REPORT

Light-induced dissociation of antenna complexes in the symbiontsof scleractinian corals correlates with sensitivity to coral bleaching

R. Hill • A. W. D. Larkum • O. Prasil •

D. M. Kramer • M. Szabo • V. Kumar •

P. J. Ralph

Received: 10 February 2012 / Accepted: 4 May 2012 / Published online: 20 May 2012

� Springer-Verlag 2012

Abstract Elevated temperatures in combination with

moderate to high irradiance are known to cause bleaching

events in scleractinian corals, characterised by damage to

photosystem II (PSII). Photoprotective mechanisms of the

symbiont can reduce the excitation pressure impinging upon

PSII. In the bleaching sensitive species, Acropora millepora

and Pocillopora damicornis, high light alone induced photo-

protection through the xanthophyll cycle, increased content of

the antioxidant carotenoid, b-carotene, as well as the disso-

ciation of the light-harvesting chlorophyll complexes. The

evidence is compatible with either the membrane-bound

chlorophyll a-chlorophyll c2-peridinin-protein (acpPC) com-

plex or the peripheral peridinin-chlorophyll-protein complex,

or both, disconnecting from PSII under high light. The acpPC

complex potentially showed a state transition response

with redistribution towards photosystem I to reduce PSII over-

excitation. This apparent acpPC dissociation/reassociation

was promoted by the addition of the xanthophyll cycle

inhibitor, dithiothreitol, under high irradiance. Exposure to

thermal stress as well as high light promoted xanthophyll de-

epoxidation and increased b-carotene content, although it did

not influence light-harvesting chlorophyll complex (LHC)

dissociation, indicating light, rather than temperature, controls

LHC dissociation. Photoinhibition was avoided in the

bleaching tolerant species, Pavona decussata, suggesting

xanthophyll cycling along with LHC dissociation may have

been sufficient to prevent photodamage to PSII. Symbionts of

P. decussata also displayed the greatest detachment of

antenna complexes, while the more thermally sensitive spe-

cies, Pocillopora damicornis and A. millepora, showed less

LHC dissociation, suggesting antenna movement influences

bleaching susceptibility.

Keywords Photoprotection � Non-photochemical

quenching � Xanthophyll cycle � State transitions �Photoinhibition � Symbiodinium

Introduction

Scleractinian corals form a symbiotic association with

dinoflagellate algae (genus Symbiodinium), which reside

within the endodermal tissue of the host. This relationship

is highly vulnerable to rising ocean temperatures with

1–2 �C above the summer average enough to induce a

bleaching response under moderate to high irradiance

where the symbionts are expelled from the host and the

white calcium carbonate skeleton underlying the cnidarian

tissue becomes visible (Fitt et al. 2001; Lesser and Farrell

2004). In response to a warming climate, mass coral

Communicated by Biology Editor Dr. Anastazia Banaszak

R. Hill � A. W. D. Larkum � M. Szabo � V. Kumar � P. J. Ralph

Plant Functional Biology and Climate Change Cluster,

School of the Environment, University of Technology, Sydney,

PO Box 123, Broadway, Sydney, NSW 2007, Australia

Present Address:R. Hill (&)

Centre for Marine Bio-Innovation and Sydney Institute of

Marine Science, School of Biological, Earth and Environmental

Sciences, The University of New South Wales, Sydney,

NSW 2052, Australia

e-mail: ross.hill@unsw.edu.au

O. Prasil

Laboratory of Photosynthesis, Institute of Microbiology,

Opatovicky mlyn, 379 81 Trebon, Czech Republic

D. M. Kramer

Plant Research Laboratory, Michigan State University,

East Lansing, MI, USA

123

Coral Reefs (2012) 31:963–975

DOI 10.1007/s00338-012-0914-z

bleaching events are becoming more intense, widespread

and frequent (Hoegh-Guldberg 1999; Hughes et al. 2003).

The combined elevated temperatures and high irradi-

ance, which results in the bleaching response in corals,

initially causes photodamage in the symbionts at a site

along the photosynthetic pathway. Considerable work has

focused on identifying the vulnerable component of the

photosynthetic apparatus, with a range of sites receiving

attention. Photosystem II (PSII), dark-reactions and thyla-

koid membrane integrity have all been suggested to play

critical roles in the loss of photosynthetic activity within

the symbionts during exposure to bleaching events,

although conflicting evidence for many of these sites has

also been collected (see review by Lesser 2011). Regard-

less of the primary site of damage, a decline in PSII pho-

tochemical efficiency has been well described (Warner

et al. 1996; Jones et al. 1998; Hill et al. 2004; Takahashi

et al. 2004) and is likely due to damage to the D1 protein of

PSII (Warner et al. 1999; Takahashi et al. 2009; Hill et al.

2011), most likely as a result of the accumulation of

reactive oxygen species (ROS) under bleaching conditions

(Lesser 1996, 1997).

Under excessive irradiance and temperatures beyond the

optimal thermal range, scleractinian corals are known to

utilise a number of structural (Salih et al. 2000; Dove 2004),

physiological (Brown et al. 1999; Jones and Hoegh-Guld-

berg 2001; Hill et al. 2005, 2010; Venn et al. 2006; Reynolds

et al. 2008) and behavioural (Brown et al. 1994, 2002)

strategies to limit long-term damage. Here, we focus on the

range of physiological photoprotective mechanisms that are

available to coral symbionts that reduce over-excitation

under excessive irradiances and that can otherwise lead to

photoinhibition. Non-photochemical quenching (NPQ)

mechanisms, the most prominent of which is the xanthophyll

cycle (Brown et al. 1999), are regulated forms of photo-

protection, which respond to environmental stressors that

have the ability to result in photoinhibition (Demmig and

Winter 1988; Niyogi 2000; Muller et al. 2001). Non-invasive

chlorophyll fluorescence techniques have been widely used

to measure NPQ, along with the other competing pathways

of energy transfer once light is absorbed by chlorophyll.

