Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Photoacclimation mechanisms of corallimorpharians on coral reefs: Photosynthetic
parameters of zooxanthellae and host cellular responses to variation in irradiance
Baraka Kuguru a,b, Yair Achituv a, David F. Gruber c, Dan Tchernov b,d,e,⁎
a
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel
The Interuniversity Institute for Marine Sciences in Eilat, P.O. Box 469, Eilat 88103, Israel
Department of Natural Sciences, City University of New York, Baruch College, P.O. Box A-0506, 17 Lexington Avenue, New York, New York 10010, United States
d
Department of Evolution, Systematics and Ecology, Hebrew University of Jerusalem, Edmund Safra Campus, Givat Ram, Jerusalem 91904, Israel
e
Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Mount Carmel, Haifa 31905, Israel
b
c
a r t i c l e
i n f o
Article history:
Received 17 March 2010
Received in revised form 7 July 2010
Accepted 8 July 2010
Keywords:
Corallimorpharians
Microhabitat
Photoacclimation
Photosynthesis
Ultraviolet radiation
Zooxanthellae
a b s t r a c t
Rhodactis rhodostoma and Discosoma unguja are the most common corallimorpharians on coral reefs in the
northern Red Sea, where individuals of R. rhodostoma form large aggregations on intertidal reef flats and
those of D. unguja occupy holes and crevices on the reef slope. Aside from these contrasting patterns of
microhabitat, little is known concerning their mechanisms of photoacclimation to environmental conditions.
We demonstrate here that different mechanisms of photoacclimation operate in both species and that these
differences explain, in part, the contrasting patterns of distribution and abundance of these common
corallimorpharians. Experimental exposure of the species' respective polyps to the synergistic effects of
ultraviolet and photosynthetically active radiation revealed that endosymbiotic zooxanthellae protected the
host R. rhodostoma from photooxidation damage. Zooxanthellae do so by reducing their chlorophyll pigment
and cellular abundance, as well as by adjusting their efficiency of light absorption and utilization according to
the level of irradiance. The host photoprotects its endosymbionts from harmful ultraviolet radiation (UVR)
by synthesizing enzymatic antioxidants against oxygen radicals. In contrast, individuals of D. unguja utilize a
behavioral mechanism of photoacclimation in which they physically migrate away from exposed areas and
towards shaded habitats and thus avoid the damaging biological effects of UVR. We conclude that a
combination of physiological and behavioral mechanisms appear to control microhabitat segregation
between these corallimorpharian species on tropical reefs. These various mechanisms of local adaptation to
environmental conditions may be largely responsible for the wide distributional ranges of some
corallimorpharians, and may enable these common reef organisms to tolerate environments that are highly
variable, both spatially and temporally.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Corallimorpharians are non-calcifying, evolutionarily important
relatives of stony corals (Medina et al., 2006), although the exact
relationship between Corallimorpharia and the Scleractinia remains
under debate (Fukami et al, 2008). Increased understanding of the
ecophysiology of corallimorpharians can provide insights into the
evolution of corals from Mesozoic to recent forms (Stanley and Fautin,
2001) and their ability to survive drastic climatic changes.
Coral reefs are among the most vital and biologically diverse
ecosystems on the planet. Despite their great value, both ecological
and socio-economical, however, coral reefs are severely threatened by
anthropogenic global climate change (IPCC, 2001; IPCC, 2007). The
⁎ Corresponding author. Marine Biology Department, The Leon H. Charney School of
Marine Sciences, University of Haifa, Mount Carmel, Haifa 31905, Israel. Tel.: + 972 4
8288790; fax: + 972 4 8282515.
E-mail address: [email protected] (D. Tchernov).
0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2010.07.007
steady rise in atmospheric CO2 has led to higher sea surface
temperatures (SST) (Hoegh-Guldberg, 1999; Hoegh-Guldberg et al.,
2002, 2007) and lower pH levels. Increasing atmospheric CO2 has
been postulated to deplete the ozone layer (Austin et al., 1992),
leading to an increase of ultraviolet radiation (UVR) on the oceans'
surfaces (Harley et al., 2006). Understanding the protective mechanisms used by marine organisms to mitigate the damage caused by UVR
is particularly urgent today, as the thinning of atmospheric ozone by
greenhouse gases has magnified the intensity of UVR reaching the sea
surface in some areas (McKenzie et al., 1998). In clear tropical
seawater, UVR penetrates to ecologically important depths (Gleason
and Wellington, 1993). UVR radiation breaks down dissolved organic
carbon (Hader et al., 2007), which is responsible for short-wavelength
absorption in the water column. In addition, oceanic warming and
acidification results in faster degradation of dissolved and particulate
organic carbon (DOC, POC), thereby enhancing the penetration of UVR
into the water column (Sinha and Hader, 2002). Short-term increases
in UVR intensity under calm, clear water conditions may expose
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B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
marine algae and invertebrates to photophysiological effects of UVR
stress (Gleason and Wellington, 1993). As a result, the productivity of
marine ecosystems may be greatly and adversely affected (Sinha and
Hader, 2002).
Tropical reef-building corals and other photosynthetic marine
invertebrates live in habitats where solar irradiance may be extremely
high (Shick et al., 1996; Lesser, 2000; Banaszak and Lesser, 2009).
Several studies have implicated exposure to elevated solar radiation,
including both photosynthetically active radiation (PAR, 400–700 nm,
Hoegh-Guldberg and Smith, 1989; Lesser and Shick, 1989) and UVR
(290–400 nm, Lesser and Stochaj, 1990; Kinzie, 1993; Baruch et al.,
2005; Lesser and Shick, 1989), as a major contributor to coral
bleaching stress. In particular, UVB (290–320 nm) suppresses photosynthesis while simultaneously increasing the risk of damage to DNA,
proteins, and membrane lipids (Greenberg et al., 1989; Lyons et al.,
1998; Baruch et al., 2005), partly through the production of reactive
oxygen species (ROS) (Lesser, 1989; Lesser and Lewis, 1996; Shick et
al., 1996; Jokiel et al., 1997; Tchernov et al., 2004). While the
photophysiological effects of UVR stress in some hexacorallians
(scleractinians, actiniarians and zoanthids) have been studied
extensively, corallimorpharians have been almost completely overlooked in this context.
