Inhibition of Calvin–Benson cycle suppresses the repair of photosystem II in Symbiodinium: implications for coral bleaching

Inhibition of Calvin–Benson cycle (CBC) activity by thermal stress has been hypothesized to cause photoinhibition of photosystem II (PSII) in zooxanthellae of reef-building corals and consequently lead to bleaching. This study tests whether the interruption of CBC by glycolaldehyde (GA) leads to photoinhibition and subsequent coral bleaching in Stylophora pistillata. When S. pistillata was incubated with GA, the O₂ evolution rate declined in a dose-dependent manner and the extent of photoinhibition, reflected by a decreased maximum quantum yield of PSII (F _v/F _m), was enhanced. The effect of GA on photoinhibition was similar to that of chloramphenicol (CAP), an inhibitor of protein synthesis in chloroplasts. When S. pistillata was incubated in weak light following a high-light-induced photoinhibitory treatment, the recovery of PSII from photoinhibition was suppressed in a similar manner to both GA- and CAP-treated samples. After incubation in moderate light at 26°C, S. pistillata showed a bleaching response only in presence of GA. These results suggest that coral bleaching-like responses are caused by interruption of the CBC activity in S. pistillata and are associated with accelerated photoinhibition through suppression of the protein synthesis-dependent repair of PSII but not to an increase in photodamage to PSII.     

Differential susceptibility to oxidative stress of two scleractinian corals: antioxidant functioning of mycosporine-glycine

This study examined the importance of mycosporine-glycine (Myc-Gly) as a functional antioxidant in the thermal-stress susceptibility of two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata. Photochemical efficiency of PSII (F_v/F_m), activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and composition and abundance of mycosporine-like amino acids (MAAs) in the coral tissue and in symbiotic zooxanthellae were analyzed during 12-h exposure to high temperature (33 °C). After 6- and 12-h exposures at 33 °C, S. pistillata showed a significantly more pronounced decline in F_v/F_m compared to P. ryukyuensis. A 6-h exposure at 33 °C induced a significant increase in the activities of SOD and CAT in both host and zooxanthellae components of S. pistillata while in P. ryukyuensis a significant increase was observed only in the CAT activity of zooxanthellae. After 12-h exposure, the SOD activity of P. ryukyuensis was unaffected in the coral tissue but slightly increased in zooxanthellae, whereas the CAT activity in the coral tissue showed a 2.5-fold increase. The total activity of antioxidant enzymes was significantly higher in S. pistillata than in P. ryukyuensis, suggesting that P. ryukyuensis is less sensitive to oxidative stress than S. pistillata. This differential susceptibility of the corals is consistent with a 20-fold higher initial concentration of Myc-Gly in P. ryukyuensis compared to S. pistillata. In the coral tissue and zooxanthellae of both species investigated, the first 6 h of exposure to thermal stress induced a pronounced reduction in the abundance of Myc-Gly but not in other MAAs. When exposure was prolonged to 12 h, the Myc-Gly pool continued to decrease in P. ryukyuensis and was completely depleted in S. pistillata. The delay in the onset of oxidative stress in P. ryukyuensis and the dramatic increase in the activities of the antioxidant enzymes in S. pistillata, which contains low concentrations of Myc-Gly suggest that Myc-Gly provides rapid protection against oxidative stress before the antioxidant enzymes are induced. These findings strongly suggest that Myc-Gly is functioning as a biological antioxidant in the coral tissue and zooxanthellae and demonstrate its importance in the survival of reef-building corals under thermal stress.     

Thermal bleaching induced changes in photosystem II function not reflected by changes in photosystem II protein content of Stylophora pistillata

Scleractinian corals exist in a symbiosis with marine dinoflagellates of the genus Symbiodinium that is easily disrupted by changes in the external environment. Increasing seawater temperatures cause loss of pigments and expulsion of the symbionts from the host in a process known as coral bleaching; though, the exact mechanism and trigger of this process has yet to be elucidated. We exposed nubbins of the coral Stylophora pistillata to bleaching temperatures over a period of 14 daylight hours. Fifty-nine percent of the symbiont population was expelled over the course of this short-term treatment. Maximum quantum yield (F _V/F _M) of photosystem (PS) II for the in hospite symbiont population did not change significantly over the treatment period, but there was a significant decline in the quantity of PSII core proteins (PsbA and PsbD) at the onset of the experimental increase in temperature. F _V/F _M from populations of expelled symbionts dropped sharply over the first 6 h of temperature treatment, and then toward the end of the experiment, it increased to an F _V/F _M value similar to that of the in hospite population. This suggests that the symbionts were likely damaged prior to expulsion from the host, and the most damaged symbionts were expelled earlier in the bleaching. The quantity of PSII core proteins, PsbA and PsbD, per cell was significantly higher in the expelled symbionts than in the remaining in hospite population over 6–10 h of temperature treatment. We attribute this to a buildup of inactive PSII reaction centers, likely caused by a breakdown in the PSII repair cycle. Thus, thermal bleaching of the coral S. pistillata induces changes in PSII content that do not follow the pattern that would be expected based on the results of PSII function.     

