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First published online January 3, 2006
Journal of Experimental Biology 209, 273-283 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.01983
Diel `tuning' of coral metabolism: physiological responses to light cues
1 Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900,
Israel
2 Israel Oceanographic and Limnological Research, Kinneret Limnological
Laboratory, Migdal 14950, Israel
* Author for correspondence at present address: The University of Queensland, Centre for Marine Studies, Cooper Rd, Gehrmann Building (Bldg. 60), St Lucia, Brisbane, QLD 4072, Australia (e-mail: o.levy{at}uq.edu.au)
Accepted 14 November 2005
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F'/Fm'). SOD and CAT activity
in the symbiotic algae also exhibited a light intensity correlated pattern,
albeit a less pronounced one. The observed rise of the free-radical-scavenger
enzymes, with a time scale of minutes to several hours, is an important
protective mechanism for the existence and remarkable success of the unique
cnidarian-dinoflagellate associations, in which photosynthetic oxygen
production takes place within animal cells. This represents a facet of the
precarious act of balancing the photosynthetic production of oxygen by the
algal symbionts with their destructive action on all living cells, especially
those of the animal host.
Key words: SOD, catalase, Photosystem II, zooxanthellae, photosynthesis, Favia favus, Plerogyra sinuosa, Goniopora lobata
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). Daily changes in
light intensity and spectrum, temperature, and nutrient and food availability
cause changes in respiration and photosynthesis, enzyme activity and pigment
composition and concentration. Increasing irradiance results in an increase in
photosynthesis rates (Falkowski and Raven,
1997). Under high irradiance levels in shallow reefs, corals
experience elevated oxygen partial pressures (PO2) in
their tissues as a result of the oxygen produced by their zooxanthellae
(Dykens and Shick, 1982),
apparently without any cellular damage. High light levels, elevated seawater
temperature and UV radiation lead to an increase in reactive oxygen species
(ROS) within the algae as well as in the host (Lesser and Shick,
1989a,b;
Dykens et al., 1992), which is
harmful to both (Lesser and Shick,
1989a,b).
Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase
function together to inactivate the harmful oxygen free radical and hydrogen
peroxide (H2O2), thereby preventing subsequent cellular
damage (Fridovich, 1986;
Asada and Takahashi, 1987).
Protective mechanisms against active oxygen species in both the animal host
and its zooxanthellae (Lesser and Shick,
1989a,b)
have been described. Responses include changes in pigment content and in the
activities of SOD, CAT and ascorbate peroxidase
(Shick et al., 1995).
The photosynthesis-irradiance relationship in corals changes in response to
ambient light (Falkowski et al.,
1990). Zooxanthellae, like most algae, photoacclimate to changes
in irradiance by adjusting light-harvesting and utilization capability (for
reviews, see Falkowski and Owens,
1980; Stambler and Dubinsky,
2004), by changing cellular pigment concentrations
(Dubinsky et al., 1984;
Porter et al., 1984).
Photoacclimation also includes changes in the respiration of the
zooxanthellae, the quantum yield and the light-saturated rate of
photosynthesis (Porter et al.,
1984). Various photoacclimation processes differ in their kinetics
(Fisher et al., 1996). While
most of these changes take longer than a day, some, such as polyp expansion
and contraction, occur within minutes
(Levy et al., 2003). Changes
in the density of the zooxanthellae and reshuffling of their species occur
within days, whereas changes in growth form of the coral colony may take
several months to years (Graus and
Macintyre, 1982; Miller,
1994; Todd et al.,
2004), and genetic selection takes thousands of years
(Falkowski et al., 1990).
In the past few decades, coral reefs have been exposed to natural and
anthropogenic stresses, such as increases in temperature and UV radiation,
which cause major bleaching events over entire coral reef systems (e.g. Glynn,
1993,
1996;
Hoegh-Guldberg, 1999;
Fitt et al., 2001;
Coles and Brown, 2003;
Jokiel, 2004). Eutrophication
and pollution are likely to act synergistically with temperature in rendering
corals more susceptible to bleaching and disease
(Dubinsky and Stambler, 1996).