Absorbed light can be utilised for one of three processes: (1)

photochemistry (Y(II)), (2) non-regulatory heat dissipation

(Y(NO): an indicator of photoinhibition) and (3) regulated

heat dissipation (Y(NPQ)) (Kramer et al. 2004). These

complementary components of energy transfer can be used

to reveal detailed shifts in photosynthetic processes under

environmental stressors (Bonfig et al. 2006).

Regulation of absorbed light energy can take place in

the light-harvesting chlorophyll complexes (LHCs), which

serve to absorb photosynthetic radiation and channel the

light energy towards the reactions centres. The LHCs of

dinoflagellates are composed of both membrane-bound

chlorophyll a-chlorophyll c2-peridinin-protein (acpPC)

complexes (Hiller et al. 1993; Iglesias-Prieto et al. 1993;

Iglesias-Prieto and Trench 1997) and peripheral peridinin-

chlorophyll a-protein (PCP) complexes (Prezelin 1976;

Iglesias-Prieto et al. 1991; Iglesias-Prieto and Trench

1997). Additional pigments within the LHCs include the

xanthophyll and b-carotene carotenoids. Pigments of the

xanthophyll cycle have been shown to provide photopro-

tection through the inter-conversion of diadinoxanthin (Dd)

to diatoxanthin (Dt) under high irradiances (Brown et al.

1999; Ulstrup et al. 2008), and the de-epoxidation of this

pigment is known to be further upregulated during expo-

sure to thermal stress, which occurs during bleaching

events in corals (Dove et al. 2006; Venn et al. 2006). The

cycling of these pigments is thought to protect PSII from

excess excitation by dissipating absorbed light energy as

heat; during ensuing darkness, Dt is converted back to Dd

(Olaizola and Yamamoto 1994; Olaizola et al. 1994).

Furthermore, an increase in the abundance of b-carotene,

which provides antioxidant defence against ROS (Burton

1989; Young and Britton 1993), has been reported in coral

symbionts exposed to bleaching conditions (Ambarsari

et al. 1997; Venn et al. 2006).

More recently, through measures of low temperature

fluorescence (77K), Reynolds et al. (2008) proposed the

dissociation of putative PCP complexes from PSII during

exposure to high irradiance in cultured Symbiodinium. To

date, though, no research has addressed the potential for

PCP detachment within in hospite symbionts under optimal

or bleaching conditions or addressed the possibility of

detachment of acpPC complexes as an alternative expla-

nation of the data. PCP detachment represents an additional

form of photoprotection as absorbed light energy is not

transferred to PSII and would assist in the prevention of

ROS production as a triplet valve, as the carotenoid triplet

has a lower energy configuration compared to the long-

lived triplet chlorophyll (Alexandre et al. 2007). Interest-

ingly, Reynolds et al. (2008) only found this peripheral

LHC detachment in one clade of Symbiodinium (although

there is no conclusive evidence to suggest it is limited to

this one clade), and no evidence of any membrane-bound

LHC dissociation was found.

The membrane-bound LHCs of higher plants and green

algae are known to rearrange and undergo state transitions

where a balance of excitation energy is achieved between

the two photosystems by redistributing the LHCs to avoid

over-excitation of one photosystem and achieve maximum

efficiency (Fork and Satoh 1986; Williams and Allen 1987;

Haldrup et al. 2001). Highly reduced PQ pools under high

irradiance lead to the detachment and/or redistribution of

LHCs from PSII to PSI, which is referred to as a state 1 to

state 2 transition. The reverse transfer of LHCs from PSI to

PSII will occur if irradiance declines to a sub-saturating

964 Coral Reefs (2012) 31:963–975

123

intensity, if there is a more even excitation of PSII and PSI,

or if PSI becomes preferentially excited by incident irra-

diance (Fork and Satoh 1986; Allen 2003). This regulated

mechanism can partly prevent over-excitation of vulnera-

ble photosystems by decreasing the functional cross-sec-

tion of PSII (in green algae by 10–30 %; see Falkowski and

Raven 2007), although to date, this form of photoprotection

has not been characterised in Symbiodinium. The capacity

for membrane-bound LHCs of Symbiodinium to undergo

state transitions has not been studied, nor has its role in

managing over-excitation under high irradiance and ele-

vated temperatures been considered in the context of corals

and climate change.

In this study, the ability to implement regulated forms of

photoprotection was evaluated in three species of coral,

which differ in their susceptibility to thermal bleaching. In

those corals that experienced a bleaching response,

photoprotective mechanisms were assessed in order to

determine whether photoinhibition occurred due to an

inability to implement adequate modes of photoprotection,

or whether strategies to reduce over-excitation were inef-

ficient at preventing a bleaching response.

Materials and methods

Coral specimens

Three species of coral, Acropora millepora (Ehrenberg),

Pocillopora damicornis (Linnaeus) and Pavona decussata

(Dana), were sampled from Heron Island lagoon, located

on the southern Great Barrier Reef of Australia (151�550E,

23�270S) during November 2009. Prior to experimentation,

coral nubbins were maintained in shaded aquaria

(\100 lmol photons m-2 s-1) at the ambient lagoon

temperature of 25 �C for 2 days.

