We provide evidence of oxidative stress followed by induction of caspase-like activity in animal host cells after an elevated temperature stress, suggesting the concomitant action of the
Trang 1stress in the symbiotic sea anemone, Anemonia viridis
Sophie Richier1, Ce´cile Sabourault1, Juliette Courtiade1, Nathalie Zucchini3, Denis Allemand1,2 and Paola Furla1
1 UMR 1112 UNSA-INRA ROSE, Nice-Sophia Antipolis University, Nice, France
2 Centre Scientifique de Monaco, Monaco
3 UMR 1112 UNSA-INRA ROSE, Sophia-Antipolis, France
Over the past several decades, symbiotic invertebrates
such as cnidarians, sponges and mollusks have been
regularly affected by a phenomenon known as
‘bleach-ing’ This event has been observed all around the world
and involves principally the mass expulsion of
unicel-lular photosynthetic symbionts from animal tissue
Bleaching phenomenon has been widely reported in the
ecologically and economically important tropical corals
reef, but several other invertebrates such as giant clams,
gorgonians and sea anemones have also been affected
Previous studies have established a causal link
between environmental stresses, such as elevated
tem-perature, ultraviolet light, pathogen infection or
pol-lution, and symbiosis disruption (reviewed in [1])
Mass symbiont expulsion has, however, been most
fre-quently associated with elevated seawater temperature,
generally considered to be the primary stress causing
worldwide bleaching [2] General mechanisms have
been proposed to explain the thermal sensitivity of symbiotic cnidarians including symbiont photoinhibi-tion [3–6], cell degradaphotoinhibi-tion [7] and cell death [8,9] However, despite the importance of the phenomenon, the underlying molecular mechanisms associated with symbiosis breakdown remained undetermined
Oxidative stress is one molecular pathway that has been suggested to cause bleaching A pro-oxidant per-iod is experienced daily by invertebrates harboring pho-tosynthetic symbionts due to the high concentration of oxygen produced throughout photosynthesis [10–13] The light-dependent hyperoxic state induces high fluxes
of reactive oxygen species (ROS) such as O2 and OH• [14] produced largely from mitochondria and chloro-plasts This increase in ROS is counterbalanced by an efficient antioxidant capacity in the host and symbiont cells [12,13,15,16] The first hypothesis of oxidative stress involvement in the bleaching event was proposed,
Keywords
apoptosis; bleaching; caspase; cnidarian;
oxidative stress
Correspondence
P Furla, UMR 1112 ROSE, Nice-Sophia
Antipolis University, Parc Valrose, BP 71,
F-06108 Nice Cedex 2, France
Fax: +33 4 92 07 65 63
Tel: +33 4 92 07 68 30
E-mail: furla@unice.fr
(Received 6 April 2006, revised 30 June
2006, accepted 11 July 2006)
doi:10.1111/j.1742-4658.2006.05414.x
Symbiosis between cnidarian and photosynthetic protists is widely distri-buted over temperate and tropical seas These symbioses can periodically breakdown, a phenomenon known as cnidarian bleaching This event can be irreversible for some associations subjected to acute and⁄ or prolonged envi-ronmental disturbances, and leads to the death of the animal host During bleaching, oxidative stress has been described previously as acting at mole-cular level and apoptosis is suggested to be one of the mechanisms involved
We focused our study on the role of apoptosis in bleaching via oxidative stress in the association between the sea anemone Anemonia viridis and the dinoflagellates Symbiodinium species Characterization of caspase-like enzymes were conducted at the biochemical and molecular level to confirm the presence of a caspase-dependent apoptotic phenomenon in the cnidarian host We provide evidence of oxidative stress followed by induction of caspase-like activity in animal host cells after an elevated temperature stress, suggesting the concomitant action of these components in bleaching
Abbreviations
AFC, 7-amino-4-trifluromethylcoumarin; CARD, caspase recruitment domain; CHO, adelhyde; DEVD, Asp-Glu-Val-Asp; IETD, Ile-Glu-Thr-Asp; ROS, reactive oxygen species; TUNEL, dUTP nick end labeling.
Trang 2and later supported, by Lesser and coworkers [17–20].
