Two inhibitors of the Calvin–Benson cycle [glycolaldehyde (GA) and potassium cyanide (KCN)] were used in cultured Symbiodinium cells and in nubbins of the coral Pocillopora damicornis to test the hypothesis that inhibition of the Calvin–Benson cycle triggers coral bleaching. Inhibitor concentration range-finding trials aimed to determine the appropriate concentration to generate inhibition of the Calvin–Benson cycle, but avoid other metabolic impacts to the symbiont and the animal host. Both 3 mmol l−1 GA and 20 μmol l−1 KCN caused minimal inhibition of host respiration, but did induce photosynthetic impairment, measured by a loss of photosystem II function and oxygen production. GA did not affect the severity of bleaching, nor induce bleaching in the absence of thermal stress, suggesting inhibition of the Calvin–Benson cycle by GA does not initiate bleaching in P. damicornis. In contrast, KCN did activate a bleaching response through symbiont expulsion, which occurred in the presence and absence of thermal stress. While KCN is an inhibitor of the Calvin–Benson cycle, it also promotes reactive oxygen species formation, and it is likely that this was the principal agent in the coral bleaching process. These findings do not support the hypothesis that temperature-induced inhibition of the Calvin–Benson cycle alone induces coral bleaching.

INTRODUCTION

Reef-building corals form an endosymbiosis with dinoflagellate algae from the genus Symbiodinium (also known as zooxanthellae), which reside within the endodermal tissue of the cnidarian. This mutualism is key to the high productivity found on coral reefs, often in oligotrophic, tropical waters, but can be highly sensitive to environmental perturbations, with a breakdown in the symbiosis a common stress response (Hoegh-Guldberg et al., 2007). Coral bleaching involves the expulsion of Symbiodinium and/or the loss of photosynthetic pigments from within algal cells, resulting in a paling, or bleaching, of the coral. Elevations in sea surface temperature, as small as 1–2°C above the summer average, in combination with high irradiance, are well known to cause mass coral bleaching events on coral reefs around the world (Hoegh-Guldberg, 1999). It is forecast that ocean temperatures will continue to increase with global warming due to anthropogenic emissions of greenhouse gasses (IPCC, 2007), placing corals more at risk of having sea temperatures exceed their upper thermal maximum. Coral bleaching events are therefore predicted to become more frequent, widespread and severe with climate change (Hoegh-Guldberg, 1999).

Breakdown in the coral symbiosis has been linked to an initial photosynthetic impairment in the endosymbiotic algae. A sustained loss of photosynthetic performance in coral symbionts is a well-defined characteristic of a thermal stress response (Jones et al., 2000). A great deal of evidence has been collected, primarily through non-invasive chlorophyll fluorescence techniques, demonstrating the loss of photosystem II (PSII) photochemical efficiency during a bleaching event (Jones et al., 1998; Warner et al., 1999; Hill et al., 2004a; Hill et al., 2004b). While such results are indicative of impacts to this early stage of the light reactions of photosynthesis, it is not clear whether inhibition of PSII activity is the initial site of damage induced by bleaching conditions, or whether it is a secondary or tertiary impact only detected following inhibition or damage at another site (Smith et al., 2005). Indeed, this initial site of impact remains elusive, with many studies aiming to identify the origin of photosynthetic impairment in Symbiodinium under simulated bleaching conditions.

Photoinhibition, which is largely an impairment of PSII caused by high irradiance, has been demonstrated in coral symbionts during thermal stress with the rate of photoinactivation of the core D1 protein exceeding the rate of repair. D1 content has been shown to drop under bleaching conditions, in correlation with declines in PSII photochemical efficiency (Warner et al., 1999; Robison and Warner, 2006; Hill et al., 2011), and this is likely due to the action of reactive oxygen species (ROS) (Lesser, 2011). The cause of this loss of D1 could be due to (1) inability of the D1 repair mechanism to match the rate of D1 damage (Hill et al., 2011), or (2) inhibition of the D1 repair mechanism (Takahashi et al., 2009). While evidence exists for both processes, the end result is the same, with PSII damage leading to photosynthetic impairment. The capacity for D1 repair has been suggested to control thermal bleaching vulnerability, with evidence indicating that bleaching-tolerant species have faster rates of repair compared with more susceptible species (Hennige et al., 2011).

While PSII inhibition has received considerable attention, other components of the photosynthetic apparatus have also been investigated, including antenna proteins (Takahashi et al., 2008; Hill et al., 2012), the oxygen evolving complex (Iglesias-Prieto, 1997; Hill and Ralph, 2008), photosystem I (Hoogenboom et al., 2012), thylakoid membrane integrity (Tchernov et al., 2004; Hill et al., 2009; Díaz-Almeyda et al., 2011) and the dark reactions (Jones et al., 1998; Leggat et al., 2004; Lilley et al., 2010; Hill et al., 2011). Thermal sensitivity of carbon fixation at bleaching-relevant temperatures has been inferred through measures of PSII chlorophyll fluorescence, where initial damage to the dark reactions resulted in an electron sink limitation and, consequently, PSII photoinhibition (Jones et al., 1998).

List of symbols and abbreviations

     
  • APX

    ascorbate peroxidase

  •  
  • FIC

    fast induction curve

  •  
  • FM

    maximum fluorescence in dark-adapted cells during saturating pulse of light

  •  
  • FO

    minimum fluorescence in dark-adapted cells

  •  
  • FV/FM

    PSII photochemical efficiency

  •  
  • GA

    glycolaldehyde

  •  
  • KCN

    potassium cyanide

  •  
  • NPQ

    non-photochemical quenching

  •  
  • PSII

    photosystem II

  •  
  • QA

    primary electron acceptor of PSII

  •  
  • QA

    reduced primary electron acceptor of PSII

  •  
  • QB

    secondary electron acceptor of PSII

  •  
  • ROS

    reactive oxygen species

  •  
  • σPSII

    functional absorption cross-section of PSII

Carbon-concentrating mechanisms have been found to be unaffected despite inhibition of photosynthesis (Leggat et al., 2004), and Rubisco protein content of in hospite symbionts has also been shown to remain constant during thermal bleaching (Hill et al., 2011). However, at higher temperatures, Rubisco activity does drop rapidly, indicating it is heat sensitive; the results of Leggat et al. (Leggat et al., 2004) were somewhat equivocal but those of Lilley et al. (Lilley et al., 2010) were well substantiated. More recently, Bhagooli (Bhagooli, 2013) demonstrated that inhibition of Calvin–Benson cycle activity in the coral Stylophora pistillata by the inhibitor glycolaldehyde (GA) causes photoinhibition and coral bleaching even at optimal temperatures. Therefore, in this species at least, there is some evidence that bleaching can be initiated by damage to the dark reactions of photosynthesis in the absence of thermal stress. To date, the literature contains no conclusive, unifying model that can explain the thermal bleaching response in corals, but rather an extensive series of independent experiments that provide detailed responses of specific components of the photosynthetic apparatus (see review by Lesser, 2011). The focus of these experiments has been predominantly on PSII (in part because of the ease of direct PSII measurements using chlorophyll fluorescence), with other photosynthetic components, such as the dark reactions, receiving less attention. The existing studies provide divergent lines of evidence of a primary site of photosynthetic impairment, which highlights the need for further detailed analyses into the mechanism of photosynthetic impairment of coral symbionts during the onset of thermal bleaching events, especially the Calvin–Benson cycle, which has recently been shown to activate bleaching when chemically inhibited (Bhagooli, 2013).

The application of chemical inhibitors can provide a useful means to manipulate the function of metabolic processes. At low concentrations, GA and cyanide have been used to inhibit the operation of the Calvin–Benson cycle (Sicher, 1984; Wishnick and Lane, 1969), essential in the fixation of organic carbon from carbon dioxide. GA is known to inhibit ribulose-1,5-diphosphate synthase in this cycle (see Miller and Canvin, 1989). However, as later reported by Salon et al. (Salon et al., 1998), GA also inhibited carbonic anhydrase in Synechococcus UTEX and at 10 mmol l−1 GA, only 20% of the total inhibition of photosynthesis could be ascribed to reduced CO2 fixation. Potassium cyanide (KCN) is also an inhibitor of the Calvin–Benson cycle, although its specific mode of action is different to that of GA. KCN inhibits CO2 fixation by binding to Rubisco activase and preventing its release from Rubisco (Wishnick and Lane, 1969; Sicher, 1984), and it also causes inhibition of plastoquinone-oxidoreductase (Buchel and Garab, 1995), ascorbate peroxidase (APX) (Ishida et al., 1998) and the scavenging of hydroxyl radicals (Jakob and Heber, 1996). The exact mechanism of cyanide action is not well described in the literature, and because Symbiodinium have Type 2 rather than Type 1 Rubisco (i.e. the Rubisco is composed of mainly two large subunits rather than eight large and eight small subunits), they may not react in the same way to cyanide, i.e. the binding of substrates and the heat sensitivity may be quite different. Furthermore, in a symbiosis such as that between corals and Symbiodinium, the effect of these inhibitors on host (animal) tissue has to be taken into account.