Experimental protocol

Nubbins of each coral species were placed in aerated and

well-mixed outdoor seawater aquaria at a depth of 20 cm at

the ambient lagoon temperature. Each coral species was

studied one at a time, with the experiments for each of the

three species run over two consecutive cloudless days.

Coral nubbins were either exposed to 25 �C (±0.2 �C) over

the 2-day period (control) or an elevated temperature

treatment (bleaching), where temperature was ramped to

31 �C (±0.2 �C) over 12 h (0.5 �C h-1) on the first day

and then maintained at this temperature using a water

heater/stirrer (Julabo Labortechnik, model EC, Germany).

Chlorophyll fluorescence measurements and 77K chloro-

phyll fluorescence emission spectra were taken at 0500

(pre-dawn), 0900, 1300, 1700 and 2100 hours (post-dusk)

on each day, while samples for symbiont pigment profile

determination (using high performance liquid chromatog-

raphy; HPLC) were taken at 0500, 1300 and 2100 hours

each day. The irradiance (in the photosynthetic active

radiation range of 400–700 nm) was measured every 5 min

using a logging photometer (Li-1400 with a Li-192

underwater quantum sensor; Li-Cor Lincoln, Nebraska).

The xanthophyll cycle inhibitor, dithiothreitol (DTT),

with a final concentration of 1.5 mM, was applied to samples

of Pocillopora. damicornis in the dark before exposure to

light to prevent symbiont diadinoxanthin de-epoxidation.

DTT is widely used as a xanthophyll cycle inhibitor in higher

plants (Bilger et al. 1989) as well as in diatoms (Olaizola

et al. 1994, Grouneva et al. 2008) and corals (Brown et al.

2002). The corals used in these DTT tests originated from

Heron Island, but had been maintained in a recirculating

500-l aquaria at the University of Technology, Sydney, for

2 months prior to use (26 �C, salinity 32 ppt, and irradiance

100 lmol photons m-2 s-1 on a 12:12 light:dark cycle). P.

damicornis nubbins were placed into aerated individual

beakers with 150 ml of seawater and exposed to 4 h of

650 lmol photons m-2 s-1 and a further 2 h of darkness at

26 �C in the presence and absence of DTT. Chlorophyll

fluorescence measurements, 77K chlorophyll fluorescence

emission spectra and symbiont pigment profiles were col-

lected at 0, 4 and 6 h. Only these DTT assays were per-

formed on corals transported to Sydney. All other

experiments were conducted on Heron Island after 2 days of

collection, and no direct physiological comparisons were

made between corals studied in the two locations.

Chlorophyll fluorescence measurements

Steady-state light curves (SSLCs) were taken on the upper,

sun-exposed surface of coral nubbins at each time point

(n = 4) using a Mini-PAM (Pulse Amplitude Modulated)

Fluorometer (Walz GmbH, Germany). Each nubbin was

placed in a dark-adaptation chamber for 15 min prior to

SSLC measurement with dark-adapted minimum fluores-

cence (FO) measured using the weak red (peak k 650 nm)

measuring light (\0.15 lmol photons m-2 s-1; gain = 12).

A saturating pulse of 0.8 s and [4,500 lmol photons

m-2 s-1 allowed for the determination of dark-adapted

maximum fluorescence (FM) and maximum quantum yield:

(FM–FO)/FM = FV/FM (Schreiber 2004). Subsequently, the

actinic light was turned on at an intensity of 180 lmol

photons m-2 s-1 and saturating pulses applied every 30 s to

determine light-adapted minimum fluorescence (Ft) and

light-adapted maximum fluorescence (FM0). Once steady

state was reached (Ft and FM0 were stable, usually after

5–10 min), the measurement was complete and effective

quantum yield (Y(II)), non-photochemical quenching yield

(Y(NPQ)) and non-regulated heat dissipation yield (Y(NO))

Coral Reefs (2012) 31:963–975 965

123

were calculated using the method of Kramer et al. (2004),

where Y(II) ? Y(NPQ) ? Y(NO) = 1.

Symbiont pigment analyses

Photosynthetic (chlorophyll a, chlorophyll c2, peridinin)

and photoprotective (diadinoxanthin, diatoxanthin and b-

carotene) pigment concentrations of the algal symbionts

were determined using reverse-phase HPLC (with slight

modification from Van Heukelem and Thomas 2001). At

each sampling time point (n = 4), a coral nubbin was

removed from the aquaria, snap frozen in liquid nitrogen

and placed in a freezer at -80 �C until further analysis.

Subsequently, each coral nubbin was placed in 10 ml of

HPLC-grade 100 % acetone, and tissue removed from the

skeleton using an ultrasonic probe (Sonic and Material Inc.

USA; Model-VC50T; 50 W, 20 kHz) for 30 s. Samples

were kept in the dark and on ice during sonication. Coral

skeletons were removed, and their surface area determined

using the paraffin wax technique (Stimson and Kinzie

1991). For HPLC analyses, the pigment absorbance spec-

trum was measured from 270 to 700 nm using a photodiode

array detector with a 4.3-nm bandwidth (Waters, Austra-

lia). Calibration and quality assurance was performed by

using external calibration standards of each pigment (DHI,

Hørsholm, Denmark). Empower Pro 2 software quantified

chlorophyll a concentrations at 665 nm, and all other

pigments at 450 nm through peak integration. Chlorophyll

a, chlorophyll c2 and peridinin pigment concentrations

were determined relative to coral surface area (lg cm-2).

b-Carotene content was normalised to chlorophyll a con-

tent to account for any bleaching response (Venn et al.