These studies demonstrated the role of ROS production
in the temperature-induced bleaching During thermal
stress, although the enzymatic antioxidant defenses are
induced [18,21–23], the additional amount of ROS
pro-duction causes a large increase in cellular damage such
as protein carbonylation [21,22,24], lipid peroxidation
[24] and DNA degradation [23]
ROS could be involved in cell death by two
path-ways: (a) they could cause oxidative stress that leads
to massive cellular damage [25] and they could be
involved in necrosis or in so-called postmitochondrial
phase of apoptosis [26]; or (b) they could be involved
in the initiation phase of apoptosis contributing to cell
death signaling [27]
Programmed cell death is known to model tissue
dur-ing embryogenesis, to remove damaged cells, protect
against pathogen infection, and regulate cell numbers
and tissue homeostasis Program cell death is
character-istic of all multicellular animals and can be extended
now to the most basal metazoan phyla as porifera and
cnidaria with occurrence of apoptosis and homologues
of caspases and Bcl2 proteins [28] Moreover, apoptosis
has been remarkably well conserved throughout
meta-zoan phyla both in terms of morphological features
and of the genes controlling the process Recently,
mor-phological indicators of programmed cell death or
apoptosis have been observed in a symbiotic sea
anem-one, Aiptasia pallida, subjected to thermal stress [8,9],
leading to the proposal of a new molecular pathway for
bleaching induction Furthermore, DNA cleavage
analysis [8,23] and increased expression of p53, a
pro-apoptotic protein expression [23], confirmed
tempera-ture-induced DNA damage in symbiotic cnidarians,
which in turn could activate the apoptotic cascade
Upstream to specific morphological modifications,
apoptosis is also characterized by activation of highly
selective cysteine aspartate-specific proteases, known as
‘caspases’, which are constitutively expressed as
pro-enzymes with little catalytic activity and are activated
following apoptotic stimulation Evidence of caspase
3-like expression in cnidarians was first obtained in
Hydra vulgarisby Cikala et al [29] with caspase
activ-ity measurements and gene characterization Recently,
evidence of caspase-like involvement in Hydractinia
echinatametamorphosis [30] and a caspase gene in the
sea anemone Aiptasia pallida [31] has been shown To
date however, no relation between heat stress and
ca-spase activity has ever been established
In this study, we examined the biological effects of
heat stress on the sea anemone Anemonia viridis, living
in symbiosis with the unicellular dinoflagellate,
Symb-iodinium sp commonly known as zooxanthellae The
first aim of this study was to characterize caspase-like activity and clone a putative caspase cDNA in sea anemone tissues In the second part of the work, we tested the effect of hyperthermal stress on antioxidant induction and on apoptotic markers (caspase-like acti-vation and⁄ or DNA degradation) in order to demon-strate the concomitant involvement of oxidative stress and apoptosis in a thermally induced bleaching event
Results
Detection of caspase-like activity in tissue extracts of A viridis
In order to test for the presence of caspase-like activity
in the symbiotic sea anemone A viridis, Asp-Glu-Val-Asp (DEVD)-dependent (Fig 1A) and Ile-Glu-Thr-Asp-Glu-Val-Asp (IETD)-dependent (Fig 1B) protease activities were tested in animal host cells (ectoderm and gastroderm) and in freshly isolated zooxanthellae For both sub-strates, high protease activities were measured in the animal host while only low activities were measured in the freshly isolated zooxanthellae extracts Moreover,
in the host extracts, IETD substrate presented a two-fold higher rate of 7-amino-4-trifluoromethylcoumarin (AFC) cleavage than DEVD-AFC substrate
Addition of the inhibitors DEVD-adelhyde (CHO) and IETD-CHO specific for the caspases 3 and 8, respectively, completely abolished both protease activit-ies in the ectodermal and gastrodermal tissue extracts (Fig 2) Table 1 summarizes the IC50 for both inhibi-tors, obtained by incubating extracts in each specific substrate In both tissue extracts, DEVD- and IETD-dependent protease activities showed the same sensitivity for the DEVD-CHO competitive substrate Surprisingly, the DEVD-CHO inhibitor had a higher effect on IETD-dependent protease activity (3 nm) than
on the DEVD-dependent protease activity (15–20 nm) For both tissue extracts and protease substrates, the IETD-CHO inhibitor showed higher IC50values with a predictably higher sensitivity of IETD-dependent activity
to this inhibitor Although inhibition of IETD-depend-ent protease activity by IETD-CHO was similar in ectodermal and gastrodermal cells, inhibition of DEVD-dependent protease activity by the same inhibitor was lower in ectodermal cells than in gastrodermal cells
Identification of a caspase-like cDNA from
A viridis
To confirm the presence of caspases in A viridis tissues, cDNA encoding a caspase 3-like protein was isolated from ectodermal cells Using a PCR
Trang 3approach with degenerate primers based on two
highly conserved caspase 3 domains, we obtained a
1627 bp sequence named AvCasp3 (accession number
DQ097195) containing an open reading frame of
1239 bp (Fig 3) The predicted amino acid sequence
of 413 amino acids (Fig 3) is highly conserved with
vertebrate caspase 3 sequences (Fig 4) and was
there-fore named caspase 3-like By homology with known
vertebrate caspases, we determined that the long form
