Oceanogr., 41(2), 1996, 271-283
0 1996, by the American Society of Limnology and Oceanography, Inc.
Limnol.
Elevated temperatures and ultraviolet radiation cause oxidative
stress and inhibit photosynthesis in symbiotic dinoflagellates
Michael P. Lesser’
Bigelow Laboratory for Ocean Sciences, McKown Point, West Boothbay Harbor, Maine 04575
Abstract
Elevated temperatures and solar ultraviolet (UV) radiation have been implicated as causes for the loss of
symbiotic algae in corals and other invertebrates with photoautotrophic symbionts (i.e. bleaching). Significantly higher cellular concentrations of superoxide radicals and hydrogen peroxide are observed when cultures
of Symbiodinium bermudense are exposed to elevated temperatures with and without exposure to UV
radiation. This increase in oxidative stress is accompanied by a reduction in the quantum yield of fluorescence
for photosystem 2 and protein-specific activities of the carboxylating enzyme, Rubisco. An increase in
antioxidant enzyme activities is unable to protect these cells from oxidative stress during exposure to UV
radiation and elevated temperatures (3 1OC).The addition of exogenous scavengers of active oxygen, however,
improves photosynthetic performance, but not to pre-exposure rates in zooxanthellae exposed to both elevated
temperature and UV radiation, confirming a role for oxidative stress in the inhibition of photosynthesis by
UV radiation and elevated temperatures. After exposure of zooxanthellae to UV radiation and elevated
temperatures, an action spectrum for the inhibition of photosynthesis shows significantly greater wavelengthdependent effects of UV radiation between 290 and 375 nm than for zooxanthellae exposed to UV radiation
alone.
an increase in the flux of harmful UV-B radiation reaching
the sea surface at high latitudes. The natural concentration of ozone is thinnest near the equator (Cutchis 1982);
as such, tropical ecosystems have a long evolutionary
history of exposure to greater fluxes of UV radiation than
do ecosystems at higher latitudes (Frederick et al. 1989).
Recent data describing global decreases in stratospheric
ozone show a highly variable trend of - 1.2 + 1.3% in the
loss of ozone over equatorial regions (5’S) and - 2 to - 4%
at 20-3O”N in the last decade (Madronich 1992), suggesting that fluxes of UV-B radiation could increase in
the future for this region. Because of the high transparency
of tropical ocean waters, UV radiation penetrates to depths
exceeding 20 m (Jerlov 1950; Fleischmann 1989; Lesser
unpubl.). These wavelengths are known to have a detrimental effect on photosynthesis and growth in zooxanthellae (Jokiel and York 1984; Lesser and Shick 1989)
and on survival of coral reef epifauna (Jokiel 1980; Siebeck 1988). The continued increase in the incidence of
coral bleaching will result in a decrease in growth and
reproduction, and an increase in mortality rates for corals
repeatedly bleached (Glynn 199 1).
To understand the underlying mechanisms that result
1 Current address and address for proofs, correspondence, and
in bleaching, one must elucidate the responses of both
reprints: Department of Zoology and Center for Marine Biology,
the host and symbiont to the proposed causes of bleachUniversity of New Hampshire, Durham 03824.
ing. Gates et al. (1992) suggested that elevated temperAcknowledgtients
atures affect the host, resulting in the loss of gastrodermal
This work was supported by the National Science Foundation
cells containing zooxanthellae. Other studies suggest that
Biological Oceanography Program (OCE 92- 16307 and OCE
the algal symbionts are the principal targets of exposure
94-96082).
to either elevated temperatures or UV radiation alone or
Special thanks to Terry Cucci and Michael Sieracki for advice
from
exposure to both simultaneously (e.g. Lesser et al.
and assistance. I also thank P. Neale for computational assis1990;
Glynn and D’Croz 1990; Iglesias-Prieto et al. 1992)
tance on the action spectra. Standards for UV-absorbing comand that damage to these symbionts results in an energetic
pounds were synthesized by Walter Dunlap and provided by
cost for the host in terms of a decrease in the translocation
Deneb Karentz.
of photosynthate or exposure to highly reactive oxygen
This is Bigelow Laboratory for Ocean Sciences contribution
radicals (Lesser and Shick 1989), for which DNA, pro95007.
271
Over the past several years, and with increasing regularity, coral reefs have been affected globally by a phenomenon known as coral bleaching, which involves either
the mass expulsion of zooxanthellae or the loss of photosynthetic pigments within individual zooxanthellae
(Glynn 199 1). These events closely follow periods of
warming that elevate seawater temperatures to 30-33°C
(Ogden and Wicklund 1988; Glynn 199 1). Field and laboratory studies on bleaching in corals and other symbiotic
cnidarians have established a causal link between temperature stress and bleaching (Jokiel and Coles 1990;
Glynn and D’Croz 1990; Fitt et al. 1993) and have also
suggested that corals are living close to their upper thermal limits (Jokiel and Coles 1990). Other studies have
also implicated UV radiation (UV-A, 320-400 nm; UVB, 290-320 nm) as a cause of bleaching, either alone
(Jokiel 1980; L esser et al. 1990; Gleason and Wellington
1993) or synergistically with elevated temperature (Lesser
et al. 1990; Glynn et al. 1993).
The decrease of stratospheric ozone from anthropogenic inputs of chlorinated fluorocarbons has resulted in
272
Lesser
teins, and membranes are all cellular targets (Fridovich
1986).
Exposure to sublethal temperature perturbation alone
(Iglesias-Prieto et al. 1992) or UV radiation alone (Lesser
and Shick 1989) does result in photoinhibition of photosynthesis in cultured zooxanthellae. Photoinhibition
occurs as a result of reduced photosynthetic electrol,
transport combined with continued high absorption of
excitation energy, leading to inactivation of or damage
to photosystem 2 (PS 2) (Osmond 198 1). The absorption
of excitation energy in the presence of oxygen leads to
production of active oxygen [singlet oxygen (lo,), superoxide radicals (O,-), and hydrogen peroxide (H202)], for
which there are many cellular targets including PS 2 and
the primary carboxylating enzyme, Rubisco (ribulose bisphosphate carboxylase/oxygenase) (Asada and Takahashi
1987). Either elevated temperatures or photodynamic
production of active oxygen could induce membrane
changes in the thylakoids that further disrupt primary
photochemistry (Asada and Takahashi 1987; Kyle 1987;
Ludlow 1987).
The photodynamic production of active oxygen occurs
when photosynthetic organisms are exposed to high visible or UV radiation in the presence of oxygen and photosensitizing components of the cell, such as chlorophyll
(Valenzeno and Pooler 1987). Symbiotic cnidarians routinely experience an elevated ~0, within their tissues as
a result of photosynthetically produced oxygen (Dykens
and Shick 1982). Physiological hyperoxia and exposure
to UV radiation act synergistically with sublethal temperature perturbations to produce active forms of oxygen
(Lesser et al. 1990). The resulting oxidative stress has
been proposed as a mechanism of photoinhibition of photosynthesis in zooxanthellae that could lead to bleaching
in symbiotic cnidarians (Lesser and Shick 1989; Lesser
et al. 1990). A loss of zooxanthellae has been observed
in a temperate sea anemone, Anthopleura elegantissima
when exposed to high fluxes of solar radiation and an
artificially elevated p02 (Dykens and Shick 1984). The
enzymes superoxide dismutase, catalase, and ascorbate
peroxidase act in concert to inactivate superoxide radicals
and hydrogen peroxide, thereby preventing formation of
the highly reactive form of reduced oxygen, the hydroxyl
radical, and subsequent cellular damage from these products of oxygen metabolism (Fridovich 1986).
The PS 2 reaction center is known to be directly damaged by UV radiation (Renger et al. 1989). One suggestion
is that this damage is of a type that also occurs with
exposure to high visible irradiance (Powles 1984). Other
work has suggested that UV-B radiation does not affect
the reaction center but affects donor or acceptor molecules
that subsequently carry out electron transport (Renger et
al. 1989). An indirect effect of exposure to visible and
UV radiation is that photodynamically produced oxygen
radicals cause damage to PS 2 at the Dl protein (Richter
et al. 1990; Tschiersch and Ohmann 1993). In particular,
the proximity of Dl to strong oxidants also makes it a
target of singlet oxygen, causing irreversible damage that
can be corrected only by enhanced synthesis and replacement of the protein (Long et al. 1994).
A direct effkct of UV-B on the specific activity and
cellular concentration of Rubisco has been demonstrated
in terrestrial plants (Strid et al. 1990), diatoms (Neale et
al. 1992), and on a free-living dinoflagellate, Prorocentrum micans (Lesser 1996). Rubisco is also inactivated
by exposure to H202, which is produced in large quantities within tLe chloroplast during oxidative stress (Asada and Takahashi 1987).
