Symbiosis-induced adaptation to oxidative stress
1 Université de Nice Sophia-Antipolis, BP 71, F-06108 Nice Cedex 02,
France
2 Centre Scientifique de Monaco, Avenue Saint-Martin, MC-98000 Monaco,
Principality of Monaco
* Author for correspondence (e-mail: allemand{at}unice.fr)
Accepted 1 November 2004
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Summary |
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Key words: cnidarians, zooxanthellae, symbiosis, oxidative stress, hyperoxia, thermal stress, SOD
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Introduction |
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The aim of the present study was twofold: (1) to determine, the role of
symbiosis in determining oxidative stress tolerance; and (2) to assess the
role of the symbiont in the expression of specific SOD isoforms in animal
compartments. We have compared the effect of environmental stress in symbiotic
and non-symbiotic species of temperate sea anemones. The Mediterranean sea
anemone, A. viridis, was chosen as the symbiotic model by virtue of
the easy separation of its two animal cells layers (ectoderm and endoderm) and
the isolation of the symbiont cells
(Bénazet-Tambutté et al.,
1996). Actinia schmidti was chosen as the non-symbiotic
model. Both animals have already been used for comparative study
(Harland et al., 1990
) on the
basis of their common classification in the Actiniidae
(Shick, 1991
) and its
relevance is also justified by their close location in the same habitat. Lipid
peroxidation and protein oxidation were chosen as biomarkers of oxidative
stress, and SOD activity as biomarker of antioxidant defence. High
PO2 tolerance was analysed during endogenous
and experimental hyperoxia (60100% O2 saturation). To test
whether O2 tolerance promotes resistance to other stress, we also
studied the effect of elevated temperatures. Interactions between host and
symbionts were observed through the expression of SOD isoforms, using two
symbiotic sea anemones (A. viridis and Aiptasia pulchella)
and the scleractinian coral S. pistillata. SOD activities of
symbiotic and aposymbiotic specimens, as well as of freshly isolated
zooxanthellae (FIZ) and cultured zooxanthellae (CZ) were analysed.
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Materials and methods |
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Specimens of the sea anemone A. pulchella were maintained at 17.0±0.1°C and illuminated under a constant irradiance of 175 µmol m2 s1, using the same type of metal halide lamps on a 12 h:12 h photoperiod. Aposymbiotic anemones were maintained at the same temperature in continual darkness.
Microcolonies of S. pistillata were propagated in the Scientific
Centre of Monaco (Tambutté et al.,
1995) where they were maintained at 27±0.5°C and 38 PSU
under the same type of metal halide lamps. Cultured zooxanthellae
(Symbiodinium sp.) were obtained from a clonal culture originally
provided by Robert Trench. The algal cultures were maintained in 500 ml
screw-top polycarbonate Erlenmeyer flasks (Corning; Acton, MA, USA) in
modified ASP-8A medium (Blank,
1987
) at pH 8.2 and incubated at 26.0±0.1°C under an
irradiance of 100 µmol photons m2 s1
provided by Sylvania Gro-Lux (Germany) and daylight fluorescent tubes, on a 12
h:12 hphotoperiod. Stock cultures were transferred monthly.
Aposymbiotic specimens correspond to bleached cnidarians in which disruption of symbiotic association has been achieved by either ambient conditions in the public aquarium of the Oceanographic Museum of Monaco (A. viridis) or by long term (>1 month) incubation in darkness (A. pulchella).
Experimental designs
Experimental hyperoxia
Specimens of A. viridis and A. schmidti were first held
(48 h) in an airtight bottle, then subjected to 10 h of100% O2 at
17.0±0.1°C under a constant irradiance of 250 µmol
m2 s1. O2 saturation of the
medium was achieved by bubbling pure O2 in seawater and was
monitored using a gas analyser (Radiometer Copenhagen ABL 30; Copenhagen,
Denmark). At the end of the stress period, a minimum of 510 tentacles
from at least four specimens of A. viridis and whole specimens of
A. schmidti (N=4) were sampled for biomarker assays.
Thermal stress
Three aquaria, each containing one specimen of A. viridis and four
of A. schmidti (designed for sampling over the kinetic), were heated
from 17°C (control temperature) to 24°C (stress temperature) of
+7°C over 2 h and maintained at this maximal temperature for 5 days. For
biomarker assays, 510 tentacles from A. viridis and a whole
specimen and/or tentacles of A. schmidti were sampled from each
aquarium day 0 (control condition) and after 1, 2 and 5 days of consecutive
thermal stress.