Comparison of stress susceptibility of in hospite and isolated zooxanthellae among five coral species

Coral species in a similar habitat often show different bleaching susceptibilities. It is not understood which partner of coral-zooxanthellae complexes is responsible for differential stress susceptibility. Stress susceptibilities of in hospite and isolated zooxanthellae from five species of corals collected from shallow water in Okinawa were compared. To estimate stress susceptibility, we measured the maximum quantum yields (F_v/F_m) of in hospite and isolated zooxanthellae after 3-h exposure to either 28 or 34 °C at various light intensities and their recovery after 12 h under dim light at 26 °C. Significant reduction in photochemical efficiency (F_v/F_m) of photosystem II (PSII) was observed in in hospite zooxanthellae exposed to high light intensity (1000 μmol quanta m⁻² s⁻¹), while PSII activity of isolated zooxanthellae decreased significantly even at a lower light intensity (70 μmol quanta m⁻² s⁻¹). The recovery of the PSII activity after 12 h was incomplete in both in hospite and isolated zooxanthellae, indicating the presence of chronic photoinhibition. The stress susceptibility of isolated zooxanthellae was more variable among species than in hospite zooxanthellae. The order of stress susceptibility among the five coral species was different between in hospite and isolated zooxanthellae. The present results suggest that the host plays a significant role in determining bleaching susceptibility of corals, though zooxanthellae from different host have different stress susceptibilities.     

Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals?

The bleaching of corals in response to increases in temperature has resulted in significant coral reef degradation in many tropical marine ecosystems. This bleaching has frequently been attributed to photoinhibition of photosynthetic electron transport and the consequent photodamage to photosystem II (PSII) and the production of damaging reactive oxygen species (ROS) in the zooxanthellae (Symbiodinium spp.). However, these events may be because of perturbations of other processes occurring within the zooxanthellae or the host cells, and consequently constitute only secondary responses to temperature increase. The processes involved with the onset of photoinhibition of electron transport, photodamage to PSII and pigment bleaching in coral zooxanthellae are reviewed. Consideration is given to how increases in temperature might lead to perturbations of metabolic processes in the zooxanthellae and/or their host cells, which could trigger events leading to bleaching. It is concluded that production of ROS by the thylakoid photosynthetic apparatus in the zooxanthellae plays a major role in the onset of bleaching resulting from photoinhibition of photosynthesis, although it is not clear which particular ROS are involved. It is suggested that hydrogen peroxide generated in the zooxanthellae may have a signalling role in triggering the mechanisms that result in expulsion of zooxanthellae from corals.     

Exposure to solar radiation increases damage to both host tissues and algal symbionts of corals during thermal stress