GA has a number of effects in animal tissues, where it interacts with several biochemical pathways and is inhibitory at a number of levels (e.g. Al-Enezi et al., 2006). KCN also inhibits mitochondrial respiration, but only at significantly higher concentrations (Barnes, 1985; Jones and Steven, 1997; Jones and Hoegh-Guldberg, 1999). Therefore, at higher concentrations of these inhibitors, multiple metabolic processes can be impacted, with specificity of the compound lost, resulting in ambiguous results that cannot provide convincing evidence of the site of action. The duration of exposure is also an important factor to consider, with a concentration- and time-dependent loss of PSII function and coral bleaching response found for GA and cyanide (Jones and Steven, 1997; Buxton et al., 2012). Therefore, the concentration-dependent action of GA and cyanide on a coral holobiont must be clearly understood in order to investigate the effect of Calvin–Benson cycle inhibition on causing coral bleaching. In this study, we ran a series of concentration- and time-dependent assays to determine the appropriate concentration of the inhibitors. Initially, cultured Symbiodinium were used for the large-scale concentration range-finding trails, with subsequent assays on intact corals used to refine and optimise the appropriate treatments for experiments on the intact symbiosis.

It has been proposed that the trigger for coral bleaching lies in the effect of elevated temperature on the photosynthetic process of the symbiotic zooxanthellae (Lesser, 2011). Two specific sites in the photosynthetic apparatus have been identified as likely targets: (1) the Calvin–Benson cycle, the site of carbon dioxide fixation (Jones et al., 1998; Lilley et al., 2010), and (2) PSII (Warner et al., 1999; Hill et al., 2011). Despite the fact that coral bleaching has emerged over the last two decades as a complex interaction of many environmental and genetic factors, operating at several levels within the coral holobiont (Buxton et al., 2012; Tolleter et al., 2013), these two potential triggers of coral bleaching are still worthy of detailed research. There has been considerable focus on impacts to PSII, with fewer studies concentrating on the operation of the dark reactions. Here we focused on the bleaching response of the common scleractinian coral Pocillopora damicornis (Linnaeus 1758) under optimal and elevated temperature while manipulating the function of the Calvin–Benson cycle using chemical inhibitors. This is the first study to investigate whether inhibition of the Calvin–Benson cycle by chemical inhibitors promotes bleaching under thermal stress. The outcomes provide a detailed insight into the role of a functional Calvin–Benson cycle in maintaining an intact symbiosis. Specifically, the aim of this work was to characterise impacts to the Calvin–Benson cycle and the consequences of its inhibition in P. damicornis under thermal bleaching conditions. We also sought to determine whether inhibition of the dark reactions induced bleaching at optimal growth temperatures in this species and whether Calvin–Benson cycle inhibition by GA or KCN accelerated thermal bleaching.

RESULTS

Inhibitor range-finding trials

GA and KCN concentration range-finding tests were performed on cultured and in hospite Symbiodinium of P. damicornis. These trials were essential for determining the appropriate concentrations to use to generate inhibition of the Calvin–Benson cycle, while avoiding other metabolic impacts to the symbiont and the animal host. In the Symbiodinium culture, a concentration-dependent decline in the ratio of variable fluorescence to maximal fluorescence (FV/FM; a measure of PSII photochemical efficiency) was found for both inhibitors between 0 and 10 mmol l−1 for GA, and between 0 and 100 μmol l−1 for KCN after 8 h light exposure (Fig. 1). Following a 12 h dark period, FV/FM continued to decline in all GA-treated samples, while recovery was found in all but the highest KCN concentration. A subsequent light period resulted in continued decline of FV/FM. These data show that in Symbiodinium cells, the application of long-term GA treatment damages the photochemical activity when the concentration is higher than 1 mmol l−1. In the case of KCN, inhibition of photosynthetic activity was largely reversible in darkness up to 50 μmol l−1. Therefore, long-term GA treatment should be restricted to low (mmol l−1) concentrations, while KCN treatment does not seem to induce irreversible side effects up to 50 μmol l−1 after a few hours of exposure time. Fig. 2 shows the fast induction curves (FICs), which are double normalized to the O and P steps (relative variable fluorescence). In the presence of both GA and KCN, the J step increased after incubation of the cells in growth light. These findings demonstrate that the reduction level of the plastoquinone pool increases as a result of partial inhibition of the Calvin–Benson cycle, which limits electron flow towards CO2, which acts as the final electron acceptor.

Fig. 1.

Photosystem II photochemical efficiency (FV/FM) of cultured Symbiodinium.FV/FM in the presence of (A) glycolaldehyade (GA; 0–10 mmol l−1) and (B) potassium cyanide (KCN; 0–100 μmol l−1) during 8h of exposure to 40 μmol photons m−2s−1, followed by 12 h darkness and a further 12 h light. The white bars indicate periods of light and the grey bars indicate darkness. Data are means ± s.d. (n=3–6).

Fig. 1.

Photosystem II photochemical efficiency (FV/FM) of cultured Symbiodinium.FV/FM in the presence of (A) glycolaldehyade (GA; 0–10 mmol l−1) and (B) potassium cyanide (KCN; 0–100 μmol l−1) during 8h of exposure to 40 μmol photons m−2s−1, followed by 12 h darkness and a further 12 h light. The white bars indicate periods of light and the grey bars indicate darkness. Data are means ± s.d. (n=3–6).

The goal of these inhibitor assays was to determine concentrations high enough to induce Calvin–Benson cycle inhibition in the algal symbionts, detectable through measures of PSII function and oxygen production, while limiting impacts to animal host condition. In the in hospite Symbiodinium of P. damicornis, FV/FM showed a concentration-dependent decline (Fig. 3) over 3 h of exposure, indicating damage to PSII function, while gross photosynthetic rates (Fig. 4) declined during exposure to 5 mmol l−1 GA, as well as 10 and 100 μmol l−1 KCN. Respiration rate was used as an indicator of coral host condition and only declined in the highest GA and KCN concentrations over the 3 h experiment, suggesting that lower concentrations did not have an inhibitory effect on animal respiration over this time period. Following these trials, 3 mmol l−1 GA and 20 μmol l−1 KCN were chosen as appropriate concentrations to apply in the subsequent experiments.

Fig. 2.

Double normalised fast induction curves (FICs) showing relative variable fluorescence [(FtFO)/(FMFO)] of cultured Symbiodinium. Relative variable fluorescence in the presence of (A,B) GA (0, 1, 3 and 5 mmol l−1) and (C,D) KCN (0, 10 and 20 μmol l−1) after 3 min exposure to inhibitors (A,C) and after a further 5 h exposure to 40 μmol photons m−2 s−1 (B,D). The control 0 h measurements are also shown for comparison with the FICs from 5 h (B,D). Averages are shown (n=5).

Fig. 2.

Double normalised fast induction curves (FICs) showing relative variable fluorescence [(FtFO)/(FMFO)] of cultured Symbiodinium. Relative variable fluorescence in the presence of (A,B) GA (0, 1, 3 and 5 mmol l−1) and (C,D) KCN (0, 10 and 20 μmol l−1) after 3 min exposure to inhibitors (A,C) and after a further 5 h exposure to 40 μmol photons m−2 s−1 (B,D). The control 0 h measurements are also shown for comparison with the FICs from 5 h (B,D). Averages are shown (n=5).

Fig. 3.

FV/FM of in hospite symbionts of Pocillopora damicornis. Treatments are as follows: (A) 0, 1 and 5 mmol l−1 GA and (B) 0, 10 and 100 μmol l−1 KCN after 0, 1 and 3 h at 26°C and under 250 μmol photons m−2s−1. Data are means ± s.e.m. (n=4).

Fig. 3.

FV/FM of in hospite symbionts of Pocillopora damicornis. Treatments are as follows: (A) 0, 1 and 5 mmol l−1 GA and (B) 0, 10 and 100 μmol l−1 KCN after 0, 1 and 3 h at 26°C and under 250 μmol photons m−2s−1. Data are means ± s.e.m. (n=4).

Quantification of bleaching

Nubbins of P. damicornis were exposed to 10 h of26, 30 or 31°C and a subsequent 14 h of darkness at 26°C. This was done in (1) the absence of any inhibitors, (2) 3 mmol l−1 GA or (3) 20 μmol l−1 KCN. Bleaching in the coral nubbins was quantified through measures of symbiont density and chlorophyll concentration (Fig. 5). After 24 h in the 26°C treatments, a significant decline in symbiont density per cm2 of coral host was only found in the presence of KCN when compared with initial symbiont densities at 0 h (one-way ANOVA, F1,6=13.520, P=0.010; Fig. 5A). No loss of symbionts was found in the absence of an inhibitor (control) or in the presence of GA at 26°C. However, after 24 h, a significant loss of symbionts was found in the 30 and 31°C treatments compared with the 26°C treatment in the absence of inhibitors (one-way ANOVA, F2,9=6.288, P=0.020) and in the presence of GA (one-way ANOVA, F2,9=10.232, P=0.005). Nubbins in all three temperature treatments in the presence of KCN experienced bleaching to a similar extent by 24 h.

