Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host
Department of Biology (Area 2), University of York, Heslington, York, YO10 5YW, UK
* Author for correspondence (e-mail: lfw3{at}york.ac.uk)
Accepted 11 June 2003
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Summary |
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Key words: Anemonia viridis, Cnidaria, dinoflagellate alga, nutrition, photosynthetic metabolism, Symbiodinium, symbiosis, zooxanthella
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Introduction |
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The nutrients translocated between the partners in a symbiosis are referred
to here as mobile compounds. This study concerns the identity of the mobile
compounds derived from the photosynthetically fixed carbon pools of the algal
cells. This topic is technically challenging for two reasons. Firstly, the
Symbiodinium cells in most associations are intracellular,
specifically located in the cytoplasm of animal cells and individually
enclosed within an animal membrane termed the symbiosomal membrane
(Wakefield et al., 2000).
Secondly, it is expected that the mobile photosynthetic compounds are
metabolized rapidly on receipt by the animal cytoplasm, thereby limiting their
backflow to the algal cells (Douglas,
1994
). It is widely accepted that the mobile photosynthetic
compounds include low-molecular-mass compounds such as glycerol, glucose and
organic acids. The principal evidence is that these are the compounds released
into the medium by Symbiodinium cells freshly isolated from certain
symbioses and incubated with extract of the host animal
(Trench, 1971a
;
Hinde, 1988
). Exceptionally,
in the tridacnid clamSymbiodinium association, the mobile
compounds can be identified relatively easily in the intact symbiosis because
the algal cells are extracellular. In this system, the dominant photosynthetic
compound released from Symbiodinium cells is glucose in the symbiosis
but glycerol in isolated cells (Streamer
et al., 1988
; Rees et al.,
1993
; Ishikura et al.,
1999
). This discrepancy suggests that it may not be justified
generally to equate the compounds released by isolated algal cells and the
mobile compounds in the intact symbiosis. Compounding these difficulties, the
Symbiodinium cells in some animal hosts, e.g. Zoanthus
robustus and Anemonia viridis, release markedly more
photosynthetic compounds to the animal tissues in the intact association than
after isolation and incubation with host homogenate
(Sutton and Hoegh-Guldberg,
1990
; L. Whitehead, J. T. Wang and A. E. Douglas,
unpublished).
Various approaches have been adopted to study photosynthate release from
Symbiodinium cells in the intact symbiosis, thereby circumventing the
limitations of studies with isolated algae. These approaches include
identification of photosynthetically fixed 14C- and
13C-labelled compounds in the animal partner
(Trench, 1971b;
Battey and Patton, 1984
;
Johnston et al., 1995
),
measurement of respiratory quotients
(Gattuso and Jaubert, 1990
) and
identification of organism-specific compounds
(Harland et al., 1991
).
Although these studies were not designed specifically to identify the mobile
photosynthetic compounds, they collectively suggest that intact lipids,
glycerol and fatty acids may be translocated from Symbiodinium cells
to the animal tissues. These data are partly, but not completely, consistent
with the candidate mobile compounds identified from studies with isolated
algal cells (see above).
The purpose of the present study was to apply the approach of `metabolite
comparisons', summarized in Fig.
1, to explore the identity of mobile photosynthetic compounds in
an intact symbiosis. The two treatments are: (1) the `control animals'
incubated with NaH14CO3 under photosynthesizing
conditions and (2) the `experimental animals' incubated with one of a panel of
14C-labelled organic substrates under non-photosynthesizing
conditions. The radioactively labelled compounds in the animal fraction are
the products of animal metabolism of the mobile compounds and of the
exogenously applied organic compounds for the control and experimental
animals, respectively. The exogenous organic substrates that generate the same
pattern of labelled metabolites in the animal fraction as that obtained with
the NaH14CO3 incubations can, consequently, be
interpreted as the mobile photosynthetic compound (or a compound metabolically
closely allied to the mobile compound). By this approach, candidate mobile
photosynthetic compounds have been identified in the symbiosis between the sea
anemone Anemonia viridis and algae of the genus
Symbiodinium. This association has been the subject of a number of
physiological and metabolic studies (e.g.