This study has several objectives. The first is to examine the photosynthetic response of zooxanthellae experimentally exposed to elevated temperatures and UV
radiation. Secl3nd, to quantitatively assess intracellular
levels of 02- and H202 - the enzymatic response (superoxide dismutase and ascorbate oxidase) to oxidative
stress-and to assessthe role of this oxidative stress on
the photophys iology of zooxanthellae. Third, to examine
PS 2 and Rubisco as targets of elevated temperature and
UV radiation. Fourth, to construct action spectra for the
photoinhibition of photosynthesis by UV radiation for
zooxanthellae exposed to normal and elevated temperatures and to assessthe functional aspects of UV-absorbing
compounds (mycosporinelike amino acids) for which a
protective role has been inferred from their UV-absorbing
properties and their logarithmic decreasein concentration
with increasing depth in corals (Dunlap and Chalker 1986).
Methods
Cultures anL?experimental conditions-Cultures of the
dinoflagellate Symbiodinium burmudense from the sea
anemone Aiptzia pallida (Banaszak et al. 1993) were
grown on a 12 : 2 L/D cycle at 25°C. Two 3-liter containers
(polycarbonate,) were fitted with a UV-transparent [50%
cutoff, -285 nm (UVT)] and UV-opaque [50% cutoff,
- 385 nm (UV-O)] Plexiglas covering. The cultures were
semicontinuous cultures kept at high optical densities in
f/2 medium (G-uillard 1975) with nitrate replaced by ammonium (NH4C1) at a final concentration of 50 PM. This
concentration ..srepresentative of the upper range for cytoplasmic .ammonium concentrations in cnidarians
(Wilkerson and Muscatine 1984). Visible radiation was
from four Vita-Lite full-spectrum fluorescent lamps providing a quantum scalar irradiance of 120 pmol quanta
m-2s-1 (photosynthetically active radiation, 400-700 nm)
as measured b.v a Biospherical Instruments QSL 100 47r
sensor immersed in a medium-filled culture vessel.
UV radiation, predominantly UV-B but including all
UV-A wavelengths, came from two aged fluorescent lamps
(FS40 Tl2-UVB, Light Sources Inc.) covered with cellulose acetate film (0.13 mm thick). The cellulose acetate
was used to atenuate the shorter wavelengths not encountered in nature (Caldwell et al. 1986) and was changed
every 3 d because its spectral properties change with age.
The UV and visible radiation sources were suspended 50
cm above the semicontinuous cultures. Representative
spectra for these experiments are available elsewhere
(Cullen and Lesser 199 1; Shick et al. 1995). Although the
photic regime tsed to acclimate these cultures is spectrally
imbalanced, if these cells and other light-dependent processesare at ir:radiance saturation then a spectral imbal-
Oxidative stress and zooxanthellae
a&e may have little effect and still provide important
mechanistic information. The treatments described here
are all at irradiances above saturation for photosynthesis
and growth in many dinoflagellates (Richardson et al.
1983).
The cultures were acclimated under these conditions
for 12 d during and after which the measurements described below were taken. The UV0 culture was subsequently split into two new UV0 and UVTsemicontinuous cultures kept under the same visible and UV radiation photic regime described above. The temperatures
of these cultures were then increased from 25 to 3 1°C
over the next 10 d, with measurements taken at day 17
(28°C) and day 22 (31°C) as described below; these temperatures are similar to the range of temperatures experienced by the host A. pallida in Bermuda (Lesser et al.
1990). On day 22, after the final measurements were taken,
ascorbate and catalase were added to a final concentration
of 500 PM and 200 U ml-l to the remaining UV0 and
UVT cultures and the cultures allowed to incubate at 3 1°C
for 1 h. Ascorbate is a well-known scavenger of both
superoxide and hydroxyl radicals (Larson 1988), and the
added catalase scavenged excess H,Oz produced during
scavenging of superoxide radicals by ascorbate. After the
1-h incubation with antioxidants, the cultures were again
measured for chlorophyll and photosynthesis.
Spectral irradiance from the UV lamps was measured
with a calibrated diode-array spectroradiometer system
(see Cullen and Lesser 199 1). Biologically effective irradiances for different treatments were determined as described by Smith and Baker (1979) and Smith et al. (1980)
using the DNA weighting function of Setlow (1974), the
generalized plant function of Caldwell (197 l), the chloroplast photoinhibition function of Jones and Kok (1966),
and the dinoflagellate photoinhibition weighting function
of Cullen et al. (1992).
Biomass, jluorescence, and growth analyses - Chlorophyll and particulate C and N were analyzed on days 12
and 22. The concentration of Chl a was measured fluorometrically with a calibrated Turner Designs lo-005R
fluorometer on triplicate samples of 1 ml collected on
Whatman GF/F filters and extracted in 10 ml of 90%
acetone and DMSO (6 : 4 vol/vol) at -4°C in the dark
for at least 24 h.
The same fluorometer was used for discrete measurements of chlorophyll fluorescence in vivo during the course
of the experiments. A qualitative assessment of damage
to PS 2 during exposure to elevated temperatures with
and without UV radiation was made based on measurements of cellular fluorescence capacity (Vincent 1980).
Fluorescence was measured after cultures had at least 30
min of adaptation in the dark and then again after they
were exposed to 3 x lO+j M DCMU [3-(3,4-dichlorophenyl)- 1,l -dimethylurea] in ethanol (Vincent 1980). The
initial fluorescence reading (Fo) was made after 15 s in
the fluorometer; DCMU was then added. Fluorescence
in the presence of DCMU (F,) was recorded after 45 s
in the fluorometer. Distilled water served as the blank.
En - F. is equivalent to variable fluorescence (F,). The
273
cellular fluorescence capacity (CFC) index was then calculated (F, : F,,) as a measure of the relative number of
functional PS 2 units or the quantum yield of fluorescence
for PS 2 (Vincent 1980).
Triplicate culture samples of 50 ml were taken at the
end of each experiment to determine particulate C and
N. These samples were filtered onto baked (450°C for 4
h) GF/F filters and stored in a desiccator. Samples were
frozen at - 50°C and freeze-dried overnight immediately
before use. Samples were combusted in a Control Equipment Corp. (Perkin Elmer) 240 XA elemental analyzer
with an automatic sampler in an air-tight box to keep the
samples dry. Acetanilide was used for a standard. Prefiltered culture medium was passed through baked filters
and used as blanks. Cell counts were obtained by flow
cytometry (seebelow). These counts were log-transformed
and used in a linear regression analysis to calculate growth
rates. The slopes (= growth rates) were then statistically
tested with a Student’s t-test.
Flow cytometry- Chlorophyll per cell was measured as
autofluorescence, with a Becton-Dickinson FACS analyzer. Single-cell measurements were made as cells passed
through a focused mercury lamp beam at an excitation
wavelength of 488 nm. The emitted fluorescent signals
were received by photomultiplier tubes fitted with 630nm long-pass filters. Chlorophyll measurements were
made on 5,000 cells to yield frequency distribution histograms. The flow cytometer was calibrated with fluorescent beads of known size and checked against cultures
of free-living phytoplankton whose cell volume and fluorescent characteristics are known.
Flow cytometry was also used to assess the level of
oxidative stress for cultures exposed to elevated temperatures with and without UV radiation (Kobzik et al. 1990).
Three specific metabolic probes were used: 2’, 7’-dichlorofluorescein-diacetate [DCF, 25 ~1 of a 1 mM solution
(in dimethylformamide) to 250 ~1 of culture incubated
for 5 min at room temperature] detects intracellular H,O,;
dihydrorhodamine 123 [DHR, 25 ~1of a 40 PM solution
(in dimethylformamide) to 250 ~1 of culture incubated
for 15 min at room temperature] is a more, sensitive indicator of H202 concentrations than DCF, and hydroethinide [HE, 25 ~1 of a 250 PM solution (in dimethylformamide) to 250 ~1 of culture incubated for 15 min at
room temperature] detects intracellular 02- formation.
The excitation wavelength for all three probes was 488
nm, and the detection wavelength was 5 15-555 nm for
DCF and DHR and 600-630 nm for HE. The fluorescent
signals of experimental cultures for these cellular probes
were collected on days 0, 1, 3, 7, 9, 12, 17, and 22. The
statistical analysis for all flow cytometry results was done
on 1,000 cells for each culture on each sampling day using
a repeated-measures ANOVA for the UV radiation experiment and the experiment with exposure to elevated
temperatures and UV radiation.