Chlorophyll a concentration per cm2 of coral host was significantly lower after 24 h at 26°C in the KCN treatment compared with the concentration at 0 h (one-way ANOVA, F1,6=7.726, P=0.032; Fig. 5B). Nubbins exposed to 24 h of 30 and 31°C in the presence of KCN experienced a loss of chlorophyll a similar to that of the 26°C treatment. In the absence of any inhibitor, chlorophyll a concentration was not affected by thermal exposure after 24 h. However, in the presence of GA, nubbins lost more chlorophyll a following 24 h at 30 and 31°C compared with the 26°C treatment. In contrast, no significant changes were found in chlorophyll c2 concentration per cm2 of coral host (Fig. 5C) or total chlorophyll per cell (Fig. 5D) over time or between thermal treatments in the presence or absence of inhibitors.

Changes in PSII photochemical efficiency

In the presence or absence of inhibitors, FV/FM declined in all three temperature treatments over time (repeated-measures ANOVAs, all P<0.001; Fig. 6). In the absence of inhibitors, FV/FM in the 30 and 31°C treatments was significantly lower than the 26°C treatment at 7 h (one-way ANOVA, F2,9=27.705, P<0.001) and 10 h (one-way ANOVA, F2,9=17.504, P=0.001; Fig. 6A). Partial recovery of FV/FM was seen in all treatments to a similar level after the 14 h recovery period, but values did not recover to 0 h levels in any temperature treatment. In the presence of GA, FV/FM was significantly higher in the 26°C treatment compared with the 30 and 31°C treatments at 10 h (one-way ANOVA, F2,9=7.050, P=0.014; Fig. 6B). At the 24 h mark, an almost complete loss of PSII photochemical efficiency occurred in the 31°C treatment, which was significantly lower than FV/FM at 26°C (one-way ANOVA, F2,9=5.630, P=0.026). In the presence of KCN, the decline in FV/FM was intermediate between the controls and the GA treatment (Fig. 6C). The 31°C treatment showed the greatest decline compared with 26°C at 10 h (one-way ANOVA, F2,9=9.817, P=0.005), and although no significant recovery was observed in FV/FM by 24 h, the 31°C treatment was lower than both the 26 and 30°C treatments at this time point (one-way ANOVA, F2,9=10.306, P=0.005).

Fig. 4.

Gross photosynthesis and respiration rate (μmol O2 h−1 cm−2) in Pocillopora damicornis. Treatments are as follows: (A) control (no inhibitors), (B) in the presence of 1 and 5 mmol l−1 GA, and (C) in the presence of 10 and 100 μmol l−1 KCN, after 0 h (black bars), 1 h (grey bars) and 3 h (white bars) of exposure. Data are means ± s.e.m. (n=4).

Fig. 4.

Gross photosynthesis and respiration rate (μmol O2 h−1 cm−2) in Pocillopora damicornis. Treatments are as follows: (A) control (no inhibitors), (B) in the presence of 1 and 5 mmol l−1 GA, and (C) in the presence of 10 and 100 μmol l−1 KCN, after 0 h (black bars), 1 h (grey bars) and 3 h (white bars) of exposure. Data are means ± s.e.m. (n=4).

Fig. 5.

Symbiont density and chlorophyll concentration in P. damicornis. (A) Symbiont density (×106cm−2); (B) chlorophyll a concentration (μg cm−2); (C) chlorophyll c2 concentration (μg cm−2); (D) total chlorophyll concentration (pg cell−1). Values were taken at 0 and 24 h in each of the three inhibitor treatments (control, GA and KCN) at 26°C (black bars), 30°C (grey bars) and 31°C (white bars). Data are means ± s.e.m. (n=4).

Fig. 5.

Symbiont density and chlorophyll concentration in P. damicornis. (A) Symbiont density (×106cm−2); (B) chlorophyll a concentration (μg cm−2); (C) chlorophyll c2 concentration (μg cm−2); (D) total chlorophyll concentration (pg cell−1). Values were taken at 0 and 24 h in each of the three inhibitor treatments (control, GA and KCN) at 26°C (black bars), 30°C (grey bars) and 31°C (white bars). Data are means ± s.e.m. (n=4).

Oxygen production and consumption

In the absence of an inhibitor, the rate of net oxygen production did not change significantly over time, nor was it different between temperature treatments, except at 24 h, where the rate of O2 production was significantly lower at 31°C compared with 26 and 30°C (one-way ANOVA, F2,9=11.628, P=0.003; Fig. 7A). The addition of GA (Fig. 7B) and KCN (Fig. 7C) resulted in temperature-dependent declines in net O2 production, with the higher temperature treatments experiencing the most rapid loss over the 24 h period (one-way ANOVAs, all P<0.001).

The respiration rate of coral nubbins in all temperatures and at all time points did not change significantly in the absence of an inhibitor (control; Fig. 7A). In comparison, in the presence of GA, respiration rate approached zero over the 24 h experimental period in all temperature treatments, with respiration rate significantly lower in the 30°C treatment at 10 h (compared with 26 and 31°C; one-way ANOVA, F2,9=12.167, P=0.003) and in the 30 and 31°C treatments at 24 h (compared with 26°C; one-way ANOVA, F2,9=67.058, P<0.001; Fig. 7B). In the presence of KCN, respiration rate remained constant across temperatures for the first 10 h. After the recovery period at 24 h, respiration rate had significantly declined at 30 and 31°C compared with 26°C (one-way ANOVA, F2,9=12.981, P=0.002; Fig. 7C).

Fast induction curves

During the application of a saturating pulse, chlorophyll fluorescence shows a polyphasic rise from minimum (FO or the O step) to maximum (FM or the P step) fluorescence, with two intermediate inflection points, the J and I steps. This OJIP curve provides detailed information on the reduction and oxidation of the primary (QA) and secondary (QB) electron acceptors of PSII. In the absence of any inhibitor, declines in the J, I and P steps were found along the FICs for each temperature treatment, indicating a loss of variable fluorescence (Fig. 8A–C). The relative decline in the J, I and P steps was the same for each curve, as verified in the double normalisation to the O and P steps, where all FICs showed a very similar pattern at each time point (supplementary material Fig. S1A–C). Application of GA resulted in a rapid decline in variable fluorescence, with the amplitude of the J, I and P steps approaching zero relative to the O step over the length of the experiment (Fig. 8D–F). Exposure to KCN showed an intermediate response with respect to the decline of the J, I and P steps during the exposure period, with the greatest loss of amplitude in the 31°C treatment (Fig. 8G–I). No change in the FICs was found during the 14 h recovery period in the KCN treatment. Calculation of the relative variable fluorescence for GA- and KCN-treated nubbins revealed an elevation of the J step over time for all temperature treatments, indicating a greater reduction of QA (supplementary material Fig. S1D–I).

Calculation of the J:P ratio of FICs revealed significant increases over time in all inhibitor and temperature treatments (repeated-measures ANOVAs, all P<0.005; Fig. 9A–C). A sharp increase was found from 0 to 10 h in the presence of both GA and KCN at 26, 30 and 31°C. By the end of the recovery period, the J:P ratio was close to 1.0 for all temperature treatments, indicating that J and P amplitudes were similar, confirming the loss of variable fluorescence between the J and P steps.

Functional absorption cross-section of PSII

Measures of the functional absorption cross-section of PSII (σPSII) provide an estimate of the fraction of absorbed light that leads to oxygen evolution. No change in σPSII was found in the absence of inhibitors in any of the temperature treatments, indicating no change to the light harvesting capabilities of PSII (Fig. 10A). In contrast, in the 26°C GA treatment, σPSII was significantly lower at 4 and 7 h compared with other time points (repeated-measures ANOVA, F4,12=3.896, P=0.030; Fig. 10B). In the 30 and 31°C GA temperature treatments, as well as all KCN temperature treatments (Fig. 10C), σPSII significantly declined after 0 h, owing to a decrease in the cross-sectional area of photosynthetic light-harvesting antennae, after which it remained stable (repeated-measures ANOVAs, all P<0.005). In the case of the GA treatment, it was not possible to record reasonable σPSII values after the 24 h treatment because of the lack of detectable variable fluorescence (e.g. Fig. 8D–F); therefore, this data point has been omitted. Significant negative correlations were found between σPSII and the J:P ratio in the 31°C GA treatment, plus all three KCN temperature treatments (Table 1). This indicates that the reduced state of QA (QA), signified by a high J:P ratio, has a direct impact on σPSII, where over-reduced QA resulted in a decrease in the cross-sectional area of photosynthetic light-harvesting antennae.

Fig. 6.

FV/FMP. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey circles) and 31°C (white circles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h (white bars). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bars). Data are means ± s.e.m. (n=4).

Fig. 6.

FV/FMP. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey circles) and 31°C (white circles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h (white bars). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bars). Data are means ± s.e.m. (n=4).