of this sequence contains a prodomain, a large (p20)
and a small (p10) subunit We identified two potential
cleavage sites at aspartate residues 164 and 172 for
cleaving the prodomain, and a potential cleavage site
at Asp306 for the cleavage between the large and
small subunits The prodomain presents a caspase
recruitment domain (CARD) consisting of six alpha
helices (Fig 3) [32] The large subunit contains highly conserved LS⁄ THG and QACXG sequences sur-rounding histine (His255) and cysteine (Cys294) resi-dues of putative active site The substrate binding site
is highly conserved and composed of Arg337, Ser343, Gln296 and Arg198 (Fig 3)
Large and small subunit sequences from various vertebrate and invertebrate caspase 3 or caspase 7 sequences were aligned with the A viridis caspase 3-like sequence The phylogenetic comparison (Fig 4) shows that AvCasp3 and other cnidarian sequences delineate a specific branch more closely related to exe-cutioner vertebrate caspases 3 or 7 than to caspases from other invertebrates models (Drosophila melano-gasterand Caenorhabditis elegans)
Effect of heat stress on apoptosis-like induction
in animal tissue of A viridis
In order to study the effect of a heat stress (+8C above ambient) on caspase-like activities, DEVD- and IETD-dependent protease activities were measured in the animal extracts of A viridis throughout the stress (7 days at 25C) In the ectodermal tissue, the DEVD-dependent protease activity decreased while IETD-dependent activity did not vary significantly (Fig 5A)
In the gastrodermal tissue (Fig 5B), both activities increased The DEVD-dependent activity was twofold higher than controls (17C) after 48 h of stress, while the IETD-dependent activity was 1.5-fold higher After
7 days at 25C, activities in both tissues were restored
to control levels
To confirm the induction of a specific caspase-like activity and not a generic protease activity, all meas-urements were also performed in the presence of
1 lm DEVD-CHO and 10 lm IETD-CHO (data not shown) In these conditions, caspase-like activity was totally abolished The induction of an apoptosis-like phenomenon in A viridis subjected to a heat stress was confirmed by the analysis of DNA fragmentation in anemone tissues, using a dUTP nick end labeling (TUNEL) assay Figure 6 shows the increase of end-labeling DNA after 48 h of thermal stress (25C) DNA fragmentation occurred largely in the gastroder-mal tissue harboring the zooxanthellae (Fig 6B)
Effect of heat stress on antioxidant defenses and bleaching in animal tissue of A viridis
The occurrence of oxidative stress in the animal tissue (ectoderm and gastroderm) and freshly isolated zoox-anthellae of A viridis was monitored during heat stress (+8C above the control temperature) by the
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Fig 1 Caspase-like activities in host epithelial tissues (ectoderm
and gastroderm) and zooxanthella extracts of A viridis maintained
in control condition (+17 C) Caspase 3-like (A) and caspase 8-like
(B) activities were assayed by fluorometric method using,
respect-ively, Ac-DEVD-AFC and Ac-IETD-AFC as substrates One hundred
and twenty-five micrograms of protein have been tested for
ecto-derm, gastroderm and zooxanthella extracts Results are expressed
as means ± SE of at least six independent tissue extractions from
distinct sea anemones.
Trang 4measurement of oxygen radical-scavenging capacities
(Fig 7) The ectodermal antioxidant capacity did not
change significantly over the stress period while the
gastrodermal antioxidant capacity increased starting at
6 h, peaking at 24 h at 2.5-fold higher than control
values and decreasing after 48 h In the zooxanthellae,
the oxygen radical-scavenging capacity decreased
signi-ficantly after 24 h
Because the stressed organisms present an evident
loss of pigmentation during the stress, concomitant
analyses have been conducted on whole tentacles of
A viridis to highlight the bleaching event Figure 8
shows a rapid decrease of chlorophyll (a+c2) content
in the first days of the stress period that became significant with a two times decrease at the end of the kinetic
Discussion
In this study, we have investigated a pathway for sym-biosis breakdown (bleaching) in the symbiotic associ-ation A viridis during an elevated temperature stress
We have also demonstrated connections between oxi-dative stress and host programmed cell death during the bleaching event
Characterization of caspase-like activities
in A viridis
In order to test for the presence of programmed cell death or apoptosis in A viridis subjected to heat stress,
we measured protease activities using mammalian caspase substrates In control conditions, we measured high specific protease activities in the animal compo-nent (ectoderm and gastroderm) of A viridis while, in freshly isolated zooxanthellae an activity was almost undetectable The presence of high caspase-like activ-ities, in animal tissues of control animals, could be
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Fig 2 Inhibition of DEVD- and IETD-dependent protease activities in animal tissue of A viridis by commercial synthetic peptide inhibitors One hundred and twenty-five micrograms of ectodermal (A,B) and gastrodermal (C,D) extracts were incubated with the fluorochromic caspase substrate Ac-DEVD-AFC (A,C) or Ac-IETD-AFC (B,D) and with the competitive inhibitors Ac-DEVD-CHO (d) or Ac-IETD-CHO (s) Results are expressed as means ± SE of at least six independent tissue extractions from distinct sea anemones.