Antioxidant enzymes- Activities of superoxide dismutase (SOD) and ascorbate peroxidase (ASPX) were
assayed spectrophotometrically as described by Lesser
274
and Shick (1989) and Matta and Trench (199 1) on days
12 and 22 and analyzed with a Student’s t-test. Assays
were performed on the supernatants of 100 ml of culture
material that had been filtered onto GF/F filters (N = 5),
resuspended in 100 mM of phosphate buffer (pH 7.0),
and sonicated for 1 min on ice to disrupt the cells. The
sonicated suspension was centrifuged for 30 min at 3,500
t-pm, and the supernatant was retained for assay of antioxidant enzymes. The protein content of the algal supernatants was measured by the Bradford (1976) method.
All enzyme assays were performed in a Shimadzu UV/
visible spectrophotometer at the temperature of the acclimation experiment. Results are expressed as units (U)
of enzyme activity per mg protein, where 1 U = 1.Opmol
substrate converted per minute.
Carbon uptake-The method of Lewis and Smith (1983)
was used to make measurements of photosynthesis as a
function of irradiance (P-I>. Samples were inoculated with
[14C]bicarbonate (final activity, - 10 PCi ml-l) in a temperature-controlled aluminum block. A range of irradiantes was provided from below through a heat filter of
circulating water and attenuated with neutral density
screens. Quantum scalar irradiance in each position was
measured with a Biospherical Instruments QSL- 100 4~
sensor with a modified collector. Incubations began within 30 min of sampling and ended after 30 min. After each
incubation, samples were immediately poisoned with 50
~1 of borate-buffered Formalin, acidified with 0.25 ml 6
N HCl, and shaken in a hood to expel inorganic leC.
Subsequently, scintillation fluid was added (Ecolume, ICN)
and dpm values were determined with a scintillation
counter using the H# method of quench correction. Subsamples (20 ~1) from the original inoculation were placed
in 4 ml of fluor (Ecolume) plus 0.2 ml of phenethylamine
to determine the amount of label added. Total CO, under
these conditions was assumed to be 2.1 mM, and all
measurements were corrected for quench. The P-I equation of Platt et al. (1980) was used to model the results,
where PiB [pg C (pg Chl a)- l h- ‘1 is the instantaneous rate
of photosynthesis normalized to Chl a at irradiance i. The
maximum capacity of photosynthesis, P,,, [pg C (pg Chl
a)-’ h-l], was calculated according to Platt et al. (1980).
P-I curves were taken on days 0, 12, 17, and 22 before
and after exposure to exogenous antioxidants.
Activities of Rubisco were measured by the method of
Glover and Morris (1979), which follows the uptake of
[14C]bicarbonate (0.25 PCi pmol-l) at the temperature of
the acclimation experiment and uses the optimal substrate conditions for the enzyme expressed on a protein
basis. Samples were taken on days 12 and 22 and analyzed
with a Student’s t-test.
Mycosporinelike amino acids (UV-absorbing compounds) -On days 12 and 22, 1OO-ml aliquots (N = 3)
from each culture were taken and filtered on GF/F filters.
These filters were then placed in 3 ml of 100% methanol
and mycosporinelike amino acids (MAAs) were extracted
according to the procedures of Dunlap and Chalker (1986).
MAAs were separated and quantified from these samples
by reverse-phase high performance liquid chromatography (HPLC) as described by Dunlap and Chalker (1986)
and later modilied by Shick et al. (1992). Individual MAAs
were separated by reverse-phase isocratic HPLC on a
Brownlee RP-8 column protected with an RP-8 guard in
an aqueous mobile phase that included 0.1% acetic acid
and 25 or 55% methanol. Detection of peaks was by UV
absorbance at 3 13 and 340 nm. Identities of peaks were
confirmed by cochromatography and concentrations calculated from standards of mycosporine-glycine, shinorine, porphyra-334, palythine, asterina-330, and palythin01.Concentrations of MAAs were normalized on a protein
basis.
Action spectra -A newly constructed analytical model
of the photosynthetic response to irradiance was used to
describe the interaction of UV and PAR in determining
the rate of photosynthesis. The model describes photosynthesis as a i’unction of PAR, and photoinhibition as a
function of PAR and biologically weighted UV radiation
(Cullen et al. 1992); it has been successfully used on microalgae that show a failure of reciprocity (Smith et al.
1980) between dose and response, as does S. bermudense
(Lesser unpubl.). Data for action spectra were collected
at the end of each experiment (days 12 and 22). Fitting
the model required data from exposures of zooxanthellae
to a broad range of PAR and UV : PAR ratios, for which
a special incubation chamber was developed. The chamber defined up to 72 radiation treatments, including eight
different UV-B : UV-A : PAR ratios (determined by longpass cutoff filters) with nine different Auence rates in each
(imposed by neutral density screens). Irradiance regime
was determineld by 5 x 5-cm Schott WG long-pass filters
and perforated nickel screens. Nominal cutoff wavelengths for the filters are 280, 295, 305, 335, 345, and
360 nm. A 400-nm cutoff long-pass filter was also included as a “no UV” control. The filter cutoff wavelengths
were chosen to define irradiance treatments containing
progressively greater amounts of first UV-A and then
UV-B added to a constant background of PAR. This
“polychromatic” approach was chosen to obtain environmentally relevant action spectra (Caldwell et al. 1986).
During the rneasurement of C fixation, UV radiation
and PAR were provided by a 1,000-W xenon arc illuminator (Oriel) and directed upward by a mirror that
reflected UV and visible radiation. High-resolution spectral irradiance in each of the 72 treatments was measured
with a diode-array spectroradiometer calibrated as described by Cullen and Lesser (199 1). Briefly, irradiance
was collected l.hrough a quartz fiber-optic probe fitted
with a diffuser suitable for UV and visible radiation; the
probe was appropriately sized for insertion into the incubation block The fiber optic was coupled to a spectrograph (monochromator) with gratings for UV and visible
regions. The output was calibrated with a Hg (Ar) source
for wavelength and a 1,000-W quartz halogen standard
source (Optronics) for absolute irradiance. Long-pass filters were used to identify and correct for small false signals
due to stray light. Photosynthesis was measured by inoculating the sample with NaH14C03 (10 mCi ml- ‘) and
275
Oxidative stress and zooxanthellae
Table 1. Biologically weighted UV irradiance (mW m-2, 290400 nm) and unweighted UV-B irradiance (mW m-2, 290-320
nm) for the cellulose acetate-filtered FS40 lamps. The weightings
tabulated by Smith et al. (1980) were used unless otherwise
noted. (NA-not applicable.)
Weighting function
UV irradiance
(mW m-2)
Treatment*
UV0
0.00
0.57
UVT
0.00
Plant
UV0
5.03
UVT
1.42
Photoinhibition
UV0
UVT
67.57
Photoinhibitionj’
0.26
UV0
UVT
12.52
Unweighted UV-B
0.00
UV0
89.58
UVT
Unweighted UV
16.79
UV0
UVT
246.32
NA
PAR : UV-B
UV0
UVT
308
* UVO- Control cultures; UVT-cultures
exposed to UV radiation.
t Determined with the biological weighting function for Prorocentrum micans (Cullen et al. 1992).
DNA
dispensing the sample into the vials. Incubation (30 min)
was stopped by adding 6 N HCl to the sample. The contents were immediately transferred to glass scintillation
vials with added Formalin, and total uptake into organic
compounds was determined by liquid scintillation count-
ing. Calculated uptake was corrected for time-zero controls.
An efficient, quantitative characterization of the spectral treatments was obtained by principal component
analysis (PCA) (Cullen et al. 1992). The reduction in the
dimensionality of the spectral n-radiances determined by
PCA (2-3 spectral components instead of the 72 spectra
with 1,024 points each) is useful for obtaining quantitative estimates of the biological weighting function and
confidence intervals by using the P-I model of Cullen et
al. (1992) and iterative, nonlinear least-squares regression
on the experimental data. The weightings obtained with
these procedures are absolute rather than relative and can
be compared directly without normalizing the action
spectrum to some nominal wavelength at which the greatest wavelength-dependent effects are observed. The model and analytical approach are described in detail by Cullen et al. (1992) and Neale et al. (1994).
Results
Biologically efective dose of UV radiation - Semicontinuous cultures of S. bermudense exposed to visible and
UV radiation experienced an unweighted UV irradiance
(Table 1) almost 15 times that received by cultures
screened from UV radiation. Biologically effective UV
irradiances were calculated with several different weighting functions (Table 1). These weighting functions, including one for the free-living dinoflagellate P. micans,
show an almost 50-fold difference in weighted UV-B irradiance between experimental and control cultures (Ta-
Table 2. Effects of UV radiation on growth, biomass, and biochemistry on Symbiodinium
bermudensecontrol cultures (UVO) and cultures exposed to UV radiation (UVT). Cultures
exposed to elevated temperatures (3 l°C) are prefixed HT. All values are expressed as mean
+ SD; growth (N = 6), chlorophyll (N = 3), C : N ratio (N = 3), particulate C and N (N = 3),
and Rubisco (N = 3). Probability (P) values are from an unpaired Student’s t-test at the 5%
significance level. (NA-not applicable.)