2006), while the de-epoxidation ratio (a measure of dia-

dinoxanthin conversion to the photoprotective diatoxan-

thin) was calculated as the diatoxanthin pool divided by the

total diatoxanthin ? diadinoxanthin pool (Dt/[Dt ? Dd]).

Chlorophyll fluorescence emission spectra at 77K

Immediately, upon removal from the experimental aquaria

at each sampling time point (n = 4), the upper, sun-

exposed tip of each coral nubbin was air brushed in 10 ml

of 0.45 lm filtered seawater to remove algal symbionts and

animal tissue from the skeleton. Then, 1 ml of this slurry

was filtered onto a 25-mm-diameter Whatman GF/F filter.

An oval disc 8 mm long and 3 mm wide was removed

from the centre of the filter using a custom-built hole-

punch. This disc was then placed into a groove at the end of

a metal shaft and immersed in a glass Dewar containing

liquid nitrogen. Surrounding the glass Dewar was a por-

table, lightweight, custom-built fluorescence spectropho-

tometer, which excluded ambient light (Prasil et al. 2009).

The sample in the Dewar was oriented at 45� to the direct

beam of a blue LED excitation light (peak k 470 nm). At

90� to the excitation light source and at 45� to the sample,

the fluorescence emission from the sample was detected

using a spectrally calibrated fibre optic spectrometer

(USB2000? UV-VIS, Ocean Optics, Florida, USA).

Fluorescence intensity (photon counts; relative units) was

detected every 0.3 nm at wavelengths [650 nm using a

long-pass filter in front of the spectrometer fibre optic.

The fluorescence spectrum (650–750 nm) was norma-

lised to a baseline of 0 and a maximum of 100 at 686 nm

and then de-convoluted into component bands (peak k at

675, 686, 701 and 721 nm) assuming a Gaussian distribu-

tion using PeakFit (SeaSolve Software Inc., Framingham,

Massachusetts, USA). The amplitude of (relative units;

0–100) and area under (expressed as a percentage) each

component band were calculated allowing for detection of

changes in the contribution of each component band to the

overall fluorescence emission spectrum.

Statistical analyses

One-way analysis of variance (ANOVA) and Tukey’s post

hoc multiple comparison tests (a = 0.05) were performed

on the independent samples to detect significant differences

over time and between treatments at each time point (SPSS

version 16). The Kolmogorov-Smirnov normality test and

Levene’s homogeneity of variance test were used to iden-

tify whether the assumptions of the parametric one-way

ANOVAs were satisfied. If these assumptions were not

met, arcsine (for ratio data) and log10 (for continuous data)

transformations were performed.

Results

Chlorophyll fluorescence

The intensity of natural photosynthetic active radiation

(PAR) incident on nubbins of three coral species over two

consecutive cloudless days for each species is shown in

Fig. 1a–c. Maximum irradiance reached approximately

1,500 lmol photons m-2 s-1 at 1200 hours on each day,

and variation in the total irradiance dose per day was

minor, with a minimum of 39.15 and maximum of

41.79 mol photons m-2. An inverse relationship between

irradiance and FV/FM was observed with FV/FM signifi-

cantly declining from a range of 0.64–0.67 at 0500 hours to

a minimum of 0.20–0.32 at 1200 hours on the first day in

all species (Fig. 1d–f) and in both temperature treatments

(P values \ 0.001). As light intensity declined on the first

day, FV/FM completely recovered, returning to a level

comparable to 0500 hours (Tukey’s post hoc comparisons).

Significant differences in species responses were observed

966 Coral Reefs (2012) 31:963–975

123

a

d

g

j

m n o

lk

h i

fe

b c

Fig. 1 a–c Changes in irradiance (lmol photons m-2 s-1), d–f FV/

FM, g–i Y(II), j–l Y(NPQ) and m–o Y(NO) for the control (closedcircles) and bleaching (open circles) treatments over the two-day

experimental period in Acropora millepora (left column), Pocillopora

damicornis (middle column) and Pavona decussata (right column). In

a–c, values beneath the light data for each day indicate the daily total

mol photons m-2. Mean ± SE (n = 4)

Coral Reefs (2012) 31:963–975 967

123

on the second day in the elevated temperature treatment with

FV/FM declining to 0.12 in A. millepora at 1300 hours and

not recovering as light intensity declined up to 2100 hours.

In P. damicornis, FV/FM declined to 0.10 at 1300 hours and

showed minimal recovery by 2100 hours with FV/FM

reaching 0.31. In contrast, the bleaching treatment had no

impact on FV/FM in Pavona decussata, with both tempera-

ture treatments showing the same diel response and a full

recovery of FV/FM by 2100 hours on the second day. FV/FM

on the second day in the control temperature treatments

in A. millepora and Pocillopora damicornis showed similar

responses to the first day with full recovery by 2100 hours

(Tukey’s post hoc comparisons).

