EFFECTS OF MODERATE HEAT STRESS AND DISSOLVED INORGANIC CARBON CONCENTRATION ON PHOTOSYNTHESIS AND RESPIRATION OF SYMBIODINIUM SP. (DINOPHYCEAE) IN CULTURE AND IN SYMBIOSIS1

Authors

  • Lucy Buxton,

    1. Department of Environmental Sciences, Institute for Water and Environmental Resource Management, University of Technology Sydney, Sydney, New South Wales 2007, Australia
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  • Murray Badger,

    1. ARC Centre of Excellence in Plant Energy Biology, Molecular Plant Physiology Group, Research School of Biological Sciences, The Australian National University, Canberra, Australian Capital Territory, Australia
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  • Peter Ralph

    1. Department of Environmental Sciences, Institute for Water and Environmental Resource Management, University of Technology Sydney, Sydney, New South Wales 2007, Australia
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  • 1

    Received 11 December 2007. Accepted 13 October 2008.

Abstract

The influence of temperature and inorganic carbon (Ci) concentration on photosynthesis was examined in whole corals and samples of cultured symbiotic dinoflagellates (Symbiodinium sp.) using combined measurements from a membrane inlet mass spectrometer and chl a fluorometer. In whole corals, O2 production at 26°C was significantly limited at Ci concentrations below ambient seawater (∼2.2 mM). Further additions of Ci up to ∼10 mM caused no further stimulation of oxygenic photosynthesis. Following exposure to 30°C (2 d), net oxygen production decreased significantly in whole corals, as a result of reduced production of photosynthetically derived oxygen rather than increased host consumption. Whole corals maintained a rate of oxygen evolution around eight times lower than cultured Symbiodinium sp. at inorganic carbon concentrations <2 mM, but cultures displayed greater levels of photoinhibition following heat treatment (30°C, 2 d). Whole corals and cultured zooxanthellae differed considerably in their responses to Ci concentration and moderate heat stress, demonstrating that cultured Symbiodinium make an incongruous model for those in hospite. Reduced net oxygen evolution, in whole corals, under conditions of low Ci (<2 mM) has been interpreted in terms of possible sink limitation leading to increased nonphotochemical energy dissipation. The advantages of combined measurement of net gas exchange and fluorometry offered by this method are discussed.

Abbreviations:
ATP

adenosine triphosphate

CCM

carbon concentrating mechanisms

Ci

inorganic carbon

CZ

cultured zooxanthellae

Fv/Fm

maximum quantum yield of PSII

NPQ

nonphotochemical quenching

O2 evol,

oxygen evolution

O2 uptk

oxygen uptake

PAM

pulse amplitude modulated

RuBP

ribulose-1,5-biphosphate

WC

whole coral

ФPSII

effective quantum yield of PSII

Corals reefs are among the most biodiverse ecosystems on earth, and maintenance of a healthy reef is of major biological and economic importance, providing income for over 500 million people and supplying ecosystem services valued globally at $447 billion per year (Costanza et al. 1997, Wilkinson 2002). Corals occurring in the photic zone maintain productivity through their symbiosis with single-celled dinoflagellates, zooxanthellae, contained within the host’s gastrodermal tissue at densities between 1 million and 5 million zooxanthellae cells per cm2. Zooxanthellae supply the cnidarian host with up to 95% of their photosynthetic production (Muscatine 1990). In turn, the host provides the zooxanthellae with respiratory-derived CO2 and nutrients. This close symbiotic relationship is believed to be the key to the success of scleratinian reef-building corals in oligotrophic waters (Muller-Parker and D’Elia 1997).