Growth
(Chl specific, d-l)
Chl a cell-’
C:N
Particulate C
(pg cell-l)
Particulate N
(pg cell - l)
Rubisco
(pg C mg protein-
1 h-1
>
UV0
UVT
HTUVO
HTUVT
UV0
UVT
HTUVO
HTUVT
UV0
UVT
HTUVO
HTUVT
UV0
UVT
UV0
UVT
HTUVO
HTUVT
UV0
UVT
HTUVO
HTUVT
0.142+0.027
0.079 kO.026
-0.038
-0.107
4.55f0.57
3.24kO.47
3.091kO.16
1.43kO.17
8.79kO.37
12.78kO.78
13.80+0.41
19.9OkO.43
53.09k2.86
54.52k4.17
7.1OkO.23
6.26kO.29
4.7OkO.07
2.95f0.35
1.83kO.29
0.84kO.05
1.65zkO.04
0.44zkO.06
P = 0.001
NA
P = 0.02
P = 0.0002
P = 0.0013
P = 0.0001
P = 0.65
P = 0.018
P = 0.001
P = 0.004
P = 0.0001
276
Lesser
Table 3. Effects of elevated temperature and UV radiation on oxidative stress for cultures
of Symbiodinium bermudense(mean&SD). Probability (I’) values are from a repeated-measures ANOVA (flow cytometry data) or an unpaired Student’s t-test (antioxidant enzyme
activities) at the 5% significance level. Assay abbreviations are defined in the text; other
abbreviations as in Table 2.
Chl fluor. cell- l
HE (H202) fluor. cell-l
DHR (H,O,) fluor. cell-l
DCF (O,-) fluor. cell I
SOD activity
(U mg protein-‘)
ASPX activity
(U mg protein-‘)
Flow cytometry
UV0
502+ 108
UVT
475f 101
HTUVO
476+ 158
HTUVT
452+171
UV0
213f 132
UVT
232f 153
HTUVO
404+ 165
HTUVT
46Ok 150
UV0
231+107
UVT
247f 106
HTUVO
399+ 142
HTUVT
416-r- 171
HTUVO
62.29+ 1.20
HTUVT
46.62k6.40
UV0
228+114
UVT
248+ 133
HTUVO
367+ 164
HTUVT
413+ 125
Enzymes
UV0
UVT
HTUVO
HTUVT
UV0
UVT
HTUVO
HTUVT
ble 1). Although the percentage of UV radiation composed of UV-B is higher than that in natural solar radiation, the PAR: UV-B for the UVT cultures is greater
than that observed for shallow Hawaiian waters (Lesser
in prep.). The higher ratio results in a high proportion of
the experimental spectral irradiance of the UV-acclimated cultures being composed of longer wavelengths involved in photorepair of UV radiation damage.
Growth rates, chlorophyll content, andparticulate Cand
N-For both UV0 and UVT cultures the linear regressions for log of cell numbers vs. time were significant
(ANOVA, P < 0.05). Comparing the slopes of those lines
revealed a significant difference (Table 2) in growth rates,
with UVT cultures showing a 45% decrease. The growth
rates of both UV0 and UVT cultures exposed to elevated
temperatures (3 l°C) showed a dramatic decreaseand were
actually negative for both treatments (Table 2). Chl a
concentrations per cell also showed a significant 29% decrease when exposed to UV radiation (Table 2). Cultures
then exposed to elevated temperatures showed a decrease
in both UV0 and UVT cultures when compared to the
previous experiment in which they were exposed to only
UV radiation. There was a significant 55% decrease of
Chl a per cell in UVT cultures compared to UV0 cultures
(Table 2). Particulate C and N expressed at C : N ratjos,
7.05+ 1.73
16.13k4.3’7
12.41+2.5(;
24.13k3.79
0.69+0.35
1.61+0.3:!
1.08+0.3:!
2.6 1f0.43
P = 0.0001
P = 0.046
P = 0.0001
P = 0.0001
P = 0.0001
P = 0.196
P = 0.014
P = 0.0001
P = 0.0002
P = 0.0025
P = 0.0004
P = 0.0025
P = 0.0003
when log-transfbrmed and analyzed, showed a significant
effect of UV radiation, with UVT cultures having higher
C : N ratios (Table 2). Elevated temperatures increased
C : N in UV0 and UVT cultures, with significant differencesbetween UV0 and UVT treatments (Table 2). These
results were attributed to changes in particulate N per cell
rather than to .?articulate C for cultures exposed to UV
radiation only and to UV radiation with elevated temperatures, although particulate C per cell also showed a
significant decrease in UVT cultures when temperatures
were elevated (Table 2).
Flow cytomel ry - Analysis of cellular chlorophyll, using
the flow cytometric analysis of fluorescence, showed a
significant effect of UV radiation and elevated temperature with UV radiation (Table 3). The magnitude of the
treatment differences is not the same as that observed for
the bulk chlorcphyll analysis, but does support the relative treatment Idifferences observed. Because the fluorescence yield of PS 2 in vivo is not constant and also reflects
the irradiance environment of the cell, pigment concentration measurements by flow cytometry and bulk extraction
are not expected to exhibit
similar
differences.
The cellular concentration of H202 and O,- measured by
fluorescent probes, increased significantly with increasing
277
Oxidative stress and zooxanthellae
UV radiation and temperature (Table 3). Additionally,
in all casesthe repeated-measure ANOVA detected a significant independent and interactive effect of time (P <
0.05) for experiments with UV radiation only that showed
an increase in the concentration of these toxic products
with time or exposure to UV radiation (= dose). The
observed increase in 02- and H202 was nonlinear, with
a lag period of -3-7 d.
cl.7
0.6
rl
0
25°C
l
UV0
27°C
z
0.5
P
-3
2. .
Lz
I
G
1
’
T
i
f
28°C
P
30°C
i
T
31°C
0.4
Antioxidant enzymes -In response to the production
of active species of oxygen, the specific activities of the
antioxidant enzymes SOD and ASPX also increased with
UV radiation and elevated temperatures. Significant differences in the specific activities of these enzymes were
observed, with UVT cultures expressing higher activities
and with UVT cultures exposed to elevated temperature
expressing still higher activities (Table 3).
In vivo jluorescence - Repeated-measures ANOVA
analysis of the cellular fluorescent capacity showed no
significant differences between UV0 and UVT cultures
(P < 0.05) over the 12-d experiment (Fig. l), suggesting
no direct or indirect effects of UV radiation, measurable
by this assay, on the quantum yield of PS 2 fluorescence.
When these cells were simultaneously exposed to elevated
temperatures and UV radiation, there was a precipitous
decline in F,, : F, (Fig. 1, after day 12), with significant
differences between UV0 and UVT cultures (P = 0.008)
and an interactive effect of treatment (UV exposure) and
time (P = 0.0025). Because the dosage rate of UV radiation was constant while temperature was increased, most
of the decline in the quantum yield of fluorescence for
PS 2 is likely the result of elevated temperature; however,
over time the cumulative dose of UV radiation also increased. The F,, : F, improved to 0.47kO.02 (UVO) and
0.32kO.02 (UVT) in cells treated with antioxidants for
1 h but were still significantly different from each other
(P < 0.05).
Rubisco activities andphotosynthesis-irradiance curvesThe specific activities of Rubisco show a significant decline in cultures exposed to UV radiation (Table 2). For
both UV0 and UVT cultures, the specific activities of
Rubisco decline further upon exposure to elevated
temperature (Table 2), with Rubisco specific activities in
UVT cultures significantly lower than those in UV0 cultures. UVT cultures have activities that are at least 50%
lower than activities in UV0 cultures when exposed to
UV radiation and activities that are at least 70% lower
when exposed to elevated temperature and UV radiation.
Photosynthesis-irradiance (P-r> curves for samples
taken on days 0, 12, 17,22, and on day 22 after exposure
to exogenous antioxidants are shown in Fig. 2. A comparison of the UV0 and UVT P-Z curves for day 12 (Fig.
2B, 25°C) shows a significant decrease in P,,,axwith UVT
cultures exhibiting a 24% decline, whereas the UV0 curve
is not statistically different from day 0 curve (Fig. 2A).