Tissue extractions
Unless otherwise specified, all chemicals were obtained from Sigma. Each
extract was prepared at 4°C in a different medium appropriate to the
analysis.
Anemonia viridis
The three cellular compartments [ectoderm, endoderm and freshly isolated
zooxanthellae (FIZ)] were extracted according to Richier et al.
(2003) thus avoiding any
contamination between FIZ protein and the endodermal host cell. `Total
extract' corresponds to the protein extract from whole tentacle
(Richier et al., 2003
)
separated from the FIZ by centrifugation (1,000 g for 3 min).
For extracts prepared under dark conditions, tentacle separation and protein
extraction were conducted under far red light to avoid photosynthesis during
the extraction steps.
Actinia schmidti
The whole animal was frozen in liquid nitrogen and powdered in a mortar.
Subsequent steps were performed in the extraction medium
(Richier et al., 2003) with
800 µl g1 of animal tissue (frozen mass). The crude
extract of A. schmidti tissues was obtained after sonication (6
x10 s) and centrifugation (12,000 g for 5 min). The
supernatant was used for subsequent assays. Extraction was also conducted on
A. schmidti tentacles to confirm whether a discrepancy exists between
whole animal and tentacle analysis.
Aiptasia pulchella
The whole animal was homogenized in a Potter-Elvehijm tissue grinder
containing chilled extraction medium at 4°C, sonicated (6 x10 s) and
centrifuged (12,000 g for 5 min). Supernatant was collected
and reserved for analysis, which constitutes the cytosolic fraction. Freshly
isolated zooxanthellae (FIZ) extraction was made according to Richier et al.
(2003).
Stylophora pistillata
Coral microcolonies were frozen in liquid nitrogen and powdered in a
mortar. Subsequent steps were performed in extraction medium containing 50
µl g1 of whole coral tissue
(Richier et al., 2003). FIZ
extraction was performed as previously described
(Richier et al., 2003
).
Chlorophyll measurement
Chlorophylls a and c2 were extracted from
whole tissues of symbiotic and aposymbiotic specimens of A. viridis
and A. pulchella in 90% acetone and measured according to Jeffrey and
Humphrey (1975).
Thiobarbituric acid assay
Malondialdehyde (MDA) has been identified as the product of lipid
peroxidation that reacts with thiobarbituric acid to give a red chromophore
absorbing at 532 nm. Measurements followed the slightly modified method of
Janero and Burghardt (1989).
Cytosolic fractions (see `Tissue extraction' method), containing 75 µg and
150 µg of protein, were analysed for A. viridis and A.
schmidti, respectively. The absorbance was read at 532 nm using a
microplate apparatus. MDA concentration of the sample was calculated using an
extinction coefficient of 1.56 x105 mol1
l1 cm1.
Protein carbonylation
Carbonyl content of the cytosolic fraction was measured using both the
ELISA assay and spectrophotometry was used according to Buss et al.
(1996). A total of 4 mg
ml1 of protein in both bovine serum albumine (BSA) standard
curves (0100% reduced BSA) and analysed extracts. The ELISA assay used
biotin-conjugated rabbit IgG polyclonal antibody raised against
dinitrophenylhydrazine component (anti-DNP 1:3000; Molecular Probes, Inc,
Cergy-Pontoise, France) and streptavidine biotinylated horseradish peroxidase
(1:2500; Amersham International; Orsay, France).
SOD activity
Both qualitative (native gel) and quantitative (spectrophotometry) analyses
were used to study SOD activity.
Qualitative analysis
SOD isoforms in each tissue compartment were monitored by 8% non-denaturing
polyacrylamide gel electrophoresis and nitro-blue tetrazolium (NBT) staining
as described by Beauchamp and Fridovich
(1971). Specific inactivation
of enzyme activity was determined by soaking gels in 10 mmol
l1 H2O2 (inhibitor of both Fe- and
CuZnSOD) or 10 mmol l1 KCN (inhibitor of CuZnSOD) 30 min
prior to the staining steps. Visualized SOD activity bands were named in
function of genus and species relative to the studied organism (AsSOD for SOD
activity bands relative to A. schmidti).