Like many marine invertebrates, some corallimorpharians harbor
endosymbiotic dinoflagellate algae, also known as zooxanthellae
(den-Hartog, 1980; LaJeunesse, 2002; Kuguru et al., 2007, 2008).
Zooxanthellae play a critical role in host nourishment via the translocation of photosynthates (Muscatine et al., 1981). Recent studies have
demonstrated that zooxanthellae are made up of eight broad clades
(designated A–H), each of which contains multiple, closely related
molecular types that exhibit a range of physiological responses and
tolerances (reviewed in Coffroth and Santos, 2005; Stat et al., 2006). The
specific Symbiodinium type(s) that an organism harbors may affect its
distribution and reaction to extreme environmental conditions (Robison
and Warner, 2006). In the Red Sea, the common corallimorpharians
Rhodactis rhodostoma and Discosoma unguja both host Symbiodinium
type C1 in shallow waters (1–6 m), and types D1a and C1 in deeper
waters (18–20 m) (Kuguru et al., 2007, 2008). A recent study in
Australia (Jones et al., 2008) indicated that both clades C1 and D1 may
confer equal thermotolerance to host corals. Both of the above
corallimorpharian species, when transplanted from their original
habitats to either shallower or deeper water, shuffle their symbiont
types, a mechanism that may contribute to their occupation of a wide
bathymetric range on coral reefs (Kuguru et al., 2008). However,
individuals of D. unguja are more susceptible to light stress than are
those of R. rhodostoma, and tend to occur in deeper, more shaded
habitats (Kuguru et al., 2008). The mechanisms employed by these two
corallimorpharians to photoacclimatize to microhabitats on the coral
reef that differ in level of irradiance (UVR + PAR) are not yet understood.
The success of corals and other reef invertebrates that have
essentially transparent tissues but manage to thrive in shallow water
indicates that they have developed effective mechanisms for UVR
protection. There are substantial differences between closely related
species in their ability to escape the damaging effects of UVR in this
high-energy waveband (Sinha and Hader, 2002). The ability of
scleractinian corals and other reef organisms to survive environmental changes depends on their physiological mechanisms of acclimatization (Gates and Edmunds, 1999). Many symbiotic cnidarians
display rapid modifications in behavior, morphology, and physiology
that enable them to photoacclimate to changing light conditions, thus
demonstrating considerable biological flexibility. Shallow reef organisms exposed to high levels of solar UVR have evolved several types of
photoacclimation mechanisms to cope with light stress, including: (1)
behavioral avoidance, such as migrating away from intense light
(Gleason et al., 2006) and into shaded microhabitats (crevices and
holes on the reef); (2) mechanisms to control internal cellular
damage, such as development of free-radical quenching agents like
carotenoids, xanthophyll pigments, and antioxidants [both enzymatic,
i.e., superoxide dismutase (SOD) and catalase; and non-enzymatic,
i.e., vitamin E (Lesser, 2006)]; (3) changes in tissue structure and
morphology (Brown et al., 1994; Loya et al., 2001; Kuguru et al., 2007;
Mass et al., 2007); and (4) sunscreens in the form of UV-absorbing
compounds (UVAC), also known as mycosporine-like amino acids
(MAAs) (Shick et al., 1999; Banaszak et al., 2000). These mechanisms
of photoacclimation vary among species of symbiotic host cnidarians,
partly because their zooxanthellae comprise a highly divergent group
of dinoflagellates (Coffroth and Santos, 2005) with a broad range of
genotypic and phenotypic responses to light (Iglesias-Prieto and
Trench, 1994, 1997; Savage et al., 2002; Robison and Warner, 2006),
which in turn influence their ecological distributions (LaJeunesse,
2002; Iglesias-Prieto et al., 2004).
In addition, variation among host species in traits such as behavior
[e.g., polyp retraction (Brown et al., 1994), and contraction/expansion
(Brown et al., 2002)], gastrodermal tissue structure (Kuguru et al.,
2007), skeletal structure (Mass et al., 2007), and light absorption by
fluorescent proteins (Salih et al., 2000), all potentially modulate the
available light and, thereby, impact the photochemical response of
their endosymbionts. While species-specific patterns of photoacclimation have been elucidated for marine algae (Huner et al., 1996),
stony corals (Falkowski and Dubinsky, 1981; Warner and Berry-Lowe,
2006), and actinian sea anemones (Stoletzki and Schierwater, 2005),
almost nothing is known about mechanisms of response to light stress
in corallimorpharians.
The corallimorpharians R. rhodostoma and D. unguja are both
common on coral reefs in some areas of the Indo-Pacific, and are
successful recolonizers of shallow habitats following disturbances
such as bleaching, which kill stony corals and other zooxanthellates
(Chadwick-Furman and Spiegel, 2000; Kuguru et al., 2004; Work et al.,
2008). They occupy contrasting microhabitats on the reef: individuals
of R. rhodostoma form large aggregations on intertidal reef flats while
those of D. unguja occupy holes and crevices deeper on the reef slope
(Muhando et al., 2002; Kuguru et al., 2008). Effects of UVR are
expected to be highly pronounced in the Red Sea, which is classified as
an oligotrophic class II water body, representing one of the most
optically clear water bodies in the world (Stambler, 2005). Thus,
understanding differences in mechanisms of UVR acclimation between these two corallimorpharians may provide insights into their
bathymetric distributional patterns and predict the extent to which
they can withstand climate change. The objective of the present study
was to experimentally assess the photoacclimation mechanisms of
these two corallimorpharian species in response to increased PAR and
UVR in terms of the photosynthetic parameters of their zooxanthellae
density and the cellular responses of their tissues.