Taylor, 1969;
Schlichter, 1978
;
Tytler and Davies, 1984
;
Stambler and Dubinsky, 1987
;
Harland et al., 1992
;
Davy et al., 1997
;
Furla et al., 1998
;
Roberts et al., 1999
) but the
mobile compounds have not been identified.
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Materials and methods |
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Replicate groups of three freshly excised tentacles of A. viridis
(taken from three separate individuals) were incubated in 1 ml seawater
buffer, comprising 420 mmol l1 NaCl, 26 mmol
l1 MgSO4, 23 mmol l1
MgCl2, 9 mmol l1 KCl, 9 mmol l1
CaCl2, 2 mmol l1 NaHCO3 and 10 mmol
l1 Hepes, pH 8.2 (Wang
and Douglas, 1997), for 30 min prior to an experiment, by which
time the tentacles had relaxed. Where appropriate, algal photosynthesis was
inhibited by 5 µmol l1 DCMU (dichlorophenyl dimethyl
urea) added 15 min before the experiment. Experiments were initiated by adding
radiolabelled substrate (details below) and, unless stated otherwise,
comprised a 5-min time course, to minimize the extent of animal metabolism of
the radioactive substrate whilst generating sufficient radioactivity for
quantification. Samples without added radioactivity were included in all
experiments as controls.
NaH14CO3 incubation under photosynthesizing and
non-photosynthesizing conditions
After the incubation with 0.37 MBq (10 µCi)
NaH14CO3 (ICN Pharmaceuticals, Costa Mesa, CA, USA), the
tentacle samples were rinsed in ice-cold 0.5 mol l1 NaCl and
homogenized in a hand-held glass homogenizer in 1 ml ice-cold 0.5 mol
l1 NaCl. The protein content of each homogenate was
quantified using the Bio-Rad protein micro-assay kit (Bio-Rad Laboratories
GmbH, Munchen, Germany) according to the manufacturer's instructions, with
bovine serum albumin as standard. The homogenate was centrifuged at 1000
g for 5 min at 4°C, and the pellet was washed twice by
centrifugation and resuspension with 1 ml 0.5 mol l1 NaCl.
The supernatants were combined to form the animal fraction, and the final
suspension of algal cells was designated the algal fraction. The cell density
in the algal fraction was quantified using an improved Neubauer haemocytometer
(six replicate counts per sample). To quantify 14C incorporation,
25 µl of each fraction was acidified with 4 mol l1 acetic
acid and shaken for 60 min to remove unfixed 14C. Scintillation
cocktail (Ultima-GoldTM XR; Packard Bioscience B.V., Gröningen, The
Netherlands) was then added, and the radioactivity quantified in a
scintillation counter (Packard Tri-Carb Scintillation Analyser) using pre-set
14C windows. The counts in the samples incubated without added
radioactivity were subtracted from values for the experimental samples.
Samples of the algal and animal fractions were incubated in 5%
trichloroacetic acid (TCA) for 15 min at room temperature and then centrifuged
at 10 000 g for 15 min. The supernatant (TCA-soluble fraction)
was extracted three times with ether to remove the TCA, evaporated to dryness
in a Speed-Vac (SC110; Savant Instruments, Holbrook, NY, USA) and then
resuspended in 80 µl and 200 µl of deionised water for algal cell and
animal samples, respectively. (The animal samples required a larger volume to
dilute the salt; ethanol extraction was not used to remove the salt because
this procedure substantially reduced the yield of radioactivity.) The lipid
fraction was extracted from the TCA-insoluble fraction with 1 ml
methanol:chloroform (2:1) for 15 min at room temperature followed by
centrifugation at 10 000 g for 15 min. The chloroform layer
containing the lipid was separated from the methanol by the addition of 0.4 ml
0.1 mol l1 KCl. The final pellet that contained protein and
nucleic acids was solubilized by incubation in 0.2 ml 1 mol
l1 NaOH at 100°C for 10 min and then neutralized with
0.2 ml 1 mol l1 HCl. Samples of each fraction were removed
for the quantification of radioactivity. The recovery of radioactive compounds
after fractionation was, on average, 90% and 65% for the algal and animal
samples, respectively. It is unclear why recovery was lower for the animal
samples but it seems unlikely to be due to the salt content as similar
recovery figures were achieved after salt removal by Streamer et al.