The complementary components of Y(II), Y(NPQ) and

Y(NO), derived from the stead state light curves, are shown

in Fig. 1g–o. Y(II) showed similar responses to FV/FM

with midday depressions and morning and evening max-

ima. In the control temperature treatments, Y(II) returned

to a level equivalent to the initial 0500 hours measure-

ments at the 2100 hours time point on both days in all three

species (0.22–0.35; Tukey’s post hoc comparisons). In the

elevated temperature treatment, however, Y(II) reached a

minimum of 0.02 by 2100 hours on day 2 in A. millepora

(significantly lower than the control; P = 0.001). At this

time point in P. damicornis, Y(II) was 0.14 (significantly

lower than the control; P = 0.008), while it recovered to

0.21 in Pavona decussata (not significantly different to the

control; P = 0.606). In all three species, Y(NO) showed an

inverse relationship with Y(II) with the elevated tempera-

ture treatment reaching 0.72 (compared to 0.23 in the

control; P = 0.005) at 2100 hours on day two in

A. millepora. Similarly, Y(NO) reached 0.51 in the ele-

vated temperature treatment in Pocillopora damicornis

(compared to the control at 0.31; P = 0.007) by

2100 hours on day two. In Pavona decussata, Y(NO)

remained consistent between the two temperature treat-

ments and reached 0.32–0.36 by 2100 hours on day two

(P = 0.635). Y(NPQ) showed little variation between

treatments or over time in all three species and reached its

lowest level of 0.26 (which was significantly lower than the

control; P = 0.032) at 2100 hours on the second day in the

bleaching treatment in A. millepora.

Photosynthetic and photoprotective pigments

In control treatments, no changes in photosynthetic

pigment content were detected in any of the three species

(P values [ 0.3), although exposure to elevated tempera-

tures in the bleaching treatment did reduce the content of

most photosynthetic pigments in A. millepora and Pocil-

lopora damicornis (Fig. 2a–i). Under bleaching conditions,

chlorophyll a, chlorophyll c2 and peridinin all significantly

declined over the 2 days in A. millepora (P = 0.022, 0.014

and 0.025, respectively), with significantly lower values

compared to the control detected consistently over the

second day, and for chlorophyll c2, from the end of the first

day (Fig. 2a,d,g). In P. damicornis, there was a significant

decrease in chlorophyll a (Fig. 2b; P = 0.005) and chlo-

rophyll c2 (Fig. 2e; P = 0.028) content over the bleaching

treatment, with lower pigment abundance observed on the

second day. The peridinin content in the P. damicornis

bleaching treatment did not significantly change over time

(P = 0.096), although at three time points throughout the

experiment, peridinin was significantly lower in the corals

under bleaching conditions compared to the controls

(Fig. 2h). In contrast to A. millepora and P. damicornis, in

Pavona decussata exposure to bleaching conditions did not

result in any change in photosynthetic pigment content

compared with controls (Fig. 2c,f,i; P values [ 0.6).

An increase in b-carotene (relative to chlorophyll a)

was detected over time in the bleaching treatments of

A. millepora (by 2100 hours on day 2; P = 0.001) and

Pocillopora damicornis (at 1300 hours on day 2;

P = 0.021). This was coupled with significantly higher

concentrations compared to the controls at 1300 and

2100 hours on day 2 for A. millepora and 2100 hours on day

2 for P. damicornis. No significant changes were found in

any control treatment (P values [ 0.05; Fig. 2j–l). In Pa-

vona decussata, the b-carotene content of the control and

bleaching nubbins were similar throughout the experiment.

The de-epoxidation ratio (Fig. 2m–o) demonstrated a

cyclic diel pattern, which corresponded with irradiance

(Fig. 1a–c). All species and all treatments showed a sig-

nificant increase in the de-epoxidation ratio on each day at

1300 hours and a lower de-epoxidation ratio each morning

(0500 hours) and evening (2100 hours) (P values \0.001). Significantly higher de-epoxidation ratios were

found in the bleaching treatment compared to the controls

in A. millepora at 1300 hours (P = 0.015) and 2100 hours

(P = 0.007) on day 2 (Fig. 2j) and at 1300 hours

(P = 0.038) in Pocillopora damicornis on day 2 (Fig. 2k).

In Pavona decussata, no differences were detected between

the nubbins under control and bleaching conditions

(Fig. 2l).

77K chlorophyll fluorescence emission spectra

Representative 77K chlorophyll fluorescence emission

spectra from 0500 and 1300 hours in the control treatment

on the first day reveal the general shifts in the shape of the

fluorescence spectra from pre-dawn to high irradiance

conditions in the three species (Fig. 3). Maximum fluo-

rescence emission was detected at 686 nm in all species, at

all time points and in all treatments. A relative increase in

the shoulder at 675 nm was detected in all three species at

1300 hours, as well as a shoulder at 701 nm, although

968 Coral Reefs (2012) 31:963–975

123

a

d

g

j

m n o

lk

h i

fe

b c

Fig. 2 a–c Changes in chlorophyll a content (lg cm-2), d–f chloro-

phyll c2 content (lg cm-2), g–i peridinin content (lg cm-2), j–l b-

carotene content (lg per lg chlorophyll a) and m–o de-epoxidation

ratio (Dt/[Dt ? Dd]) for the control (black bars) and bleaching (whitebars) treatments over the two-day experimental period in Acropora

millepora (left column), Pocillopora damicornis (middle column) and

Pavona decussata (right column). Mean ? SE (n = 4). Asterisksindicate statistically significant differences between control and

bleaching treatments at each time point (a = 0.05)

Coral Reefs (2012) 31:963–975 969

123

variation in the amplitude of these shifts was found

between species.