2. Materials and methods
2.1. Study site and polyp collection
This study was conducted during the period January–August 2007
at the Interuniversity Institute for Marine Sciences (IUI), in Eilat,
Israel, in the northern Red Sea (29º30′N, 34°55′E). Polyps of the
corallimorpharians R. rhodostoma and D. unguja were collected on
coral reefs adjacent to the IUI at depths of 3–20 m, and attached to
polyvinyl chloride (PVC) bases using underwater epoxy. Care was
taken to ensure that the replicate polyps were located at least 10 m
apart from each other, to avoid collecting individuals that originated
asexually from the same parent colonies. Following a one-month
acclimation period in outdoor aquaria (irradiance of 300 μmol photons m− 2 s− 1 (PAR), equivalent to that at 18–20 m depth, flowthrough seawater at 120 L h− 1), the polyps were transferred to
experimental aquaria for treatments (see below, modified after
Kuguru et al., 2007, 2008).
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
55
2.2. Experimental design
We assayed three major types of characteristics related to host and
algal responses to light stress (increased PAR and UVR) in these
corallimorpharians: (1) physiological parameters reporting photosynthetic activity (zooxanthella density, chlorophyll a (chl a) pigment
biomass, and chl a fluorescence), to indicate whether metabolic
processes (photosynthesis and oxidative phosphorylation) were
subject to photoinhibition; (2) host-lipid peroxide (LPO) levels, to
indicate whether the structural integrity of host cells was challenged;
and (3) host enzymatic activity, specifically copper/zinc superoxide
dismutase (Cu/ZnSOD), to indicate whether there was an enzymatic
response to oxidative stress. These parameters were selected because
they reflect cellular physiological functions that provide evidence for
identifying the stressors in terms of PAR, UVR, or both (Dunlap and
Chalker, 1986; Kana, 1992; Iglesias-Prieto and Trench, 1994; Downs et
al., 2002).
In this study, we performed a laboratory experiment under
controlled conditions to understand the physiological acclimatization
mechanisms of both photosymbiont and host cells of the two species
of corallimorpharians when exposed to changes in environmental
conditions. The setup of the experiment was based on results obtained
from our previous studies (Kuguru et al., 2007, 2008). This
experiment was performed using twelve 9-L aquaria, each supplied
with ambient (26 °C) through-flow seawater at a constant flow rate of
2 L min− 1 and a mixing pump of 230 L h− 1. A filter (100 μm) was
fitted to the 20-mm supply pipe connected to a 50-mm main seawater
supply pipe to filter out bivalve shells while allowing phytoplankton
and zooplankton to pass through. The filter was cleaned every two
days to ensure a uniform flow rate to each unit.
Every aquarium contained one polyp each of R. rhodostoma and D.
unguja. Six individuals of each species were photoacclimated using
layers of plastic-netting shade for one month in either of two outdoor
treatments: (1) high light (HL), 50% shade, with a maximum midday
irradiance of 700 μmol photons m− 2 s− 1, equivalent to irradiance at
approximately 5 m water depth; and (2) low light (LL), 90% shade,
with a maximum midday irradiance of 350 μmol photons m− 2 s− 1,
equivalent to irradiance at approximately 20 m depth. Thus, there
were six aquaria for each treatment, totalling 12 aquaria. Light (PAR;
in μmol photons m− 2 s− 1) was measured using a quantum sensor (LI190SA, LiCor, USA). The experiment was designed to mimic the
natural light conditions during summer months on coral reefs in Eilat,
based on PAR measurements.
After one month of pre-acclimation in LL and HL, the organisms
were covered with two different cut-off filters: PAR alone (700 nm ≤
λ ≥ 400 nm), hereafter called UVO; or PAR + UVR (700 ≤ λ ≥ 295 nm),
hereafter called UVT; each with 90% spectral irradiance transmission
(Fig. 1). Thus, four PAR/UV treatments were generated: (1) high light
with UVT (HLUVT); (2) high light without UV (HLUVO); (3) low light
with UVT (LLUVT); and (4) low light without UV (LLUVO). There were
three polyps of each species in each of these four treatments.
Irradiances (UVR + PAR; in μW cm− 2) in each treatment were
quantified using a radiometer (Biospherical PRR-800, San Diego,
USA). We used a ‘preacclimatization’ stage before exposing the
corallimorpharians to the four treatments in consultation with results
from our previous study (Kuguru et al., 2008), which showed that when
exposed to high irradiance under laboratory conditions or in field
transplants for about a month, members of both corallimorpharian
species hosted Symbiodinium type C1. Thus, in the present study, we
pre-acclimatized all polyps for one month to ensure that all hosted only
one Symbiodinium clade to prevent clade-based variation in their
tolerance/acclimatization mechanisms.
At the end of the above experiment on light/UV exposure (i.e.,
30 days), the stress responses of polyps of R. rhodostoma and D.
unguja to the synergistic effects of UVR and PAR were assessed.
First, a chlorophyll a fluorescence reading was taken 30 min after
Fig. 1. Irradiance spectrum of natural solar radiation over outdoor experimental aquaria
at the Interuniversity Institute for Marine Science in Eilat, Israel, and of 290 nm and
400 nm cut-off filters deployed to create experimental treatments (see text for details).
sunset using the fluorescence induction and relaxation system FIRe
[Satlantic Instrument, Canada (Kolber et al., 1998), see below for
further explanation]. Then, at midday the following day, a small
amount of tissue (approximately 0.1 g) was removed from the oral
disc of each polyp and immediately snap-frozen in liquid nitrogen to
minimize handling effects and stop enzymatic activities. Collection
took place between 12:00 and 13:00 h to ensure that explants were
sampled at the highest light intensities. Samples were then stored at
−80 °C.