DISCUSSION

Inhibitors of the Calvin–Benson cycle and considerations for the coral holobiont

Here we tested the hypothesis that inhibition of the Calvin–Benson cycle induces the release of endosymbionts and/or loss of photosynthetic pigments from a bleaching-sensitive coral species under thermal stress. We used two inhibitors of the Calvin–Benson cycle that have been used with corals before: GA (Buxton et al., 2012; Bhagooli, 2013) and KCN (Chalker and Taylor, 1975; Barnes, 1985; Jones and Steven, 1997; Jones and Hoegh-Guldberg, 1999). They have also been widely used for studies on higher plants (Bishop and Spikes, 1955) and microalgae (Whittingham, 1952; Takahashi and Murata, 2005). With both these inhibitors, care was taken to ensure that concentrations affected only (or at least predominantly) the Calvin–Benson cycle. It was therefore necessary to carry out experiments on the effect of GA and KCN at a range of concentrations on algal photosynthesis and animal host respiration. GA and KCN concentrations were carefully chosen following a series of range-finding assays on cultured and in hospite Symbiodinium (Figs 1, 2, 3 and 4). The goal was to apply GA and KCN in concentrations high enough to induce inhibition of photosynthesis in the algal symbionts, but not to interfere with the functioning of host metabolism. Indeed, our quantifications of photosynthetic damage in the preliminary experiments highlighted the concentration-dependent decline in FV/FM over time that was due to the inhibitory effect of GA and KCN on mechanisms such as photosynthetic dark reactions (Fig. 1). In addition, we also probed for signs of damage to the animal host through measures of oxygen consumption in the dark. Gross primary production and respiration rate declined at 5 mmol l−1 GA and 100 μmol l−1 KCN over 3 h, but neither process was affected by 1 mmol l−1 GA or 10 μmol l−1 KCN. An intermediate concentration of 3 mmol l−1 GA and 20 μmol l−1 KCN was therefore chosen for subsequent experiments as it was expected to cause inhibition to the Calvin–Benson cycle (as probed via measures of FV/FM and oxygen production; Figs 1, 2 and 3), but have little impact on coral host function (Fig. 4). Nevertheless, it should also be kept in mind that GA and KCN at the chosen concentrations may affect other enzymes or processes.

During subsequent experimentation, GA and KCN exposure lasted for 24 h and measures of respiration rate suggested that GA did have some effect on host metabolism that was accelerated by thermal stress (Fig. 7). KCN also impacted host respiration, but only at 31°C after 24 h of exposure. As the bleaching response was consistent in the presence and absence of GA (Fig. 5), we suggest that although the host experienced damage from the chemical inhibitor, it did not alter the bleaching process. In comparison, while damage to host respiration was only found after 24 h in the highest temperature treatment during KCN exposure, bleaching was accelerated in all treatments by 24 h. Therefore, host condition may not have played a crucial role in the bleaching response.

The effect of Calvin–Benson cycle inhibition on coral bleaching

Pocillopora damicornis is a bleaching-sensitive coral species known to live close to its upper thermal threshold (Marshall and Baird, 2000; Loya et al., 2001; Hill et al., 2004a; Hill et al., 2004b). Bleaching is well characterised in this species around the world, and as previously reported, we found that thermal stress was sufficient to induce a bleaching response characterised by the expulsion of symbionts, rather than a loss of intracellular photosynthetic pigments (Fig. 5) (Hill et al., 2011). In this work we were able to confirm that in the absence of any Calvin–Benson cycle inhibitor, both symbiont density and chlorophyll concentration (per cm2 of coral tissue) declined from 0 to 24 h in the 30 and 31°C treatments, while there was no change in the 26°C treatment.

GA at 3 mmol l−1 did not affect the severity of bleaching, nor did it induce bleaching in the absence of thermal stress. Clearly, then, this evidence does not support the hypothesis that inhibition of the Calvin–Benson cycle is a key factor in initiating coral bleaching. However, at higher concentrations, GA did have profound effects that were related to an effect on respiration or the integrity of the animal host. While previous research has shown the effects of GA on coral bleaching (Bhagooli, 2013), these experiments were conducted at a higher concentration of GA and only investigated effects on photosynthesis and not respiration, and should therefore be treated with caution. To gain evidence on coral bleaching at 3 mmol l−1 GA, we were obliged, for comparison with others' experiments, to carry out our experiments for up to 24 h, a time frame that is a common practice for coral bleaching experiments (e.g. Hill et al., 2012), and it was clear (Figs 5, 6) that at this time point there were effects other than inhibition of the Calvin–Benson cycle. GA, when applied at a similar concentration (2.5 mmol l−1) in the cyanobacterium Synechocystis PCC 6803, induced a 16% loss of PSII activity in 4 h, an effect assigned to the inhibition of the repair of photodamaged PSII centres (Takahashi and Murata, 2005). However, the consequences of long-term GA treatment in photosynthetic systems have not been investigated previously. Therefore, after 24 h, it is probable that other effects of GA, on respiration and metabolism of the coral host, would have interfered with the coral bleaching mechanism and prevented the expulsion of symbionts. However, it should be noted that at earlier time points, no evidence was found for coral bleaching, unlike in the presence of KCN, and therefore we conclude that GA did not induce coral bleaching conditions at any time.

Fig. 7.

Production (net primary production; left side of figure) and respiration rate (right side of figure) in μmol O2 h−1 cm−2 of P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black bars), 30°C (grey bars) and 31°C (white bars). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment. At the 10 h time point, all corals were given a 14 h recovery period at 26°C. Corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h and then 14 h of darkness. Data are means ± s.e.m. (n=4).

Fig. 7.

Production (net primary production; left side of figure) and respiration rate (right side of figure) in μmol O2 h−1 cm−2 of P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black bars), 30°C (grey bars) and 31°C (white bars). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment. At the 10 h time point, all corals were given a 14 h recovery period at 26°C. Corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h and then 14 h of darkness. Data are means ± s.e.m. (n=4).

KCN, a known inhibitor of CO2 fixation in higher plants and microalgae (Whittingham, 1952; Bishop and Spikes, 1955), has been linked with coral bleaching under field conditions (Jones and Steven, 1997) and in laboratory experiments (Jones et al., 1998; Jones and Hoegh-Guldberg, 1999). In our experiments, exposure to 20 μmol l−1 KCN not only strongly inhibited photosynthetic oxygen production (without having an impact on respiration rate), as did 3 mmol l−1 GA, but, unlike GA, also promoted bleaching, with symbiont density and chlorophyll concentration declining significantly in all three temperature treatments, with no temperature-dependent effect. Thus we have the interesting situation that of these two inhibitors of the Calvin–Benson cycle, only KCN induced bleaching. This suggests that KCN might have other effects besides its effect on the Calvin–Benson cycle.

An early explanation for the coral bleaching effect of KCN was that with the inhibition of the Calvin–Benson cycle, electrons would back up into PSI and PSII and this would generate large amounts of ROS, offset to some extent by the flow of electrons into the Mehler ascorbate peroxidase pathway (Jones et al., 1998; Jones and Hoegh-Guldberg, 1999). However, the fact that GA inhibits the Calvin–Benson cycle but does not induce bleaching seems to require some further explanation for the effect of KCN. We suggest that this is the action of KCN in inhibiting APX (Shigeoka et al., 2002) and stimulating OH (hydroxyl radical) production, in a Fenton reaction from H2O2 (Ishida et al., 1998; Jakob and Heber, 1996). In addition, it has been reported that KCN also inhibits the catalase enzyme in intact chloroplasts of higher plants, which leads to enhanced production of H2O2 (Forti and Gerola, 1977). Therefore, in contrast with GA, KCN would induce a much greater production of ROS and this could explain the marked difference in bleaching response. Overall, therefore, this evidence supports a strong role for ROS in inducing bleaching (Lesser, 2011). Furthermore, KCN did not significantly enhance bleaching under thermal stress compared with 26°C, as the extent of bleaching was the same in all temperature treatments (Fig. 5), and no different to the extent of bleaching in nubbins exposed to 30 and 31°C in the absence of any inhibitors. This suggests that the inhibition caused by KCN at an optimal temperature (26°C) applied the same bleaching pressure as when thermal stress was applied at 30 and 31°C, regardless of whether KCN was present or absent. If our hypothesis is correct, that ROS is driving the bleaching process, then KCN at 26°C must produce the same degree of ROS as 31°C in the absence of KCN, i.e. ROS production in the presence of KCN is saturating at 26°C.

Fig. 8.

FICs of P. damicornis. Treatments are as follows: (A–C) in the absence of any inhibitor (control), (D–F) in the presence of 3 mmol l−1 GA and (G–I) in the presence of 20 μmol l−1 KCN at 26°C (A,D,G), 30°C (B,E,H) and 31°C (C,F,I). Averages are shown (n=4).

Fig. 8.

FICs of P. damicornis. Treatments are as follows: (A–C) in the absence of any inhibitor (control), (D–F) in the presence of 3 mmol l−1 GA and (G–I) in the presence of 20 μmol l−1 KCN at 26°C (A,D,G), 30°C (B,E,H) and 31°C (C,F,I). Averages are shown (n=4).

The concentration of KCN that has been used in our studies (20 μmol l−1) is comparable to that used in other experiments on the inhibition of the Calvin–Benson cycle in algal and coral bleaching studies (Wishnick and Lane, 1969; Sicher, 1984; Jones and Hoegh-Guldberg, 1999). The concentrations of KCN, which have been used to inhibit algal APX, have traditionally been higher than here, viz. 1 mmol l−1 (Shigeoka et al., 2002). However, a structural study by Hill et al. (Hill et al., 1997) showed that APX had a binding (dissociation) constant to the enzyme of Kd=11.6 μmol l−1 and it is entirely reasonable that APX was inhibited in our experiments.