Elevated seawater temperatures have long been accepted as the principal stressor causing the loss of symbiotic algae in corals and other invertebrates with algal symbionts (i.e., bleaching). A secondary factor associated with coral bleaching is solar irradiance, both its visible (PAR: 400–700 nm) and ultraviolet (UVR: 290–400 nm) portions of the spectrum. Here we examined the synergistic role of solar radiation on thermally induced stress and subsequent bleaching in a common Caribbean coral, Montastraea faveolata. Active fluorescent measurements show that steady-state quantum yields of photosystem II (PSII) fluorescence in the zooxanthellae are markedly depressed when exposed to high solar radiation and elevated temperatures, and the concentration of D1 protein is significantly lower in high light when compared to low light treatments under the same thermal stress. Both photosynthetic pigments and mycosporine-like amino acids (MAAs) are also depressed after experimental exposure to high solar radiation and thermal stress. Host DNA damage is exacerbated under high light conditions and is correlated with the expression of the cell cycle gene p 53, a cellular gatekeeper that modulates the fate of damaged cells between DNA repair processes and apoptotic pathways. These markers of cellular stress in the host and zooxanthellae have in common their response to the enhanced production of reactive oxygen species during exposure to high irradiances of solar radiation and elevated temperatures. Taking these results and previously published data into consideration, we conclude that thermal stress during exposure to high irradiances of solar radiation, or irradiances higher than the current photoacclimatization state, causes damage to both photochemistry and carbon fixation at the same time in zooxanthellae, while DNA damage, apoptosis, or necrosis are occurring in the host tissues of symbiotic cnidarians.Abbreviations _PSII Functional absorption cross-section for PSII - F_o, F_m Minimum and maximum yields of chlorophyll a fluorescence measured after dark acclimation (relative units) - F_v Variable fluorescence after dark acclimation (=F_m–F_o), dimensionless - F_v/F_m Maximum quantum yield of photochemistry in PSII measured after dark acclimation, dimensionless - F, F_m Steady-state and maximum yields of chlorophyll a fluorescence measured under ambient light (relative units) - F/F_m Quantum yield of photochemistry in PSII measured at steady state under ambient light Communicated by R.C. Carpenter     

Response of two species of Indo-Pacific corals, Porites cylindrica and Stylophora pistillata, to short-term thermal stress: The host does matter in determining the tolerance of corals to bleaching

The role of both host and dinoflagellate symbionts was investigated in the response of reef-building corals to thermal stress in the light. Replicate coral nubbins of Stylophora pistillata and Porites cylindrica from the GBR were exposed to either 28 °C (control) or 32 °C for 5 days before being returned to an ambient reef temperature (28 °C). S. pistillata was found to contain either Symbiodinium genotype C1 or C8a, while P. cylindrica had type C15 based on ITS genotyping. Analysis of the quantum yield of photosystem (PS) II fluorescence of the symbionts in P. cylindrica showed that light-induced excitation pressure on the C15 Symbiodinium was significantly less, and the steady state quantum yield of PSII fluorescence at noon (ΔF/Fm′) greater, than that measured in C1/C8a Symbiodinium sp. from S. pistillata. Immunoblots of the PS II D1 protein were significantly lower in Symbiodinium from S. pistillata compared to those in P. cylindrica after exposure to thermal stress. The biochemical markers, heat-stress protein (HSP) 70 and superoxide dismutase (SOD), were significantly greater in P. cylindrica before the experiment, and both species of coral increased their biosynthesis of HSP 70 and SOD when exposed to thermal stress. Concentrations of MAAs, glycerol, and lipids were not significantly affected by thermal stress in these experiments, but DNA damage was greater in heat-stressed S. pistillata compared to P. cylindrica. There was minimal coral mucus, which accounts for up to half of the total energy budget of a coral and provides the first layer of defense for invading microbes, produced by S. pistillata after heat stress compared to P. cylindrica. It is concluded that P. cylindrica contains a heat resistant C15 Symbiodinium and critical host proteins are present at higher concentrations than observed for S. pistillata, the combination of which provides greater protection from bleaching conditions of high temperature in the light.     

Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Photosystem II recovery in the presence and absence of chloroplast protein repair in the symbionts of corals exposed to bleaching conditions

Increased seawater temperature causes photoinhibition due to accumulation of photodamaged photosystem II (PSII) in symbiotic algae (genus Symbiodinium) within corals, and it is assumed to be associated with coral bleaching. To avoid photoinhibition, photosynthetic organisms repair the photodamaged PSII through replacing the PSII proteins, primarily the D1 protein, with newly synthesised proteins. However, in experiments using cultured Symbiodinium strains, the PSII repair of Symbiodinium has been suggested not to be related to the synthesis of the D1 protein. In this study, we examined the relationship between the recovery of PSII photochemical efficiency (F _V/F _M) and the content of D1 protein after high-light and high-temperature treatments using the bleaching-sensitive coral species, Pocillopora damicornis and Acropora millepora, and the bleaching-tolerant coral species, Montipora digitata and Pavona decussata. When corals were exposed to strong light (600 µmol photons m^?2 s^?1) at elevated temperature (32 °C) for 8 h, significant bleaching occurred in bleaching-sensitive coral species although an almost similar extent of reduced PSII function was found across all coral species tested. During a subsequent 15-h recovery under low light (10 µmol photons m^?2 s^?1) at optimal temperature (22 °C), the reduced F _V/F _M recovered close to initial levels in all coral species, but the reduced D1 content recovered only in one coral species (Pavona decussata). D1 content was therefore not strongly linked to chloroplast protein synthesis-dependent PSII repair. These results demonstrate that the recovery of photodamaged PSII does not always correspond with the recovery of D1 protein content in Symbiodinium within corals, suggesting that photodamaged PSII can be repaired by a unique mechanism in Symbiodinium within corals.     

