Ammonium excretion by a symbiotic sponge supplies the nitrogen requirements of its rhodophyte partner
1 School of Biological Sciences, A08, University of Sydney, New South Wales
2006, Australia
2 School of Biological Sciences and Biotechnology, Murdoch University,
Murdoch, Western Australia 6150, Australia
* Author for correspondence at present address: Institute of Marine Studies, University of Plymouth, Plymouth PL4 8AA, UK (e-mail: sdavy{at}plymouth.ac.uk)
Accepted 13 August 2002
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Summary |
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Key words: symbiosis, sponge, rhodophyte, Haliclona cymiformis, Ceratodictyon spongiosum, nitrogen flux
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Introduction |
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The HaliclonaCeratodictyon symbiosis is locally common in
shallow (<4 m) tropical reef waters of the Indo-Pacific region
(Trautman et al., 2000);
neither partner is known to occur alone in the field. The profusely branching
macroalga forms a dense network, with the intercellular encrusting sponge
spreading around and between the algal branchlets and covering most of the
alga; only branchlets at the apex of the thallus are free of sponge tissue.
The whole association is heavily branched, dark green in colour and may form
clumps of up to 1 m across (Fromont,
1993
). The close proximity of the autotrophic and heterotrophic
partners in this symbiosis would certainly facilitate nutritional exchanges
but, until now, this has never been investigated.
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Materials and methods |
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Nitrogen flux
To determine whether the association can take up exogenous ammonium and
nitrate, spongealga branches (N=3 or 5), each from a different
clump, were cleaned of debris and placed in glass beakers containing glass
microfibre (Whatman GF/C) filtered sea water (FSW; 200 ml and 600 ml for
ammonium and nitrate experiments, respectively). Controls consisted of FSW
alone (N=3). Ammonium levels were increased to 20 µmol
l-1 by spiking with NH4Cl; existing nitrate levels (2-5
µmol l-1) were sufficient for the experiments. All beakers were
aerated regularly and kept at an irradiance of 1000 µmol photons
m-2 s-1 (using quartz halogen lamps) and at ambient
temperature (mean 23.5°C and 27.5°C in winter and summer,
respectively). Known volumes (5-10 ml) of water were removed from each beaker
at regular intervals, sterilised using a 0.2 µm filter and stored at
-20°C in acid-washed tubes. Samples were analysed within one week of
storage, using the phenolhypochlorite method for ammonium and the
cadmium-reduction method for nitrate; nitrate samples were corrected for
nitrite content (Parsons et al.,
1984). All values were normalised to branch dry mass, which was
determined after drying to constant mass at 60°C. One-way analysis of
variance (ANOVA) was used to determine if ammonium and nitrate concentrations
changed significantly over time.
To determine the rate of ammonium excretion by the sponge and how excretion is affected by algal photosynthesis, the release of ammonium in the dark by branches maintained previously in darkness for 4 h, 8 h or 24 h was measured. This was carried out as above, except that 11 of FSW was used, experiments were performed in darkness, and 40 ml samples were collected after 0 h, 2 h, 4 h and 6 h (N=5 for each time point).
Enrichment with nitrate is known to enhance photosynthesis and respiration
in nitrogen-deficient macroalgae (Littler
et al., 1988; Littler and
Littler, 1992
). To test the physiological effects of enrichment
with nitrate in the laboratory, clumps of the association were collected and
placed in aquaria containing sea water (controls) or sea water plus 10 µmol
l-1 NaNO3 for 24 h (N=10 for each treatment). A
branch (5-10 g wet mass) was then cut from each clump, and dark respiration
and maximal rate of photosynthesis (Pmax) were measured
with a Radiometer oxygen electrode (Copenhagen, Denmark; N=3 for each
branch). Field experiments were performed the same way, except that the clumps
were incubated in sea water or 10 µmol l-1 NaNO3/sea
water at the site of collection in sealed 31 plastic bags.
Pmax and respiratory rate were then measured in the
laboratory. Photosynthesis and respiration by freshly collected branches,
which had not been enclosed in plastic bags, were also measured.