Hypotheses for the mechanism of bleaching include (1) elevated oxygen tension
in the symbionts and the host, leading to internal damage by ROS
(Shick et al., 1996), (2)
reduction of the photosynthesis/respiration ratio, producing an energetic
imbalance (Jokiel and Coles,
1990), and (3) free-radical-induced damage to Photosystem II
(PSII) of the zooxanthellae (Lesser,
1996), triggering a membrane lipid mediated apoptosis cascade
(Tchernov et al., 2004) and in
some cases pathogen infection (see
Rosenberg and Loya, 2004).
The aim of the present study was to document some responses of corals and their symbionts to the diel patterns of solar radiation. The study of concomitant changes in metabolism, oxygen evolution, fluorescence yields, pigments, and protective enzymes (SOD, CAT) correlated or tracking the course of sunlight, will afford a better understanding of the processes occurring in the host and in the zooxanthellae, allowing corals to optimize the benefits of photosynthesis while minimizing the inevitable risks of concurrent oxidative damage.
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) of Favia favus Forskal, Plerogyra
sinuosa Dana and Goniopora lobata Milne Edwards and Haime of
similar size (
10 cm diameter) were collected from a depth of 4-5 m. These
corals were chosen because we have information on some aspects of their
energetics, such as prey capture, photosynthesis and their
expansion/contraction behavior (see Levy et al.,
2003,
2004, 2005a,b). The daily
metabolism (photosynthesis and respiration) and chlorophyll fluorescence
measurements were performed during July-August 2001. From December 2001 until
March 2002, additional colonies of the same species, 30 cm in diameter,
were collected from 4-5 m-depth next to the Interuniversity Institute for
Marine Sciences of Eilat (the H. Steinitz Marine Biology Laboratory), Israel.
Three mother colonies from each species were divided into six subcolonies of
5 cm diameter each and placed at 4-5 m-depth for 75 days for `healing'
and acclimation, before experimentation. One subcolony from each species was
placed in the respirometer (see below) for 24-h measurements. After
measurement, these subcolonies were frozen in liquid nitrogen. Parallel to the
respirometer experiment, every two hours during daytime (08.00-16.00 h) and
before dawn (05.00 h), three subcolonies from each species were sampled and
immediately frozen in liquid nitrogen. The samples were stored for 1-2 weeks
at -70°C before analysis of the biomass parameters and enzyme
activity.
Measurement of the light spectrum and UV
The light spectrum was measured at 5 m-depth (in the Gulf of Eilat, Red
Sea, in front of the Interuniversity Institute for Marine Science).
Measurements were conducted on several cloudless days during 15-19 February
2001. A spectral scan was performed using a Li-Cor LI1800 scanning
spectroradiometer (Lincoln, NE, USA). Readings were conducted every 30 min at
2 nm interval spans between 300 and 750 nm (units are W m-2
nm-1). Only data from 15 February are presented since that day was
considered representative for a cloudless day. The data represent only UV
measurements between 300 and 400 nm, which include UVA + UVB.
Photosynthesis and dark respiration
The in situ oxygen flux data were obtained using twin,
three-chamber submersible respirometers (AIMS, Townsville, QLD, Australia).
This instrument is equipped with UV-transparent chambers, each with an oxygen
sensor (Kent EIL galvanic type ABB, Stonehouse, Gloucestershire, UK), one
light meter (Li-Cor 4
underwater quantum sensor), a temperature probe and
a data logger. A centrifugal pump flushes the water in the chambers at
programmable intervals. Twenty-minute intervals were used for these
experiments (Fabricius and Klumpp,
1995). Prior to the incubation period, the colony surfaces were
carefully cleaned of epiphytes and other debris using a small toothbrush. The
respirometers were deployed at 4-5 m-depth in front of the H. Steinitz Marine
Biology Laboratory (near the incubation site). Data processing was performed
using the AIMS `Respiro' program for calibrating and normalizing the data.