Quantitative analysis
Activity was measured after preparing each sample in a modified extraction
medium (50 mmol l1 potassium phosphate, 0.1 mmol
l1 EDTA; method by
McCord and Fridovich, 1969).
This method is based on the reduction of ferricytochrome c by
O2, generated during the sequential oxidation of
xanthine by xanthine oxidase. The reaction mixture contained 10 µmol
l1 ferricytochrome c, 50 µmol
l1 xanthine, 20 µg ml1 catalase and 50
nmol l1 xanthine oxidase to produce a rate of reduction of
ferricytochrome c at 550 nm of 0.025 absorbance unit per min.
Measurements were made after adding a determined quantity of protein to 1 ml
of reaction solution at 25°C. In control conditions, standard curves were
generated for total extract of each sea anemone (A. viridis, A.
schmidti and A. pulchella) and the three compartments (ectoderm,
endoderm and FIZ) of A. viridis to determine the quantity of protein
resulting in 50% inhibition of cytochrome c reduction. In this
context, the amount of superoxide dismutase required to inhibit of 50% the
rate of reduction of cytochrome c is defined as 0.33 unit of
activity. Absorbance was measured at 550 nm.
Presentation of results
Results are presented as means ± S.E.M. and normalized by
the protein content of each fraction. Protein content was determined by the
Biorad protein assay using BSA as the standard protein. The results were
validated by one-way ANOVA with Fisher post-hoc test or Student's
t-test and were considered statistically significant when
P<0.05.
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Results |
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Response to environmental stress
Effects of experimental hyperoxia on SOD activities
Qualitative analysis of SOD activity on native gel revealed no change in
SOD isoform expression within the animal compartments of A. viridis
during endogenous (photosynthetic) hyperoxia (results not shown) or
experimental hyperoxia (Fig.
2A). Spectrophotometric measurement shows a significant 1.5-fold
increase in SOD activity in the ectodermal fraction. No change occurred in
endodermal cells (Table 1).
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In the zooxanthellae, qualitative analysis of SOD activities shows an unaltered electrophoretic pattern during hyperoxic stress. However, a significant 1.9-fold increase in activity was quantified by spectrophotometric measurements (Table 1).
After 10 h incubation at high PO2, A. schmidti expresses a new SOD activity band, AsSODd, identified as CuZnSOD by KCN native gel incubation (Fig. 2B). A weaker AsSODa activity band was observed under hyperoxia (Fig. 2B). Spectrophotometric measurements of total extract of A. schmidti show a slight decrease in SOD activity after 10 h incubation at high PO2 (Table 1).
Effects of experimental hyperoxia on damage biomarkers
In the symbiotic sea anemone A. viridis, damage biomarkers
(protein carbonyl or MDA concentration) detected no significant variation
during either endogenous hyperoxia (daily light/dark cycle, results not shown)
or experimental hyperoxia (Fig.
3A,B). By contrast, significant differences were observed in
A. schmidti tissues during experimental hyperoxia. Protein
carbonylation was sixfold higher in treated tissues than in control tissues
(Fig. 3C). However, MDA values
did not differ significantly after similar treatment
(Fig. 3D). Basal values of
damage biomarkers, either for MDA level or carbonyl content were higher in
symbiotic specimens than in non-symbiotic specimens
(Fig. 3).
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Effects of thermal stress on SOD activities
The overall SOD pattern of A. viridis did not change in animal
compartments even after 5 days of incubation at a temperature 7°C above
normal (Fig. 4AC).
Quantitative measurements detected a slight increase after 24 h incubation in
ectodermal and no significant change in endodermal animal cells
(Table 1). In zooxanthellae,
two or more SOD activity bands, identified as FeSOD, appeared as soon as the
stress period begins. Quantitative analysis by spectrophotometry revealed a
1.5-fold increase in SOD activity in this compartment
(Table 1).
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A. schmidti SOD activities responded strongly to the experimental treatment (Fig. 4D). The CuZnSOD activity bands (AsSODa and AsSODb) disappeared after 2 days and a novel activity band (AsSODe) appeared (Fig. 4D). After 5 days of incubation, another CuZnSOD activity band (AsSODf) was expressed, and a high-molecular mass smear appeared (Fig. 4D). Pharmacological identification of SOD activity bands is presented in Fig. 4E. Parallel measurements by spectrophotometer show a global decrease in SOD activity during 5 days of incubation at elevated temperature (Table 1).