Upon entering the aqueous environment, atmospheric CO2 reacts with water, forming carbonic acid, bicarbonate, and carbonate, collectively known as the dissolved inorganic carbon (DIC) pool. To support the high level of primary productivity, inorganic carbon (Ci) acquisition, Ci transport, and CO2-concentrating mechanisms (CCM) are fundamental processes identified in free-living algae that ultimately provide RUBISCO, the enzyme responsible for carbon fixation during photosynthesis, with an intracellular source of CO2. Unlike free-living microalgae, those that exist in symbiosis do not have direct access to the relatively constant seawater DIC pool (2.2 mM). Zooxanthellae located in the metabolically active host cytoplasm (within the endodermal cell) are able to utilize CO2 produced by host respiration (Muller-Parker and D’Elia 1997). Additionally, corals have been shown to posses active carbon transport mechanisms that preferentially take up external HCO3 as a substrate for photosynthesis (Al Moghrabi et al. 1996, Goiran et al. 1996). However, their physical isolation leaves the symbiont dependent upon the Ci transport mechanisms of the host to provide sufficient Ci for photosynthesis.

In the last 100 years, human activities have contributed considerably to atmospheric CO2 emissions. CO2 is already recognized as a major greenhouse gas, and clear causal links have now been drawn between atmospheric CO2 and increases in global mean temperatures (Brown 1997, Walther et al. 2002). Rising sea-surface temperatures are known to have negative impacts on coral reef ecosystems (Jokiel and Coles 1990, Gattuso et al. 1996, Hoegh-Guldberg 1999). Mean sea-surface temperatures have increased 1°C–2°C above their average summer maxima, and these changes have been linked to loss of symbiotic zooxanthellae cells from the cnidarian host, or a decrease in chl pigment, commonly known as coral bleaching.

However, high atmospheric CO2 levels have recently been shown to cause chemical changes in oceanic surface waters (Caldeira and Wickett 2003, Raven et al. 2005), including increased concentrations of dissolved CO2, decreased carbonate ion concentration (CO32−) (Zeebe and Wolf-Gladrow 2001), with an associated pH decrease of up to 0.25 units by 2100 (IPCC 2001, Raven et al. 2005). Impacts of anthropogenic CO2 on marine DIC are in addition to that of rising sea-surface temperature alone, and the negative synergistic effects of these two factors on oceanic flora and fauna will be substantial (Feely et al. 2004, Raven et al. 2005, Miles et al. 2007). Among the systems likely to be impacted by these biochemical changes, coral reef ecosystems have been identified as especially vulnerable because of their complex carbon budgets linked to photosynthesis, calcification, and respiration (Reynaud et al. 2003, Raven et al. 2005).

In response to this global issue, coral research has investigated the effects of increased pCO2 on calcification (Riebesell 2004, Orr et al. 2005, Kleypas et al. 2006), and the effects of temperature on coral primary productivity (Iglesias-Prieto et al. 1992, Goulet et al. 2005). To our knowledge, no studies have so far focused on the combined effects of DIC and temperature on coral photosynthesis. While it has been generally assumed that 2.2 mM inorganic carbon occurring in seawater is sufficient to saturate coral photosynthesis (Burris et al. 1983, Goiran et al. 1996, Gattuso et al. 1999), studies on the effects of varied seawater DIC concentration have had conflicting results (Burris et al. 1983, Herfort et al. 2008), and definite conclusions on the combined effects of increasing DIC concentrations and temperature are confounded by variability across studies and species.

The interactions of temperature and Ci concentration remain poorly understood because physiological responses have been examined by manipulating one parameter at a time. This may have been exacerbated by the fact that previous investigations have relied on either exogenous oxygen evolution rates or measurement of effective quantum yield of PSII as an indicator of photosynthetic functionality. While both methodologies are acknowledged indicators of photosynthetic functionality, the complexities of oxygen exchange between the host and algal symbiont in the coral symbiosis may have contributed to some observed inconsistencies between studies. Here, we present results using combined measurement photosynthesis determined by O2 evolution, O2 uptake, and chl a fluorometry of the branching coral Pocillopora damicornis exposed to a range of Ci concentrations (0.1–15 mM) at 26°C and 30°C. Used for the first time on corals, membrane inlet mass spectrometry (MIMS) allows the continuous measurement of gas exchange (O2 evol and O2 uptk) and chl a florescence to occur noninvasively. To examine the physiological variation of respiration (O2 uptk) and photosynthesis (O2 evol) of Symbiodinium sp. in and out of symbiosis, two tissue models were used: whole coral pieces with zooxanthellae in hospite and those free-living in culture (CZ).