Biomass ratios
The ratio of sponge to algal biomass was estimated by cutting sections
(approximately 1 cm long) from the tips of branches and from 8-10 cm below the
tip (N=20 for each branch position, with each branch coming from a
different clump). The sections were blotted dry and cut in half
longitudinally, and each piece was wet weighed. One piece from each section
was then stripped of sponge material by teasing apart, shaking in a 1:5 sodium
hypochlorite:water solution and rinsing in water. The whole and `cleaned'
pieces were dried to constant mass at 60°C. The proportion of the
association consisting of each partner was calculated from the dried masses of
the whole and `cleaned' pieces, adjusted for their original wet masses.
Stable isotope analysis (15N)
For stable isotope analysis, branches from four different clumps were
squeezed in FSW to liberate sponge cells. The resultant cell suspension was
centrifuged (1000 g for 15 min), the supernatant was decanted, and
the cells were resuspended in 20% FSW and pelleted again. Algal samples were
isolated from 10 clumps by teasing apart and shaking in several changes of 10%
FSW (which lysed and removed the sponge cells). Sponge and algal samples were
dried to constant mass at 60°C, ground to a fine powder and analysed by
mass spectrometry (analysis performed by CSIRO Division of Land and Water,
Adelaide, Australia). This gave values for percentage nitrogen and
15N (in
).
Nitrogen sufficiency
The nitrogen status of the alga was assessed by measuring the extent to
which ammonium enhanced the rate of dark carbon fixation (Cook et al.,
1992,
1994
). Within 24 h of
collection, branches (N=3) were teased apart to obtain clean samples
of alga. Pieces of alga were then blotted dry, and 5-6 mg of each algal sample
were weighed into a 5 ml vial containing either 0.6 ml FSW or 0.6 ml 20
µmol l-1 NH4Cl in FSW (N=2 for each
condition). Three `background' vials contained alga and 0.6 ml 10% formalin in
FSW. NaH14CO3 stock (30 µl containing 18.5 kBq) was
then added to each vial in a dark room. The formalin vials were immediately
sampled (50 µl) for added radioactivity, and all the vials were then sealed
and left in darkness at 22°C. After 2 h, the vials were acidified with 1
mol l-1 HCl and dried using a heating block. Distilled water (0.6
ml) and scintillation fluid (5 ml) were then added, and radioactivity was
measured by liquid scintillation counting. The ammonium enhancement ratio was
calculated (Cook et al., 1992
)
following correction for background activity and normalization of dark
carbon-fixation rates to algal weight.
The nitrogen status was also measured after the supply of nitrogen had been varied. This could only be done with the cultured alga, as the whole association could not be kept alive long enough for use in such experiments. Cultured alga was maintained for 3 weeks in medium containing 100 µmol l-1 NH4Cl; the medium was replaced every 2 days. Cultures were then switched to nitrogen-free medium for up to 6 weeks, and their ammonium enhancement ratio was measured (N=2 for each of three separate cultures) at a series of time points. Three further cultures were kept continuously in 100 µmol l-1 NH4Cl and analysed, as above, at the conclusion of the experiment.
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Results |
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While the HaliclonaCeratodictyon symbiosis could take up DIN from sea water, elevated levels of nitrate did not significantly enhance Pmax or respiratory rates (one-way ANOVA, P>0.1, N=10 in all experiments; Fig. 2).
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Excretion of DIN by the sponge and its potential importance to the
alga
Given the relatively low levels of DIN in the sea water and the failure of
added DIN to enhance algal metabolism in the intact association significantly,
waste material from the feeding activity of the sponge (i.e. excretory
ammonium) may be an important nitrogen source for the algal partner. When we
prevented algal photosynthesis by pre-incubating the association in darkness
for 24 h, the sponge excreted a significant amount of ammonium to the external
medium during a further 6 h in the dark (one-way ANOVA, P<0.0001,
N=5; Fig. 3). This
release translated to approximately 4.6 µg ammonium-N g-1 dry
mass h-1 (0.110 mg ammonium-N g-1 dry mass
day-1). By contrast, branches that had been pre-incubated for 8
h in the dark released little ammonium (one-way ANOVA, P>0.05,
N=5), suggesting that ammonium assimilation can, potentially,
continue throughout the night.
|
To assess the potential importance of waste ammonium to the alga, it is
necessary to consider the nitrogen required for algal growth. The whole
spongealga association grows at a rate of 0.83% day-1 in the
field (Trautman et al., 2000)
and, on a dry mass basis, the alga comprises 70±7.5% of the symbiosis
and contains 1.9±0.1% nitrogen (compared with 6.7±0.2% nitrogen
for the sponge). Therefore, assuming that the growth rate of the whole
association is representative of the growth rates of the symbiotic partners,
we estimate that 0.108 mg N g-1 association dry mass
day-1 is used to support algal growth. Interestingly, this rate
almost exactly matches the maximum measured rate of ammonium-N release from
the sponge (Fig. 4).
