenous carbon compounds (Fig. 3). The most
parsimonious explanation for the results of this study is that
organic carbon, whether derived from algal photosynthate or
exogenous sources, promotes the assimilation of ammonium
2.2
2.2
2.1
2.1
2.0
2.0
1.9
1.9
1.8
1.8
1.7
1.7
1.6
1.6
1.5
1.5
1.4
3.03.0 3.53.5 4.0
4.0 4.5
4.5
5.0
5.0
5.5
6.0
log10(ammonium concentration)
log10(glutamine synthetase activity)
Nitrogen metabolism in an alga–invertebrate symbiosis 2451
1.4
6.5
6.5
log10(algal density)
Fig. 5. Regression analysis of glutamine synthetase activity (䊏) and
ammonium concentration (䊐) of the animal fraction on algal density
(cells mg−1 animal protein), for alga-depleted Aiptasia pulchella
incubated in a 12 h:12 h L:D regime for 12 weeks. The regression
equations are: log(glutamine synthetase activity)=0.114log(algal
density)+1.09 (r=0.90, P<0.05) and log(ammonium concentration)=
−0.120log(algal density)+2.43 (r=0.76, P<0.05).
(Fig. 1Biv) and/or depresses the production of ammonium in
the animal tissues (Fig. 1Biii).
This study has obtained strong evidence for the promotion
of ammonium assimilation in the animal by organic carbon
compounds, principally via glutamine synthetase, an enzyme
that catalyses the ATP-dependent amination of glutamate to
produce glutamine. The treatments that resulted in an
elevated ammonium concentration (darkness, depletion of
the algal population) also depressed glutamine synthetase
activity (Table 1B), and the organic carbon supplements
enhanced glutamine synthetase activity (Fig. 3). Further, the
finding that the regressions of glutamine synthetase activity
and ammonium concentration on algal density had closely
similar slopes of opposite sign (Fig. 5) suggests that
glutamine synthetase may play an important role in
mediating the impact of algal density on the ammonium
content of the animal. It is unlikely, however, that algaderived photosynthate contributes to the substrate pool of
glutamate for glutamine synthetase in the animal tissues,
because the principal fates of algal photosynthate
translocated to the animal are respiration and incorporation
into the lipid fraction, not the amino acid or protein fractions
(e.g. Trench, 1971a; Battey and Patton, 1987; Wang, 1998).
Perhaps the algal photosynthate and exogenous carbon
compounds enhance the respiration rates of the animal, and
the resultant increase in ATP content and energetic status of
the animal tissues may result in increased glutamine
synthetase activity. Consistent with this proposal, both
feeding and the receipt of algal photosynthate are known to
enhance rates of respiratory oxygen consumption in Cnidaria
(Muller-Parker, 1984: Tytler and Davies, 1984; Harland and
Davies, 1995), and a favourable energetic status promotes
glutamine synthetase activity in some algae (e.g. GarciaFernandez et al. 1995). Further research is required to test
this interpretation because, at present, nothing is known
about the regulation of glutamine synthetase activity in
Cnidaria beyond the expectation that, as with all other
eukaryotes, it is not controlled by the complex
adenylylation/deadenylylation system found in most bacteria
(White, 1995).