Table 1 IC50of the competitive substrate inhibitors Ac-DEVD-CHO
and Ac-IETD-CHO on the DEVD-dependent and IETD-dependent
protease activities in the two animal tissue extracts of A viridis.
Tissue extracts
Protease
substrates
Ac-DEVD-CHO
IC50(n M )
Ac-IETD-CHO
IC50(n M )
Trang 5related to the high regeneration ability of cnidarians.
In fact, the role of apoptosis in development and
regeneration has been determined not only in
verte-brates (i.e., bone regeneration; reviewed in [33]) but
also in invertebrates such as cnidarians and flatworms
[34–36] Apoptosis is considered a necessary
character-istic of all self-renewing tissues and its presence has
been detected not only in stressed organisms but also
in healthy ones Mire and Venable [34] reported that
up to 10% of cells from the sea anemone Haliplanella
lineata contained TUNEL-labeled nuclei even under control conditions
Moreover, protease activities related to animal extracts display several properties characteristic of caspases, the critical central molecules of apoptotic pathways First, they were activated by two polypep-tides, DEVD and IETD, which are used to distinguish some of the caspase classes in mammalian cells DEVD-AFC is generally cleaved by caspase 3, which belongs to executioner caspases [37] while, IETD-AFC
Fig 3 Nucleotide and deduced amino acid sequence of caspase 3-like cDNA of A viridis (AvCasp3) Putative prodomain sequence appears
in italic characters, the small subunit in regular type The large (p20) subunit in bold and the small subunit fit between the two domains Residues boxed are the component of substrate binding site Asterisks indicate the His and Cys residues of the putative active site located
in the large subunit The six a-helix components of the prodomain are underlined.
Trang 6is cleaved by caspase 8, an initiator caspase [38] In
control specimens of A viridis, IETD-dependent
prote-ase activity was two times higher than the
DEVD-dependent one, suggesting a predominantly caspase
8-like activity in the animal tissue Secondly, the
spe-cificity of detected caspases has been tested using their
respective competitive substrates: DEVD-CHO and
IETD-CHO [39] The inhibition of the two protease
activities by competitive substrates strengthens the
involvement of a caspase-like activity, in animal tissue
of A viridis, avoiding interference by generic
proteas-es In the two animal cell layers, the effect of the two
inhibitors was similar Although we measured a higher
efficiency of DEVD-CHO for IETD-dependent activity
in both tissues of A viridis (Table 1), in the literature
this inhibitor was found to be highly effective on both
caspase 3 and caspase 8 activities but still more
speci-fic to caspase 3 [39] DEVD peptide, which was
devel-oped as a caspase 3 inhibitor, is also a fairly potent
inhibitor of caspases 1, 4 and 7, and is not
conse-quently selective for a particular caspase [40] This
seems to indicate that tetrapeptide-based inhibitors are
unlikely to achieve the specificity required to allow
selective inhibition of caspases However, compared to
results related to inhibitor specificity performed on
mammalian cells, we can conclude that there are at
least two original caspase-like activities in the animal
tissue of A viridis
Previous work has already highlighted the presence
of a caspase-like activity in the hydrozoan Hydra vul-garis using DEVD substrate and DEVD-CHO inhib-itor The presence of such an enzyme in cnidarians was confirmed by gene sequencing first, in H vulgaris with 3A and 3B Casp, sharing a high degree of identity with, respectively, C elegans CED3 and human Casp 3, respectively [29] More recently a caspase 3-like cDNA has been sequenced in both the anemone
A pallidasharing high identity with 3B casp of H vul-garis [31] and in Hydractinia vulgaris [30] We also confirmed the presence of a caspase 3-like cDNA in
A viridishost tissues We obtained a full-length cDNA sequence from ectodermal tissue with a deduced amino acid sequence that is closely related to vertebrate exe-cutioner caspases 3⁄ 7 All of the conserved residues involved in the catalytic mechanism of caspases are present in AvCasp3, as well as cleavage sites identified
by homology with vertebrate caspases However, this sequence also possess a long prodomain homologous
to the CARD domain [41], mostly similar to the initi-ator caspases 2, 8, 9 and 10 [42] The characteristics have been described in Acasp from the sea anemone
A pallida[31] Acasp (large and small subunits) shares
an 81% identity and 91% similarity with AvCasp3 but only a 40% identity and 57% similarity with the caspase 3B from H vulgaris As cnidarian caspase 3-like sequences shared both characteristics of
Rattus norvegicus Casp3 Mus musculus Casp3 Homo sapiens Casp3 Gallus gallus Casp3 Xenopus laevis Casp3 Danio rerio Casp3 Salmo salar Casp3B Xenopus laevis Casp7 Rattus norvegicus Casp7 Homo sapiens Casp7
Hydra vulgaris Casp3B Aiptasia pallida Casp3-like
Anemonia viridis Casp3-like
D melanogaster Casp3
Homo sapiens Casp8 Mus musculus Casp8
C elegans Casp3
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Fig 4 Phylogenetic comparison of A viridis
caspase with caspase sequences from
ver-tebrates, invertebrates caspase 3 and from
vertebrates caspase 7 Vertebrate caspase 8
sequences have been used as an outgroup.