Respiration was measured as oxygen uptake in the dark. Parameters for the
photosynthesis (P) versus energy (E) curves were
calculated from a non-linear curve-fitting, including 
(initial slope).
Pmax (light-saturated photosynthesis rate and compensation
light levels), Eopt (optimum irradiance),
Ecom (compensation intensity) and Ek
(saturating intensity) were calculated from a non-linear curve-fitting based
on theoretical models of a hyperbolic tangent equation
(Ben-Zion and Dubinsky,
1988).
FRR fluorescence measurements of the quantum
Diel changes in chlorophyll fluorescence and fluorescence characteristics
of the three symbiotic corals were monitored for three consecutive days for a
diurnal cycle from 31 July to 2 August 2001, using the SCUBA-based Fast
Repeating Rate Fluorometer (FRRF; Gorbunov
et al., 2000). The FRRF measures the chlorophyll fluorescence
yield using a sequence of flashlets that gradually close the PSII reaction
centers, leading to an increase in chlorophyll fluorescence
(Kolber and Falkowski, 1993).
The maximum quantum yield of photochemistry in PSII was determined in a
dark-adapted state as the ratio of
Fv/Fm=(Fm-Fo)/Fm,
according to Butler (1972),
where Fv is variable fluorescence, Fm
and Fo are maximum and minimum yields of chlorophyll
a fluorescence measured in a dark-adapted state (relative units). The
steady-state quantum yield was measured under illumination by ambient light
and defined by
F'/Fm'=(Fm'-F')/Fm'
(see Gorbunov et al., 1999,
2000;
Lesser and Gorbunov, 2001).
The quantum yield of irradiance-stimulated thermal dissipation [i.e.
non-photochemical quenching (NPQ)] is assessed from the ratio of
(Fm-Fm')/Fm
(Gorbunov et al., 2001). The
instrument was placed on a tripod at 5 m-depth, and measurements were taken
automatically from the top part of the coral colony
(Lesser and Gorbunov, 2001) at
10-min intervals. Cross section was calculated
(Gorbunov et al., 2001). The
data presented show mean measurements for each hour.
Biomass parameters
Tissue homogenates were prepared by removing all tissue by an airbrush
method, which is similar to the Water-Pik method
(Johannes and Wiebe, 1970) but
uses airjet to strip the tissue of the coral skeleton into a few ml of 100
mmol l-1 phosphate buffer (pH 7.0). The volume of the homogenate
was measured and subsamples taken for determination of: (1) zooxanthellae
density, from direct counts on a Neubauer hemacytometer; (2) chlorophyll
a concentration, measured spectrophotometrically on a Cary
spectrophotometer (Varian, Palo Alto, CA, USA) in 90% acetone
(Jeffrey and Humphrey, 1975)
and (3) enzyme activity, quantified using the methods described below. The
surface area was calculated based on a digital photograph using the program
Image tool (Uthscsa Image Tool for Windows, v. 2.00, University of Texas, San
Antonio, TX, USA).
Enzyme assays
The homogenate was centrifuged twice at 1500 g for 15 min.
The supernatant was used for analysis of animal protein and enzyme activity.
The pellets containing the zooxanthellae were suspended in 2 ml 100 mmol
l-1 phosphate buffer (pH 7.0). One milliliter was taken for HPLC
analysis, and an additional 1 ml was dissolved by sonication (Misonix 3000;
4x15 s pulses at 35 W). The sonicated suspension was replaced by a 0.05%
Triton X-100 solution in 1 ml 100 mmol l-1 phosphate buffer (pH
7.0). After incubation for 10 min, the suspension was centrifuged at 14 000
g for 30 min and used for protein and enzyme analyses. The
enzymes were examined in the cytosoluble fraction of protein extractions. SOD
activity was assayed spectrophotometrically as described by Elstner and Heupel
(1976) and Oyanagui
(1984). Standards were
prepared using bovine erythrocyte SOD (Sigma) for each set of samples.