Effects of thermal stress on damage biomarkers
After 1 day of thermal stress, the symbiotic sea anemone had a threefold
increase of carbonyl content in its three compartments
(Fig. 5A). Under the same
conditions, MDA content did not vary significantly
(Fig. 5B).
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In the non-symbiotic A. schmidti, carbonyl content responded immediately and significantly. A sixfold increase of the basal value occurred after one day of exposure (Fig. 5C). After 5 days, carbonyl content increased a tenfold relative to the control. Lipid peroxidation increased threefold after 5 days (Fig. 5D).
Symbiotic regulation of SOD expression
Symbiont regulation of SOD expression within the host
Bleached specimens of A. viridis and A. pulchella exhibit
a global decrease in SOD activities and a loss of some SOD activity bands
(Fig. 6). Bleaching was
confirmed by the measured a decrease in chlorophyll content
(Table 2). The analysis of the
three compartments (ectoderm, endoderm and FIZ) from A. viridis
enabled a more precise localization of modifications in the SOD pattern
(Fig. 6A). While the
electrophoretic pattern for ectodermal cells were unchanged, the endodermal
host cells lost its major SOD activity bands, representing MnSOD and FeSOD
classes as identified by Richier et al.
(2003). Spectrophotometric
measurements confirm five- and threefold decreases of SOD activity in the
ectodermal and endodermal compartments, respectively
(Table 3). In the A.
pulchella total extract, three SOD activity bands (pharmacologically
identified as MnSOD) disappear from the bleached anemone
(Fig. 6B). However a MnSOD band
and a CuZnSOD band remain in both symbiotic and aposymbiotic specimens
(Fig. 6A,B). The CuZnSOD class
was neither observed in FIZ from A. viridis
(Richier et al., 2003
) nor in
those of A. pulchella, S. pistillata and Galaxea
fascicularis (results not shown). This would suggest an animal cell
specificity of this isoform.
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Host regulation of SOD expression within zooxanthellae
Fig. 7 compares the SOD
patterns of FIZ and cultured zooxanthellae (CZ) from both A. viridis
and S. pistillata symbiotic specimens. Despite their common genotype
clade A (D. Forcioli, personal communication), FIZ and CZ showed specific
electrophoretic patterns depending on their animal host origin. Native gels
revealed that CZ from both cnidarians showed additional specific bands
compared with their respective FIZ. These additional bands were identified as
MnSOD in CZ from A. viridis, and as MnSOD and FeSOD in CZ from S.
pistillata.
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Discussion |
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Symbiotic adaptation to the hyperoxic state
Under control conditions, the SOD electrophoretic patterns are more
diversified in animal cells living in association with photosynthetic
dinoflagellates than in the non-symbiotic sea anemone. Three SOD classes
(FeSOD, CuZnSOD and MnSOD) and up to seven activity bands were observed in
A. viridis symbiotic host cells
(Richier et al., 2003). Only
two classes are generally present in animals
(Halliwell and Gutteridge,
1999
) as was observed for the non-symbiotic A. schmidti.
This high diversity in the symbiotic example is similar to that observed in
plants, which can display up to seven isoforms
(Halliwell and Gutteridge,
1999
; Alscher et al.,
2002
). A similar high diversity (six SOD activity bands) is
observed in zooxanthellae, a symbiotic chlorophyll-containing protist.
Furthermore, as has been previously observed
(Shick and Dykens, 1985
), the
symbiosis-dependant SOD diversity in activity bands is associated with a
global increase in SOD activity (Table
1). The comparison of SOD expression between these two sea
anemones suggests that the diversification of SOD activity in animal host cell
could be a consequence of the endosymbiont presence's rather than a unique
feature of the phylum Cnidaria.
Naturally, dealing with oxidative stress while avoiding membrane damage
represents an inherent challenge to all aerobic life forms. Lipid peroxidation
and protein oxidation are universal responses
(Ernster and Hochstein, 1994).