The tree was derived from alignments of
p10 and p20 domains excluding the
pro-domain.
Trang 7tioner (caspases 3 and 7) and initiator caspases
(CARD domain, caspases 2, 8, 9, 10), this suggests
that cnidarian caspase 3-related enzymes may be
considered as potential ancestors of other metazoan and vertebrate executioner caspases [31] This has also been suggested for H vulgaris caspase 3-like [29] and
by Wiens et al [43] for sponge caspase 3-like enzymes Furthermore, the caspase 3-like gene we described in
A viridis appears more related to vertebrates than to other invertebrate biological models such as nematodes and flies [44] This could be explained by the basal position of cnidarians on the metazoan tree and by the extensive gene loss in protostomes
Caspase-like activity and thermal stress After 2 days of heat stress, the increase in at least two caspase-like activities detected in the animal tissue and the DNA fragmentation induction in the gastrodermal cells suggest the involvement of apoptotic events dur-ing the first hours of high temperature treatment It also confirms previous work, which has already dem-onstrated the induction of apoptosis in cnidarians sub-jected to heat stress [8,9] Because the present data constitute the first evidence of caspase activation under heat stress in cnidarian, further experiments are required to exclude the hypothesis of involvement of this latter enzyme in mechanisms other than the apop-totic cascade Nonapopapop-totic functions of caspase 3 have been described recently in human nervous tissue [45,46] Dunn et al [8,9] have recently reported an increase in morphological apoptotic indicators in the sea anemone Aiptasia sp incubated at high tempera-ture These authors reported a high frequency of cells with apoptosis-like morphology predominantly in the gastrodermal host cells and from the first hour of exposure A similar time-dependent pattern has been
Ectoderm
Gastroderm
control condition (17°C) stress condition (25°C)
20 µm
Mesoglea
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Fig 6 Temperature-induced DNA fragmen-tation in tentacle tissue of A viridis DNA fragmentation in specimen maintained at
17 C (A, control condition) or incubated at
25 C for 48 h (B, stress condition) was revealed by TUNEL staining with DAB ⁄ H 2 O2 substrate Arrows indicate the different cell layers.
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Fig 5 Temperature-induced protease activity in host epithelial
tis-sue of A viridis One hundred and twenty-five micrograms of
ecto-dermal (A) and gastroecto-dermal (B) tissue extracts were incubated
with the Ac-DEVD-AFC (Caspase 3; black bars) and Ac-IETD-AFC
(Caspase 8, white bars) substrates The assays were performed
during the increase temperature treatment (25 C) and are
expressed as percentage of control (temperature incubation
17 C) ± SE of at least five independent tissue extractions from
dis-tinct sea anemones Asterisks indicate significant differences
between control and stress conditions (P < 0.05; ANOVA ).