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The supply of DIN by the sponge raises the question of how this influences
the nitrogen status of the alga. The enhancement of dark carbon fixation in
the presence of ammonium is inversely related to nitrogen limitation
(Cook et al., 1992). In the
case of freshly harvested C. spongiosum, the enhancement ratio (the
ratio of dark carbon fixation in sea water plus ammonium to dark carbon
fixation in sea water alone) was approximately 1.4. Similar values were
obtained when cultured C. spongiosum was grown for up to 9 weeks in
medium containing 100 µmol l-1 ammonium (one-way ANOVA,
P>0.05, N=3). However, enhancement ratios as high as 2.6
were observed when cultured C. spongiosum was grown in nitrogen-free
medium over several weeks (Fig.
5).
|
Supply of nitrogen to the sponge
From Fig. 4, it can be seen
that <2% of waste nitrogen is surplus to algal growth requirements. This
surplus may be recycled back to the sponge as amino acids (A. Grant, personal
communication), although the possible amount recycled is very small. We
therefore propose that almost all of the nitrogen demands of the association
must be met from external sources. Even though the sponge contributes only
30±7.5% of the biomass of the association, its higher nitrogen content
(6.7±0.2% versus 1.9±0.1% on a dry mass basis) means
that it uses more nitrogen for growth (0.167 mg N g-1 association
dry mass day-1) than the alga (0.108 mg N g-1
association dry mass day-1). Therefore, to meet the nitrogen
demands of both the alga and the sponge, at least 0.275 mg N g-1
dry mass must be supplied to the symbiosis from external sources each day
(Fig. 4). If, as in
cnidariaalga symbioses (Crossland et
al., 1980), DON is secreted into the water as mucus, more nitrogen
will be required.
Additional evidence for the source of nitrogen comes from the stable
isotope composition (15N), which has been used to elucidate
nutritional sources in a variety of marine symbioses (e.g.
Muscatine and Kaplan, 1994
;
Kline and Lewin, 1999
).
15N was +2.23±0.18
in freshly collected
C. spongiosum and +4.88±0.28
in its sponge partner,
H. cymiformis.
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Discussion |
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Sponges are highly efficient filter-feeders
(Reiswig, 1971;
Pile et al., 1996
), and H.
cymiformis is known to filter bacteria and picoplankton from the water
column in One Tree Lagoon (A. Grant, personal communication). Indeed, feeding
upon particulate organic matter, which tends to be enriched in 15N,
no doubt contributes to the relatively high
15N value of the
sponge. At present, the
15N value of the sponge's prey is
unknown, as is the
15N value of any substrates that may be
translocated from the algal partner, so our data must be interpreted with
caution. However, the sponge value of +4.88
is very similar to the
value of +4.74
reported for the non-symbiotic coral Tubastrea
coccinea and is higher than the values of +2.79-4.11
reported for
a range of shallow-water symbiotic corals
(Muscatine and Kaplan, 1994
).
These corals are known to receive small quantities of nitrogenous compounds
from their zooxanthellae (Muscatine and Cernichiari, 1969;
Markell and Trench, 1993
).
Clearly, the feeding efficiency of sponges means that the acquisition of
PON is not a problem, even in relatively unproductive reef waters, and that a
sponge is an ideal partner for an alga in a nutrient-poor habitat. The
15N value measured for C. spongiosum
(+2.23
) was within the range of +0.95-3.54
reported for
zooxanthellae from a variety of shallow-water coral species
(Muscatine and Kaplan, 1994
).
It was also similar to values of +2.2-3.1
reported for the
non-symbiotic, tropical rhodophyte Halymenia dilatata
(Kline and Lewin, 1999
).
Therefore, our data are consistent with the alga acquiring its nitrogen as DIN
from sea water and/or the sponge.
The uptake of excretory nitrogen presumably enhances the nutritional status
of C. spongiosum. Indeed, this uptake may explain why exogenous
nitrate did not increase the Pmax and the respiratory rate
of the association (Fig. 2) and
why dark carbon fixation by the alga only increased slightly in the presence
of ammonium (Fig. 5). In
comparison, when nitrate is supplied to free-living macroalgae on coral reefs,
it generally elevates Pmax and, sometimes, the respiratory
rate (Littler et al., 1988;
Littler and Littler, 1992
).