Nevertheless, in the present study, both damage basal values and SOD
activities remain unchanged during natural light-induced hyperoxia (results
not shown). This suggests a host adaptation to symbiont photosynthesis,
possibly involving a constitutive expression of several SOD activity bands, as
is the case for plants (Gillham and Dodge,
1987
). During daytime, however, symbiotic sea anemones have higher
basal MDA and carbonyl concentrations than do the non-symbiotic organisms.
This would implicate continuous ROS production from zooxanthellae
photosynthesis (Dykens and Shick,
1984
; Dykens et al.,
1992
). A high basal value for damage biomarkers has also been
shown in photosynthetic organisms. For example, plant leaves are unable to
escape photooxidative damage due to their exposure to bright light, while they
produce O2 (Asada and Takahashi,
1987
).
To test whether natural transitions between hyperoxia and anoxia contribute
to oxidative stress adaptation, both SOD activity and damage biomarkers were
further analysed under experimental hyperoxia. In the non-symbiotic sea
anemone A. schmidti, experimental hyperoxia (100% O2)
leads to qualitative changes in SOD. Evidence for this is the appearance of
new isoforms (CuZnSOD activity band, Fig.
2B). However, such a high PO2 seems
to be deleterious for this antioxidant enzyme, since SOD activity decreased
during the stress period (Table
1). Under conditions of elevated prooxidative challenge, depletion
of antioxidant defenses has been reported as a common pathway of toxicity in
several marine invertebrates (Winston and
DiGiulio, 1991; Regoli and
Principato, 1995
). In the present study, an increase in cellular
damage was observed as a 10-fold increase in protein oxidation
(Fig. 3C). These results agree
with those previously reported for animal cells
(Ahmed et al., 2003
).
By contrast, neither qualitative changes in SOD pattern (Fig. 2) nor significant increases in damage biomarkers (Fig. 3) were observed in the symbiotic sea anemone A. viridis, under imposed hyperoxia. Meanwhile, in response to the hyperoxic stress, SOD activity increased 1.6-fold relative to the control in the ectodermal compartment, presumably to counterbalance prooxidant period during hyperoxia. This differential response to hyperoxic stress in the two cnidarians supports the hypothesis of the influence of the photosynthetic protist on the animal cell to deal with ROS.
Does symbiosis contribute host adaptation to environmental stressors, such as increase temperatures? Similar to the response to hyperoxic stress, A. schmidti responds to thermal stress with a decrease in global SOD activity and the expression of a novel stress-specific SOD isoform (the thermal stress-inducible isoform, CuZnSOD). Thermal stress on A. schmidti resulted in a time-dependent increase in both damage biomarkers (a threefold increase in MDA production and a 10-fold increase in protein oxidation) after 5 days under stress. By contrast, in the symbiotic species A. viridis, similar thermal stress had no effect on lipid peroxidation or resulted in less than 3.5-fold increase in protein oxidation after 5 days (Fig. 6) No modification of the SOD activity pattern occurred. In conjunction with the SOD activity response to hyperoxic stress, the symbiotic sea anemones also have a progressive increase in ectodermal compartment during thermal stress.
This comparative study demonstrates that, while the non-symbiotic species
appeared sensitive to thermal stress, the sympatric symbiotic species was
tolerant. These observations suggest that the symbiotic state plays a role in
host cell adaptation to thermal stress. A similar relationship has already
been demonstrated in associations between fungi and plants. In this symbiosis,
the host plant acquires a thermotolerance because the fungal endophyte
produces cell wall melanin that may dissipate heat and/or complex with oxygen
radicals generated during heat stress. Alternatively, the endophyte may act a
`biological trigger' allowing symbiotic plants to activate stress-response
systems more rapidly and strongly than non-symbiotic plants. Furthermore, this
mutualism may involve other benefits (e.g. nutrient acquisition by the fungus;
Redman et al., 2002).
Moreover, a recent study on the Mediterranean symbiotic sponge Petrosia
ficiformis shows comparable adaptation, with antioxidant changes in
response to photosynthetically produced ROS
(Regoli et al., 2004
).
The present results support the hypothesis that environmental stress seems
to enhance antioxidant defenses to limit and/or avoid cellular damage in the
symbiotic A. viridis. However, in the non-symbiotic sea anemone, SOD
responds differently to stress. The qualitative analysis shows diversification
of SOD isoforms, and quantitative analysis shows repression of global activity
that might lead to an increase in damage biomarkers (Figs
3C,D, and
5C,D). Regoli et al.