Catalase activity was assayed spectrophotometrically by monitoring
H2O2 depletion at 240 nm. All assays were conducted at
25°C and expressed as enzyme activity units (U) per mg protein. SOD and
CAT activities were averaged and presented as a percentage of the total
maximum activity. Protein content was determined by the Bradford assay
(Bradford, 1976).
Pigment analysis (HPLC)
For pigment analysis, frozen subsamples were extracted in 1 ml cold 100%
acetone, vortexed and centrifuged. The supernatant was transferred to a test
tube and kept in the dark at 4°C for approximately 1 h. The acetone
extract was then filtered onto a GF/F filter, and 0.3 ml 1 mol l-1
ammonium acetate was added in order to facilitate the separation of the
hydrophilic components of the extract
(Zapata et al., 1987). The
reverse-phase HPLC system consisted of a multiple solvent delivery pump
(CM4000 LDC-Milton-Roy, Riviera Beach, FL, USA), an injector (Rheodyne)
equipped with a 100 µl loop and a C-18, 25x4.6 mm (Alltech,
Deerfield, IL, USA) Spherisorb column. The pigments were detected with a
variable-wavelength spectrophotometer (LDC-Milton-Roy) set at 436 nm. Data
were recorded and processed by a digital-analog converter and software
(Jasco-Borwin, La Fontanil, France). Two solvents were used in the system:
solvent A consisted of 30% 1 mol l-1 ammonium acetate (Sigma) in
double-distilled deionized water and 70% methanol (HPLC grade; Bio Lab,
Jerusalem, Israel); solvent B consisted of 30% ethylacetate (HPLC grade, Bio
Lab, Israel) and 70% methanol. The solvent program exhibited a linear increase
in solvent B from 20 to 60% in 7 min, a plateau at 60% for 5 min, a linear
increase in solvent B from 60 to 100% from 12 to 20 min and was then
maintained at 100% solvent B for 20 min.
Pigment identification was facilitated by the use of ChromaScope (BarSpec,
Rehovot, Israel), a spectral peak analyzer that allows scanning of pigments
separated by the HPLC system in the range of 360-700 nm. The spectral data of
the separated peaks and their retention times were used as the parameters for
peak identification using published data
(Rowan, 1989;
Jeffrey et al., 1997) and were
compared to standards (Yacobi et al.,
1996). The major pigments found in this study were isolated
online; their concentrations were determined and subsequently used for
quantification. Quantification of the chromatograms was facilitated by
injection of known pigment concentrations into the HPLC system and calculation
of the response factor based on the area under the peak. All pigment
concentration calculations are presented as the mean of duplicate
measurements. Individual measurements did not differ by more than 10%. The
pigment data collected by HPLC were expressed in relation to moles of
chlorophyll a (Chl a).
Statistical analysis
Differences between SOD and CAT activities were estimated using one-way
analysis of variance (ANOVA) and the Student-Newman-Keuls (SNK) test at the 5%
significance level. The means of enzyme activity for each coral species were
converted using the arcsine function. The Pearson correlation factor was used
in order to determine the relationship between light intensity and enzyme
activity (Sokal and Rohlf,
1981). Values are represented as means ± s.d.
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2000 µmol
quanta m-2 s-1. The intensity values of UVA + UVB
radiation in February 2001 at the 5-m depth is presented in
Fig. 1B. The net oxygen
evolution rate tracked the diurnal course of solar energy flux
(Fig. 1A), while respiration
rates did not change significantly during the night. Typical photosynthesis
versus irradiation curves for colonies of Favia favus, Plerogyra
sinuosa and Goniopora lobata are presented in
Fig. 1C-E. The optimum light
intensity (Eopt) at which photosynthesis is at a maximum
without exhibiting photoinhibition was similar for all three species,
approximately 1100 µmol quanta m-2 s-1, which is
close to the maximum radiation intensity at this depth. The compensation point
(Ecom) at which photosynthesis and respiration rates are
equal was reached at approximately 10-15% of the optimum light intensity,
between 119 and 155 µmol quanta m-2 s-1. Favia
favus and P. sinuosa were light-saturated near 700 µmol
quanta m-2 s-1, while G. lobata reached almost
950 µmol quanta m-2 s-1. The maximum photosynthesis
rate of F. favus colonies (0.11±0.03 µmol O2
cm-2 min-1) was lower than that of both G.