For example, when nitrate was supplied to various species of the green algal
genus Halimeda, Pmax was elevated by up to 1.7 times
(Littler et al., 1988
).
Ammonium enhancement experiments have demonstrated a clear link between animal
feeding and nitrogen status of the algal partner in zooxanthellate sea
anemones and corals (Cook et al.,
1992
,
1994
). For example, ammonium
enhancement ratios for zooxanthellae from the reef coral Madracis
mirabilis were approximately 1.2 in well-fed corals, as opposed to
1.7 in starved corals (Cook et al.,
1994
).
It should be recognised, however, that, as in cnidariaalga symbioses
(Rees, 1986;
Wang and Douglas, 1998
), the
provision of photosynthetically fixed carbon to a symbiotic sponge may
stimulate a degree of ammonium assimilation by the sponge itself. This would
no doubt reduce the availability of waste ammonium to the alga, although
evidence to date suggests that this flux of photosynthate in spongealga
symbioses may be relatively small. For example, cyanobacterial symbionts in a
range of reef sponges translocated only 1-5% of their photosynthate
(Wilkinson, 1983
), and
preliminary evidence from the HaliclonaCeratodictyon symbiosis
suggests a similarly low translocation rate (A. Grant, personal
communication). In any case, it seems that the
HaliclonaCeratodictyon symbiosis forms a coherent unit, where
carbon skeletons generated in the alga during photosynthesis enable the uptake
of excretory ammonium from the sponge during both day and night. This
interaction, in turn, may enhance the nutrient status of the macroalgal
partner in an otherwise nutrient-poor environment.
By contrast, while it is known that the amino acids alanine, leucine,
glutamate, threonine and aspartate are translocated by C. spongiosum
(A. Grant, personal communication), our results suggest that there is a
limited potential for nitrogen recycling in the
HaliclonaCeratodictyon symbiosis. Whether nitrogen recycling
is important in cnidariaalga symbioses is debatable. In their nitrogen
budget for the reef coral Stylophora pistillata, Rahav et al.
(1989) estimated that
zooxanthellae need only 10% of the excretory nitrogen from the host for their
growth and, potentially, recycle 90% back to the host. However, while
zooxanthellae may release amino acids, such as alanine (Muscatine and
Cernichiari, 1969), and nitrogen-containing glycoconjugates
(Markell and Trench, 1993
),
the material they translocate consists predominantly of nitrogen-poor
compounds, such as glycerol and glucose
(Muscatine, 1967
;
Trench, 1971
). In addition, as
demonstrated in the zooxanthellate sea anemone Aiptasia pulchella,
cnidarian hosts can assimilate ammonium
(Wang and Douglas, 1998
).
Hence, nitrogen recycling in cnidariazooxanthella symbioses may not be
as significant as initially thought.
Our findings therefore reveal that sponge-alga symbioses may not only be
autotrophic (Wilkinson, 1983)
but may also benefit from the transfer of nitrogen between the symbiotic
partners. Similarly, in associations between massive root-fouling sponges
(Tedania ignis and Haliclona implexiformis) and the red
mangrove (Rhizophora mangle), the sponge obtains organic carbon from
the roots of the mangrove, and the growth of the mangrove is enhanced by the
uptake of excretory nitrogen from the sponge
(Ellison et al., 1996
).
However, study of a wider range of spongealga symbioses is still
required to determine how general such interrelationships are, while the
direct demonstration of nitrogen transfer between the sponge and alga requires
the use of a tracer, such as 15N. More information is also required
concerning the sites and mechanisms involved in ammonium assimilation, the
quantities and identities of metabolites passing between the symbiotic
partners, and the temporal and spatial variability of the various nutritional
fluxes. The roles of symbiotic bacteria, which are harboured by numerous
marine sponges (Wilkinson,
1984
), also await clarification. For example, nitrifying bacteria
may enhance local concentrations of nitrate
(Corredor et al., 1988
).
Clearly though, it is evident that to understand the growth and success of
coral reefs fully, we need to understand the biology and ecology not just of
corals but of all symbiotic organisms and, in particular, sponges.
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Acknowledgments |
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