For biochemical analysis, frozen samples (approximately 0.1 g
fresh weight) were homogenized with 2 mL of ice-cold 50 mM
potassium phosphate buffer (pH 7.4). To prevent oxidation of
samples, 10 μL 0.5 M butylated hydroxytoluene (BHT) in acetonitrile
was added to 1 mL of tissue homogenate. For separation of
zooxanthellae and host cells, the resulting slurry from homogenized
tissue was centrifuged for 5 min at 6000 rpm at 4 °C. The pellet
(zooxanthella cells) was re-suspended in 2 mL 50 mM potassium
phosphate buffer (pH 7.4), aliquoted in 1000 μL and stored at −80 °C.
The supernatant (host cells) was placed in a new eppendorf tube and
further centrifuged for 10 min at 10,000 rpm at 4 °C to remove host
tissue. The clear supernatant was aliquoted in 400 μL and stored at
−80 °C. The aliquots of zooxanthella cells were used for zooxanthella
abundance and chlorophyll a pigment assessment. Host cells aliquots
were used for Cu/ZnSOD and LPO assessment.
In zooxanthella-containing fractions from the corallimorpharians
(see above), we measured (1) chl a content (overnight extraction in
90% acetone), with absorbance readings at 630, 664, and 750 nm
(Jeffrey and Humphrey, 1975); and (2) zooxanthella abundance (cells
counted under a light microscope using a haemocytometer) after
Kuguru et al. (2007). Algal pigment and abundance were normalized
per milligram of total protein. Normalizing data per wet tissue mass
(Kuguru et al., 2007) was not possible because all collected samples
were frozen immediately in liquid nitrogen to minimize handling
effects and to stop enzymatic activities.
To determine the synergistic effects of PAR and UVR, the activity
of antioxidant enzymes (Cu/ZnSOD) due to oxidative stress in host
cells was measured using a spectrophotometric kit assay for
superoxide dismutase (Cu/ZnSOD-525, Catalog number 21010 from
Oxis International, Inc., USA) according to the manufacturer's
instructions. Each assay was done in triplicate in each of the four
irradiance treatments. Results were expressed as Cu/ZnSOD U mg− 1
soluble host protein.
To test whether oxidative and cell damage was associated with
lipid peroxidation (LPO) due to the synergistic effects of PAR and UVR
on host cells, malondialdehyde (MDA) was assayed spectrophotometrically using a lipid peroxidation assay kit (Oxford Biomedical
56
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
Research MDA-586, Catalog number 21025, from Oxford Biomedical
Research, USA) according to the manufacturer's instructions. Each
assay was done in triplicate in each of the four irradiance treatments.
Results were expressed as μM LPO mg− 1 soluble host protein.
The level of protein concentration in the crude extracts of samples
(n = 3) in all irradiance treatments was determined spectrophotometrically (Bradford, 1976) using a protein assay kit [Bovine serum
albumin (BSA) standard set, Catalog number 500-0207 from Bio-Rad,
USA] according to the manufacturer's instructions.
Comparison of the synergistic effects of UVR and PAR on the
photosynthetic performance of algae associated with the polyps in the
four treatments was performed at the end of PAR/UVR acclimation,
i.e., after one month of incubation. Chlorophyll fluorescence yields
were measured using the fluorescence induction and relaxation
system (FIRe; Satlantic Instrument) as an indication of the number of
photosystem II (PSII) units (Kolber et al., 1998). FIRe fluorometry
measures chlorophyll fluorescence transients using a controlled series
of subsaturating flashes that cumulatively saturate PSII within
~ 100 μs, i.e., a single photochemical turnover (Kolber and Falkowski,
1993; Kolber et al., 1998). The FIRe technique can, therefore, measure
several fluorescence parameters. Fo and Fm are minimum and
maximum yields of chl a fluorescence, measured in a dark-adapted
state. Fv is variable fluorescence (Fo − Fm), and Fv/Fm (−(Fm − Fo)/Fm)
is the maximum quantum yield of photochemistry in PSII as measured
in a dark-adapted state. In addition to these parameters, the FIRe
fluorometer instantaneously measures the functional absorption cross
section of PSII (σPSII), which characterizes the capacity to absorb and
utilize visible radiation (Kolber et al., 1998) via the fluorescence
signature of the O2-evolving complex, PSII. Cross sections derived
from conventional O2 evolution measurements were similar to σPSII
(Falkowski et al., 1988). Using a pulse amplitude-modulated fluorometer (Diving PAM, Walz, Germany), light pressure or excitation
pressure on photosystem II (Qm) was determined according to Kuguru
et al. (2007). Differences in Fv/Fm, σPSII, and excitation pressure on PS
II between symbionts within some corals provide a predictive
measure of how coral species and algal symbionts respond to natural
thermal stress (Falkowski and Chen, 2003).
A separate laboratory experiment was conducted to assess the
behavioral responses of both corallimorpharian species to UVR stress.
An outdoor 100-L flow-through aquarium supplied with running
seawater at a constant flow rate of 120 L h− 1 was set up under an
overhanging roof (not exposed to direct sunlight), with a maximum
midday irradiance of approximately 85% μmol photons m− 2 s− 1
(equivalent to irradiance at approximately 20–30-m depth). This
treatment mimicked irradiance levels at the deeper end of the depth
range of these two species (Chadwick-Furman and Spiegel, 2000;
Kuguru et al., 2007). Five polyps of each species were affixed to a PVC
base, and then the bases were attached to a 4 × 30-cm plastic plate.
Two plates, each with five polyps of either species of corallimorpharian, were acclimated in the aquarium for one year. Observations were
made on the behavioral responses of the corallimorpharians in terms
of their positions on the bases.