Inhibition of PSII function

The two inhibitors of the Calvin–Benson cycle, GA and KCN, would be expected to have indirect effects on PSII through a limitation on electron transport and ROS production. GA has been shown to inhibit the PSII repair cycle (Takahashi and Murata, 2005), which could be induced by the suppression of D1 protein synthesis at the translation elongation step because of the accumulation of 1O2 in the absence of electron transfer towards CO2 as the final electron acceptor (Nishiyama et al., 2001; Nishiyama et al., 2004). In addition, the effect of superoxide could also contribute to the inhibition of D1 repair in the presence of GA (Takahashi and Murata, 2005). KCN is also expected to enhance 1O2 production as a result of over-reduction of electron transport components at the acceptor side of PSII in the absence of electron flow towards the Calvin–Benson cycle, while the inhibitory effect of KCN on catalase (Forti and Gerola, 1977) would enhance production of H2O2, which is also an inhibitor of D1 protein synthesis, and therefore of the PSII repair cycle (Nishiyama et al., 2001). Our current findings obtained with the coral symbiosis provide further evidence for the differential effect of inhibiting the process of CO2 uptake by different inhibitors of the Calvin–Benson cycle reported earlier in other microalgae and higher plants.

In contrast to GA and KCN, enhanced temperatures may have a direct effect on one or more functions of PSII (Warner et al., 1999; Hill et al., 2011). In this study, FV/FM (Fig. 6) and the amplitude of FIC steps (Fig. 8) showed a temperature-dependent decline even in the absence of inhibitors, with the 31°C treatment experiencing the greatest decrease in FV/FM and the J, I and P steps. The initial decline in FV/FM and the loss of variable fluorescence in FICs in the 26°C treatment in the absence of inhibitors indicates that the light intensity applied (600 μmol photons m−2 s−1) caused some photoinactivation of PSII. Prior to experimentation, corals were maintained in aquaria at 100 μmol photons m−2 s−1 and we conclude that the sixfold increase in irradiance was sufficient to lower photosynthetic efficiency in the absence of thermal stress. With the application of thermal stress, a greater loss of FV/FM and amplitude of J, I and P steps was found in all inhibitor treatments compared with the 26°C treatment, with photoinactivation only partially reversible in the absence of GA and KCN after 14 h of recovery in darkness. The decline in FIC amplitude was primarily the result of changes to the J step. This was visualised in supplementary material Fig. S1, which shows the relative variable fluorescence. In the presence of GA or KCN, the non-reversible rise in the J step over time indicates a greater reduction of QA owing to closure of PSII reaction centres (Strasser et al., 1995; Hill et al., 2004a; Ulstrup et al., 2005). The loss of variable fluorescence between the J and P steps (Fig. 9) matches the greatly inhibited FV/FM values in the presence of GA by 24 h in all three temperature treatments. The low FV/FM readings at 24 h indicate an almost complete loss of variable fluorescence, and by comparing the J:P ratio and FV/FM, we can conclude that the small variable fluorescence that remained was due to increases from the O phase to the J phase. After the 3 ms time point in the FICs, no further change in fluorescence occurred. This indicates that there was still some minor capacity for reduction of QA to QA at this time point, but no potential for QB or plastoquinone pool reduction (Strasser et al., 1995; Hill et al., 2004a). Together, these impacts on PSII function suggest that net PSII repair was able to partially counter photoinactivation when the Calvin-Benson cycle was still functional. However, exposure to GA and KCN prevented any net repair to PSII, with no recovery of FV/FM or FIC steps found following 14 h of darkness.

Fig. 9.

J:P ratio of FICs in P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey squares) and 31°C (white triangles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h (white bar). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bar). Data are means ± s.e.m. (n=4). Values close to 1 indicate J=P.

Fig. 9.

J:P ratio of FICs in P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey squares) and 31°C (white triangles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2s−1 for the initial 10 h (white bar). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bar). Data are means ± s.e.m. (n=4). Values close to 1 indicate J=P.

The elevated excitation pressure on PSII as a consequence of over-reduced QA can be judged from the OJIP FICs along with the σPSII data. The negative correlations between the J:P ratio and σPSII (Table 1) demonstrated how the development of over-reduced QA led to a smaller PSII cross-sectional area, which is a potential photoprotective mechanism to reduce excitation pressure on PSII reaction centres (Falkowski and Owens, 1980; Gorbunov et al., 2001; Suggett et al., 2004). A similar response was observed in the green alga Chlamydomonas reinhardtii, where inhibition of Calvin–Benson cycle by GA triggered enhanced non-photochemical quenching (NPQ) as a photoprotective mechanism (Takahashi and Murata, 2005). Interestingly, the correlation between J:P and changes in σPSII in GA treatments was poor at 26°C but improved in the 31°C heat treatments, indicating that the correlation between the reduction state of QA and changes in cross-sectional area was stronger when the heat stress was more severe during the GA treatment. Nevertheless, a clear tendency of σPSII and J:P as a function of temperature could not be given. The fact that in the absence of inhibitors J:P was much smaller and no significant change in σPSII was observed indicates that rapid changes in functional cross-sectional area of PSII is not the major photoprotective mechanism of in hospite Symbiodinium, which is in agreement with earlier findings (Lesser and Farrell, 2004; Hennige et al., 2011). Changes in σPSII becomes relevant only during the inhibitor treatments, when excitation pressure on PSII increases as a consequence of an over-reduced state of QA (elevated J:P ratio) that is triggered by impaired CO2 fixation via the Calvin–Benson cycle. Based on the σPSII and FIC data, the effect of GA and KCN was similar, but not the same. The lack of correlation between over-reduced QA and changes in σPSII at 26 and 30°C in the presence of GA, but the significant correlations at these temperatures in the presence of KCN, suggests that mechanisms specific to each inhibitor might be responsible for the downregulation of photochemical efficiency and are unrelated to Calvin–Benson cycle activity. In the case of GA, loss of photochemical efficiency may be due to increased NPQ (Takahashi and Murata, 2005), from changes in the light harvesting antenna complexes (Gorbunov et al., 2001; Hill et al., 2012) or reaction centre inactivation (Hennige et al., 2011). Takahashi and Murata (Takahashi and Murata, 2005) suggest that although NPQ increases in Chlamydomonas sp. in the presence of GA, the photochemical quenching was unaltered under this treatment, indicating that the reduction state of QA does result in engagement of the photoprotective processes in all cases. Although our observation in Symbiodinium sp. indicates that QA becomes reduced in the presence of GA based on the elevated J:P ratio (Fig. 9) a clear correlation between the amount of QA and the value of σPSII cannot be given (Table 1), in agreement with the finding of Takahashi and Murata (Takahashi and Murata, 2005). The effect of KCN appeared to be somewhat different, with a strong correlation found. In the presence of KCN, the reduction state of QA can be linked directly to decreased σPSII, possibly as a result of KCN blocking APX. A recent study showed that APX-deficient mutants of rice (Oryza sativa) exhibited enhanced excitation energy pressure on PSII because of the accumulation of hydrogen peroxide and hydroxyl radicals under light stress conditions (Caverzan et al., 2014). Although there is no similar detailed study available of the physiological impact of the absence of APX activity in Symbiodinium, we propose that the differential response of KCN and GA is related to the additional inhibitory effect KCN has on APX activity and thereby the strong correlation between the reduction state of the plastoquinone pool and σPSII.

Fig. 10.

Functional absorption cross-section of photosystem II (σPSII) of P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey circles) and 31°C (white circles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2 s−1 for the initial 10 h (white bars). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bars). Data are means ± s.e.m. (n=4).

Fig. 10.

Functional absorption cross-section of photosystem II (σPSII) of P. damicornis. Treatments are as follows: (A) in the absence of any inhibitor (control), (B) in the presence of 3 mmol l−1 GA and (C) in the presence of 20 μmol l−1 KCN at 26°C (black circles), 30°C (grey circles) and 31°C (white circles). Temperatures were ramped from 26°C to treatment temperature over the first 2 h of the experiment and corals were exposed to 600 μmol photons m−2 s−1 for the initial 10 h (white bars). At the 10 h time point, all corals were given a 14 h recovery period at 26°C in darkness (grey bars). Data are means ± s.e.m. (n=4).

The changes in FV/FM for control, GA and KCN treatments were mirrored in the rates of net oxygen production (Fig. 7), except that instead of recovery at the 24 h time point in the absence of GA and KCN, oxygen production continued to decline, with the greatest loss in the 31°C treatment. While in this treatment FV/FM showed potential for recovery, photosynthetic oxygen production showed a significant decline in the thermally treated corals (31°C), as considerable bleaching had occurred and the symbiont density and chlorophyll concentration per cm2 of coral skeleton declined (Fig. 5). The temperature-dependent reduction in O2 production in the light by GA and KCN indicates a loss of photosynthetic electron transport, which is confirmed by the FV/FM data. Interestingly, however, there was often a large mismatch between FV/FM and the rate of oxygen production, particularly in the KCN treatments. Photochemical efficiency was retained in the presence of KCN in the 30°C and 31°C after 3 h of exposure, as well as at 26°C after 24 h. A similar result was seen in the absence of inhibitors after 24 h at 31°C. This indicates that although PSII is still functional when probed with a saturating pulse following dark adaptation, the electron transport chain and dark reactions are inhibited during illumination, resulting in a greatly depressed rate of oxygen production compared with FV/FM. This suggests that while PSII experiences some level of damage, more severe inhibition is likely occurring at the site of the Calvin–Benson cycle under thermal stress. Alternatively, this mismatch could be related to the bleaching of nubbins, which would affect oxygen production rates, but not FV/FM, or an inhibition to the oxygen evolving complex that is more severe than damage to the PSII core complex [although this latter possibility is unlikely (see Hill and Ralph, 2008)].