Trang 8observed in our study, however, the apoptotic events
appear later in the heat stress (with a high activity
reported at 48 h) The difference between the two
stud-ies could be related to a specstud-ies-specific sensitivity or
to the temperature range Moreover, the later
induc-tion of necrotic events observed by Dunn et al [9]
could be correlated with the caspase-like activity
decrease measured in A viridis gastrodermal tissue
after 7 days of treatment The decrease in ectodermal
caspase-like activity observed after 6 h was, however,
not related to necrotic cell death because it concerned
only DEVD-dependent protease activity It could be
the result of caspase inhibition by a still undetermined
mechanism Implication of different molecules is
suggested, such as inhibitor of apoptosis protein and heat shock proteins [47,48]
Caspase-like activity and oxidative stress After the finding of caspase-like activities and a response by these enzymes to heat stress, parallel ana-lyses were conducted to follow the occurrence of oxi-dative stress in the stressed organisms Variations of antioxidant defenses and caspase activities were then compared
An increase in caspase 8-like activity appears after
24 h of gastrodermal antioxidant defense induction High protein damage has been shown in previous stud-ies and supports the occurrence of an oxidative stress period in heat stressed A viridis [24] We suggest that apoptosis induction could be the consequence of the previous oxidative stress event, a phenomenon that is well established in vertebrates [27] Increases in ROS are the consequence of electron transport chain impair-ments, principally in mitochondria and chloroplasts and can directly and⁄ or indirectly cause caspase activa-tion (reviewed in [25,27,49]) The delay observed in our study between the antioxidant induction and the caspase-like activation largely supports this hypothesis
Bleaching event and oxidative stress Several studies have underlined the effect of thermal stress on symbiotic cnidarians [1,2] and several hypo-theses have been suggested to explain the mechanisms
of symbiosis breakdown, focused on the respective implication of both partners in the phenomenon In the zooxanthella, heat stress has been demonstrated to reduce the photosynthetic rate by decreasing the effi-ciency of the photosystem II [4–6] and⁄ or by causing damage to the Calvin cycle [50,51] Moreover, in situ degeneration of zooxanthellae has been reported in corals [52–54] and sea anemones [8,9,55] Heat stress was also documented to induce host cell degeneration [8,9,53,56] and⁄ or gastrodermal detachment [7,52,57] Nevertheless, independently of the resulting effect of the thermal stress, all mechanisms point to the involve-ment of oxidative stress in the early stages of symbiosis breakdown In fact, several authors have suggested the involvement of ROS production in the zooxanthella photoinhibition (reviewed in [1]), gastrodermal cell detachment [7], and host and symbiont degeneration [9,23] However, molecular mechanisms linked to ther-mal stress induction, production of ROS and its phy-siological consequences (e.g., photoinhibition and cell degeneration) are still unclear In previous work, Richier et al [24] showed the occurrence of oxidative
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Fig 7 Temperature-induced antioxidant activity in tentacle tissue
of A viridis Relative antioxidant activities were measured in 1 lg
of ectodermal (black bars), gastrodermal (grey bars) and
zooxanthel-la (white bars) extracts by fluorometric assay during the increase
temperature treatment (25 C) and are expressed relative to control
condition (temperature incubation 17 C) ± SE of at least five
inde-pendent tissue extractions from distinct sea anemones Asterisks
indicate significant differences between control and stress
condi-tions (P < 0.05; ANOVA ).
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Fig 8 Chlorophyll a and c 2 content in A viridis total tentacle extract
during thermal stress (25 C) Results are expressed as means ± SE
of at least three independent chlorophyll extractions from distinct
sea anemone tentacles Asterisks indicate significant differences
between control and stress conditions (P < 0.05; ANOVA ).
Trang 9stress in the animal host and its time course of
appear-ance While symbiotic sea anemones seem to be more
resistant to thermal stress than nonsymbiotic species,
there was nonetheless an increase in oxidative attack
on proteins, as evidenced by the carbonylation of
pro-tein in A viridis after a thermal increase of 8C in
gastrodermal cells where zooxanthellae are housed
[24] The present study supports these previous results
and shows the induction of antioxidant defenses