2.3. Statistical analyses
The combined effects of PAR and UVR on zooxanthella abundance,
chlorophyll-pigment concentration per cell, Fv/Fm, Qm, Sigma, SOD,
and LPO were examined using two-way ANOVA. The raw data were
used following examination of the homogeneity of the variances
(Levene's test) and the normality of the data (normal probability
plots). Proportional data were arcsine-transformed prior to statistical
analysis. Significant differences between levels within the factors
were examined post-hoc with Student-Newman-Keuls (SNK) tests.
The behavioral response (habitat preference) by corallimorpharians
was evaluated using the Strauss (1979) selectivity index (L).
3. Results
The irradiance spectra of the UVR cut-off filters used in this study
show that these filters screened UVR effectively (Fig. 1). A comparison
of the irradiance quantities measured at sea and in each experimental
laboratory treatment revealed that the corallimorpharian polyps
under the HLUVT treatment experienced PAR and UVR (313 nm)
equal to radiation experienced by polyps at approximately 2-m depth
in the sea (Table 1). Polyps under LL treatment experienced PAR equal
to approximately 23-m depth, but UVR (313 nm) in the LLUVT was
equivalent to approximately 5-m depth. Polyps situated under the
hanging roof experienced PAR equal to approximately 40-m depth
and UVR (313 nm) equal to approximately 10-m depth (Table 1).
Polyps of the two corallimorpharian species differed in their
physiological responses to solar radiation effects (PAR and/or UVR
only). The zooxanthella abundance and chlorophyll a concentrations
of both species decreased with increasing levels of irradiance
(HLUVT N HLUVO N LLUVT N LLUVO; Fig. 2a, b). However, individuals of
D. unguja had significantly higher levels of chlorophyll a concentration
(2-way ANOVA: F1, 24 = 7.66, p = 0.014) and lower levels of zooxanthella abundance (2-way ANOVA: F1, 24 = 37.65, p = 0.00001) in their
tissues than did individuals of R. rhodostoma (Fig. 2a, b). Despite
these trends, chlorophyll a pigment per zooxanthella cell did not differ
significantly between the two species among treatments (2-way
ANOVA, F3, 24 = 1.24, p = 0.33; Fig. 2c). There was no significant interaction effect between species and treatment group for chlorophyll
pigment (2-way ANOVA: F3, 24 = 3.06, p = 0.058), zooxanthella abundance (2-way ANOVA F3, 24 = 1.16, p = 0.4), and chlorophyll concentration per algal cell (2-way ANOVA, F3, 24 = 1.36, p = 0.3; Fig. 2a, b, c).
Measurements of chlorophyll a fluorescence parameters (Fv/Fm,
σPSII, and Qm) revealed that higher irradiance levels reduced
photosynthetic processes for both species of corallimorpharians
(Fig. 3). The impact was most vivid in the HLUVT treatment, where
high irradiance levels reduced Fv/Fm (2-way ANOVA: F1, 24 = 21.03,
p = 0.0003; σ PSI, 2-way ANOVA: F1, 24 = 4.9, p = 0.04), and also
caused a significant increase in Qm (2-way ANOVA: F1, 24 = 16.93,
p = 0.001) levels in polyps of D. unguja compared to R. rhodostoma
(Fig. 3). There was a significant interaction effect between species and
treatment group for Fv/Fm (2-way ANOVA: F3, 24 = 8.22, p = 0.002),
σPSII (2-way ANOVA: F3, 24 = 5.48, p = 0.01), and Qm (2-way ANOVA:
F3, 24 = 9.28, p = 0.001; Fig. 3). Student-Newman-Keuls (SNK) posthoc tests at P b 0.05 revealed that polyps of D. unguja exposed to
HLUVT irradiance levels had significantly lower values of Fv/Fm and
σPSII than did polyps of D. unguja exposed to the other three irradiance
Table 1
Irradiance (UVR + PAR) levels from radiometer (Biospherical PRR-800, San Diego, USA)
measurements under field and laboratory conditions in the present study.
A. Irradiance (μWcm− 2) attenuation in the field on coral reefs at Eilat, northern
Red Sea.
Depth (m)
UVB (313 nm)
UVA (340 nm)
PAR (400-700 nm)
0
1
3
5
15
20
23
17.5
9.3
2.9
0.7
0.1
0.0
0.0
56.6
34.5
16.2
6.4
2.4
0.8
0.5
155.5
94.2
47.0
29.8
19.4
11.5
9.0
B. Irradiance levels (μWcm− 2) within each laboratory treatment.
Irradiance
treatment
HL
LL
UVT
UVO
UVT
UVO
UVB (313 nm)
UVA (340 nm)
PAR
6.7
0.0
0.7
0.0
23.9
0.0
2.4
0.0
89.8
89.5
9.0
9.0
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
57
Fig. 2. Variation in zooxanthella and chlorophyll parameters among laboratory treatments in polyps of the corallimorpharians Rhodactis rhodostoma and Discosoma unguja. Data
shown are after 30 days of exposure to four laboratory treatments: high light (daily maximum 700 μmol photons m− 2 s− 1) with UV radiation (HLUVT), high light with no UV
radiation (HLUVO), low light (daily maximum 350 μmol photons m− 2 s− 1) with UV radiation (LLUVT), and low light with no UV radiation (LLUVO). Data are means ± SE, n = 3
polyps per treatment. Treatments marked with different superscript letters are significantly different at P b 0.05 (Student-Newman-Keuls post-hoc tests, see text for details).
treatment levels (HLUVO, LLUVT/-UVO). In contrast, polyps of R.
rhodostoma demonstrated equal values of Fv/Fm (2-way ANOVA: F3, 24 =
1.86, p = 0.2) and σPSII (2-way ANOVA: F3, 24 = 10.85, p = 0.5) at all
four irradiance treatment levels, indicating that UVR did not induce
stress responses in the algal symbionts of this host species (Fig. 3a, b).