Materials and methods

Coral and Symbiodinium sp. growth conditions.  Colonies of the branching coral P. damicornis were collected from Heron Island lagoon (<2 m deep) (152°06′ E, 20°29′ S) (Great Barrier Reef Marine Park Authority collection permit number G05/14083.1) and transported to the University of Technology, Sydney, Australia. Colonies were acclimated for 2 months in a 500 L recirculating aquarium using artificial seawater (Red Sea Coral Pro sea salt mix made up in reverse osmosis water [carbonate 140 ppm, salinity 33 ppm] 26 ± 1°C and 250 μmol photons · m−2 · s−1). A 2 cm3 coral branch was removed from one of each of five separate P. damicornis colonies and maintained at the Australian National University, Canberra, in a 20 L aquarium with the same water chemistry 2 weeks prior to the experiments. Genetic analysis of zooxanthellae isolated from P. damicornis collected from Heron Island by our laboratory has identified the population as clade C1 (R. Hill, K. Ulstrup, and P. Ralph, unpublished data), which is in agreement with other studies (Magalon et al. 2007).

Populations of cultured Symbiodinium sp. CS-156 were originally isolated from the coral Montipora verrucosa and identified as being clade C (CSIRO Collection of Living Microalgae) (Carlos et al. 1999, Baillie et al. 2000, Hill 2008). Cells were grown in f/2 culture media (Guillard and Ryther 1962) made up in artificial seawater (as above) and maintained under an irradiance of 100 μmol photons · m−2 · s−1 at 26°C. To remove the influence of culture age on physiological responses, all samples were taken from cultures in the exponential growth phase (21 d of growth). Cells were concentrated by centrifugation (5 min at 280g; model Z200A; Hermke Labortechnik GmbH, Wehingen, Germany) and used immediately.

For experiments conducted at elevated temperature (30°C), aquarium temperatures were ramped 1°C · 12 h−1 over 2 d, then left at 30°C for 2 d before use in experiments. A temperature of 30°C was selected to provide thermal stress without inducing bleaching. Temperature ramping of cultures was performed in the same manner but in a water bath (AR620 Aqua One; Ingleburn, New South Wales, Australia). Irradiance was maintained at 250 and 100 μmol photons · m−2 · s−1 for whole corals and cultures, respectively.

Measurement of gas exchange.  Simultaneous measurements of oxygen uptake and evolution as well as pulse-amplitude-modulated (PAM) chl a fluorescence were performed using a temperature-controlled stirred chamber attached to a membrane inlet mass spectrometer (Micromass IsoPrime EA, Manchester, UK) as previously described (Badger and Andrews 1982, Badger et al. 1985, Sultemeyer et al. 1995, Franklin and Badger 2001) allowing measurement of 16O2 (mass 32), 18O2 (mass 36), and CO2 (mass 44). Actinic light was supplied from a halogen source (KL 1500; Schott, Mainz, Germany) through the top of the chamber, which was also fitted with a fiber optic allowing simultaneous measurement of chl fluorescence (PAM 101/103; Walz GmbH, Effeltrich, Germany). To confirm that actinic light levels stimulated sufficient photosynthetic activity in both whole corals and cultured zooxanthellae for measurement with both instruments, irradiance curves (0–2,000 μmol photons · m−2 · s−1) were conducted prior to commencement of experimental assays (Ralph and Gademann 2005). Net O2 evolution and ФPSII was measured after stabilizing at a given irradiance (5 min). Experimental light intensity used was selected at the irradiance saturation point, Ek, where photosynthetic rate (O2 evol) reached a plateau and before increased irradiance caused a decrease in ФPSII.