lobata and P. sinuosa (0.22±0.05 and 0.22±0.13
µmol O2 cm-2 min-1, respectively). The
highest Pg/R24h ratio was found in
F. favus (4.37±0.68), while P. sinuosa
(3.78±0.18) and G. lobata (3.48±0.23) exhibited lower
values (one-way ANOVA; P<0.05;
Table 1). Net oxygen production
followed ambient light intensity at the same depth of 5 m, and the peak of
oxygen evolution was reached at midday. Photoinhibition was not observed even
at noon (Fig. 2).
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FRR fluorescence
In situ measurements of chlorophyll fluorescence yields revealed a
diel variation in response to ambient illumination. The variable fluorescence
F'/Fm' declined from 0.40
(arbitrary units, a.u.) in the early morning to 0.07 at midday for P.
sinuosa, 0.25 for F. favus and 0.1 for G.
lobata (Fig. 2). Decreases
in F'/Fm' values were found
during the midday hours (between 10.00 and 14.00 h) in F. favus and
G. lobata (Fig. 2A,C),
while in P. sinuosa the values were low until late afternoon (16.00
h; Fig. 2B). In all three
species from early afternoon, there was an increase in
F'/Fm' values as light
intensity decreased. In all three species,
F'/Fm' values were inversely
correlated to the net oxygen production
(Fig. 2A-C). The functional
absorption cross section of PSII (
PSII') decreased
slowly with increasing irradiance, with minimum values being observed between
11.00 and 14.00 h in all coral species examined
(Fig. 3; data not presented for
G. lobata). The recovery in PSII' occurred in
late afternoon, when light intensity decreased down to less than 500
µmol quanta m-2 s-1
(Fig. 3). The
PSII' values did not change significantly during the
night and remained constant (data not presented). The irradiance-induced
decrease in F'/Fm' was
accompanied by an increase in the quantum yield of NPQ in all corals (Figs
2,
3). In F. favus
(Fig. 2A) and G.
lobata (not presented),
F'/Fm' decreased to a level of
0.1-0.25 at a maximum light intensity of 2000 µmol quanta
m-2 s-1, while in P. sinuosa
F'/Fm' values were less than
0.1 (Fig. 2B). In F.
favus, NPQ maximum values reached 0.4, whereas P. sinuosa
exhibited higher NPQ values of 0.65 when light intensity was above
1800 µmol quanta m-2 s-1
(Fig. 3).
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Enzyme activity
Superoxide dismutase (SOD) activity in the animal tissue of the three
species exhibited a similar trend (Fig.
4) ofincreasing activity with irradiance and net photosynthesis
productivity (one-way ANOVA for F. favus, P<0.0001; for G.
lobata P<0.05; for P. sinuosa P<0.001). In tissues of
P. sinuosa and F. favus, SOD activity values observed
between 10.00 and 14.00 h were significantly higher than during the rest of
the day and night (SNK, P=0.006 and P=0.009, respectively).
SOD activity in G. lobata coral tissue was significantly higher only
in the afternoon (16.00 h) and during the night (05.00 h) (SNK,
P<0.05; Fig. 4B).
SOD activity in the isolated zooxanthellae of the three coral species did not
differ significantly during day or night (one-way ANOVA, P>0.05),
although a clear trend of increasing activity with increased light intensity
could be observed (Fig.
4A-C).