Gene expression profiles during short‐term heat stress in the red sea coral Stylophora pistillata

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide‐spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic ‘r’ strategist. It can thrive in relatively unstable environments and is considered a stress‐tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short‐term (13‐day) heat stress. The results allowed us to identify a two‐step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential ‘early warning genes’ and ‘severe heat‐related genes’. Our findings suggest that during short‐term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER‐unfolded protein response (UPR) and ER‐associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.     

The effects of marine heatwaves on acute heat tolerance in corals

Scleractinian coral populations are increasingly exposed to conditions above their upper thermal limits due to marine heatwaves, contributing to global declines of coral reef ecosystem health. However, historic mass bleaching events indicate there is considerable inter- and intra-specific variation in thermal tolerance whereby species, individual coral colonies and populations show differential susceptibility to exposure to elevated temperatures. Despite this, we lack a clear understanding of how heat tolerance varies across large contemporary and historical environmental gradients, or the selective pressures that underpin this variation. Here we conducted standardised acute heat stress experiments to identify variation in heat tolerance among species and isolated reefs spanning a large environmental gradient across the Coral Sea Marine Park. We quantified the photochemical yield (F_v/F_m) of coral samples in three coral species, Acropora cf humilis, Pocillopora meandrina, and Pocillopora verrucosa, following exposure to four temperature treatments (local ambient temperatures, and + 3°C, +6°C and + 9°C above local maximum monthly mean). We quantified the temperature at which F_v/F_m decreased by 50% (termed ED50) and used derived values to directly compare acute heat tolerance across reefs and species. The ED50 for Acropora was 0.4–0.7°C lower than either Pocillopora species, with a 0.3°C difference between the two Pocillopora species. We also recorded 0.9°C to 1.9°C phenotypic variation in heat tolerance among reefs within species, indicating spatial heterogeneity in heat tolerance across broad environmental gradients. Acute heat tolerance had a strong positive relationship to mild heatwave exposure over the past 35 years (since 1986) but was negatively related to recent severe heatwaves (2016–2020). Phenotypic variation associated with mild thermal history in local environments provides supportive evidence that marine heatwaves are selecting for tolerant individuals and populations; however, this adaptive potential may be compromised by the exposure to recent severe heatwaves.     

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES1

The photosynthetic efficiency and photoprotective capacity of the sea‐ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (?1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen‐replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (F_V/F_M). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (F_M) could only be obtained by adding 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). reoxidation curves showed the presence of Q_B nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (Q_A) to the secondary (Q_B) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of Q_B nonreducing PSII centers within the photosystem. On a physiological level, the formation of Q_B nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient‐limiting conditions.     

In situ measured seasonal variations in F v/F m of two common Red Sea corals

Pulse amplitude modulated (PAM) fluorometry has been suggested as a tool for estimating environmental stresses on corals. However, information regarding natural changes in maximal quantum yields (F _v/F _m) of corals during “normal” (i.e. non-bleaching) years has been limited. In this study, seasonal variations in F _v/F _m for Stylophora pistillata and Favia favus, measured in situ, correlated with seasonal changes in solar irradiance but not in sea temperature. Interactions between sea temperature and irradiance were further studied by growing these corals and Pocillopora damicornis under controlled conditions. Exposure to high light with normal or high temperatures resulted in lower F _v/F _m values than exposure to low light at both temperatures. Thus, high irradiances may cause decreased F _v/F _m values in corals at least as much as, if not more than, high temperatures. Such seasonal variations should be taken into account when using PAM fluorometry as a diagnostic tool for predicting coral bleaching.     