Assays were conducted in 6 mL nitrogen-sparged incubation buffer (20 mM bis-Tris propane [BTP] buffered artificial seawater [Red Sea Coral Pro sea salt mix made up in reverse osmosis water], herein abbreviated to 20 mM BTP-ASW), pH 8.0, flushed with nitrogen >4 h in a thermostatted cuvette (26°C and 30°C). Before commencement of experiments, 20 mM BTP was incubated with coral tissue, which had no effect on coral photosynthesis or respiration (L. Buxton, M. Badger, and P. Ralph, unpublished data). Cultured Symbiodinium sp. (CS-156) was concentrated by centrifugation and resuspended in 1 mL sparged 20 mM BTP-ASW (pH 8.0), to achieve a chl a concentration between 0.8 and 1.5 μg chl a · mL−1 (conversion factor nmol O2 · μg chl a−1 · mL−1 · min−1 to nmol O2 · cell−1 · mL−1 · min−1 = 2.89 × 106 for whole coral samples and 6.72 × 106 for Symbiodinium sp.). Calculation and standardization of rates to chl content are described by Franklin and Badger (2001). For whole coral assays, subcolony fragments were washed twice with sparged incubation buffer immediately before use and then supported in the chamber on wire mesh over a magnetic stir bar. The 18O2 uptake was measured through the introduction of an 18O2 gas bubble to the stirred medium injected via syringe through a port in the closed MIMS chamber. The excess 18O2 bubble was removed when the total oxygen concentration reached 250 μM. O2-exchange and fluorescence measurements proceeded as follows. Samples in the low Ci (<0.2 mM) 18O2–enriched medium were dark adapted for 5 min and then exposed to a saturating pulse to obtain minimum and maximum fluorescence (Fo, Fm), respectively (Schreiber 2004). At the commencement of the assay, actinic light was supplied for 5 min (WC 250 μmol photons · m−2 · s−1, CZ 100 μmol photons · m−2 · s−1) before applying another saturating pulse to obtain the light-adapted quantum efficiency of PSII (ΦPSII), which was calculated as (Fm′–Fs)/Fm′, where Fm′ is the maximal fluorescence yield during irradiation with actinic light, and Fs is the steady state of fluorescence during irradiation (Genty et al. 1989). Prescribed concentration of Ci was added using either 0.1 or 1 M NaHCO3 stocks using a syringe through the cuvette port. Fluorescence and O2 exchange were recorded until the rate of photosynthesis became stable (up to 8 min). Measurements were initiated at ∼0.1 mM Ci, before further bicarbonate additions were added sequentially to the same sample to achieve concentration from 0.1 to 15 mM Ci. The Ci level in the cuvette at each stable point was estimated directly from the CO2 level in the cuvette (mass 44). CO2 was used as a direct measure of Ci by calibration of the CO2/Ci ratio by injection of known amounts of NaHCO3 into the reaction buffer. There was little disequilibrium between CO2 and HCO3 during CO2 fixation, and thus CO2 gave an accurate estimate of total Ci. The effective quantum yield was measured after each Ci addition.

Analysis of photosynthetic pigments.  On completion of each experiment, whole coral pieces were removed from the MIMS cuvette, and the zooxanthellae isolated according to methods of Masuda et al. (1993). Briefly, coral pieces were gently brushed with a toothbrush in 15 mL 20 mM BTP-ASW. The mixture was filtered through 20 μm mesh, centrifuged at 280g, 26°C for 5 min, and the pellet was washed with 8 mL 20 mM BTP-ASW. The homogenate was centrifuged again as before, resuspended in a final volume of 3 mL of 20 mM BTP-ASW, and used immediately in the experiment. The supernatant was removed, and the final pellet resuspended in 5 mL 90% acetone for 20 h at 4°C. For cultured zooxanthellae, the whole cell suspension was removed from the MIMS cuvette, spun at 300g, and resuspended in 5 mL 90% acetone (20 h at 4°C). After removal of cells by centrifugation (5 min at 1,000g), an absorption spectrum (400 to 800 nm) of the acetone supernatant was measured using a spectrophotometer (Cary 50 Bio UV-Vis Spectrometer; Varian Inc., Palo Alto, CA, USA). Chl a content was calculated according to the equations of Jeffrey and Humphrey (1975).