To investigate detailed changes in the shape of the fluo-

rescence spectra, curves normalised at 686 nm were de-

convoluted and component bands found at 675, 686, 701 and

721 nm. Amplitude and area at the four wavelengths in both

the control and bleaching treatments over the experimental

period is displayed in Fig. 4. In all cases, the two temperature

treatments showed corresponding changes in both the

amplitude and area data over time, which oscillated on a diel

cycle. In A. millepora and P. damicornis, the amplitude and

area of the 675, 701 and 721 nm bands, relative to 686 nm,

showed a positive relationship with light intensity towards

1300 hours, which relaxed as incident irradiance decreased

each day. The significant increases in these component bands

towards 1300 hours were matched by a significant decrease

in the area of the 686-nm band (P values \ 0.01). In

P. decussata, the amplitude and area of the 675-nm

band significantly increased with increasing irradiance

(P values \ 0.01), and this was paralleled by a significant

decrease in the area of 686-nm band (P values \ 0.001).

There were no significant changes in the amplitude or area of

the 701- or 721-nm bands in P. decussata (P values [ 0.05),

and no significant changes in the amplitude of the 686-nm

band in all three species (P values [ 0.05). Comparisons

between the three species revealed that P. damicornis had the

greatest contribution at 701 and 721 nm under high irradi-

ance, whereas P. decussata had the greatest contribution

from 675 nm. The response of A. millepora was intermediate

between the previous two species.

DTT inhibition of the xanthophyll cycle

FV/FM significantly declined following 4 h of high light

exposure and showed no recovery after a further 2 h of

darkness in both the absence (P = 0.013) and presence

(P \ 0.001) of DTT (Fig. 5a). However, the reduction was

much greater in the ?DTT treatment, suppressing FV/FM

to 0.37, compared to 0.59 in the control, at the 4-h time

point. In the control, the de-epoxidation ratio significantly

increased following 4 h of high light exposure (due to Dd

de-epoxidation to Dt), but returned to the initial state

after 2 h of recovery (reversal of pigment conversion;

P \ 0.001). In contrast, the de-epoxidation ratio remained

consistent at the three time points in the presence DTT as

Dd de-epoxidation to Dt was blocked (P = 0.266; Fig. 5a).

In the absence (Fig. 5b) and presence (Fig. 5c) of DTT,

the chlorophyll fluorescence emission spectra at 77K

showed two peaks at 0 h, one at 675 and a higher peak at

686 nm (Fig. 5b). Following 4 h of exposure to high light,

the amplitude of the 675-nm peak exceeded the peak at

686 nm. After two hours of recovery in darkness, a partial

relaxation of the 675-nm peak was seen. At both 4 and 6 h,

DTT was found to greatly increase the shoulder at 701 nm,

compared to the control.

Discussion

In this paper, we have used three species of scleractinian

coral with varying susceptibility to high light and bleaching

temperatures to test the hypothesis that a major protective

mechanism, in addition to other known strategies, is the

redistribution of absorbed energy by detachment of LHCs

from PSII and/or the reassociation with PSI.

Inter-species thermal sensitivity

The patterns of the effect of high light and bleaching

conditions on the three species studied are consistent with

previous findings. The loss of PSII photochemical effi-

ciency (indicated by FV/FM and Y(II); Fig. 1) was greatest

in A. millepora, which showed no recovery following

2 days of exposure to bleaching conditions. P. damicornis

showed an intermediate response under bleaching condi-

tions, while P. decussata displayed no significant deviation

a b c

Fig. 3 Representative fluorescence emission spectra (650–750 nm)

at 77K, normalised to 686 nm, at 0500 hours (solid line) and

1300 hours (dashed line) in the control treatment on the first day for

a Acropora millepora, b Pocillopora damicornis and c Pavonadecussata. The difference spectrum is indicated by the dotted line

970 Coral Reefs (2012) 31:963–975

123

from controls. This same pattern was found when com-

paring the photosynthetic pigment content from the three

species, with A. millepora showing the fastest and greatest

loss, P. damicornis showing an intermediate response, and

P. decussata remaining unchanged (Fig. 2). These findings

are consistent with past research quantifying pigment loss

during bleaching (Ambarsari et al. 1997; Dove et al. 2006;

Venn et al. 2006), as well as studies which have ranked

species by their bleaching vulnerability. Acroporids, such

as A. millepora are classed as being severely susceptible to

bleaching (Marshall and Baird 2000; Loya et al. 2001),

while pocilloporids, such as P. damicornis have been

identified as having a high to severe susceptibility (Jones

et al. 1998; Marshall and Baird 2000; Fitt et al. 2001; Loya

et al. 2001; Hill et al. 2004). In contrast, P. decussata has

been reported to have mixed to tolerant thermal bleaching

characteristics (Marshall and Baird 2000; McClanahan

et al. 2001; McClanahan 2004).

a

d

g

j lk

h i

fe

b c

Fig. 4 The amplitude (relative units between 0–100; circles) and area

(%; squares) of component bands in the fluorescence emission spectra

at 77K (normalised to 686 nm) at a–c 675, d–f 686, g–i 701 and

j–l 721 nm. Changes in the amplitude and area are shown for the

control (closed symbols) and bleaching (open symbols) treatments

over the two-day experimental period in Acropora millepora (leftcolumn), Pocillopora damicornis (middle column) and Pavonadecussata (right column). Mean ± SE (n = 4)

Coral Reefs (2012) 31:963–975 971

123

The dynamic channelling of light energy absorbed by

LHCs between the three energy pathways varied over a diel

cycle, between species and between treatments (Fig. 1).