HL irradiance treatments significantly increased Qm values in the
Fig. 3. Variation in chlorophyll a fluorescence parameters among four laboratory treatments in polyps of the corallimorpharians Rhodactis rhodostoma and Discosoma unguja. Data
shown are after 30 days of exposure to four laboratory treatments: high light (daily maximum 700 μmol photons m− 2 s− 1) with UV radiation (HLUVT), high light with no UV
radiation (HLUVO), low light (daily maximum 350 μmol photons m− 2 s− 1) with UV radiation (LLUVT), and low light with no UV radiation (LLUVO). Data are means ± SE, n = 3
polyps per treatment. Treatments marked with different superscript letters are significantly different at P b 0.05 (Student-Newman-Keuls post-hoc tests, see text for details).
58
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
zooxanthellae of both host species as compared to LL irradiance
treatments (2-way ANOVA: F3, 24 = 2.73, p = 0.08). In contrast, D.
unguja was significantly more sensitive to UVR than was R.
rhodostoma (2-way ANOVA: F1, 24 = 16.93, p = 0.001; Fig. 3c) in that
the zooxanthellae of D. unguja had significantly higher values of Qm
when exposed to UVT versus UVO treatments, while those of R.
rhodostoma did not (SNK post-hoc tests at P b 0.05; Fig. 3c.)
Exposure to high levels of irradiance induced increased host
cellular (enzymatic) Cu/ZnSOD and LPO activity in both R. rhodostoma
and D. unguja, but the magnitude of these responses differed between
the host species (2-way ANOVA: F1, 12 = 16.13, p = 0.004; Fig. 4a), and
LPO (2-way ANOVA, F1, 24 = 24.83, p = 0.0001; Fig. 4b). For the HLUVT
treatment, higher irradiance levels caused significantly lower Cu/
ZnSOD activities (2-way ANOVA: F 3, 12 = 351.05, p = 0.0001; Fig. 4a)
and higher LPO (2-way ANOVA: F1, 24 = 24.83, p = 0.000; Fig. 4b)
(host cellular degradation) in D. unguja as compared to R. rhodostoma.
In addition, all treatments with UVT, but not UVO, caused significantly
higher host cellular (protective) Cu/ZnSOD enzymes (2-way ANOVA:
F3, 12 = 351.05, p = 0.0001; Fig. 4a). In both LL Cu/ZnSOD treatments,
samples were inadvertently mistreated and, therefore, no data were
obtained.
The behavioral response experiment revealed D. unguja to be lightsensitive [Strauss (1979) selectivity index L = −0.5; Table 2]. All five
polyps of D. unguja moved from the upper exposed surfaces to the
more shaded sides of their PVC bases within 6–8 weeks (Fig. 5b). In
contrast, the polyps of R. rhodostoma did not relocate at all during the
12-month period [Strauss (1979) selectivity index L = 0.5; Table 2],
and remained on top of their bases, fully exposed to ambient irradiance
(Fig. 5a).
Fig. 4. Variation in levels of superoxide dismutase (Cu/ZnSOD) and lipid peroxidation
(LPO) of the corallimorpharians Rhodactis rhodostoma and Discosoma unguja among
laboratory treatments. Data shown are after 30 days of exposure to four laboratory
treatments: high light (daily maximum 700 μmol photons m− 2 s− 1) with UV radiation
(HLUVT), high light with no UV radiation (HLUVO), low light (daily maximum
350 μmol photons m− 2 s− 1) with UV radiation (LLUVT), and low light with no UV
radiation (LLUVO). Data are means ± SE, n = 3 polyps per treatment. Treatments
marked with different superscript letters are significantly different at P b 0.05 (StudentNewman-Keuls post-hoc tests, see text for details).
Table 2
Proportions of corallimorpharian polyps maintaining their position on plastic bases (R),
the relative contribution of polyps of both corallimorpharian species (two plates
pooled) (P), and the Strauss (1979) Linear Selectivity Index (L). NB: Selectivity Index
(L) values range from − 1 to 1, with positive values indicating preference and negative
values indicating avoidance; ** = L very significant from 0.
Rhodactis rhodostoma
Discosoma unguja
R
P
L = R−P
L=0
1.00
0.00
0.50
0.50
0.50
− 0.50
**
**
4. Discussion
We demonstrate here that two common corallimorpharians on
Indo-Pacific coral reefs utilize a variety of contrasting mechanisms of
photoacclimation to mitigate the damaging effects of high solar
irradiation. Their defensive mechanisms, when experimentally exposed to the synergistic effects of UVR + PAR, included: (1) reductions
in the abundance of zooxanthellae and concentration of chlorophyll a
(Fig. 2); (2) quenching of the light excitation energy, as revealed by
variations in Qm and σPSII (Fig. 3b, c); (3) host-mediated synthesis of
enzymatic antioxidants (Fig. 4a, b); and (4) behavioral responses to
high irradiance via polyp migration to shaded microhabitats (Fig. 5).
Our observations that both chlorophyll concentration and zooxanthella abundance in these corallimorpharians decrease with level
of irradiance (Fig. 2a, b) are similar to patterns observed in other
cnidarians with variations in depth, irradiance, and temperature
(Fagoonee et al., 1999; Fitt et al., 2000; Kuguru et al., 2007; Mass et al.,
2007). These changes apparently prevent host cellular damage from
the oxidative stress induced by elevated sea temperature, PAR, and/or
UVR (Baruch et al., 2005; Lesser and Shick, 1989), and also allow the
host to adjust its photosynthate supply to nutritional demands. The
amount of chl a per symbiont cell in these corallimorpharians was not
affected by light regime, in contrast to the stony coral Stylophora
pistillata, which increases its pigment per symbiont cell with
decreasing light (Falkowski and Dubinsky, 1981). However, patterns
in the corallimorpharians are consistent with the effects of ambient
UVR on the stony coral Montipora verrucosa zooxanthellae, in which
equal values of chl a occur per algal cell at both low and high
irradiance levels (Kinzie, 1993). Gleason and Wellington (1993)
concluded that coral bleaching induced by UVR results from
reductions in zooxanthella densities rather than the combined effects
of zooxanthella expulsion and decreases in chlorophyll concentration
per algal cell, as has been noted for temperature-related bleaching
(Glynn and Dcroz, 1990). We did not quantify levels of accessory
pigments other than chlorophyll in the zooxanthellae, but these also
may have responded differently to the irradiance treatments.