Table 1.

Pearson correlation coefficients between the J:P ratio and the functional absorption cross-section of PSII (σPSII) over time for the control, glycolaldehyde (GA) and potassium cyanide (KCN) treatments at 26, 30 and 31°C

Pearson correlation coefficients between the J:P ratio and the functional absorption cross-section of PSII (σPSII) over time for the control, glycolaldehyde (GA) and potassium cyanide (KCN) treatments at 26, 30 and 31°C
Pearson correlation coefficients between the J:P ratio and the functional absorption cross-section of PSII (σPSII) over time for the control, glycolaldehyde (GA) and potassium cyanide (KCN) treatments at 26, 30 and 31°C

Inhibition of CO2 fixation as a trigger of coral bleaching

Considerable research effort has focused on understanding the photosynthetic trigger that results in coral bleaching (Lesser, 2011). Here, we used chemical compounds (GA and KCN) known to inhibit the Calvin-Benson cycle to probe their effect on bleaching in coral nubbins exposed to three different temperature regimes. We found that while bleaching was not detected at 26°C in the presence of GA, an almost complete loss of photosynthetic function was found. This supports the conclusion that inhibition of photosynthetic CO2 fixation was not an immediate cause of bleaching in P. damicornis. The results from the KCN experiments are in stark contrast to this, with bleaching occurring in all treatment temperatures. We hypothesise that KCN-driven ROS production was the driver of the bleaching response. Bhagooli (Bhagooli, 2013) found that bleaching did occur at the growth temperature of 26°C in Stylophora pistillata when CO2 fixation was inhibited by GA. This difference between our study and that of Bhagooli (Bhagooli, 2013) may be due to: (1) the higher concentration of GA used in Bhagooli's study (5 mmol l−1 GA compared with 3 mmol l−1 GA used in our study), which may have had additional impacts on the symbiosis or which may induce a more rapid and/or chronic response, or (2) the different coral species used, a factor that is believed to play an important role in determining the site of primary photosynthetic impairment during thermal bleaching (Buxton et al., 2012). Thus we suggest that in P. damicornis, elevated ROS production from KCN exposure causes dysfunction to the symbiosis and results in bleaching. The suggestion that in coral bleaching KCN acts by inhibiting APX, thereby stimulating ROS production, needs further testing.

MATERIALS AND METHODS

Symbiodinium cultures

The CS-156 Symbiodinium culture was obtained from the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Australian National Algae Culture Collection, and grown in f/2 medium at 25°C and 40 μmol photons m−2 s−1, with a light:dark ratio of 12 h:12 h. Measurements were performed on cultures with ~7 μg chlorophyll (a + c2) ml−1 at 25°C.

Coral specimens

Four colonies of Pocillopora damicornis were collected from Heron Island lagoon, located on the southern Great Barrier Reef of Australia (151°55′E, 23°27′S), and transported to the University of Technology, Sydney, where they were placed in recirculating artificial seawater at 26°C and 100 μmol photons m−2 s−1. Nubbins ~1 cm in length were broken off from each colony for subsequent use in experimental treatments 1–3 days later.

Inhibitor range-finding trials

Gross photosynthesis, dark respiration, maximum quantum yield (FV/FM), chlorophyll fluorescence fast induction curves (FICs), and chlorophyll and zooxanthellae density were measured as detailed below.

Initial experiments were performed on Symbiodinium cultures where temperature was kept constant during experimentation and irradiance was applied for 8 h, followed by 12 h darkness and a further 12 h of light (40 μmol photons m−2s−1). Photo synthetic activity was assessed by measuring the so-called OJIP transient of variable chlorophyll fluorescence during application of a 2 s saturating pulse (Strasser et al., 1995; Hill et al., 2004a). FV/FM was obtained by calculating (FMFO)/FM, where FO and FM represent the minimum fluorescence in dark-adapted cells and the maximal fluorescence yield under continuous saturating light, respectively. These measurements were performed using the FL-3000 Fluorometer (Photon Systems Instruments, Brno, Czech Republic), which is designed to measure algal cells in suspension.

Following these concentration range-finding tests on Symbiodinium cultures, in hospite Symbiodinium of P. damicornis were tested at 0, 1 and 5 mmol l−1 GA and 0, 10 and 100 μmol l−1 KCN. One nubbin from each of the four colonies was placed in an individual 500 ml beaker filled with 250 ml of 0.2 μm filtered seawater and gently mixed during exposure to 600 μmol photons m−2 s−1 (white LED panels; IronHorse WLED80, Arlec, Blackburn, Australia). FV/FM was measured on a Mini-PAM fluorometer (Walz Effeltrich, Germany) after 10 min of dark adaptation (0.8 s saturating pulse of >4500 μmol photons m−2s−1, gain=8) at 0, 1 and 3 h. A 6 mm diameter glass fibre optic probe, connected to the Mini-PAM, was used to measure the photosynthetic surface of the coral nubbins. Gross photosynthesis rates were measured using a temperature-controlled oxygen electrode cuvette (3–5 ml capacity; Rank, Bottisham, UK) and a Firesting optical oxygen sensor (PyroScience, Aachen, Germany).

Experimental protocol

Nubbins of P. damicornis were placed in individual beakers as described above for 24 h. For the first 10 h, nubbins were exposed to 600 μmol photons m−2 s−1, and then 14 h of darkness. During the first 2 h of the light treatment, nubbins were ramped from 26°C to their experimental temperature treatments of 26 (control), 30 and 31°C. At the end of the light period (10 h mark), temperatures were gradually reduced to 26°C over 2 h and maintained at this temperature until 24 h. In each temperature treatment, four nubbins were exposed to seawater only (control), 3 mmol l−1 GA or 20 μmol l−1 KCN (n=4). At the 10 h mark, a 100% water change (containing GA or KCN in the inhibitor treatments) was given to each nubbin.

Four nubbins were harvested at 0 h and another four at 24 h from each of the three temperature treatments and three inhibitor treatments for determination of symbiont density and chlorophyll a and c2 concentration. Nubbins were placed in 15 ml of filtered seawater and airbrushed to remove all coral tissue from the skeleton. The host and endosymbiont slurry was centrifuged at 1000 g for 10 min and the supernatant was discarded. The algal pellet was resuspended in 4 ml FSW and 100 μl removed for symbiont counting on haemocytometer slides. Eight replicate counts were performed per sample to determine symbiont density. The remaining 3.9 ml was centrifuged again at 1000 g for 10 min. The supernatant was discarded and the algal pellet was resuspended in 4 ml 90% acetone. Twenty-four hours later, the samples were centrifuged at 1000 g for 10 min and the absorbance of the supernatant was measured at 630 and 664 nm on a UV-Vis spectrophotometer (Shimadzu, USA). Chlorophyll a and c2 concentrations were then determined using the equations of Jeffrey and Humphrey (Jeffrey and Humphrey, 1975). Symbiont density and chlorophyll a and c2 concentration were normalised to nubbin skeleton surface area, which was determined using the paraffin wax technique (Stimson and Kinzie, 1991). Total chlorophyll (a and c2) per symbiont cell was also calculated.

At 0, 2, 4, 7, 10 and 24 h, FV/FM of the in hospite symbionts was measured after 10 min of dark adaptation using a Mini-PAM fluorometer (as described above), followed by the functional absorption cross-section of PSII (aPSII) on a FastTracka II Fast Repetition Rate fluorometer (FRRf; Chelsea Technologies Group Ltd, West Molesey, Surrey, UK). A preprogrammed single turnover acquisition was used for all measurements controlled by FASTPro software (version 2.0). One measuring sequence included both saturation and relaxation phases. For saturation, 60μ1 μs flashlets were given with an interval of 3 μs between each flashlet. These settings resulted in a 240 μs measuring cycle for saturation. For relaxation, 50μ1 μs flashlets were given, initially with an interval of 100 μs between each flashlet, which increased by 10% in length for each consecutive flashlet interval. This resulted in a 116 ms measuring cycle for the relaxation phase. To improve the signal-to-noise ratio, four sequences were averaged per acquisition, separated by 100 ms. aPSII was calculated using FASTPro software according to the biophysical model of Kolber et al. (Kolber et al., 1998) and is expressed in nm2 quanta−1 using the flash calibration factor for the instrument. The excitation wavelength was 470±20 nm and the fluorescence emission was detected at 685±10 nm. The LED intensity and the photomultiplier voltage were adjusted according to the manufacturer's recommendations (see Oxborough et al., 2012) to optimize the FO levels and the rate of PSII closure during the train of flashlets for the coral nubbins. Upon completion of aPSII determination, a double-modulation fluorometer (Photon Systems Instruments, FL-3300) was used to measured FICs during a 3700 μmol photons m−2 s−1 5 s flash from red (640 nm) and blue (455 nm) LEDs (see Hill and Ralph, 2006). Chlorophyll fluorescence measurements were recorded every 10 μs for the first 2 ms, every 1 ms up until 1 s, and then every 500 ms up to 5 s. Curves were normalised to the O step (FO) at 0.05 ms (see Hill et al., 2004a). Relative variable fluorescence of the FICs was calculated using the formula (Ft−FO)/(FMFO), where Ft is chlorophyll fluorescence at time t (Strasser et al., 1995; Hill et al., 2004a; Ulstrup et al., 2005). The ratio of the amplitude of the J step (inflection at 3 ms) to the P step (FM) was also calculated.