(1988).
The TCA-soluble fraction was further separated into neutral, acidic and
basic fractions using cation (Dowex 50 x 8 H+; Sigma-Aldrich,
Dorset, UK) and anion (Dowex 1 x 8; Sigma-Aldrich) exchange columns,
following the procedure of Quick et al.
(1989). To identify individual
radiolabelled compounds, samples of the TCA-soluble fraction were separated by
thin layer chromatography/thin layer electrophoresis, as in Wang and Douglas
(1997
), for organic acids,
phosphate esters, sugars and sugar alcohols and as in Wang and Douglas
(1999
) for amino acids. The
chromatography/electrophoresis plates were exposed to Kodak X-ray film for
46 weeks, and radioactive spots were identified by comparison with
authentic compounds. The identification of the sugars, sugar alcohols and
amino acids was verified by reverse-phase HPLC using the procedures of Ashford
et al. (2000
) and Karley et al.
(2002
).
14C organic substrate incubations
The analyses were performed as for NaH14CO2
incubations with the following modifications. All experiments were performed
under non-photosynthesizing conditions (in the presence of 5 µmol
l1 DCMU) in autoclaved seawater buffer supplemented with the
antibiotics ampicillin (1 mg ml1) and streptomycin (50 µg
ml1) to inhibit the metabolism of any contaminating bacteria
(Wang and Douglas, 1998).
Organic substrates {[U-14C]glucose, [U-14C]glycerol,
[2,3-14C]succinate, [2,3-14C]fumarate (Sigma-Aldrich),
[U-14C]malate and [1,5-14C]citrate (Amersham Pharmacia
Biotech UK Ltd, Amersham)} were supplied at a concentration of 1 mmol
l1 and 0.11 MBq ml1 for single substrate
incubations and 0.5 mmol l1 and 0.06 MBq
ml1 for double substrate incubations (i.e. giving a total
concentration of 1 mmol l1 and 0.11 MBq
ml1). The chemical purity of radiochemical compounds was
>99%, as determined by thin layer chromatography. The experimental design
was developed following preliminary experiments on the uptake of organic
substrates by tentacle samples over time from 5 s to 5 min, which revealed
minimal nonspecific binding of 14C with >98% recovery of
radioactivity from the anemone fraction, i.e. <2% in the algal cells. Algal
cells and animal tissue were therefore not separated in the definitive
experiments. The experimental material was homogenized in ice-cold deionised
water, and the TCA-soluble fractions, which were essentially salt-free, were
resuspended in 80 µl volumes after evaporation.
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Results |
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Incorporation of radioactivity into the principal chemical fractions of the algal cells and animal tissue after 5-min incubations is shown in Fig. 2. The distribution of radioactivity (expressed as d.p.m. mg1 protein) across the chemical fractions varied significantly between the algal cells and animal tissues and was significantly altered by the DCMU treatment (statistical analysis shown in legend to Fig. 2). The two principal factors contributing to this variation were: (1) the greater incorporation of radioactivity into the TCA-soluble fraction of animal tissues (94% of the total) compared with algal cells (59%) under photosynthesizing conditions and (2) the disproportionate reduction in 14C incorporation into the lipid fraction in the DCMU-treated samples relative to those under photosynthesizing conditions. The radioactivity in the neutral, acidic and basic fractions of the TCA-soluble fraction is shown in Fig. 3, with statistical analysis in the legend. For the samples under photosynthesizing conditions, the distribution of radioactivity differed significantly between the algal cells and animal tissues, with recovery from the neutral, acid and basic fractions in approximate ratios of 2:1:2 for algal cells and 1:2:1 for the animal tissues. The distribution of radioactivity was also significantly altered by DCMU treatment.
|
|
Various compounds were radioactively labelled in both the algal cells and animal tissue of samples incubated with NaH14CO3 under photosynthesizing conditions (Table 1). Differences between the algal cells and the animal tissue included the detection of 14C-labelled glycerol in the algal cells but not in the animal tissue, and intense labelling of malate in the animal tissue as opposed to citrate in the algal cells. The 14C content of DCMU-incubated samples was so low that identification of individual radiolabelled compounds was not feasible.