Ocean acidification has no effect on thermal bleaching in the coral Seriatopora caliendrum

The objective of this study was to test whether elevated pCO₂ predicted for the year 2100 (85.1 Pa) affects bleaching in the coral Seriatopora caliendrum (Ehrenberg 1834) either independently or interactively with high temperature (30.5 °C). Response variables detected the sequence of events associated with the onset of bleaching: reduction in the photosynthetic performance of symbionts as measured by maximum photochemical efficiency (F _v/F _m) and effective photochemical efficiency (ΔF/F _m′) of PSII, declines in net photosynthesis (P ^net) and photosynthetic efficiency (alpha, α), and finally, reduced chlorophyll a and symbiont concentrations. S. caliendrum was collected from Nanwan Bay, Taiwan, and subjected to combinations of temperature (27.7 vs. 30.5 °C) and pCO₂ (45.1 vs. 85.1 Pa) for 14 days. High temperature reduced values of all dependent variables (i.e., bleaching occurred), but high pCO₂ did not affect Symbiodinium photophysiology or productivity, and did not cause bleaching. These results suggest that short-term exposure to 81.5 Pa pCO₂, alone and in combination with elevated temperature, does not cause or affect coral bleaching.     

Functional plasticity of photosynthetic apparatus and its resistance to photoinhibition in <Emphasis Type="Italic">Plantago media</Emphasis>

Morphological and functional characteristics of Plantago media L. leaves were compared for plants growing at different light regimes on limestone outcrops in Southern Timan (62°45′N, 55°49′E). The plants grown in open areas under exposure to full sunlight had small leaves with low pigment content and high specific leaf weight; these leaves exhibited high photosynthetic capacity and elevated water use efficiency at high irradiance. The maximum photochemical activity of photosystem II (F _v/F _m) in leaves of sun plants remained at the level of about 0.8 throughout the day. The photosynthetic apparatus of sun plants was resistant to excess photosynthetically active radiation, mostly due to non-photochemical quenching of chlorophyll fluorescence (qN). This quenching was promoted by elevated deepoxiation of violaxanthin cycle pigments. Accumulation of zeaxanthin, a photoprotective pigment in sun plant leaves was observed already in the morning hours. The plant leaves grown in the shade of dense herbage were significantly larger than the sun leaves, with pigment content 1.5–2.0 times greater than in sun leaves; these leaves had low qN values and did not need extensive deepoxidation of violaxanthin cycle pigments. The data reveal the morphophysiological plasticity of plantain plants in relation to lighting regime. Environmental conditions can facilitate the formation of the ecotype with photosynthetic apparatus resistant to photoinhibition. Owing to this adjustment, hoary plantain plants are capable of surviving in ecotopes with high insolation.     

Antioxidant plasticity and thermal sensitivity in four types of Symbiodinium sp.

Warmer than average summer sea surface temperature is one of the main drivers for coral bleaching, which describes the loss of endosymbiotic dinoflagellates (genus: Symbiodinium) in reef‐building corals. Past research has established that oxidative stress in the symbiont plays an important part in the bleaching cascade. Corals hosting different genotypes of Symbiodinium may have varying thermal bleaching thresholds, but changes in the symbiont's antioxidant system that may accompany these differences have received less attention. This study shows that constitutive activity and up‐regulation of different parts of the antioxidant network under thermal stress differs between four Symbiodinium types in culture and that thermal susceptibility can be linked to glutathione redox homeostasis. In Symbiodinium B1, C1 and E, declining maximum quantum yield of PSII (F_v/F_m) and death at 33°C were generally associated with elevated superoxide dismutase (SOD) activity and a more oxidized glutathione pool. Symbiodinium F1 exhibited no decline in F_v/F_m or growth, but showed proportionally larger increases in ascorbate peroxidase (APX) activity and glutathione content (GSx), while maintaining GSx in a reduced state. Depressed growth in Symbiodinium B1 at a sublethal temperature of 29°C was associated with transiently increased APX activity and glutathione pool size, and an overall increase in glutathione reductase (GR) activity. The collapse of GR activity at 33°C, together with increased SOD, APX and glutathione S‐transferase activity, contributed to a strong oxidation of the glutathione pool with subsequent death. Integrating responses of multiple components of the antioxidant network highlights the importance of antioxidant plasticity in explaining type‐specific temperature responses in Symbiodinium.     