The reduction in glutamine synthetase activity in Cnidaria
deprived of their algae has been reported previously only in
the freshwater hydra–Chlorella symbiosis (Rees, 1986), and
our data serve to generalise those findings from freshwater
to marine systems and across two classes (Hydrozoa to
Anthozoa). The impact of algae on the glutamine synthetase
activity in hydra was considered exclusively in terms of its
proposed function to reduce the availability of ammonium to
the algal cells (Fig. 1Bii). As described above, data from this
study on A. pulchella indicate that glutamine synthetase is
modulated by alga-derived photosynthate (Fig. 1Biv) and not
by the presence of algae per se. [The reduced glutamine
synthetase activity of hydra bearing Chlorella, which release
relatively little photosynthate (Rees, 1986), suggests that this
interpretation may apply to the hydra association.] The
implication of our data is that the translocation of algal
photosynthate would tend, via increased glutamine
synthetase activity, to depress the availability of ammonium
to the algal cells. If ammonium were the principal, or sole,
nitrogen source utilised by the algae, then nitrogen limitation
could be a direct consequence of the translocation of
photosynthate to the animal tissues. However, the relative
importance of ammonium and organic nitrogen compounds
to the nitrogen nutrition of the algae in symbiosis is
uncertain; Symbiodinium can certainly assimilate both
ammonium (see Introduction) and amino acids (e.g. Carroll
and Blanquet, 1984; Bester et al. 1997) efficiently from low
external concentrations. The data in Fig. 4 provide
circumstantial evidence that the algal cells in symbiosis
utilise animal-derived ammonium. The significantly lower
intercept for the regression of ammonium concentration on
glutamine synthetase activity in control animals than in algadepleted animals suggests that the former assimilate
ammonium by a route, additional to animal glutamine
synthetase, that is absent from the latter. This may be the
algal cells. Symbiodinium is known to be capable of sustained
ammonium assimilation, even in darkness (McAuley and
Smith, 1995, and references cited therein).
In developing the nitrogen conservation hypothesis, Rees
and Ellard (1989) emphasized the importance of algal
photosynthate as an alternative respiratory substrate to the
2452 J.-T. WANG AND A. E. DOUGLAS
amino acid pool of the animal (Fig. 1Biii). This study
provides only qualified evidence for this interpretation. The
expectation that darkness and depletion of the algal
population would enhance animal transaminase activity (see
Introduction) was borne out for one of the two transaminases
examined (Table 1B; Fig. 2B,C), and the expectation that
algal depletion would depress the free amino acid
concentrations was confirmed for the protein amino acids, but
not for total amino acids, in the free amino acid pool of the
animal tissues (Table 1A). This latter result may reflect the
role of free amino acids in the regulation of cell volume in
sea anemones (Shick, 1991), such that the total free amino
acid content of the animal tissues is constrained to vary
within very narrow limits, in medium of a given salinity. As
with some other sea anemone species (e.g. Male and Storey,
1983; Deaton and Hoffmann, 1988; Herrera et al. 1989), the
principal osmolyte in A. pulchella may be taurine, an amino
acid that cannot, generally, be utilised as a respiratory
substrate by animals (Huxtable, 1992). The concentration of
protein amino acids (but not total amino acids) in the animal’s
free amino acid pool is, therefore, the most appropriate test
of the prediction (above) that algal photosynthate may
enhance the free amino acid concentration in the animal
tissues. Consistent with this prediction of the nitrogen
conservation hypothesis, the protein amino acids contribute
42 % and 50 %, respectively, of the total free amino acid pool
in control animals and in alga-depleted animals incubated
with an exogenous carbon compound, but just 20 % of the
total in alga-depleted animals in routine culture.
In conclusion, this study has revealed the crucial
importance of organic carbon to the nitrogen metabolism of
the animal tissues in the Aiptasia pulchella–Symbiodinium
symbiosis. Whether derived from algal photosynthate or
exogenous sources, this organic carbon may both promote
animal assimilation of ammonium via glutamine synthetase
(Fig. 1Biv) and reduce the rates of ammonium production by
providing an alternative respiratory substrate to amino acids
(Fig. 1Biii). The impact of alga-derived photosynthate on
ammonium production and consumption by the animal
confounds the interpretation of experimental designs
traditionally used to explore the role of the algae in nitrogen
recycling. As a result, the relative contributions of nitrogen
conservation and nitrogen recycling to the overall nitrogen
relations of alga–invertebrate symbioses remain unresolved.
The key issue to establish is the relative strength of the
ammonium assimilatory capabilities of algal and animal cells.
This may vary with environmental circumstance and between
different associations, with substantial implications for both
the regulation of algal population increase and the nitrogen
nutrition of the animal.
We thank Dr K. Soong (National Sun Yat-sen University,
Taiwan) for providing us with Aiptaisia pulchella and Dr T.
L. Wilkinson for his constructive comments on the
manuscript. This research was funded by a studentship to
J.T.W. provided by the Ministry of Education, Taiwan.
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