At 0, 3, 10 and 24 h, the rate of gross primary production and respiration was determined using the Rank oxygen cuvette setup described earlier. Nubbins were placed in 5 ml of treatment seawater and held at the treatment temperature. They were exposed to 5 min of darkness, followed by 5 min of 400 μmol photons m−2 s−1 from a quartz iodine light source. The rate of oxygen consumption or production was expressed per cm2 of nubbin surface area (μmol O2 h−1 cm−2), determined as described above.

Statistical analyses

One-way ANOVA tests were used to identify differences between inhibitor treatments, temperature treatments or changes over time in independent samples. Repeated-measures ANOVA was applied in the analysis of samples that were measured continuously throughout the experiment. In cases where significant differences were detected, Tukey's post hoc comparisons identified the significantly different treatments. Pearson's correlation tests were used to detect significant correlations between σPSII and the J:P ratio. A significance level of 0.05 was applied throughout these analyses and the Kolmogorov-Smirnov normality test and Levene's homogeneity of variance test were used to determine whether assumptions of the parametric analyses were satisfied. SPSS statistical software (version 21, 2012) was used to perform these analyses.

FOOTNOTES

Funding

This work was supported by the Australian Research Council [grant number DP120101360 to R.H.] and the Hungarian Granting Agency [grant number OTKA K-101433 to I.V. and A.R.].