Day 17 (Fig. 2C, 28°C) curves show a significant difference
in enax between UV0 and UVT. The UV0 curve exhibits
a Q10of 2.25 for P,,, at 28°C compared to the 25°C UV0
0.3
0.2
T
9
I
I
I
1
4
8
I
I
I
I
I
1
10
12
16
17
19
22
Fig. 1. Effects of elevatedtemperatureand UV radiation on
in vivo fluorescence(Fv: F,) for cultures of Symbiodinium bermudense (mean + SD). UVT-Cultures
exposed to UV radiation; UVO-cultures
not exposed to UV radiation.
curve, whereas the 28°C UVT curve shows a significant
decline in photosynthetic capacity compared to the 25°C
UVT curve. Day 22 curves (Fig. 2D) show the most dramatic decrease in P,,,, with measurements for UVT cultures significantly lower (63%) than those of UV0 cultures. This decline in P,,, is associated with declines in
Rubisco activities, particulate N, and the quantum yield
of PS 2 fluorescence.
Samples of cultures from day 22 were then exposed to
exogenous antioxidants (ascorbate and catalase) for 1 h
and the P-I curves repeated. This second set of curves at
3 1°C (Fig. 2E) showed an improvement in photosynthetic
capacity of 24% for UV0 and 37% for UVT cultures
relative to day 22 cultures without antioxidants (Fig. 2D).
A significant difference in maximum rates of photosynthesis remained between the two treatments after exposure to antioxidants, although P,,, for the UV0 cultures
exposed to elevated temperatures recovered - 50% of its
photosynthetic capacity after antioxidants were added.
No statistical differences were found for light-limited C
fixation (CU)
or sensitivity to higher irradiances (p) between
UV0 and UVT curves; however, there is a trend of declining light-limited C fixation with increasing temperature for UVT cultures.
MAAs (UV-absorbing compounds) -Quantitative chromatography showed the presence of two different MAA
compounds in S. bermudense (Fig. 3). Cultures not exposed to UV radiation showed the presence of only mycosporine-glycine, but in cultures exposed to UV radiation both mycosporine-glycine and shinorine were detected. Concentrations of mycosporine-glycine (P =
0.0006) and shinorine (P = 0.002) were significantly higher in UVT cultures (Fig. 3). Total concentration of MAAs
was significantly (P = 0.0002) higher in UVT (68.8k4.2
nmol mg-l protein) than in UV0 (34.8k3.4 nmol mg-l
protein) cultures. For cultures exposed to both elevated
temperature and UV radiation, the concentrations of
MAAs declined, with UVT cultures maintaining higher
278
Lesser
concentrations of mycosporine-glycine (P = 0.005) and
shinorine (P = 0.002). Total concentration of MAAs was
significantly (P = 0.0007) higher in UVT (49.1 k 3.9 nmol
mg-l protein) .than in UV0 (26.0k3.3 nmol mg-l protein) cultures at 3 1°C.
0-l
0
500
1000
1500
2000
2500
=
3000
B
.c
.
.
.
.
.
UV0
0
. . . . . . . . .e .._.____” _,_,,,_,,__._,____,.,,.,.,.,..
..“..
0
I
0 I
0
500
1000
1500
UVT
2000
2500
3000
4 ,
I
Action spectra -The action spectra (biological weighting functions) for UVT and UV0 cultures at normal or
elevated temperatures exhibited similar shapes with no
specific shape change in any portion of the action spectra
attributable to the accumulation and high absorbance of
MAAs in the UV portion of the spectrum (Fig. 4). The
action spectra for the UV0 and UVT cultures were not
statistically difkrent from one another, but UV0 cultures
showed an inc::ease in the wavelength dependence of inhibition of photosynthesis by UV radiation. These cultures also had significantly lower total concentrations of
MAAs than did UVT cultures. The weighting fknction
for UV0 cultures at elevated temperatures showed a
greater effect of UV radiation on photosynthesis than did
UV0 and UVT cultures at normal temperatures, but this
weighting function was not statistically different from the
functions at normal temperatures. Only the weighting
function from cultures exposed to elevated temperatures
and UV radiation, which exhibits the greatest wavelength-dependlznt effects of UV radiation, was statistically
different from all other weighting functions between 290
and 375 nm (Fig. 4).
Discussion
0
500
1000
1500
2000
2500
3000
Exposure of’ cultured zooxanthellae to elevated temperatures and UV radiation causes a depression of photosynthesis associated with changes in primary photochemistry and C fixation. The slow rise in temperature,
from 25-26 to 30-3 1°C (a 4-6°C increase), used in these
t
~:~
__. . . . . . . . . . . . . . . .. . . . "0 . . . . . . . ..." . . . .
0
500
1000
____,__,_ D
p,.
2000
1500
2500
3000
E
^
500
1000
Irradiance
1500
(pm01
quanta
2000
m - '5
2500
3000
- ')
Fig. 2. A. Photosynthesis-irradiance curve from day 0 of
experiments. Values (*SD) for PmaX= 3.19 I!Z0.54, (x = 0.025
kO.009, and ,f3= -0.0007 kO.0002. B. Curves from UV0
(opaque to UV radiation) and UVT (transparent to UV radiation) for day 12 (25°C) of the experimental cultures. Values for
RIax WV0 = 3.08 f 0.37; UVT = 2.19 f 0.22) were significantly different from one another (t-test, P < 0.05). (Y(UV0 =
0.0 13 -t 0.005; UVT = 0.026 + 0.003) and fl (UV0 = --0.0005
* 0.0001; UV’T = -0.0002 IL 0.0001) were not significantly
different from one another. C. Curves for day 17 (28°C) of the
experimental cultures. Values for P,,, (UV0 = 3.93 + 0.43;
UVT = 2.10 + 0.15) were significantly different from one another (t-test, P <: 0.05). a (UV0 = 0.022 + 0.004; UVT = 0.0 11
+ 0.002) and p (UV0 = -0.0009 + 0.0002; UVT = -0.0009
k 0.00003) were not significantly different from one another.
D. Curves for d,2y 22 (3 1OC)of the experimental cultures. Values
for P,,,,, (UV0 = 2.20 + 0.35; UVT = 0.81 -t 0.09) were
significantly di:Yerent from one another (t-test, P < 0.05). (Y
(UV0 = 0.024 + 0.005; UVT = 0.014 f 0.002) and p (UV0
= -0.0004 t- 0.00006; UVT = -0.00009 -C0.00004) were not
significantly diRerent from one another. E. Curves for day 22
(3 l°C) I h after the addition of ascorbate and catalase:.Values
for P,,, (UVC = 2.9 1 + 0.50; UVT = 1.29 -t 0.36) were
significantly different from one another (t-test, P < 0.05). (x
(UV0 = 0.025 + 0.014; UVT = 0.009 t- 0.003) and p (UV0
= -0.0006 + 13.00008;UVT = -0.0009 -t 0.0005) were not
significantly diiferent from one another.
279
Oxidative stress and zooxanthellae
_._._._
HT,,VO
lE-06
!
290
I
310
I
330
8
350
Wavelength
Treatment
1
370
I
390
(nm)
Fig. 3. Effects of elevated temperature and UV radiation on
the concentration of mycosporinelike amino acids for cultures
of Symbiodinium bermudense (mean + SD). UVO-Cultures
not exposed to UV radiation; UVT-cultures
exposed to UV
radiation; HTUVO-cultures
exposed to elevated temperatures
but not to UV radiation; HTUVT-cultures
exposed to elevated
temperatures and UV radiation.
Fig. 4. Action spectra (biological weighting function) for culturcs of Symbiodinium bermudense; E(X)= (mW m-2)-1. UVOCultures not exposed to UV radiation; UVT-cultures exposed
to UV radiation; HTUVO-cultures
exposed to elevated temperatures but not to UV radiation; HTUVT-cultures
exposed
to elevated temperatures and UV radiation. There were significant differences (no overlap between 95% C.I.) between HTUVT
and the remaining action spectra between 290 and 375 nm.
experiments is similar to that seen in Bermuda, the Caribbean, and El Nifio-Southern Oscillation (Glynn 199 1)
before the onset of bleaching events. Chl a concentrations
per cell declined upon exposure to UV radiation and decreased again in zooxanthellae exposed to elevated temperatures and UV radiation. These results are similar to
those in natural and experimental bleaching events (Kleppel et al. 1989; Lesser et al. 1990) and may partly explain
changes in photosynthetic performance. This bleaching
of photosynthetic pigments is an indication that similar
types of stress are being imposed on these culture experiments as occur during natural bleaching events. Again,
although a spectral imbalance may exist in these experiments, the cellular targets responsible for the decrease
in photosynthesis would be the same for exposures to
natural solar radiation and elevated temperatures.