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Pigment analysis
The pigment suite of zooxanthellae revealed in the HPLC chromatograms
demonstrated the typical components of dinoflagellates
(Table 3). The major pigments
were Chl c2, peridinin, dinoxanthin, diadinoxanthin (DD),
Chl a and ß-carotene. Chlorophyllide a (Chld
a) was present in all chromatograms, and its concentrations were
pooled with those of Chl a. Even though the samples were preserved in
liquid N2 immediately after collection of the coral, we were not
able to detect diatoxanthin, which is a signature pigment for the
photoprotective response. It is known that this pigment vanishes within
minutes if algal cells are allowed to relax in low light
(Goericke and Welschmeyer,
1992). The statistical analyses did not detect any significant
differences (one-way between pigment compositions ANOVA, P>0.05)
(Table 3). The three coral
species were almost uniform in their pigment concentration relative to Chl
a throughout the day (Table
3).
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; Barnes and Chalker,
1990). Changes in enzymatic activity and behavioral responses,
such as variations in tentacle expansion and contraction
(Levy et al., 2003), are all
light dependent and have been studied for many years
(Brown et al., 2002a;
Stambler and Dubinsky, 2004).
Hoegh-Guldberg and Jones
(1999), Lesser and Gorbunov
(2001) and Gorbunov et al.
(2001) monitored diurnal
in situ chlorophyll fluorescence yields. Brown et al.
(1999) followed the diurnal
changes in photochemical efficiency and xanthophyll concentrations.
The present study highlights the dynamics of the diurnal cycle of corals
in situ and their mechanism of continuous `tuning' in relation to the
course of light and UV radiation and their penetration into shallow water. The
three corals studied, Favia favus, Plerogyra sinuosa and
Goniopora lobata, are common reef-building corals in the Red Sea,
from the surface to a depth of 30 m. The distributed three species exhibit a
diurnal course in oxygen evolution parallel to that of light intensity.
Different Pg/R24h ratios were shown to
occur as a result of polyp expansion/contraction, as described by Fabricius
and Klumpp (1995) for soft
corals. Their data reveal that colonies with expanded tentacles have higher
Pg/R24h values (1.3±0.02) than
colonies with contracted tentacles (1.14±0.02). The
Pg/R24h ratios presented in
Table 1 for the three species
range from 3.48 to 4.37. Generally,
Pg/R24(h) ratios in hard corals range
between 2 and 4. Scleractinian corals have much higher photosynthetic rates
than soft corals (Fabricius and Klump,
1995), which may be related to a far higher density of
zooxanthellae cell in host tissue in scleractinians (usually in the range of
0.5-1x106 zooxanthellae cm-2) than in most
octocorallians (with the possible exception of Tubipora musica),
which is reflected in the higher host respiration/symbiont photosynthesis
ratio. The stony corals F. favus and P. sinuosa expand their
tentacles nocturnally and remain open until sunrise, whereas G.
lobata remains expanded continuously
(Levy et al., 2003). P.
sinuosa can also expand and contract its photosynthetic vesicles in
response to varying light intensities, as described by Vareschi and Fricke
(1986). Therefore, the
different ratios of oxygen evolution and respiration observed by us are likely
to reflect polyp behavior such as expansion or contraction of the tentacles
(Levy et al., 2003).
Differences in metabolism, especially in respiration rate, can also be
influenced by feeding behavior. Porter
(1976) suggested that corals
with large polyps depend on heterotrophy more than those with small polyps
(see Lesser et al., 2000);
however, no support for this hypothesis was found in subsequent studies
(Haramati et al., 1997).
Goniopora lobata polyps are small (5 mm) compared with those of
F. favus (11 mm) and P. sinuosa (up to 20 mm).
However, G. lobata has a greater tentacular surface for feeding than
is found in other small polyp corals
(Sebens et al., 1997). From
the Pg/R24(h) ratios measured, it
seems that G. lobata exhibits a lower ratio with a small polyp size.
This can be related to the changes in tentacle state. The tentacles can be
expanded or contracted during daytime, which can influence photosynthetic rate
and reduce the ratio of Pg/R24h (Levy
et al., 2005a). Plerogyra sinuosa, with the largest polyp size, had
similar Pg/R24h values
(Table 1) to those of G.
lobata. This results from daytime expansion of the photosynthetic
vesicles.