Statistical analysis.  For each temperature treatment (26°C, 30°C, = 5 for each treatment), oxygen evolution and consumption rates were compared using analysis of covariance (ANCOVA). In all cases, < 0.05 was accepted as indicating a significant difference between Ci concentrations and temperature. If no covariance was detected, analysis of variance (ANOVA) was used to define significant differences caused by either Ci treatment (low <1 mM, ambient 1–3 mM, high >3 mM), or temperature (26°C, 30°C). Relative maximum rates of oxygen evolution or uptake were calculated from the rate expressed in the control (26°C at 2 mM Ci).

Results

Figure 1 shows that Ci concentration has a significant effect on the rate of oxygen evolution at 26°C (= 0.037) in whole corals, where photosynthesis increases by 40% in the presence of Ci between 1 and 4 mM (2.2–4.0 nmol O2 · μg chl a−1 · mL−1 · min−1), at which point, the rate saturates and no further change was observed with carbon addition (6–15 mM Ci). Nonphotochemical quenching rapidly decreased with addition of Ci at 26°C (Fig. 2); NPQ was 0.54 at 0.09 mM Ci and 0.05 at 4 mM Ci (< 0.005). Although there is also a positive trend in ΦPSII on addition of Ci up to 2 mM (Fig. 2), this is not significant. Elevated temperature had a significant effect on the rate of O2 evol (< 0.005) (Fig. 1); at 30°C the saturated rate of O2 evol decreased by 50% when compared to 26°C (4.13 compared to 2.16 nmol O2 · μg chl a−1 · mL−1 · min−1 at 2 mM Ci, 26°C and 30°C, respectively).

Figure 1.

 Effect of temperature and Ci concentration on net oxygen production (O2 evol, O2 uptk) in the whole coral Pocillopora damicornis at 26°C and 30°C. Data show means (= 5); error bars represent standard error. Dotted vertical line at 2.2 mM inorganic carbon represents concentration of seawater. The dashed horizontal line represents O2 uptk in the dark (−3.04 ± 0.63 and −2.67 ± 0.33 nmol O2 · μg chl a−1 · mL−1 · min−1, at 26°C and 30°C, respectively).

Figure 2.

 Effect of temperature and Ci concentration on photosynthetic efficiency and nonphotochemical quenching (NPQ) in the whole coral Pocillopora damicornis at 26°C and 30°C. Data show means (= 5); error bars represent standard error. Dotted vertical line at 2.2 mM inorganic carbon represents concentration of seawater.

There was no significant effect of temperature on ΦPSII (Fig. 2). However, NPQ at 2 mM Ci was higher at 30°C than 26°C. While not significant (= 0.063), addition of Ci above 2 mM caused a further increase in ФPSII and a decrease in NPQ at 30°C. Neither Ci concentrations (= 0.361) nor temperature (= 0.097) had a significant effect on the rate of O2 uptk (Fig. 1). In whole corals, PSII electron flow was maintained between 71.1 and 76.9 μmol · m−2 · s−1 at 26°C and decreased by 15% (60.2–72.9 μmol · m−2 · s−1) at 30°C (Fig. 3a).

Figure 3.

 (A and B) Rate of PSII electron flow as a function of oxygen evolution in the whole coral Pocillopora damicornis and cultured Symbiodinium sp., respectively, at 26°C and 30°C. Data show mean (= 5); error bars represent standard error.