This study shows that cultures of a symbiotic dinoflagellate, grown in an environment that attempts to simulate in hospite (within the host) light and N conditions
and subsequent exposure to elevated temperatures with
and without exposure to UV radiation, exhibit a significant decline in maximum photosynthetic capacity partly
due to the presence of active forms of oxygen. The observed decrease in photosynthetic capacity translates into
a significant decrease in cellular growth rates (Table 2).
Similar decreases in the growth of zooxanthellae have
been observed in cultures exposured to UV radiation.
Lesser and Shick (1989) showed a significant 30% decline
in the growth of cells exposed to UV radiation (solar
simulator), and Jokiel and York (1982) observed an 80%
decrease in growth rates in zooxanthellae exposed to natural solar irradiance. The 45% decrease in growth rates
described here compares well with these studies, all of
which used cultured zooxanthellae and different spectral
regimes. Additionally, the range of growth rates reported
here is similar to in hospite growth rates, measured as
mitotic indices, for zooxanthellae from A. pallida (Cook
et al. 1988).
The effect of UV radiation on C : N was significant, but
the values are still in line with C : N of zooxanthellae in
hospite (Cook ct al. 1988). The C : N ratios of zooxanthellae exposed to both elevated temperature and UV
radiation arc extremely high and consistent with nutrient
limitation. The significant decreasesin particulate N suggest a decrease in the uptake of inorganic N because UV
radiation has been shown to decrease inorganic N uptake
in phytoplankton (Diihler 1986).
Active oxygen is produced during hyperoxia subsequent to the photosynthetic production of oxygen (Asada
and Takahashi 1987). Zooxanthellae exposed to elevated
temperatures under which bleaching is known to occur
do not completely cease to photosynthesize (InglesiasPrieto et al. 1992; this study). Therefore, the oxygen concentration of zooxanthellae in vivo will be higher than
that of the surrounding medium (i.e. hyperoxic). The production of active oxygen can be caused indirectly through
photodynamic action during exposure to both UV-A and
UV-B wavelengths (Asada and Takahashi 1987; Valenzeno and Pooler 1987). An additional increase in the rates
of active oxygen production, due to temperature-enhanced metabolic rates, occurs primarily in the mitochondria of host and symbiont, adding to the cellular
concentration of reduced oxygen intermediates (Burdon
et al. 1990).
Higher fluxes of O,- and H,02 will lead to the production of highly reactive hydroxyl radicals without a
robust defense in the form of antioxidants. Symbiotic
cnidarians and their zooxanthellae, upon illumination,
produce hydroxyl radicals (Dykens et al. 1992), and previous work (Lesser and Shick 1989) and the work described here suggest that the increased activities of antioxidant enzymes are insufficient to scavenge the increase
in the cellular flux of 02- and H202. The addition of
exogenous antioxidants in illuminated cells at elevated
temperatures, with and without exposure to UV radiation, resulted in the partial recovery of photosynthetic
performance, suggesting that the fluxes of reduced oxygen
intermediates were causing damage to the photosynthetic
apparatus. The role of oxidative stress in the irreversible
injury to the photosynthetic apparatus is not readily discernible; however, the experiments described here also
would have led to the production of other forms of active
oxygen (i.e. 102) that would not have been scavenged by
the antioxidants used in this study and result in nonreversible injury caused by oxidative stress (see below).
PS 2 photochemistry improved after the addition of
antioxidants, and zooxanthellae exposed to only UV radiation also may have exhibited reversible photoprotection (= nonphotochemical quenching), which depresses
energy conversion to protect PS 2 reaction centers under
photoinhibitory conditions (Osmond 198 1; Krause and
Weis 1991). Cells simultaneously exposed to elevated
temperatures and UV radiation showed a significant decline in the quantum yield of PS 2 fluorescence. Most of
this decline was attributable to a decrease in variable
fluorescence (data not shown). A decline in variable fluorescence indicates that nonphotochemical quenching
rather than a dissociation of the light-harvesting complex
from the reaction center may explain the observed changes
in CFC during exposure to UV radiation and elevated
temperatures. The decline in photosynthetic capacity was
caused by the decrease in Rubisco activity and primary
photochemistry, the effects of which are further supported
by the significantly higher wavelength-dependent effects
of UV radiation on photosynthesis observed in the action
spectrum of the cultures exposed to both elevated temperatures and UV radiation.
My data show that Rubisco and PS 2 are sites of damage
when cells are exposed to both elevated temperature and
UV radiation. This damage is not completely reversed
by exogenous antioxidants, as shown in the P-Z curves
and the slight improvement in CFC. Damage to the Dl
protein of PS 2 has been shown to involve active oxygen
(Richter et al. 1990; Tschiersch and Ohmann 1993) as
has the inactivation of Rubisco (Asada and Takahashi
1987). Tschiersch and Ohmann (1993) showed that adding exogenous antioxidants to an illuminated culture of
Euglena gracilis not only improved variable fluorescence
and photosynthetic performance, but also decreased the
rate of membrane-lipid peroxidation; however, full recovery of the culture to preexposure levels of photosynthesis was not attained, as observed in this study. Inglesias-Prieto et al. (1992) also showed a decline in photosynthetic rates and variable fluorescence in cultures of
Symbiodinium microadriaticum under imposed hyperoxia and elevated temperatures. Additionally, photosynthetic rates in hypoxic cultures at 30°C were - 37% higher
than in hyperoxic cultures, and variable fluorescence was
lower in hyperoxic cultures at all temperatures tested (Inglesias-Prieto et al. 1992). Without a statistical analysis,
they discounted the role of hyperoxia in their interpretation of the data. Both the Dl protein of PS 2 (Renger
et al. 1989) and Rubisco (Neale et al. 1992; Strid et al.
1990) are also directly affected by UV-B radiation, which
can result in a new equilibrium between damage and
replacement of these important proteins and lower rates
of photosynthesis (Lesser et al. 1994).
Despite the available experimental evidence that zooxanthellae and many other biological systems are affected
by oxygen toxcity, Goreau and Hayes (1994) discounted
the role of UV radiation as a cause and oxygen toxicity
as an underlying mechanism for bleaching. Certainly, elevated temperatures have been shown to be the principal
environmental cause of bleaching, and UV radiation can
also cause bleal:hing in corals despite the presenceof MAAs
(Gleason and Wellington 1993); however, at the cellular
level, oxygen oxicity has been demonstrated here to be
a mechanism for the physiological effects observed in
zooxanthellae because exposure to elevated temperature
of UV radiation causes an increase in the flux of these
reduced oxygen products.
When cultures are exposed to UV radiation, an increase
in the level of’ oxidative stress in host tissues occurs simultaneously with the induction of antioxidant enzymes
(Dykens and Shick 1984; Dykens et al. 1992). Oxidative
stress in the host tissues occurs independently as a result
of photodynarnic action, but also could occur as a result
of oxidative slress in the algal symbionts because H,Oz
can readily difuse through biological membranes (Asada
and Takahashj 1987). Peroxides and oxygen radicals can
affect cellular proteins (Davies 1987) -potentially cytoskeletal and adhesion-which
may lead to the loss of
symbiotic dinoflagellates via expulsion or complete loss
of gastrodermal cells, as described by Gates et al. (1992).
Within both the algal symbionts and host, the production
of reduced oxygen intermediates due to UV radiation and
elevated temperatures produces stress that results in a
decrease in photosynthesis that is compounded by the
direct effects of UV radiation. Whether oxidative stress
or a reduction in photosynthesis ultimately initiates the
cellular events leading to bleaching is not known and
direct linkage cannot be inferred from this study. Bleached
coral colonies clearly exhibit a decrease in photosynthesis
(Porter et al. 1989), but experimental work with intact
corals will be reeded to demonstrate that photosynthetic
rates decline prior to actual bleaching.
Additionally, there are likely to be species-specific
(Trench and Blank 1987; Rowan and Powers 199 1) responses of Symbiodinium sp. to temperature stress and
exposure to UV radiation; however, an action spectrum
for the sun-loving symbiont S. microadriaticum (Lesser
unpubl.) shows no significant differences when compared
to the action spectrum for S. bermudense. Additionally,
the irradiance history of laboratory cultures and natural
Oxidative stress and zooxanthellae
samples will affect not only the shape but also the magnitude of the wavelength-dependent effects observed for
an action spectrum (Neale et al. 1994). For the free-livingdinoflagellate P. micans, the effects of UV radiation are
not ameliorated with the induction and accumulation of
UV-absorbing compounds (Lesser 1996), as is the case
with S. bermudense, which has cellular concentrations of
MAAs greater than those of P. micans. It has been suggested that for free-living microalgae and cyanobacteria
the functional aspects of accumulating MAAs may depend directly on the optical pathlengths-on the order of
microns-for these cells (Garcia-Pichel 1994). For symbiotic dinoflagellates in corals and other invertebrates,
the optical pathlengths would be much longer, and presumably would result in an increase in the absorbance of
UV radiation by MAAs which occur predominantly in
the host’s tissues, but presumed to be synthesized by the
symbiotic zooxanthellae (Lesser et al. 1990; Shick et al.