|
Uptake and assimilation of 14C-labelled organic substrates
into the animal tissue
Tentacles of A. viridis incubated under non-photosynthesizing
conditions with each of the six organic substrates (glucose, glycerol, malate,
citrate, succinate and fumarate) accumulated radioactivity equivalent to
between 2.74 nmol substrate mg1 total protein and 4.20 nmol
substrate mg1 total protein over the 5-min incubation period
(Table 2). The pattern of
incorporation of radioactivity into the principal chemical fractions was very
similar for all the organic substrates tested. Radioactivity was recovered
predominantly from the TCA-soluble fraction (99%), with a small amount in the
protein/nucleic acid fraction (approximately 0.8%) and barely detectable
amounts (0.2%) in the lipid fraction. For all the organic substrates, the
distribution of radioactivity between the neutral, acidic and basic fractions
of the TCA-soluble fraction was different to that recorded from the control
experiments where the symbiosis was supplied with
NaH14CO3 under photosynthesizing conditions
(Fig. 4). For every substrate,
radioactivity was detected almost exclusively in just two of the three
TCA-soluble fractions: little radioactivity was recovered from the neutral
fraction (0.7% of total 14C in the TCA-soluble fraction) when
an organic acid was supplied or from the basic fraction when glucose (4% of
the total) or glycerol (5% of the total) was supplied. For most organic
substrates tested, the most strongly radiolabelled fraction corresponded to
that of the substrate supplied. Exceptionally, 58% of the total radioactivity
derived from exogenous 14C-labelled malate was detected in the
basic fraction.
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|
Individual radioactively labelled compounds recovered from tentacles that had been incubated in the various organic substrates are shown in Table 3, which includes the control data obtained for tentacles incubated with NaH14CO3 (Table 1) for comparison. For all substrates tested, four 14C-labelled compounds malate and three sugar phosphates were detected in common with the control. The most strongly labelled compounds were those corresponding to the substrate supplied and to glucose 6-phosphate. In general, when an organic acid was supplied, a greater number of 14C-labelled compounds were detected in common with the control compared with when glucose and glycerol were supplied. However, certain 14C-labelled compounds detected in the control samples (notably proline) were only detected in the experimental samples supplied with 14C-labelled glucose.
|
When the tentacle samples were incubated with two 14C-labelled substrates simultaneously, the total uptake varied between 4.25 nmol mg1 total protein and 5.21 nmol mg1 total protein (Table 2). Incorporation of radioactivity into the principal fractions was very similar to single substrate incubations, with 99% of the radioactivity recovered from the TCA-soluble fraction. The pattern of incorporation that most closely resembled the control incubations with NaH14CO3 was obtained with glucose and either succinate or fumarate as substrates (Fig. 5).
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The profile of labelled compounds in double substrate incubations (Table 4) was identical to the combined profile of compounds labelled in previous experiments using the same substrates applied singly (Table 3). For the incubations with glucose and succinate or glucose and fumarate, the radiolabelled compounds were very similar to those labelled in the control experiments incubated with NaH14CO3. The products of double substrate incubations that included glycerol were also similar to the results from the control experiments, but two of the most strongly labelled compounds, glycerol and an unknown amino acid, in these double substrate incubations were not detected in the control incubations.
|
In summary, the profile of metabolites in the animal tissues derived from exogenous glucose and succinate/fumarate, but no other exogenous compounds tested, was closely similar to the equivalent profiles derived from photosynthetically fixed compounds translocated from the algal cells. This comparison of metabolite profiles (see Fig. 1) suggests that glucose and a dicarboxylic acid (probably succinate and/or fumarate), or metabolically allied compounds, are important mobile photosynthetic compounds in the Anemonia viridisSymbiodinium symbiosis.
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Discussion |
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To our knowledge, the photosynthetic mobile compounds in the A.
viridis symbiosis have not previously been identified. This may be
because although a number of studies have demonstrated carbon translocation in
the intact symbiosis (Stambler and
Dubinsky, 1987; Davy et al.,
1997
), the algal cells isolated from A. viridis do not
release photosynthetically fixed carbon, even in the presence of host
homogenate (L. F. Whitehead and A. E. Douglas, unpublished results). The
conclusion that glucose and succinate/fumarate are the likely photosynthetic
mobile compounds in A. viridis is partly consistent with previous
reports, in that these compounds have been cited as mobile compounds in a
range of symbioses involving Symbiodinium (e.g.