Our measurements of σPSII revealed that the mechanisms of
physiological acclimation differed significantly between R. rhodostoma
and D. unguja (Fig. 3b). σPSII measures the efficiency of excitation energy
transfer from the antenna pigments to the PSII reaction center and
represents the amount of light energy usable for photochemical
reactions (Kolber et al., 1998). It strongly depends on environmental
conditions such as Qm/light pressure (Kolber et al., 1990). Qm values
showed higher excitation energy in D. unguja zooxanthellae than in
those of R. rhodostoma (Fig. 3c). In addition, a significant decline in σPSII
values during HLUVT treatment in D. unguja zooxanthellae (Fig. 3b)
suggests that they were highly susceptible to the synergistic effects of
high irradiance and UVR. Lower values of both σPSII and Fv/Fm in HL
compared to LL treatments in D. unguja (Fig. 3a, b) most likely resulted
from a combination of photodamage and photoprotective processes
(Warner et al., 2002) and correlated with long-term photoinhibition (qI)
values (Brown et al., 1999; Falkowski and Chen, 2003; Gorbunov et al.,
2001). In contrast, both σPSII and Fv/Fm values did not change in R.
rhodostoma zooxanthellae regardless of the irradiance treatment,
suggesting that these symbionts were not physiologically compromised,
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
59
Fig. 5. Photographs of representative polyps of the corallimorpharians Rhodactis rhodostoma and Discosoma unguja, showing their behavioral responses to irradiance under: (a, b)
laboratory conditions and (c, d) field conditions, on coral reefs in the Red Sea.
possibly because they possess effective mechanisms for the dissipation
of excess light excitation energy. The cellular mechanisms underlying
these physiological responses may involve differential gene expression
induced by changes in irradiance, leading to antenna systems with
different σPSII values (Mortainbertrand et al., 1990; Smith et al., 2005).
This resilience to photooxidative stress can also result from other
underlying processes including sustained PSII repair, compensatory
electron flow through remaining functional reaction centers (Behrenfeld
et al., 1998) and structural differences in thylakoid membranes
(Tchernov et al., 2004).
Under laboratory conditions at low light irradiance, R. rhodostoma
harbors symbiont type C1 while D. unguja harbors symbiont type D1a.
However, at high irradiance, both R. rhodostoma and D. unguja harbor
Symbiodinium type C1 (Kuguru et al., 2008). A recent study in
Australia (Jones et al., 2008) indicates that both clades C1 and D1 may
confer equal thermo-tolerance to host corals. Hence, the differences in
zooxanthella photosynthetic performance observed here were most
likely due to microenvironment effects (Coffroth and Santos, 2005)
provided by the host rather than to genetic differences among their
zooxanthellae. Host-mediated photoacclimation mechanisms (Lesser
and Shick, 1989; Ambarsari et al., 1997; Salih et al., 2000; Brown et al.,
2002) may enhance photosynthetic rates in resident zooxanthellae
(Gates and Edmunds, 1999). Furthermore, inconsistencies between
the phylogenetic and physiological attributes of zooxanthellae
(Savage et al., 2002; Tchernov et al., 2004; Robison and Warner,
2006; Kuguru et al., 2008), and the physiological responses of isolated
versus in hospite Symbiodinium (Bhagooli and Hidaka, 2003; Goulet et
al., 2005), suggest that the host could be the determinant factor for the
fitness of the holobiont.
Levels of oxidative stress in the host were exacerbated when the
corallimorpharians were exposed to the synergistic effects of PAR +
UVR versus either PAR or UVR alone (Fig. 4). Host cellular responses
(Fig. 4a) indicate that individuals of D. unguja have higher levels of ROS
than do those of R. rhodostoma. In other cnidarians, high levels of ROS
correlate with exposure to UVR radiation, which causes inhibition in
the photosynthetic machinery of PSII (Iwanzik et al., 1983) by
degrading the D1/32 kDa protein complex (Melis et al., 1992; Malanga
et al., 1997). UVR radiation also causes ROS and high levels of SOD in
green algae and diatoms (Lesser, 2006). Generation of photosynthesismediated ROS occurs in the chloroplast through several mechanisms
associated with photosystem I- and photosystem II-catalyzed electron
transfer, the most notable being the Mehler reaction and the
generation of hydrogen peroxide by the oxygen-evolving complex
(Kana, 1992; Downs et al., 2002). H2O2 may diffuse out of the
chloroplasts through the algal symbiont into the host cytoplasm,
where it can be neutralized by either an enzymatic or non-enzymatic
antioxidant pathway or catalyzed through the Fenton reaction to
hydroxyl (OH) radical (Downs et al., 2002). Above a threshold level,
the presence of OH will cause the host to either expel or destroy the
zooxanthellae as a defense mechanism against oxidative stress (Lesser
and Shick, 1989). Perez and Weis (2006) later suggested that ROS
released from heat/UV-stressed photosystems in algal cells might
induce nitric-oxide (NO) production in host cells. They specifically
hypothesized that NO converts to cytotoxic peroxynitrite upon
interaction with superoxide in the host cell, leading to cell death and
bleaching. In our case, the significantly higher Cu/ZnSOD and LPO
levels (Fig. 4a, b) in D. unguja host cells in the HLUVT treatment (Fig. 4)
indicate that the enzymatic and non-enzymatic oxidants within these
cells were unable to compensate for and ameliorate the destructive
capacity of the ROS. On the other hand, low values of LPO and Cu/
ZnSOD in the host cells of R. rhodostoma demonstrate that they more
effectively suppress the ROS produced by zooxanthellae.