1995).
Will this increase in the concentration of MAAs and
optical pathlengths result in a decrease in the inhibition
of photosynthesis by UV radiation and in the synergistic
effects of elevated temperatures and UV radiation (Lesser
et al. 1990; Glynn et al. 1993)? Some evidence suggests
that this may be the case. MAA-rich colonies of the coral
Acropora microphthalma show no UV inhibition of photosynthesis evident in zooxanthellae freshly isolated from
this coral (Shick et al. 1995). Similar results have been
obtained for the mushroom coral Fungia sp. (Masuda et
al. 1993). However, the action spectrum for inhibition of
photosynthesis by UV radiation in the coral Pocillopora
damicornis from Hawaii shows a significant decrease in
the wavelength-dependent effects of UV-A radiation, but
not UV-B radiation below 3 10 nm, when compared to
action spectra available for zooxanthellae and phytoplankton (Lesser and Lewis 1996). The increased sensitivity under 3 10 nm is below the predominant absorbance
of MAAs. This surprising result, combined with the data
reported here, suggests that corals may be at their upper
limit of tolerance to the direct and indirect effects of UV
radiation and that any subsequent increase in the flux of
UV radiation could affect the productivity and health of
shallow-water corals.
References
M. TAKAHASHI.
1987. Production and scavenging of active oxygen in photosynthesis, p. 228-287. In
D. J. Kyle et al. [eds.], Photoinhibition. Elsevier.
BANASZAK, A. T., R. IGLESIAS-PRIETO, AND R. K. TRENCH. 1993.
Scrippsiella velellae sp. nov., Peridiniales and Gloedinium
viscum sp. nov., Phytodiniales, dinoflagellate symbionts of
two hydrozoans (Cnidaria). J. Phycol. 29: 5 17-528.
BRADFORD, M. M. 1976. A rapid and sensitive method for
the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
ASADA, K., AND
72: 248-254.
R. H., V. GILL, AND C. EVANS. 1990. Active oxygen
and heat shock protein induction, p. 19-25. In M. J. Schlesinger et al. [eds.], Stress proteins. Springer.
CALDWELL, M. M. 197 1. Solar ultraviolet radiation and the
BURDON,
281
growth and development of higher plants, p. 13l-l 77. In
A. C. Giese [ed.], Photophysiology. Academic.
-,
L. B. CAMP, C. W. WARNER, AND S. D. FLINT. 1986.
Action spectra and their key role in assessing biological
consequences of solar UV-B radiation change, p. 87-l 11.
In R. C. Worrest and M. M. Caldwell [eds.], Stratospheric
ozone reduction, solar ultraviolet radiation and plant life.
Springer.
COOK, C. B., C. F. D’ELIA, AND G. MULLER-PARKER.
1988.
Host feeding and nutrient sufficiency for zooxanthellae in
the sea anemone Aiptasia pallida. Mar. Biol. 98: 253-262.
CULLEN, J. J., AND M. P. LESSER. 199 1. Inhibition of phytoplankton photosynthesis by ultraviolet radiation as a function of dose and dosage rate. Mar. Biol. 111: 183-190.
-,
P. J. NEALE, AND M. P. LESSER. 1992. Biological
weighting function for the inhibition of phytoplankton photosynthesis by ultraviolet radiation. Science 258: 646-65 1.
CUTCHIS, P. 1982. A formula for comparing annual damaging
ultraviolet (DUV) radiation doses at tropical and mid-latitude sites, p. 2 13-228. In J. Calkins [ed.], The role of solar
ultraviolet radiation in marine ecosystems. Plenum.
DAVIES, K. J. A. 1987. Protein damage and degradation by
oxygen radicals. J. Biol. Chem. 262: 9895-9901.
D~HLER, G. 1986. Impact of UV-B radiation on [15N] ammonia and [15N] nitrate uptake of Dictylum brightwellii.
Photochem. Photobiophys. 11: 115-l 2 1.
DLJNLAP, W. C., AND B. E. CHALKER.
1986. Identification and
quantitation of near-UV absorbing compounds (S-320) in
a hermatypic scleractinian. Coral Reefs 5: 153-l 59.
DYKENS, J. A., AND J. M. SCHICK. 1982. Oxygen production
by endosymbiotic algae controls superoxide desmutase activity in their animal hosts. Nature 297: 579-580.
1984. Photobiology of the symbiotic sea
-,
AND -.
anemone, Anthopleura elegantissima: Defenses against
photodynamic effects, and seasonal photoacclimatization.
Biol. Bull. 167: 683-697.
C. BENOIT, G. R. BUETTNER, AND G. W.
WINSTON. ’ 1992. Oxygen radical production in the sea
anemone Anthopleura elegantissima and its endosymbiotic
algae. J. Exp. Biol. 168: 219-241.
FITT, W. K., H. J. SPERO, J. HALAS, M. W. WHITE, AND J. W.
PORTER. 1993. Recovery of the coral Montastrea annularis in the Florida Keys after the 1987 Caribbean “bleaching event.” Coral Reefs 12: 57-64.
FLEISCHMANN, E. M. 1989. The measurement and penetration
of ultraviolet radiation into tropical marine water. Limnol.
Oceanogr. 34: 1623-l 629.
FREDERICK, J. E., H. E. SNELL, AND E. K. HEYWOOD.
1989.
Solar ultraviolet radiation at the earth’s surface. Photothem. Photobiol. 50: 443-450.
FRIDOVICH, I. 1986. Biological effects ofthe superoxide radical.
Arch. Biochem. Biophys. 247: l-l 1.
GARCIA-PICHEL,
F. 1994. A model for internal self-shading in
planktonic organisms and its implications for the usefulness
of ultraviolet screens. Limnol. Oceanogr. 39: 1704-l 7 17.
GATES, R. D., G. BAGHDASARIAN, AND L. MUSCATINE.
1992.
Temperature stress causes host cell detachment in symbiotic cnidarians: Implications for coral bleaching. Biol. Bull.
182: 324-332.
D. F., AND G. M. WELLINGTON.
1993. Ultraviolet
radiation and coral bleaching. Nature 365: 836-838.
GLOVER, H. E., AND I. MORRIS.
1979. Photosynthetic carboxylating enzymes in marine phytoplankton. Limnol. Oceanogr. 24: 510-519.
GLYNN, P. W. 199 1. Coral reef bleaching in the 1980s and
GLEASON,
282
Lesser
possible connections with global warming. Trends Ecol.
Evol. 6: 175-179.
and L. D’CROZ. 1990. Experimental evidence for high
temperature stress as the cause of El Niiio-coincident coral
mortality. Coral Reefs 8: 18 l-l 9 1.
-,
R. IMAI, K. SAKAI, Y. NAKANO, AND K. YA.MAzATO.
1993. Experimental responses of Okinawan (Ryukyu Islands, Japan) reef corals to high sea temperature and UV
radiation, p. 27-37. In Proc. 7th Coral Reef Symp. Univ.
Guam.
GOREAU,T. J., AND R. L. HAYES. 1994. Coral bleaching and
ocean “hot spots.” Ambio 23: 176-l 80.
GUILLARD, R. R. L. 1975. Culture of phytoplankton for feeding
marine invertebrates, p. 29-60. In W. L. Smith and M. H.
Chanley [eds.], Culture of marine invertebrate animals. Plenum.
IGLESIAS-PRIETO, R., J. L. MATTA, W. A. ROBINS, AND R. K.
TRENCH.
1992. Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc. Natl. Acad. Sci. 89: 1030210305.
JERLOV, N. G. 1950. Ultra-violet radiation in the sea. Nature
116: 111-112.
JOKIEL, P. L. 1980. Solar ultraviolet radiation and coral reef
epifauna. Science 207: 1069-l 07 1.
-,
AND S. L. COLES. 1990. Responses of Hawaiian and
other Indo-Pacific reef corals to elevated temperatures. Coral Reefs 8: 155-162.
-,
AND R. H. YORK, JR. 1982. Solar ultraviolet photobiology of the reef coral Pocillopora damicornis and symbiotic zooxanthellae. Bull. Mar. Sci. 32: 301-315.
. 1984. Importance of ultraviolet radiation
-,ANDin photoinhibition of microalgal growth. Limnol. Oceanogr.