Under carbon-saturating conditions (>2.2 mM) at 26°C, whole corals maintained rates of oxygen evolution some eight times lower than free-living Symbiodinium sp. (Figs. 1 and 4) (∼4 compared to ∼32 nmol O2 · μg chl a−1 · mL−1 · min−1 at 2 mM Ci for WC and CZ, respectively). Exposure to moderate heat stress caused a 70% reduction in O2 evol when compared to 26°C in cultured Symbiodinium sp. (Fig. 4) (16.53 compared to 4.95 nmol O2 · μg chl a−1 · mL−1 · min−1 at 2 mM Ci, 26°C and 30°C, respectively, < 0.005). Similarly, ΦPSII decreased at 30°C compared to the control (0.69 at 26°C, 0.43 at 30°C at 2 mM) (Fig. 5). PSII electron flow was similar in cultured Symbiodinium sp. at 26°C compared to whole corals (70.7–72.9 compared to 71.1–76.9 μmol · m−2 · s−1). However, following mild heat stress, it was significantly reduced in cultures (39.4–49.3 μmol · m−2 · s−1, < 0.005) (Fig. 3, a, b).

Figure 4.

 Effect of temperature and Ci concentration on net oxygen production (O2 evol, O2 uptk) in cultured Symbiodinium CS-156 at 26°C and 30°C. Data show means (= 5); error bars represent standard error. Dotted vertical line at 2.2 mM inorganic carbon represents concentration of seawater. The dashed horizontal line represents O2 uptk in the dark (−19.51 ± 8.79 and −9.90 ± 0.84 nmol Oμg chl a−1·mL−1·min−1, at 26°C and 30°C, respectively).

Figure 5.

 Effect of temperature and Ci concentration on photosynthetic efficiency and nonphotochemical quenching (NPQ) in cultured Symbiodinium CS-156 at 26°C and 30°C. Data show means (= 5); error bars represent standard error. Dotted vertical line at 2.2 mM inorganic carbon represents concentration of seawater.

Discussion

Seawater concentrations of DIC (∼2.2 mM) have long been considered saturating for photosynthesis; however, there has been growing speculation that DIC supply may be a limiting factor for a number of marine phototrophs (Raven 1993, Beer and Rehnberg 1997, Mercado et al. 2001, 2003, Herfort et al. 2008). Although some investigations have speculated about carbon limitation of photosynthesis in corals (Muscatine et al. 1989, Weis et al. 1989, Lesser et al. 1994), the results need to be considered with caution because, as stated above, using exogenous oxygen evolution rates or effective quantum yield of PSII alone does not reflect the complexities of oxygen exchange between the host and algal symbiont in the coral symbiosis. Anticipated increases in atmospheric carbon dioxide concentrations will certainly affect carbon assimilation and metabolism to some degree in marine autotrophs. Efficient photosynthesis under these conditions will require maintaining a balance between absorbed light energy, heat dissipation, and photochemistry. Results here show that under carbon-limiting conditions (<2 mM, 26°C), whole corals exhibited significant depression in net O2 production, accompanied by increased levels of NPQ. The reduction in net O2 production was caused by a decrease in photosynthetically derived oxygen (O2 evol) rather than increased host consumption (O2 uptk). Increased temperature did not significantly affect host respiration as seen by O2 uptk in Figure 1. Furthermore, the rate of O2 uptk in the dark in whole corals is significantly greater than under irradiance, demonstrating that algal-derived oxygen production contributes to host respiratory demand under irradiance. Results show that Ci concentration has a significant negative effect on the rate of PSII electron flow and O2 evol (Fig. 3, a, b). The relationship between electron flow and O2 evol is temperature dependent, and sensitivity varies between cultured Symbiodinium sp. and those in hospite. Additionally, there is a synergistic effect of temperature and Ci on the rate of electron transport in both tissue models (cultured Symbiodinium sp. or in hospite), and results show a greater sensitivity to subsaturating Ci at high temperatures. As the terminal electron acceptor of photosynthesis, carbon dioxide availability partially controls the reduction state of the electron transport chain (Durchan et al. 2001). Under subsaturating pCO2 conditions, the Calvin cycle is down-regulated, causing overreduction of the electron transport chain, therefore encouraging activation of NPQ (Durchan et al. 2001). Reduced linear electron transport can induce cyclical electron transport, therefore reducing the ATP pool (Ergorova and Bukhov 2006). Given that thermal stress on coral results in a similar reduction in Calvin cycle activity (Jones et al. 1998), we suggest that under subsaturating pCO2, NPQ pathways act to dissipate unutilized energy, reducing electron flow and causing an overall decrease in O2 evol, as seen in Figures 1 and 2.