Although Y(II) showed an inverse relationship with irra-

diance (Brown et al. 1999), Y(NO), which is an indicator of

PSII photoinhibition, displayed a positive correlation, with

more energy transferred to this non-regulatory heat dissi-

pation pathway around midday (Kramer et al. 2004; Bonfig

et al. 2006). Measures of NPQ showed an expected diel

pattern in response to light with the proportion of energy

going to Y(NPQ) declining under high irradiance. An

increase in NPQ has been observed in corals at midday,

especially when exposed to irradiance under bleaching-

relevant temperatures (Warner et al. 1996; Hill et al. 2005).

Photoprotective properties of pigments

A number of photoprotective strategies are available to

corals to cope with variable and often over-saturating irra-

diances. Within the symbionts of corals, xanthophyll

cycling, as shown here, is a regulated and effective means of

dissipating excess absorbed light energy as heat (Brown et al.

1999; Venn et al. 2006; Ulstrup et al. 2008), while abundance

of the carotenoid, b-carotene, appears to act as an effective

antioxidant (Burton 1989; Young and Britton 1993; Venn

et al. 2006). The findings of this research confirm diel xan-

thophyll cycling (Brown et al. 1999; Ulstrup et al. 2008) and

the increase of the de-epoxidation ratio in corals experi-

encing bleaching (Venn et al. 2006). In all three coral spe-

cies, the inter-conversion of xanthophyll pigments was used

throughout the day in response to increasing irradiance with

excess absorbed light energy dissipated as heat (Fig. 2).

Furthermore, our findings suggest that this de-epoxidation

was correlated with species vulnerability to thermal

bleaching. The most bleaching sensitive species, A. mille-

pora, showed most upregulation of xanthophyll cycling

under bleaching with greater de-epoxidation at midday on

day 2 in the bleaching treatment, which was sustained until

2100 hours. A similar response was found in P. damicornis

at 1300 hours, while the diel changes in de-epoxidation were

independent of temperature in P. decussata. Complementing

these findings are the changes in b-carotene content (Fig. 2),

which has previously been reported to increase during ther-

mal stress in corals (Ambarsari et al. 1997; Venn et al. 2006).

The proportion of b-carotene relative to chlorophyll a was

also correlated to the bleaching sensitivity of the coral spe-

cies, with the greatest abundance found in A. millepora,

followed by P. damicornis. This suggests that in corals

experiencing bleaching through symbiont expulsion, the

antioxidant properties of b-carotene were used to detoxify

potentially damaging ROS (Burton 1989; Young and Britton

1993; Venn et al. 2006). However, this conclusion may not

be supported if the primary mode of bleaching was via

photosynthetic pigment degradation within symbiont cells,

rather than symbiont expulsion. As symbiont density was not

determined for this experiment, it is possible that the increase

in the b-carotene:chlorophyll ratio may not represent a

strategy to enhance protection against ROS and that instead

chlorophyll degradation was occurring due to photobleach-

ing (Takahashi et al. 2008) or that a rapid form of photo-

acclimation was underway in the corals to reduce absorption

of incident irradiance (Robison and Warner 2006).

a

b

c

Fig. 5 a FV/FM (left y axis; represented by the lines and circles) and

de-epoxidation ratio (Dt/(Dt ? Dd); right y axis; represented by the

bars) in the absence (black circles and black bars) and presence

(white circles and white bars) of DTT (mean ± SE (n = 4)).

Chlorophyll fluorescence emission spectra (650–750 nm) at 77K,

normalised to 686 nm, in the b absence and c presence of DTT at 0, 4

and 6 h. Curves represent the average of 4 traces

972 Coral Reefs (2012) 31:963–975

123

Photoprotection through LHC dissociation

An additional means of protecting PSII from high irradi-

ance and thermal stress is through the dissociation of LHCs

from PSII and PSI and perhaps re-arrangement between the

two photosystems. Evidence of changes in membrane-

bound and peripheral LHC attachments can be inferred

from chlorophyll fluorescence spectra at 77K (Fig. 3).

Shifts in the shape of these spectra were observed over a

diel cycle with a peak at 686 nm and clear shoulders

appearing at 675 and 701 and 721 nm. Fluorescence

emissions at 675 nm have been proposed as a result of the

dissociation of putative PCP complexes, emissions at

686 nm as energy channelled into PSII reaction centres

(including that from attached PCP), and wavelengths

longer than 700 nm (i.e. 701 and 721 nm) as energy

channelled into PSI reaction centres (including attached

LHCs) (Reynolds et al. 2008). The interpretations here of

the 77K fluorescence emission spectra are based upon these

assumptions, except we have also allowed for a role of the

membrane-bound acpPC in the emission at 675 nm.

Initial 0500 hours measurements, with the principal

fluorescence peak at 686 nm, indicated both the mem-

brane-bound and peripheral LHCs were fully connected to

PSII (Figs. 3, 4; c.f. Reynolds et al. 2008); that is, transfer

of energy from PCP to PSII was highly efficient, so that no

PCP fluorescence was detected. Shifts in the amplitude of,

and area under peaks, suggested detachment of photosyn-

thetic pigments following light exposure, and this disso-

ciation seemed tightly linked to elevations in incident

irradiance, rather than exposure to thermal stress (Fig. 4).