29: 192-l 99.
JONES, L. W., AND B. KOK.
1966. Photoinhibition of chloroplast reactions. 1. Kinetics and action spectra. Plant Physiol. 41: 1037-1043.
KLEPPEL, G. S., R. E. DODGE, AND C. J. REESE. 1989. Changes
in pigmentation associated with the bleaching of stony corals. Limnol. Oceanogr. 34: 1331-1335.
KOBZIK, L., J. J. GODLESKI, AND J. D. BRAIN. 1990. Oxidative
metabolism in the alveolar macrophage: Analysis by flow
cytometry. J. Leukocyte Biol. 47: 295-303.
KRAUSE, G. H., AND E. WEIS. 199 1. Chlorophyll fluorescence
and photosynthesis: The basics. Annu. Rev. Plant Physiol.
Mol. Biol. 42: 3 13-349.
KYLE, D. J. 1987. The biochemical basis for photoinhibition
of photosystem 2, p. 197-226. In D. J. Kyle et al. [eds.],
Photoinhibition. Elscvier.
LARSON, R. A. 1988. The antioxidants of higher plants. Phytochemistry 27: 969-978.
LESSER, M. P. 1996. Acclimation of phytoplankton to UV-B
radiation: Oxidative stress and photoinhibition of photosynthesis are not prevented by UV absorbing compounds
in the dinoflagellate, Prorocentrum micans. Mar. Ecol. Prog.
Ser. In press.
-,
J. J. CULLEN, AND P. J. NEALE. 1994. Photoinhibition
of photosynthesis in the marine diatom Thalassiosira pseudonana during acute exposure to ultraviolet B radiation:
Relative importance of damage and repair. J. Phycol. 30:
183-192.
-,
AND S. LEWIS. 1996. Action spectrum for the effects
of UV radiation on photosynthesis in the hermatypic coral,
Pocillopora damicornis. Mar. Ecol. Prog. Ser. In press.
1989. Effects of irradiance and
-,
AND J. M. SCHICK.
ultraviolet radiation on photoadaptation in the zooxan-
-,
thellae of Ai$asia pallida: Primary production, photoinhibition, and enzymic defenses against oxygen toxicity. Mar.
Biol. 102: 243-255.
W. R. STOCHAJ,D. W. TAPLEY, AND J. M. SHICK. 1990.
Bleaching in coral reef anthozoans: Effects of irradiance,
ultraviolet radiation and temperature on the activities of
protective enzymes against active oxygen. Coral Reefs 8:
225-232.
M. R., A>ID J. C. SMITH. 1983. A small volume, shortincubation-r ime method for measurement of photosynthesis as a funs;tion of incident irradiance. Mar. Ecol. Prog.
Ser. 13: 99-102.
LONG,S. P.,S. HUMPHRIES,AND
P.G. FALKOWSKI. 1994. Photoinhibition of photosynthesis in nature. Annu. Rev. Physiol. Plant Mol. Biol. 45: 633-662.
LUDLOW,M. M. 1987. Light stress at high temperature, p. 89110. In D. J-. Kyle et al. [eds.], Photoinhibition. Elsevier.
MADRONICH, S. 1992. Implications of recent total atmospheric
ozone measurements for biologically active ultraviolet radiation reaching the earth’s surface. Geophys. Res. Lett. 19:
LEWIS,
37-40.
K., M. GOTO, T. MARWAMA, AND S. MIYACHI. 1993.
Adaptation of solitary corals and their zooxanthellae to low
light and UV radiation. Mar. Biol. 117: 685-691.
MATTA, J. L., AND R. K. TRENCH.
199 1. The enzymatic response of the symbiotic dinoflagellate Symbiodinium microadriatictim (Freudenthal) to growth in vitro under varied
oxygen tensions. Symbiosis 11: 31-45.
NEALE, P. J., M. P. LESSER, AND J. J. CULLEN.
1994. Effects
of ultraviolc=t radiation on the photosynthesis of phytoplankton in the vicinity of McMurdo Station, Antarctica.
Antarct. Res. Ser. 62: 125-142.
-AND J. GOLDSTONE.
1992. Detecting
UV-induced inhibition of photosynthesis in antarctic phytoplankton. Antarct. J. 27: 122-124.
OGDEN, J., AN-DR. WICIU~ND.
1988. Introduction, p. l-9. In
Mass bleacl-ing of corals in the Caribbean: A research strategy. Ress Rep. 88-2. NOAA Undersea Res. Program.
OSMOND, C. B. 198 1. Photorespiration and photoinhibition;
some implications for the energetics of photosynthesis. Biothem. Biophys. Acta 639, p. 77-98.
PLATT, T., C. L. GALLEGOS, AND W. G. HARRISON.
1980. Photoinhibition of photosynthesis in natural assemblages of
marine phytoplankton. J. Mar. Res. 38: 687-701.
PORTER, J. W., W. K. FITT, H. J. SPERO, AND C. S. ROGERS.
1989. Bleaching in reef corals: Physiological and stable
isotopic responses. Proc. Nat]. Acad. Sci. 86: 9342-9346.
POWLES,S. B. 1984. Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35: 15MASUDA,
44.
G., A~[D OTHERS. 1989. On the mechanism of photosystem 2 deterioration by UV-B irradiation. Photochem.
Photobiol. 49: 97-105.
RICHARDSON, K.., J. BEARDALL, AND J. A. RAVEN.
1983. Adaptation of’ unicellular algae to irradiance: An analysis of
strategies. New Phytol. 93: 157-l 9 1.
RICHTER, M., PI. R~~HLE,AND A. WILD. 1990. Studies on the
mechanism of photosystem 2 photoinhibition. 1. The involvement of toxic oxygen species. Photosynth. Res. 24:
RENGER,
237-243.
R., AND D. A. POWERS. 199 1. Molecular genetic identification o r symbiotic dinoflagellates (zooxanthellae). Mar.
Ecol. Prog. Ser. 71: 65-73.
SETLOW, R. B. 1974. The wavelengths in sunlight effective in
producing skin cancer: A theoretical analysis. Proc. Natl.
Acad. Sci. 71: 3363-3366.
ROWAN,
Oxidative stress and zooxanthellae
SHICK, J. M., W. C. DUNLAP, B. E. CHALKER,
AND T. K. ROSENZWEIG.
1992. Survey
A. T. BANASZAK,
of ultraviolet radiation-absorbing mycosporine-like amino acids in organs
of coral reef holothuroids. Mar. Ecol. Prog. Ser. 90: 139148.
AND OTHERS. 1995. Depth-dependent responses to sola; ultraviolet radiation and oxidative stress in the zooxanthellate coral Acropora microphthalma. Mar. Biol. 122: 4 l51.
SIEBECK, 0. 1988. Experimental investigation of UV tolerance
in hermatypic corals (Scleractinia). Mar. Ecol. Prog. Ser.
43: 95-103.
SMITH, R. C., AND K. S. BAKER.
1979. Penetration of UV-B
and biologically effective dose-rates in natural waters. Photochem. Photobiol. 29: 3 1l-323.
0. HOLM-HANSEN, AND R. S. OLSON. 1980.
Photoinhidition of photosynthesis in natural waters. Photochem. Photobiol. 31: 585-592.
STRID, A., W. S. CHOW, AND J. M. ANDERSON.
1990. Effects
of supplementary ultraviolet-B radiation on photosynthesis
in Pisum sativum. Biochem. Biophys. Acta 1020, p. 260268.
283
R. K., AND R. J. BLANK. 1987. Symbiodinium microadriaticum Fredenthal, S. Goreau sp. nov., S. Kawagut2
sp. nov., and S. pilosum sp. nov.: Gymnodinoid dinoflagellate symbionts of marine invertebrates. J. Phycol. 23:
469-481.
TSCHIERSCH, H., AND E. OHMANN.
1993. Photoinhibition in
Euglena gracilis: Involvement of reactive oxygen species.
Planta 191: 3 16-323.
VALENZENO, D. P., AND J. P. POOLER. 1987. Photodynamic
action. Bioscience 37: 270-276.
VINCENT, W. F. 1980. Mechanisms of rapid photosynthetic
adaptation in natural phytoplankton communities. 2.
Changes in photochemical capacity as measured by DCMUinduced chlorophyll fluorescence. J. Phycol. 20: 20 l-2 11.
WILKERSON, F. P., AND L. MUSCATINE.
1984. Uptake and assimilation of dissolved inorganic nitrogen by a symbiotic
sea anemone. Proc. R. Sot. Lond. Ser. B 221: 71-86.
TRENCH,
Submitted: 5 January 1995
Accepted: 8 August 1995
Amended: 11 September 1995