(2000) reported similar
responses to oxidative stress in the symbiotic demosponge Petrosia
ficiformis.
Our results also show that different isoforms of CuZnSOD appear
stress-sensitive in the non-symbiotic sea anemone A. schmidti. New
isoforms are expressed following hyperoxia (AsSODd) or thermal stress (AsSODe,
AsSODf), and certain isoforms are repressed following high
PO2 (AsSODa) or thermal increase (AsSODa and
AsSODb). Although no qualitative changes are observed in A. viridis,
quantitative modifications for CuZnSOD are still possible given that total SOD
activity increased during the experiment. This last result supports several
recent field studies on tropical scleractinian corals
(Brown et al., 2002;
Downs et al., 2002
) that
document an increase in total SOD content by ELISA assay following thermal
stress.
Following thermal or hyperoxic stress, zooxanthellae seem to adapt to O2 variation in a manner comparable to the animal cells of A. viridis. Global SOD activities increase (Table1) and cellular damages are minimized (Figs 3A,B, and 5A,B). After 5 days of incubation at 25°C, the increases in SOD activity observed by spectrophometry may be correlated with the appearance on native gels of FeSOD isoforms (Fig. 4C).
Hostsymbiont interactions in antioxidant defence
One of the key questions arising from these results is whether SOD activity
is sensitive to the symbiotic state. To answer this question, we compared
dissociated partners (aposymbiotic animal and cultured zooxanthellae) and
intact symbiotic associations. As expected, we observed a consistent decrease
in SOD activity (in aposymbiotic tissue extracts of A. viridis and
Aiptasia pulchella). Interestingly, activity bands common to host and
symbionts disappeared. Furthermore, the effect of dissociation of the
symbiosis is observed not only in the endodermal compartment but also in the
ectodermal compartment (which never harbours zooxanthellae). SOD activity
decreased fivefold upon bleaching.
Decrease of host defence upon bleaching may be the result of a decrease of
oxidative stress following the reduction in the photosynthetic process
(`oxidative stress-regulated SOD expression') or a consequence of disruption
of the symbiotic association (`symbiosis-regulated SOD expression'). These
possibilities will be tested in further experiments. Similarly, a lower SOD
activity in aposymbiotic cells compared with symbiotic one was also described
in the symbiotic sea anemone Anthopleura elegantissima by Dykens and
Shick (1982) and Dykens
(1984
), who originally
proposed that photosynthetic hyperoxia necessitates higher antioxidant
defenses in the host.
Cultured zooxanthellae, revealed a persistent increases in SOD activity and
the appearance of new activity bands. Similar increases in antioxidant defence
(SOD and catalase) in cultured zooxanthellae compared with FIZ were also
observed in another symbiotic sea anemone, A. pallida, by Lesser and
Shick (1989). Cultured
zooxanthellae may, therefore, require additional endogenous antioxidant
protection compared with the in hospite state and may depend on the
animal to play a protective role. This conclusion is in agreement with Brown
et al. (2002
), who suggested
such protection in the host Goniastrea aspera following thermal
stress.
This work provides evidence for an acute resistance against oxidative stress of the symbiotic species of sea anemone compared to the non-symbiotic one. This resistance could be the result of the presence of the symbiont that acclimates the animal cell to high PO2 on a daily basis. The greater diversity of isoforms induced by the presence of the photosynthetic symbionts within animal tissue could contribute to this adaptation. Hyperoxic adaptation may also be a preconditioning step that could prevent cellular damage during thermal stress, implying that symbiosis may confer host resistance to multiple stress. Moreover, interactions at the antioxidant defence level between both symbiotic partners could also be a basis for protecting cells following environmental changes. As a result of physical interactions or possible molecular communication between both species, the presence of the zooxanthellae may induce increases in SOD activity and may contribute to the adaptation to stress encountered by the animal partner. Further molecular characterization of common SOD isoforms shared by two phylogenetically different species would yield important information concerning the origin of the unusual SOD diversity present within animal tissues (endoderm) and its relevance to the evolution of antioxidant enzymes in both host and symbionts.
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Acknowledgments |
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