exclu-sively localized within the animal compartment and
more precisely in gastrodermal cells These results
sug-gest the induction of antioxidant defenses in the
gastro-dermal compartment in order to counteract the
increase in cellular damage By contrast, a decrease in
global antioxidant defenses in the zooxanthellae was
observed during the experiment Previous results
obtained by Lesser and coworkers [18,19] and Richier
et al [24] showed a slight increase in the activity of the
antioxidant enzymes superoxide dismutase and
ascor-bate peroxidase in zooxanthellae Nevertheless, a
glo-bal decrease in zooxanthellae antioxidant defenses
during thermal stress would suggest two hypotheses:
(a) a dysfunction of zooxanthella metabolism induced
by necrosis or programmed cell death as suggested by
Dunn et al [8,9]; or (b) a decrease in antioxidant
def-enses following a chlorophyll decrease as demonstrated
by Shick et al [58] In this thermal stress experiment
(+8C), we have observed a significant decrease of
chlorophyll content after 7 days incubation Moreover,
visible bleaching of the experimental animals occurred
as the heat stress incubation progressed This result
supports the induction of a bleaching event as a
conse-quence of an oxidative stress period
In conclusion, our results contribute to the
under-standing of the mechanisms involved in coral bleaching
events in host cells of symbiotic cnidarians
Gastroder-mal cells appear to be the predominant location of
thermal stress impact In the first hour of stress, the
gastrodermal cells undergo oxidative stress, which is
rapidly followed by apoptotic events and completed by
occurrence of bleaching The gastrodermal cell death is
then hypothesized to be responsible of zooxanthella
expulsion and⁄ or gastrodermal cell detachment
Fur-ther investigation will, however, be necessary to link
gastrodermal cell death with zooxanthella
photoinhibi-tion and expulsion Finally, this work contributes to
the investigation of the evolutionary conservation and
the role of apoptosis in basal metazoans
Experimental procedures
Unless otherwise specified, all chemicals were obtained
from Sigma-Aldrich (St Louis, MO)
Biological materials Specimens of the Mediterranean sea anemone, Anemonia viridis (Forska˚l), were collected in Villefranche-sur-mer (France) and maintained in a closed-circuit seawater aquar-ium at 17 ± 1C Half of the aquarium seawater was changed every week A metal halide lamp (HQI-TS, 400 W; Philips, Eindhoven, the Netherlands) provided light, at a constant saturating irradiance of 250 lmolÆquanta m)2Æs)1
on a 12 h light⁄ 12 h dark regime
Experimental designs Three tanks, each containing one anemone, were heated from 17 ± 1C (control condition) to 25 ± 1 C (stress condition) over 2 h and maintained at 25C for 7 days During the course of the experiment, all tanks were main-tained under identical illumination conditions (250 lmolÆ quanta m)2Æs)1, 12 h⁄ 12 h L ⁄ D) and the sea anemones were not fed Five to ten tentacles from each specimen were sam-pled from each aquarium after 6, 24, 48 and 168 h after the initial temperature increase The experiment was repeated twice with distinct sea anenome specimens
Tissue extractions The three cellular compartments of the symbiotic associ-ation (host ectoderm, host gastroderm and zooxanthellae) were extracted from tentacles according to Richier et al [13], avoiding any contamination between zooxanthellae protein and the host gastrodermal cell Each extract was prepared at 4C in the appropriate medium for the subse-quent analyses
Oxygen radical-scavenging assay The oxygen radical-scavenging activities of different tissue extracts were determined using a fluorometric assay accord-ing to Naguib [59] Tissue extractions were performed in a medium containing 50 mm phosphate buffer (pH 7.0),
2 mm phenylmethylsulfonyl fluoride and 10 lgÆml)1 prote-ase inhibitor cocktail (P8340, Sigma) The volume corres-ponding to 1 lg of protein extract was then incubated with
75 mm phosphate buffer (pH 7.0), 15 nm fluorescein as fluorescent probe, 3 mm 2,2¢-azobis (2-amidino-propane) dihydrochloride as the peroxyl radical generator, and 1 lm Trolox as antioxidant standard The decay of fluorescence signal was recorded by a spectrofluorometer (Safas, Monaco) at an excitation⁄ emission wavelength of
520⁄ 495 nm every minute for a total duration of 45 min
Caspase assay Caspase 3-like and caspase 8-like activities were assayed fluorometrically using the specific substrates
Trang 10Ac-DEVD-AFC
(N-acetyl-Ile-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin) and Ac-IETD-AFC
(N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin), respectively (Biosource
International, Cliniscience, Montrouge, France) Tissue
extractions were performed in a medium containing 25 mm