60
B. Kuguru et al. / Journal of Experimental Marine Biology and Ecology 394 (2010) 53–62
Both our laboratory (Fig. 5a, b) and field observations (Fig. 5c, d)
indicate that individuals of D. unguja exhibit behavioral mechanisms
to avoid the damaging effects of high solar radiation (UVB).
Corallimorpharians locomote at slow rates of b10 mm/month due to
their lack of basilar muscles on the pedal disc (Chadwick and Adams,
1991). Thus, we observed polyps changing their positions over a
period of weeks rather than hours or days. In shallow water on coral
reefs, D. unguja occupies shaded areas such as holes, crevices, and the
branches of corals (Fig. 5c). In the foliose coral Turbinaria mesenteria,
the vertical orientation of the plates leads to self-shading of N50% and
is an effective mechanism of photoprotection to avoid photoinhibition
in shallow areas (Hoogenboom et al., 2008). Accordingly, individuals
of D. unguja that live among the branches of T. mesenteria (Fig. 5c)
protect themselves from high irradiance. Furthermore, polyps of D.
unguja rapidly withdraw into coral and rock crevices when physically
disturbed, and also expand their oral discs upon exposure to low
levels of irradiance but contract when encountering excessive
irradiance (B. L. Kuguru, personal observation). This is similar to the
behavioral reactions of some stony corals (Levy et al., 2006). Many
types of host cnidarians adopt behavioral photoprotective mechanisms under high-light conditions (Brown et al., 1994, 2002). Tissue
retraction also has been reported in the sea anemone Anthopleura
elegantissima as a response to intense sunlight, which not only limits
exposure to high irradiance but also reduces the production of
harmful oxygen radicals (Shick and Dykens, 1984). Marked retraction
when exposed to high irradiance may be triggered by high levels of
photosynthetically generated O−
2 in actinian sea anemones (Shick et
al., 1991) and by photodynamic processes in the tissues of corals
(Dykens and Shick, 1984). Such avoidance behavior in anemones
(Shick and Dykens, 1984), corals (Brown et al., 1994), and
corallimorpharians (Kuguru et al., 2007) may be likened to the
movement of zooxanthellae away from the mesoglea in the
corallimorpharian R. rhodostoma (Kuguru et al., 2007), as well as
chloroplast movements in plants (Brugnoli and Bjorkqman, 1992) and
seagrasses (Sharon and Beer, 2008), where pronounced reductions in
chlorophyll fluorescence have been noted during leaf-folding chloroplast re-orientation at high irradiance. In-situ experimental studies on
the effects of UVR on the behavior and recruitment of the stony coral
Porites asteoides showed that many larvae swam and settled in
habitats where UVR was reduced (Gleason and Wellington, 1995;
Gleason et al., 2006). Thus, behavioral avoidance of habitats with
biologically damaging levels of UVR may be a major factor
contributing to the successful recruitment of coral larvae. As a driving
force for extensive behavioral, physiological, and cellular adaptations
in benthic marine invertebrates, UVR may be a more important factor
than previously recognized in determining community structure in
aquatic ecosystems (Hader et al., 2007). It appears that the two
corallimorpharians examined here respond to UVR by exerting major
control over their patterns of microhabitat use and depth distributions
on reefs.
Some of the differences that we observed in photosynthetic
performance between these corallimorpharians may be due to the
differences in the mechanisms to adjust photoprotective versus nonphotosynthetic pigments in their light-harvesting antennae (IglesiasPrieto and Trench, 1997). The ratio of light-protective pigments in the
xanthophyll cycle, diadinoxanthin (DD), to the sum of DD and
diatoxanthin (Dt), shows linear correspondence with σPSII, i.e., as DD
de-epoxidizes (forms a quencher) to DT, the σPSII decreases (Kolber
and Falkowski, 1993). Expression of low values of σPSII and high Qm at
HLUVT suggests that D. unguja zooxanthellae exhibited a deficiency in
either de-epoxidation or epoxidation of their xanthophyll pigments
(Niyogi et al., 1997). The inability of D. unguja zooxanthellae to
dissipate excessive light energy apparently caused a reduction in their
Fv/Fm at HLUVT, and higher values of host LPO and ROS at HL/LLUVT,
indicating that UVR, especially UVB, caused oxidative damage to both
the photosynthetic apparatus and host cells. The presence in R.
rhodostoma polyps of efficient mechanisms for quenching excitation
energy in their photosynthetic apparatus, and amelioration of ROS in
symbiont and host cells, all cause members of this species to be less
susceptible to high irradiance.
We conclude that exposure to UVR is a major factor controlling the
depth-related distributional patterns of these two common reef
cnidarians. Further studies will be needed to fully understand the
underlying cellular mechanisms and biochemical pathways used by
corallimorpharians during adjustment of their photosystems (PSI and
PSII) and photoprotective pigments in response to irradiance on coral
reefs.
Acknowledgements
We gratefully acknowledge the staff and students of Dan Tchernov
and Maoz Fine of the Interuniversity Institute for Marine Sciences in
Eilat for technical assistance in various aspects of field and laboratory
studies. We also thank Gal Dishon for spectral irradiance measurements and Beverly Goodman for assistance in manuscript preparation.
This research was supported by funds from the Bar-Ilan University,
Auburn University, City University of New York, Baruch College, the
Interuniversity Institute for Marine Sciences in Eilat and Israel Science
Foundation grant (# 981/05). We also wish to thank the US National
Science Foundation grant (# 0920572). This research was submitted
in partial fulfilment of the requirements for a doctoral degree by B.K.
at Bar-Ilan University. Experiments performed in this study complied
with the current laws of Israel. [SS]
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