References

Al-Enezi
K. S.
,
Alkhalaf
M.
,
Benov
L. T.
(
2006
).
Glycolaldehyde induces growth inhibition and oxidative stress in human breast cancer cells
.
Free Radic. Biol. Med.
40
,
1144
-
1151
.
Barnes
D. J.
(
1985
).
The effects of photosynthetic and respiratory inhibitors upon calcification in the staghorn coral Acropora formosa
. In
Proceedings of the 5th International Coral Reef Congress
(ed.
Gabrie
C.
,
Toffart
J. L.
,
Salvat
B.
), pp.
161
-
166
.
Moorea
:
Antenne du Museum-Ephe
.
Bhagooli
R.
(
2013
).
Inhibition of Calvin-Benson cycle suppresses the repair of photosystem II in Symbiodinium: implications for coral bleaching
.
Hydrobiologia
714
,
183
-
190
.
Bishop
N. I.
,
Spikes
J. D.
(
1955
).
Inhibition by cyanide of the photochemical activity of isolated chloroplasts
.
Nature
176
,
307
-
308
.
Buchel
C.
,
Garab
G.
(
1995
).
Evidence for the operation of a cyanide-sensitive oxidase in chlororespiration in the thylakoids of the chlorophyll c contaming alga Pleurichloris meiringensis (Xanthophyceae)
.
Planta
197
,
69
-
75
.
Buxton
L.
,
Takahashi
S.
,
Hill
R.
,
Ralph
P. J.
(
2012
).
Variability in the primary site of photosynthetic damage in Symbiodinium sp. (Dinophyceae) exposed to thermal stress
.
J. Phycol.
48
,
117
-
126
.
Caverzan
A.
,
Bonifacio
A.
,
Carvalho
F. E. L.
,
Andrade
C. M. B.
,
Passaia
G.
,
Schünemann
M.
,
Maraschin
F. S.
,
Martins
M. O.
,
Teixeira
F. K.
,
Rauber
R.
, et al. 
. (
2014
).
The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice
.
Plant Sci.
214
,
74
-
87
.
Chalker
B. E.
,
Taylor
D. L.
(
1975
).
Light-enhanced calcification, and the role of oxidative phosphorylation in calcification of the coral Acropora cervicornis
.
Proc. R. Soc. B
190
,
323
-
331
.
Díaz-Almeyda
E.
,
Thomé
P. E.
,
El Hafidi
M.
,
Iglesias-Prieto
R.
(
2011
).
Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium
.
Coral Reefs
30
,
217
-
225
.
Falkowski
P. G.
,
Owens
T. G.
(
1980
).
Light-shade adaptation: two strategies in marine phytoplankton
.
Plant Physiol.
66
,
592
-
595
.
Forti
G.
,
Gerola
P.
(
1977
).
Inhibition of photosynthesis by azide and cyanide and the role of oxygen in photosynthesis
.
Plant Physiol.
59
,
859
-
862
.
Gorbunov
M. Y.
,
Kolber
Z. S.
,
Lesser
M. P.
,
Falkowski
P. G.
(
2001
).
Photosynthesis and photoprotection in symbiotic corals
.
Limnol. Oceanogr.
46
,
75
-
85
.
Hennige
S. J.
,
McGinley
M. P.
,
Grottoli
A. G.
,
Warner
M. E.
(
2011
).
Photoinhibition of Symbiodinium spp. within the reef corals Montastraea faveolata and Porites astreoides: implications for coral bleaching
.
Mar. Biol.
158
,
2515
-
2526
.
Hill
R.
,
Ralph
P. J.
(
2006
).
Photosystem II heterogeneity of in hospite zooxanthellae in scleractinian corals exposed to bleaching conditions
.
Photochem. Photobiol.
82
,
1577
-
1585
.
Hill
R.
,
Ralph
P. J.
(
2008
).
Impact of bleaching stress on the function of the oxygen evolving complex of zooxanthellae from scleractinian corals
.
J. Phycol.
44
,
299
-
310
.
Hill
A. P.
,
Modi
S.
,
Sutcliffe
M. J.
,
Turner
D. D.
,
Gilfoyle
D. J.
,
Smith
A. T.
,
Tam
B. M.
,
Lloyd
E.
(
1997
).
Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase
.
Eur. J. Biochem.
248
,
347
-
354
.
Hill
R.
,
Larkum
A. W. D.
,
Frankart
C.
,
Kühl
M.
,
Ralph
P. J.
(
2004a
).
Loss of functional photosystem II reaction centres in zooxanthellae of corals exposed to bleaching conditions: using fluorescence rise kinetics
.
Photosynth. Res.
82
,
59
-
72
.
Hill
R.
,
Schreiber
U.
,
Gademann
R.
,
Larkum
A. W. D.
,
Kühl
M.
,
Ralph
P. J.
(
2004b
).
Spatial heterogeneity of photosynthesis and the effect of temperature-induced bleaching conditions in three species of corals
.
Mar. Biol.
144
,
633
-
640
.
Hill
R.
,
Ulstrup
K. E.
,
Ralph
P. J.
(
2009
).
Temperature induced changes in thylakoid membrane thermostability of cultured, freshly isolated and expelled zooxanthellae from scleractinian corals
.
Bull. Mar. Sci.
85
,
223
-
244
.
Hill
R.
,
Brown
C. M.
,
DeZeeuw
K.
,
Campbell
D. A.
,
Ralph
P. J.
(
2011
).
Increased rate of D1 repair in coral symbionts during bleaching is insufficient to counter accelerated photoinactivation
.
Limnol. Oceanogr.
56
,
139
-
146
.
Hill
R.
,
Larkum
A. W. D.
,
Prášil
O.
,
Szabó
M.
,
Kumar
V.
,
Ralph
P. J.
(
2012
).
Light-induced dissociation of antenna complexes in the symbionts of scleractinian corals correlates with sensitivity to coral bleaching
.
Coral Reefs
31
,
963
-
975
.
Hoegh-Guldberg
O.
(
1999
).
Climate change, coral bleaching and the future of the world's coral reefs
.
Mar. Freshw. Res.
50
,
839
-
866
.
Hoegh-Guldberg
O.
,
Mumby
P. J.
,
Hooten
A. J.
,
Steneck
R. S.
,
Greenfield
P.
,
Gomez
E.
,
Harvell
C. D.
,
Sale
P. F.
,
Edwards
A. J.
,
Caldeira
K.
, et al. 
. (
2007
).
Coral reefs under rapid climate change and ocean acidification
.
Science
318
,
1737
-
1742
.
Hoogenboom
M. O.
,
Campbell
D. A.
,
Beraud
E.
,
Dezeeuw
K.
,
Ferrier-Pagès
C.
(
2012
).
Effects of light, food availability and temperature stress on the function of photosystem II and photosystem I of coral symbionts
.
PLoS ONE
7
,
e30167
.
Iglesias-Prieto
R.
(
1997
).
Temperature-dependent inactivation of photosystem II in symbiotic dinoflagellates
. In
Proceedings of the 8th International Coral Reef Symposium
(ed.
Lessios
H. A.
,
Macintyre
I. G.
), pp.
1313
-
1318
.
Panama
:
Smithsonian Tropical Research Institute
.
IPCC
(
2007
).
Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change
(ed.
Core Writing Team
Pachauri
R. K.
,
Reisinger
A.
).
Geneva
:
IPCC
.
Ishida
H.
,
Shimizu
S.
,
Makino
A.
,
Mae
T.
(
1998
).
Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves
.
Planta
204
,
305
-
309
.
Jakob
B.
,
Heber
U.
(
1996
).
Photoproduction and detoxification of hydroxyl radicals in chloroplasts and leaves and relation to photoinactivation of photosystems I and II
.
Plant Cell Physiol.
37
,
629
-
635
.
Jeffrey
S. W.
,
Humphrey
G. F.
(
1975
).
New spectrophotometry equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton
.
Biochem. Physiol. Pflanzen
167
,
191
-
194
.
Jones
R. J.
,
Hoegh-Guldberg
O.
(
1999
).
Effects of cyanide on coral photosynthesis: implications for identifying the cause of coral bleaching and for assessing the environmental effects of cyanide fishing
.
Mar. Ecol. Prog. Ser.
177
,
83
-
91
.
Jones
R. J.
,
Steven
A. L.
(
1997
).
Effects of cyanide on corals in relation to cyanide fishing on reefs
.
Mar. Freshw. Res.
48
,
517
-
522
.
Jones
R. J.
,
Hoegh-Guldberg
O.
,
Larkum
A. W. D.
,
Schreiber
U.
(
1998
).
Temperature-induced bleaching of corals begins with impairment of the CO2 fixation metabolism in zooxanthellae
.
Plant Cell Environ.
21
,
1219
-
1230
.
Jones
R. J.
,
Ward
S.
,
Yang
A. A.
,
Hoegh-Guldberg
O.
(
2000
).
Changes in quantum efficiency of photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event
.
Mar. Freshw. Res.
51
,
63
-
71
.
Kolber
Z. S.
,
Prasil
O.
,
Falkowski
P. G.
(
1998
).
Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols
.
Biochim. Biophys. Acta
1367
,
88
-
106
.
Leggat
W.
,
Whitney
S.
,
Yellowlees
D.
(
2004
).
Is coral bleaching due to the instability of the zooxanthellae dark reactions?
Symbiosis
37
,
137
-
153
.
Lesser
M. P.
(
2011
).
Coral bleaching: causes and mechanisms
. In
Coral Reefs: an Ecosystem in Transition
(ed.
Dubinsky
Z.
,
Stambler
N.
), pp
405
-
419
.
Dordrecht
:
Springer
.
Lesser
M. P.
,
Farrell
J. H.
(
2004
).
Exposure to solar radiation increases damage to both host tissues and algal symbionts of corals during thermal stress
.
Coral Reefs
23
,
367
-
377
.
Lilley
R. M.
,
Ralph
P. J.
,
Larkum
A. W. D.
(
2010
).
The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short-term thermal stress of the alga
.
Plant Cell Environ.
33
,
995
-
1004
.
Loya
Y.
,
Sakai
K.
,
Yamazato
K.
,
Nakano
Y.
,
Sambali
H.
,
van Woesik
R.
(
2001
).
Coral bleaching: the winners and the losers
.
Ecol. Lett.
4
,
122
-
131
.
Marshall
P. A.
,
Baird
A. H.
(
2000
).
Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa
.
Coral Reefs
19
,
155
-
163
.
Miller
A. G.
,
Canvin
D. T.
(
1989
).
Glycolaldehyde inhibits CO2 fixation in the cyanobacterium Synechococcus UTEX 625 without inhibiting the accumulation of inorganic carbon or the associated quenching of chlorophyll a fluorescence
.
Plant Physiol.
91
,
1044
-
1049
.
Nishiyama
Y.
,
Yamamoto
H.
,
Allakhverdiev
S. I.
,
Inaba
M.
,
Yokota
A.
,
Murata
N.
(
2001
).
Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery
.
EMBO J.
20
,
5587
-
5594
.
Nishiyama
Y.
,
Allakhverdiev
S. I.
,
Yamamoto
H.
,
Hayashi
H.
,
Murata
N.
(
2004
).
Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803
.
Biochemistry
43
,
11321
-
11330
.
Oxborough
K.
,
Moore
C. M.
,
Suggett
D. J.
,
Lawson
T.
,
Chan
H. G.
,
Geider
R. J.
(
2012
).
Direct estimation of functional PSII reaction center concentration and PSII electron flux on a volume basis: a new approach to the analysis of fast repetition rate fluorometry (FRRf) data
.
Limnol. Oceanogr. Methods
10
,
142
-
154
.
Robison
J. D.
,
Warner
M. E.
(
2006
).
Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (Pyrrhophyta)
.
J. Phycol.
42
,
568
-
579
.
Salon
C.
,
Li
Q.
,
Canvin
D. T.
(
1998
).
Glycolaldehyde inhibition of CO2 transport in the cyanobacterium Synechococcus UTEX 625
.
Can. J. Bot.
76
,
1
-
11
.
Shigeoka
S.
,
Ishikawa
T.
,
Tamoi
M.
,
Miyagawa
Y.
,
Takeda
T.
,
Yabuta
Y.
,
Yoshimura
K.
(
2002
).
Regulation and function of ascorbate peroxidase isoenzymes
.
J. Exp. Bot.
53
,
1305
-
1319
.
Sicher
R. C.
(
1984
).
Glycolaldehyde inhibition of photosynthetic carbon assimilation by isolated chloroplasts and protoplasts
. In
Advances in Photosynthesis Research
, Vol.
3
(ed.
Sybesma
C.
), pp.
413
-
416
.
The Hague
:
W. Junk
.
Smith
D. J.
,
Suggett
D. J.
,
Baker
N. R.
(
2005
).
Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals?
Glob. Chang. Biol.
11
,
1
-
11
.
Stimson
J.
,
Kinzie
R. A.
(
1991
).
The temporal pattern and rate of release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus) under nitrogen-enriched and control conditions
.
J. Exp. Mar. Biol. Ecol.
153
,
63
-
74
.
Strasser
R. J.
,
Srivastava
A.
,
Govindjee
(
1995
).
Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria
.
Photochem. Photobiol.
61
,
32
-
42
.
Suggett
D. J.
,
MacIntyre
H. L.
,
Geider
R. J.
(
2004
).
Evaluation of biophysical and optical determinations of light absorption by photosystem II in phytoplankton
.
Limnol. Oceanogr. Methods
2
,
316
-
332
.
Takahashi
S.
,
Murata
N.
(
2005
).
Interruption of the Calvin-Benson cycle inhibits the repair of photosystem II from photodamage
.
Biochim. Biophys. Acta
1708
,
352
-
361
.
Takahashi
S.
,
Whitney
S.
,
Itoh
S.
,
Maruyama
T.
,
Badger
M.
(
2008
).
Heat stress causes inhibition of the de novo synthesis of antenna proteins and photobleaching in cultured Symbiodinium
.
Proc. Natl. Acad. Sci. USA
105
,
4203
-
4208
.
Takahashi
S.
,
Whitney
S. M.
,
Badger
M. R.
(
2009
).
Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species
.
Proc. Natl. Acad. Sci. USA
106
,
3237
-
3242
.
Tchernov
D.
,
Gorbunov
M. Y.
,
de Vargas
C.
,
Narayan Yadav
S.
,
Milligan
A. J.
,
Häggblom
M.
,
Falkowski
P. G.
(
2004
).
Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals
.
Proceed Proc. Natl. Acad. Sci. USA
101
,
13531
-
13535
.
Tolleter
D.
,
Seneca
F. O.
,
DeNofrio
J. C.
,
Krediet
C. J.
,
Palumbi
S. R.
,
Pringle
J. R.
,
Grossman
A. R.
(
2013
).
Coral bleaching independent of photosynthetic activity
.
Curr. Biol.
23
,
1782
-
1786
.
Ulstrup
K. E.
,
Hill
R.
,
Ralph
P. J.
(
2005
).
Photosynthetic impact of hypoxia on in hospite zooxanthellae in the scleractinian coral Pocillopora damicornis
.
Mar. Ecol. Prog. Ser.
286
,
125
-
132
.
Warner
M. E.
,
Fitt
W. K.
,
Schmidt
G. W.
(
1999
).
Damage to photosystem II in symbiotic dinoflagellates: a determinant of coral bleaching
.
Proc. Natl. Acad. Sci. USA
96
,
8007
-
8012
.
Whittingham
C. P.
(
1952
).
Inhibition of photosynthesis by cyanide
.
Nature
169
,
838
-
839
.
Wishnick
M.
,
Lane
M. D.
(
1969
).
Inhibition of ribulose diphosphate carboxylase by cyanide. Inactive ternary complex of enzyme, ribulose diphosphate, and cyanide
.
J. Biol. Chem.
244
,
55
-
59
.

Competing interests

The authors declare no competing financial interests.

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