Heat stress stimulates nitric oxide production in Symbiodinium microadriaticum: a possible linkage between nitric oxide and the coral bleaching phenomenon

Nitric oxide (NO) is a gas displaying multiple physiologicalfunctions in plants, animals and bacteria. The enzymes nitratereductase and NO synthase have been suggested to be involvedin the production of NO in plants and algae, but the implicationof those enzymes in NO production under physiological conditionsremains obscure. Symbiodinium microadriaticum, commonly referredto as zooxanthellae, is a marine microalga commonly found insymbiotic association with a cnidarian host including reef-buildingcorals. Here we demonstrate NO production in zooxanthellae uponsupplementation of either sodium nitrite or L-arginine as asubstrate. The nitrite-dependent NO production was detectedelectrochemically and confirmed by the application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO), a specific NO scavenger. Cells stained with the diaminofluorescein,DAF-2 DA, an NO fluorescent probe, showed an increase in fluorescenceintensity upon supplementation of both sodium nitrite and L-arginine.Microscopic observations of DAF-stained cells verified thatNO was produced inside the cells. NO production in S. microadriaticumwas found to increase upon exposure of cells to an acute heatstress which also caused a decline in the photosynthetic efficiencyof PSII (F_v/F_m). This study provides substantial evidence toconfirm that zooxanthellae can synthesize NO even when theyare not in a symbiotic association with a coral host. The increasein NO production at high temperatures suggests that heat stressstimulates the microalgal NO production in a temperature-dependentmanner. The implications of these findings are discussed inthe light of the coral bleaching phenomenon which is associatedwith elevated sea surface temperature due to global warming.     

Seasonal Mesophotic Coral Bleaching of Stylophora pistillata in the Northern Red Sea

Coral bleaching occurs when environmental stress induces breakdown of the coral-algae symbiosis and the host initiates algae expulsion. Two types of coral bleaching had been thoroughly discussed in the scientific literature; the first is primarily associated with mass coral bleaching events; the second is a seasonal loss of algae and/or pigments. Here, we describe a phenomenon that has been witnessed for repeated summers in the mesophotic zone (40–63 m) in the northern Red Sea: seasonal bleaching and recovery of several hermatypic coral species. In this study, we followed the recurring bleaching process of the common coral Stylophora pistillata. Bleaching occurred from April to September with a 66% decline in chlorophyll a concentration, while recovery began in October. Using aquarium and transplantation experiments, we explored environmental factors such as temperature, photon flux density and heterotrophic food availability. Our experiments and observations did not yield one single factor, alone, responsible for the seasonal bleaching. The dinoflagellate symbionts (of the genus Symbiodinium) in shallow (5 m) Stylophora pistillata were found to have a net photosynthetic rate of 56.98–92.19 µmol O₂ cm⁻² day⁻¹. However, those from mesophotic depth (60 m) during months when they are not bleached are net consumers of oxygen having a net photosynthetic rate between −12.86 - (−10.24) µmol O₂ cm⁻² day⁻¹. But during months when these mesophotic corals are partially-bleached, they yielded higher net production, between −2.83–0.76 µmol O₂ cm⁻² day⁻¹. This study opens research questions as to why mesophotic zooxanthellae are more successfully meeting the corals metabolic requirements when Chl a concentration decreases by over 60% during summer and early fall.     

Temporal patterns in effective quantum yield of individual zooxanthellae expelled during bleaching

Bleaching is a worldwide phenomenon affecting coral reefs. During elevated temperature and light conditions (bleaching), expelled zooxanthellae show distinct patterns in photosynthetic health. An innovative new device was used to collect individual expelled zooxanthellae, when a coral was exposed to bleaching conditions. This has provided new insight into the photosynthetic condition and abundance of expelled zooxanthellae. It has been assumed that expelled zooxanthellae were dead or moribund; however, we have found individual cells can have healthy effective quantum yields (?_PSII) >0.65 after 8 h of bleaching conditions (500 μmol photons m⁻² s⁻¹, 33 °C). The population of expelled zooxanthellae from Cyphastrea serailia and Pocillopora damicornis showed distinct patterns in the frequency distribution of ?_PSII over time and between locations (sun versus shade) within a colony. During the first 4 h of exposure to bleaching conditions, only 5% of expelled individual cells from P. damicornis were photosynthetically inactive (?_PSII<0.05), whereas for C. serailia, this was 30%. The overall photosynthetic health of expelled zooxanthellae from C. serailia was better than P. damicornis (0.53±0.13 and 0.38±0.13 after 8 h, respectively). This was generally reflected by the in hospite measurement of the coral, yet, the in hospite cells always had a higher ?_PSII than expelled cells, suggesting that host tissue provided added photoprotection for the zooxanthellae.