Respirometer data on coral metabolism in shallow waters do not indicate any
photoinhibition during the midday hours of July-August 2001
(Fig. 2A-C) and March 2002
(Fig. 1A-E). A similar finding
was noted by Goiran et al.
(1996) for the symbiotic coral
Galaxea fascicularis and by Muller-Parker
(1984) for the sea anemone
Aiptasia pulchella. Absence of photoinhibition in both intact coral
colonies and whole reefs is common and predictable: while branch tips and
surface zooxanthellae are exposed to the full, measured irradiance, most of
the symbionts in branched colonies and deep-seated algae in massive species
are still light limited. The result is that photoinhibition of
high-light-exposed cells is masked by the status of most of the zooxanthellae,
a situation akin to that of entire reefs, where surfaces may be
photoinhibited, while light in reef crevices and shaded parts of colonies
never reaches saturation levels.
Despite the fact that oxygen measurements did not show photoinhibition, the
FRRF measurements demonstrate a clear pattern of midday depression in
F'/Fm' associated with
increasing light intensity accompanied by an increase in non-photochemical
quenching. As ambient light intensity increased, both steady-state
(F') and maximum (Fm') fluorescence
yields decreased due to non-photochemical quenching (NPQ) of chlorophyll
fluorescence (Lesser and Gorbunov,
2001; Levy et al.,
2004). Again, this difference between the data integrated from
whole colonies, measured by the respirometer, and those of the FRRF, which
observes only a small section of the colony at its exposed surface, is to be
expected and underscores the benefits inherent in combining both methodologies
concomitantly. NPQ is one of the protective mechanisms against oxidative
damage that is associated with the ability to dissipate excess energy as heat
(Falkowski and Raven, 1997).
The heat from the NPQ process is partly dissipated via the
xanthophyll cycle, in which diadinoxanthin is converted into diatoxanthin; in
this way, corals can keep excess light energy from damaging components of the
photosynthetic pathway (Ambarsari et al.,
1997; Brown et al.,
1999,
2002b). The diurnal changes in
the chlorophyll fluorescence cycle (Fig.
2) are similar to those obtained in earlier studies by Brown et
al. (1999), Hoegh-Guldberg and
Jones (1999), Lesser and
Gorbunov (2001) and Gorbunov
et al. (2001). The higher
values of NPQ found in P. sinuosa as compared with F. favus
(Fig. 3) indicate that this
species is capable of dissipating excess excitation energy more efficiently
through the xanthophyll cycle, and by doing so, this coral might be less prone
to bleaching events (see Warner et al.,
1999). Diel periodicity may lead to differences in the pigment
concentrations due to circadian rhythm, as reported for phytoplankton
(Marra, 1980;
Owens et al., 1980;
Post et al., 1984). In the
present study, photosynthetic pigments did not exhibit temporal patterns in
concentration (Table 3). Again,
it may well be that such changes did take place but went unnoticed because of
the different exposure of the zooxanthellae to light on a mm-to-cm scale,
which could not be resolved by us.
High oxygen levels within the host tissues of symbiotic cnidarians are a
result of the high rate of oxygen production in zooxanthellae exposed to high
irradiance (Lesser et al.,
1989a,b).
Photosynthetic oxygen evolution by the zooxanthellae in hospice can
raise tissue oxygen concentrations to 
200% saturation
(Dykens and Shick, 1982;
Kuhl et al., 1995;
De Beer et al., 2000). Such
levels of photosynthetically produced molecular oxygen act synergistically
with sunlight, especially UV radiation, in the presence of photosensitizing
agents (e.g. flavins and chlorophylls) to produce active harmful forms of
oxygen (Foote, 1976;
Asada and Takahashi, 1987).