In A. millepora and P. damicornis, elevated incident irra-

diance resulted in an increase in fluorescence emission at

701 and 721 nm relative to 686 nm (Figs. 3, 4). This could

indicate a redistribution of LHCs away from PSII towards

PSI, that is, a typical state transition response as observed

in higher plants and green algae (Fork and Satoh 1986;

Demmig-Adams et al. 1987; Williams and Allen 1987). In

P. damicornis treated with DTT, high irradiance resulted in

a large increase in those regions of the fluorescence spectra

indicative of LHCs attached to PSI reaction centres (701

and 721 nm; Fig. 5c). DTT is a xanthophyll cycle inhibitor,

and the lack of de-epoxidation in the DTT-treated corals,

compared to the controls, is evidence of its inhibitory

activity (Fig. 5a). The enhanced loss of FV/FM in the

presence of DTT also indicates a greater decline in PSII

photochemical efficiency. We therefore suggest that once

the capacity for excess energy dissipation through xan-

thophyll cycling is exceeded, acpPC detachment from PSII

occurs with LHCs moving from the overexcited PSII

reaction centres possibly towards PSI. We suggest the

membrane-bound LHC as the mechanism, since there is no

suggestion that PCP can redistribute from PSII to PSI.

This response is akin to a state transition; however, further

work is needed to confirm the redistribution of LHCs and

excitation energy between the two photosystems.

Increase in the amplitude and percentage area at 675 nm

in the 77K chlorophyll fluorescence emission spectra has

been taken to indicate a disconnection of excitation energy

transfer from the extrinsic light-harvesting peridinin-

chlorophyll-protein (PCP) complex to PSII (Iglesias-Prieto

et al. 1993; Iglesias-Prieto and Trench 1996; Polivka et al.

2006; Reynolds et al. 2008), although this region would

also probably respond in the same way if membrane-

intrinsic light-harvesting proteins (acpPC) were discon-

nected. Reynolds et al. (2008) obtained evidence indicating

a decrease in excitation energy transfer to PSII from

putative PCP after high light exposure in cultured clade A

Symbiodinium. Detaching PCP and/or acpPC complexes

from photosystems could provide photoprotection by

reducing the delivery of excitation energy to PSII, hence

preventing over-excitation of PSII. In addition, these

complexes could also play a role in the prevention of ROS

production by shunting energy from long-lived triplet

chlorophyll to the carotenoid triplet (lower energy config-

uration), thus preventing the generation of singlet oxygen

(Alexandre et al. 2007), which would ordinarily cause

photosynthetic damage (Lesser 1996, 1997). Indeed, our

findings highlight a link between LHC detachment and

bleaching sensitivity. We suggest that the PCP and/or ac-

pPC detachment process is likely to contribute to heat

dissipation (i.e. photoprotection) as P. decussata, the most

bleaching resistant species with no signs of photodamage,

displayed the greatest level of detachment, along with no

evidence of greater xanthophyll cycling or b-carotene

content under thermal stress. Perhaps, this detachment is a

very efficient means of preventing PSII over-excitation and

hence aids in the avoidance of photodamage.

The detachment of PCP and/or acpPC was found here to

be tightly controlled by irradiance, with no significant

influence from elevations in temperature (Fig. 4). Perhaps,

the maximum potential for LHC detachment was reached

under the irradiance levels applied, and no further detach-

ment was possible, or the feedback mechanisms that control

the detachment process is light dependent only, with no

trigger from temperature elevations above the optimal

threshold. The redox state of plastoquinone (PQ) is believed

to control LHC dissociation in higher plants and some algae,

with highly reduced PQ pools promoting a transition from

state 1 to state 2 (Fork and Satoh 1986; Williams and Allen

1987; Haldrup et al. 2001). A similar biochemical trigger

may be present in Symbiodinium, although this is yet to be

confirmed and the details of any possible PCP and/or acpPC

detachment are as yet unknown.

Interestingly, we found PCP and/or acpPC detachment

from PSII occurred in all three coral species, which,

Coral Reefs (2012) 31:963–975 973

123

according to past studies on Heron Island, are known to

harbour clade C Symbiodinium, with A. millepora often

also containing clade A (LaJeunesse et al. 2003; Ulstrup

et al. 2008; Hill et al. 2009). This contrasts to Reynolds

et al. (2008) who used cultured Symbiodinium where

putative PCP detachment was only found in clade A, and

not clade B or C. A likely explanation for such disparity is

the presence of the intact symbiotic relationship in our

study, which is integral to the regulation of the dinofla-

gellate symbionts to environmental perturbations (Baird

et al. 2009). We suggest that the PCP and/or acpPC

detachment process is likely to be an effective photopro-

tective strategy of in hospite Symbiodinium, as P. decuss-

ata, the most bleaching resistant species with no signs of

photodamage, displayed the greatest level of detachment

and experienced little membrane-bound LHC redistribu-

tion, along with no evidence of greater xanthophyll cycling

or b-carotene content under thermal stress. Perhaps, this

detachment is a very efficient means of preventing PSII

over-excitation and hence aids in the avoidance of

photodamage.

Acknowledgments We thank Marlene Zbinden for assistance with

extraction of algal pigments and running of the HPLC. This work was

supported by Australian Research Council grant number

DP110105200. The research of O.P. was supported by projects

GAAV IAA601410907 and Algatech (CZ.1.05/2.1.00/03.0110). This

research was performed under the Great Barrier Reef Marine Park

Authority permit number G08/27673.1.

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