Trench, 1971a
;
Rees et al., 1993
;
Wang and Douglas, 1997
).
However, glycerol, which is accepted widely as a dominant mobile
photosynthetic compound in many symbioses involving Symbiodinium, is
apparently not translocated in the A. viridis system. If glycerol
were mobile in A. viridis, it would be expected that
14C-labelled glycerol would be detected in the animal fraction
after incubation in NaH14CO3, because exogenous glycerol
was not metabolized rapidly in the animal tissues [also noted by Ishikura et
al. (1999
) in tridacnid
clams]. Contrary to this expectation, glycerol was not recovered as a
radioactively labelled metabolite in the animal tissues in any of the many
NaH14CO3 incubation experiments conducted here. This
result is open to at least three alternative explanations. First, as suggested
by the study of Ishikura et al.
(1999
), glycerol may not
generally be an important mobile compound in symbiosis. Its dominance among
the photosynthetic compounds released from isolated algal cells incubated with
host homogenate may reflect a general shift in metabolism associated with the
physiological responses either to isolation from the symbiosis or to the host
homogenate (Goiran et al.,
1997
). Second, the symbiosis in A. viridis may be
metabolically different from many symbioses, linked to its exclusively high
latitude distribution (Manuel,
1988
) or its genetically distinctive symbiotic algal partner
(Savage et al., 2002
).
Finally, the identity of the mobile photosynthetic compounds may vary with
environmental conditions. For example, Gattuso et al.
(1993
) concluded that glycerol
is released from the algal cells to the animal tissues of the coral
Stylophora pistillata only under high light conditions, whereas the
experiments described here on A. viridis were conducted under
relatively low light conditions. Further research is required to discriminate
between these possibilities. For the present, we note that although glycerol
is incorporated preferentially into lipid in other symbioses
(Trench, 1971b
;
Schmitz and Kremer, 1977
), we
found no evidence for this in A. viridis and that the generally lower
lipid content of temperate symbioses, including A. viridis, compared
with tropical species (Harland et al.,
1991
) is consistent with glycerol not being an important mobile
photosynthetic compound in the A. viridis symbiosis.
Other data obtained in this study support the possibility that the fate of
photosynthetic carbon in the A. viridis symbiosis may differ from
that in low-latitude symbioses. In particular, photosynthetic carbon is
preferentially incorporated into acidic compounds in the TCA-soluble fraction
of the animal tissue in A. viridis
(Fig. 3B), but very little
radioactivity is recovered from this fraction in low-latitude symbioses,
including Zoanthus flos marinus and Condylactis gigantea
(von Holt and von Holt, 1968).
A second distinctive feature of the data obtained here is the low
incorporation of photosynthetically fixed carbon into the lipid fraction of
both the algal cells (16%) and the animal tissue (3%), relative to other
symbioses (3590% and 2060%, respectively;
von Holt and von Holt, 1968
;
Trench, 1971b
;
Gattuso et al., 1993
). This may
reflect the low total lipid content of A. viridis compared with
tropical anemones and corals (11% and 50% of dry mass, respectively;
Harland et al., 1992
), which
has been hypothesised to result from the lower light and lower temperature in
temperate environments (Harland et al.,
1992
; Muller-Parker and Davy,
2001
). However, further research on the impact of season, light
and temperature on the incorporation patterns of photosynthetic carbon is
required to resolve this issue.
In conclusion, the metabolite comparison method developed here to study the identity of mobile photosynthetic compounds in symbioses involving Symbiodinium has potential value to investigate a range of issues that are currently not readily tractable to study. These include comparisons among symbioses from different habitats (e.g. temperate versus tropical) involving different algal genotypes and maintained under various environmental conditions, e.g. irradiance and temperature. With appropriate modifications, the approach may also contribute to the study of nutritional interactions in other symbioses between microorganisms and plants or animals, especially where, as with the Symbiodinium systems, alternative methods to study nutrient translocation in the intact association are not fully developed.
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Acknowledgments |
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