Hepes (pH 7.5), 5 mm MgCl2, 5 mm 1,4-dithiothreitol,
2 mm phenylmethylsulfonyl fluoride and 10 lgÆml)1
prote-ase inhibitor cocktail (P8340, Sigma) A quantity of 125 lg
of ectodermal, gastrodermal and zooxanthella extracts was
incubated in a reaction buffer containing 50 lm specific
probe, 100 mm Hepes (pH 7.5), 10% (v⁄ v) sucrose, 0.1%
(v⁄ v) CHAPS, 10 mm dithiothreitol and dimethylsulfoxide
The AFC fluorescence was measured in a
spectrofluorome-ter (Safas, Monaco) at an excitation⁄ emission wavelength
of 400⁄ 505 nm every 3 min for 21 min for animal
compart-ments and extended to 60 min for zooxanthellae extracts
Caspase-like activities were expressed in picomol of AFC
cleavage per minute, according to a standard curve
obtained from AFC (A8401, Sigma) For inhibition
experi-ments, the competitive peptide (inhibitor) Ac-DEVD-CHO
or Ac-IETD-CHO were added to the reaction buffer prior
the addition of the fluorometric substrate
RNA extraction
RNA from the ectodermal compartment was extracted
from six tentacles of A viridis according to a modified
pro-tocol of Trizol extraction (Invitrogen, Carlsbad, CA) The
tentacles were isolated and immediately dried with blotting
paper The gastrodermal cells were scraped at 4C with
forceps in order to effectively separate the ectodermal cell
layer, which was dissolved in 2 mL Trizol using a glass
ho-mogenizer Homogenate was then processed in accordance
with Invitrogen recommendations, followed by final
addi-tional treatment using chloroform After the precipitation
step using 70% ethanol, the RNA pellet was finally air
dried and dissolved in RNAse-free water The RNA was
quantified by measuring the absorbance at 260 nm (Safas
UVmc2 spectrophotometer)
RT-PCR
Total RNA from the ectodermal fraction of A viridis was
reverse transcribed using oligodT primer and Superscript II
reverse transcriptase (Invitrogen) Degenerate primers were
designed from two highly conserved regions of caspase 3
amino acid sequences from phylogenetically different
organisms (Hydra vulgaris AAF98012, Homo sapiens
AAH15799, Aiptasia pallida DQ218058): CniCaspF,
5¢-CAYGGNGARGARGGRAT-3¢ and CniCaspR 5¢-AT
NGANGGDATYTGYTTYTT-3¢) A volume of 0.5 lL of
ectodermal cDNA was PCR amplified using CniCaspF
(300 nm), CniCaspR (300 nm) and Platinum TAQ
poly-merase (Invitrogen) PCR products were analyzed by 2%
(w⁄ v) agarose gel electrophoresis, then subcloned into
pGEM-T Easy vector (Promega, Madison, WI) and se-quenced (Macrogen Inc, Seoul, South Korea)
Rapid amplification of cDNA ends
To further obtain the full-length cDNA sequence of A vir-idis caspase, 5¢ ⁄ 3¢ RACE-PCR kit and Expand Long Template DNA Polymerase (both from Roche, Mannheim, Germany) were used For 3¢-RACE, the gene specific prim-ers were: AvCasp1F (5¢-CTTGGCGAAACTCAGTCAAT GG-3¢) and AvCasp2F (5¢-CTGCTGACAATGATGACG AGAG-3¢) For 5¢-RACE, the gene specific primers were: AvCasp1R (5¢-GTCAGCAGATCTGTGGTTTTG-3¢), AvCasp2R (5¢-CCATTGACTGAGTTTCGCCAAG-3¢) PCR products were cloned into pGEM-T Easy vector and sequenced
Sequence analysis The blast sequence analysis program [60] was used for ini-tial comparisons Multiple alignment of large (p20) and small (p10) caspase 3 domains from vertebrates (Homo sapiens P42574, Mus musculus P70677, Rattus norvegicus NP037054, Gallus gallus AF083029, Danio rerio AAH78310, Salmo salar AAY28972, Xenopus laevis P55866) and invertebrates (Hydra vulgaris AAF98012, Aiptasia pallida DQ218058, Drosophila melanogaster AAD54071, Caenorhabditis elegans P42573), of vertebrate caspase 7 sequences (Homo sapiens AAH15799, Rattus norvegicus NP071596, Xenopus laevis AAH78049) and of vertebrate caspase 8 sequences as an outgroup (Homo sap-iensQ14790, Mus musculus AF067834) was performed with clustalx program [61] A phylogenetic tree was derived from alignments using the Neighbour Joining method and the mega3 software [62] Secondary structure (alpha heli-ces) of the corresponding peptide was predicted using the PSIPRED server [63]
TUNEL assay DNA fragmentation was identified in situ by terminal de-oxynucleotidyl transferase mediated dUTP nick end labe-ling (TUNEL) labelabe-ling [64] Tentacle bags of A viridis [65] were fixed with 4% (w⁄ v) paraformaldehyde in a fixation buffer (450 mm NaCl, 10 mm KCl, 58 mm MgCl2, 100 mm Hepes pH 7.8) overnight at 4C Tissues were then dehy-drated in ethanol series, cleared with toluene and embedded
in paraplast Sections of 8 lm-thick were attached to Silane-Prep slides, deparaffinized with xylene, and
rehydrat-ed in ethanol series Sections were successively incubatrehydrat-ed in proteinase K (1 ngÆml)1 in TE buffer) for 15 min at room temperature and in TdT buffer (140 mm cacodylate, 1 mm cobalt chloride, 30 mm Tris pH 7.4) for 5 min End-labeling was carried out in 45 lL TUNEL-labeled dUTP (Roche