Increased SOD and CAT activity is associated with increased light and UV
intensity (Fig. 3;
Table 2), suggesting higher
O2- radical and H2O2 production
(Levy et al., 2005b). Therefore, a diurnal change in SOD/CAT activity in the
host corresponds to the diurnal patterns of solar radiation both in the
visible and the UV domains, acting as a protective mechanism against oxygen
radicals. It is likely that the animal host is more susceptible to oxidative
damage than a plant, where high oxygen tension is the norm; indeed, SOD
activity in the symbiotic algae did not show a significant diurnal pattern,
although it did correlate with light intensity (Figs
3,
4;
Table 2). The fact that SOD/CAT
activity was higher in the morning suggests that, as light increases ahead of
any photoacclimation processes, the algae are susceptible at that time to the
increasing irradiance and UV penetration. Oxidative stress and apoptosis occur
in both the animal and the zooxanthellae
(Dunn et al., 2002;
Lesser and Farrell, 2004).
However, we still believe that oxidative stress is primarily an animal
response, aggravated by the presence of symbiotic algae that can cause
hyperoxia (Nii and Muscatine,
1997). The work done by Nii and Muscatine
(1997) on sea anemones
suggested that exposure to chronic stress may cause symbionts to release
oxidants directly and that oxidative stress in the sea anemone A.
pulchella is primarily an animal response. Downs et al.
(2002) proposed that
algae-generated H2O2 could diffuse from the algal
symbionts into the coral cytoplasm. Once inside the coral cytosol,
H2O2 can be neutralized by protective enzymes or
converted into the hydroxyl radical, which can eventually lead to coral
bleaching (Levy et al., 2005b). Therefore, the rise in free-radical-scavenger
enzymes in the host is likely to be a defense reaction to the accumulation of
free radicals within the host tissue. The difference in enzymatic activities
between species indicates that although the acclimation process depends on
environmental conditions, genetic variation among species controls the
function of these enzymes and, thus, plays a role in the survival and the
depth distribution of different corals in the coral reef.
Excessively high photosynthetically available radiation (PAR) is
potentially detrimental to photosynthetic organisms. The photosynthetic
apparatus saturates rapidly at relatively low flux levels
(Hoegh-Guldberg and Jones,
1999). Above these levels, light energy damages the photosynthetic
machinery (Walker, 1992;
Foyer et al., 1994;
Osmond, 1994). Short
wavelengths (290-400 nm) and elevated temperature may act synergistically with
intense radiation, causing destructive effects in both partners (host and
symbiotic algae; Jokiel, 1980;
Shick et al., 1996). These
environmental stresses may lead to photoinhibition, a reduction in
photosynthesis and an increase in respiration, and to a change in pigment
content. The outcome of these environmental effects leads to the evolution of
several protective mechanisms. These include the production of
mycosporine-like amino acids for which a UV-absorbing function has been
inferred (Dunlap and Chalker,
1986; Lesser and Shick,
1989b; Lesser,
2000; Shick and Dunlap,
2002) and the development of active oxygen scavenging systems
(Shick et al., 1996). The
present study indicates that shallow water corals respond rapidly to the
prevailing internal and external conditions and accomplish fine-tuned
adjustments to ensure that they continue to function effectively.
In this fine-tuning, the coral host plays the role of an important
guardian, as can be observed from the increase in the activity of two
free-radical scavenging enzymes, SOD and CAT. The adjustments occur within a
few minutes to several hours, while changes in the pigment content may occur
on a scale of days. In addition, increasing NPQ while reducing the cross
section of PSII are important mechanisms, protecting both algae and host from
harmful excess energy. We suggest that the symbiotic dinoflagellates within
the host live in an environment that protects them from chronic
photoinhibition (Gorbunov et al.,
2001). This symbiotic association owes its success partially to
the effectiveness of the host's anti-oxidative defenses and partially to the
photoacclimative plasticity of the zooxanthellae. Since corals have evolved in
stable tropical environments, we assume that even slight changes in the
environment, such as rising sea-surface temperatures, increased UV load,
lowering of ambient pH and bacterial infection, occurring on time scales too
short to allow the necessary genetic adaptation and extending outside the
phenotypic ranges covered by acclimation, can upset this unique system.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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