O2 evol increased rapidly with addition of Ci up to 2 mM, accompanied by a rapid decrease in NPQ, suggesting that increased ATP consumption via photochemical pathways can dissipate the transthylakoid proton gradients allowing NPQ to decrease. Additions of Ci above 2 mM did not cause any further increase in net O2 production, indicating that photosynthesis in corals is saturated at ∼2 mM Ci in agreement with other studies (Burris et al. 1983, Goiran et al. 1996). High ФPSII values (0.6–0.7) are not unusual in healthy P. damicornis samples (Ralph et al. 2005), yet this is also indicative of samples being exposed to subsaturating irradiance. Should the experiment have been performed under higher irradiance, the effects of Ci limitation on O2 evol may have been exacerbated and enhanced the levels of NPQ.

Elevated temperature is known to cause photoinhibition in corals, characterized by a decrease in maximal and effective quantum yields of PSII (Lesser 1997, Warner et al. 1999, Hill et al. 2005). Indeed, the overall levels of net O2 production were significantly lower, and NPQ higher, in whole corals at 30°C. Results show that the rate of O2 evol is more sensitive to Ci concentration than PSII electron flow at 26°C (Fig. 3, a, b). The discrepancy between O2 evol and ФPSII (Figs. 1 and 2) results may be associated with fluorometry measurements having less sensitivity to subtle changes in ATP and NADPH production as a result of heat stress, which highlights the advantages of MIMS combined measurements. Indeed, changes to the activation state of RUBISCO as a result of heat exposure are known to precede and show greater sensitivity than measurements of maximum quantum yield of PSII (Crafts-Brandner and Law 2000). MIMS, therefore, allows a unique insight into the effects of host and zooxanthellae metabolism. The sensitivity of this nondestructive technique demonstrates the ability to examine host versus algal responses to temperature and Ci concentration, and the advantage of O2 evol rates and fluorometery measurements alone.

The thermal range of cultured zooxanthellae has been suggested to lie between 20°C and 30°C, over which algal respiration rates remained constant while photosynthesis increased, as measured by oxygen evolution (Iglesias-Prieto et al. 1992). Contrary to these results, our experiments show significant depression in both algal respiration (O2 uptk) (P = <0.005) and photosynthesis (O2 evol and ФPSII) (= 0.001) from 26°C to 30°C (Figs. 4 and 5). The temperature-induced change in O2 evol rate in cultured zooxanthellae was greater than observed in either whole corals, indicating that cultured zooxanthellae are more sensitive to 30°C at 100 μmol photons · m−2 · s−1 than those in hospite exposed to 250 μmol photons · m−2 · s−1. This finding may be because host tissue may provide a more stable environment for zooxanthellae, absorbing some light or heat from the surrounding seawater and dissipating it through the tissue and skeleton, allowing photosynthesis to occur at higher temperatures than those experienced by their free-living counterparts (Dunlap and Shick 1998, Salih et al. 2000). The cnidarian host is also known to provide enzymatic defenses in response to solar ultraviolet radiation or elevated temperature, protecting the algal symbionts from oxidative stress (Shick et al. 1995, Lesser 1997, Downs et al. 2002, Trapido-Rosenthal et al. 2005). Zooxanthellae in hospite are thought to utilize CO2 (aq) and HCO3, while cultured Symbiodinium sp. are shown to preferentially utilize HCO3 (Goiran et al. 1996), which may lead to differences in substrate competition between Symbiodinium sp. in and out of symbiosis. However, despite a greater relative decrease in O2 evol following exposure to heat stress in free-living Symbiodinium sp., their final rate of O2 evol under carbon-saturating conditions at 30°C was still greater than that of whole corals at 26°C (∼13 compared to ∼4 nmol O2 · μg chl a−1 · mL−1 · min−1 at 2 mM Ci for CZ at 30°C and WC at 26°C, respectively). Other studies have recorded similar levels of O2 evol by algae (Badger et al. 2000, Woodger et al. 2003), a further indication that photosynthesis by Symbiodinium sp. in hospite is tightly coupled to the ability of the host to supply carbon for photosynthesis and that these mechanisms may be impaired by temperature.

Investigations were performed on whole corals and free-living Symbiodinium sp. to draw environmentally relevant conclusions about the combined effects of Ci concentration and temperature on Symbiodinium sp. in and out of symbiosis. However, it should be remembered that direct comparisons of carbon uptake in cultured zooxanthellae to those in hospite are not strictly comparable. The volume of solution surrounding cells in an aqueous environment is large with respect to those in hospite. Therefore, the diffusional distance of Ci is considerably larger and leads to increased resistance of CO2 uptake in free-living cells under low Ci concentrations (Espie and Colman 1987). Despite this, cultured zooxanthellae were able to maintain a rate of O2 evol around eight times greater than whole corals, even at low Ci concentrations (<2 mM) (Fig. 4). This observation would suggest that, unlike whole corals, photosynthesis in cultured zooxanthellae is carbon saturated even at low carbon concentrations. As shown in the cyanobacteria Synechococcus sp., zooxanthellae grown in culture may have the capacity to up-regulate their CCM to efficiently utilize all forms of inorganic carbon in the seawater pool (CO2(aq), HCO3 and CO3−2) (Woodger et al. 2005), factors possibly contributing to increased photosynthetic efficiency and increased yield of O2 evol observed here. Additionally, the rate of photosynthesis of in hospite cells may be tightly regulated by host factors, such as metabolically available dissolved O2/CO2 from the host, nutrient availability, and light (Sutton and Hoegh-Guldberg 1990, Gates et al. 1999).

Coral symbionts are critical components of coral reef ecosystems, and the relationship between corals and zooxanthellae is an area of considerable interest in coral biology research. We acknowledge that the different responses of cultures in vivo and in vitro to Ci and temperature seen here may be a reflection of either subcladal specific physiological or genetic variation (Robison and Warner 2006, Sampayo et al. 2008). Despite this, cultured Symbiodinium sp. cells are widely used in laboratory experiments, and results are commonly inferred as a model for those in hospite (Iglesias-Prieto et al. 1992, Rodriguez-Roman and Iglesias-Prieto 2005). The results presented here show that extrapolation between cell cultures and those in hospite should be treated with care. Measurement of net oxygen evolution in cultured Symbiodinium sp. is distinct from whole corals by orders of magnitude, and their sensitivity to inorganic carbon and temperature are markedly different, making them unsuitable models for whole coral symbioses.

In conclusion, this investigation suggests that the growth and living environment (culture or in hospite) of zooxanthellae showed a significant effect on their response to both low Ci conditions (those below ambient seawater, 2.2 mM) and heat stress. Thermal stress had less effect on zooxanthellae in hospite than those in culture. Conversely, zooxanthellae in hospite exhibited greater sensitivity to limited Ci compared to free-living cells. Exposure to Ci concentrations above ambient seawater caused no further increase in photosynthetic efficiency or net O2 production in either cultured Symbiodinium sp. or those in symbiosis, supporting the theory that coral photosynthesis is carbon saturated at present seawater concentrations.

Acknowledgments

The authors would like to thank Dr. Martina Doblin for editorial comments. This research was made possible through the financial support of the Hermon Slade Foundation, the Australian Research Council, and performed under GBRMPA permit number G05/14083.1.

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