Warm preconditioning protects against acute heat-induced respiratory dysfunction and delays bleaching in a symbiotic sea anemone.

Preconditioning to non-stressful warming can protect some symbiotic cnidarians against the high temperature-induced collapse of their mutualistic endosymbiosis with photosynthetic dinoflagellates (Symbiodinium spp.), a process known as bleaching. Here, we sought to determine whether such preconditioning is underpinned by differential regulation of aerobic respiration. We quantified in vivo metabolism and mitochondrial respiratory enzyme activity in the naturally symbiotic sea anemone Exaiptasia pallida preconditioned to 30°C for >7 weeks as well as anemones kept at 26°C. Preconditioning resulted in increased Symbiodinium photosynthetic activity and holobiont (host+symbiont) respiration rates. Biomass-normalised activities of host respiratory enzymes [citrate synthase and the mitochondrial electron transport chain (mETC) complexes I and IV] were higher in preconditioned animals, suggesting that increased holobiont respiration may have been due to host mitochondrial biogenesis and/or enlargement. Subsequent acute heating of preconditioned and 'thermally naive' animals to 33°C induced dramatic increases in host mETC complex I and Symbiodinium mETC complex II activities only in thermally naive E. pallida These changes were not reflected in the activities of other respiratory enzymes. Furthermore, bleaching in preconditioned E. pallida (defined as the significant loss of symbionts) was delayed by several days relative to the thermally naive group. These findings suggest that changes to mitochondrial biogenesis and/or function in symbiotic cnidarians during warm preconditioning might play a protective role during periods of exposure to stressful heating.

Temperature moderates the infectiousness of two conspecific Symbiodinium strains isolated from the same host population.

Symbioses between cnidarians and symbiotic dinoflagellates (Symbiodinium) are ecologically important and physiologically diverse. This diversity contributes to the spatial distribution of specific cnidarian-Symbiodinium associations. Physiological variability also exists within Symbiodinium species, yet we know little regarding its relevance for the establishment of symbiosis under different environmental conditions. Two putatively conspecific Symbiodinium strains (both ITS2-type A4) were isolated from the sea anemone Exaiptasia pallida and placed into unialgal culture. Thermal tolerance of these cultures was compared following heating from 26°C to 33.5°C over 18 days. Photosystem II function was negatively impacted by heating in one strain while PSII function in the other showed little response to elevated temperature. Additionally, infection of Symbiodinium cells into aposymbiotic anemones was assessed for both strains at 26°C and 30.5°C. The heat-sensitive strain had greater infection success at 26°C, while there was no difference in infection between the two strains at the higher temperature. Results from this work suggest that variability in thermal optima or -tolerance within Symbiodinium spp. has relevance for early stages of host-Symbiodinium interactions. Thus, varying infectiousness among differentially heat-sensitive Symbiodinium strains could provide a mechanism for the emergence of novel and potentially resilient cnidarian-Symbiodinium associations in a rapidly warming environment.

Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress.

Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

Nitric oxide mediates coral bleaching through an apoptotic-like cell death pathway: evidence from a model sea anemone-dinoflagellate symbiosis.

Coral bleaching (involving the loss of symbiotic algae from the cnidarian host) is a major threat to coral reefs and appears to be mediated at the cellular level by nitric oxide (NO). In this study, we examined the specific role of NO in bleaching using the sea anemone Aiptasia pulchella, a model system for the study of corals. Exposure of A. pulchella to high-temperature shock (26-33°C over <1 h) or an NO donor (S-nitrosoglutathione) resulted in significant increases in host caspase-like enzyme activity. These responses were reflected in the intensities of bleaching, which were significantly higher in heat- or NO-treated specimens than in controls maintained in seawater at 26°C. Notably, the inhibition of caspase-like activity prevented bleaching even in the presence of an NO donor or at elevated temperature. The additional use of an NO scavenger controlled for effects mediated by agents other than NO. We also exposed A. pulchella to a more ecologically relevant treatment (an increase from 26 to 33°C over 6-7 d). Again, host NO synthesis correlated with the activation of caspase-like enzyme activity. Therefore, we conclude that NO's involvement in cnidarian bleaching arises through the regulation of host apoptotic pathways.

Nitric oxide and coral bleaching: is peroxynitrite generation required for symbiosis collapse?

The temperature-induced collapse ('bleaching') of the coral-dinoflagellate symbiosis is hypothesised to result from symbiont oxidative stress and a subsequent host innate immune-like response. This includes the production of nitric oxide (NO), which is involved in numerous microbial symbioses. Much of NO's cytotoxicity has been attributed to its conversion, in the presence of superoxide (O2(-)), to highly reactive peroxynitrite (ONOO(-)). However, ONOO(-) generation has yet to be observed in either a lower invertebrate or an intracellular mutualism. Using confocal laser scanning microscopy with the fluorescent ONOO(-) indicator aminophenyl fluorescein (APF), we observed strong evidence that ONOO(-) is generated in symbiotic Aiptasia pulchella under conditions known to induce thermal bleaching. However, a role for ONOO(-) in bleaching remains unclear as treatment with a peroxynitrite scavenger had no significant effect on thermal bleaching. Therefore, while ONOO(-) may have a potential for cytotoxicity, in vivo levels of the compound may be insufficient to affect bleaching.

Nitric oxide production and tolerance differ among Symbiodinium types exposed to heat stress.

Nitric oxide (NO) is a ubiquitous molecule and its involvement in metazoan-microbe symbiosis is well known. Evidence suggests that it plays a role in the temperature-induced breakdown ('bleaching') of the ecologically important cnidarian-dinoflagellate association, and this can often lead to widespread mortality of affected hosts. This study confirms that dinoflagellates of the genus Symbiodinium can produce NO and that production of the compound is differentially regulated in different types when exposed to elevated temperature. Temperature-sensitive type B1 cells under heat stress (8°C above ambient) exhibited significant increases in NO synthesis, which occurred alongside pronounced photoinhibition and cell mortality. Tolerant type A1 cells also displayed increases in NO production, yet maintained photosynthetic yields at levels similar to those of untreated cells and displayed less dramatic increases in cell death. Type C1 cells displayed a down-regulation of NO synthesis at high temperature, and no significant mortality increases were observed in this type. Temperature-induced mortality in types A1 and B1 was affected by the prevailing level of NO and, furthermore, photosynthetic yields of these temperature-tolerant and -sensitive types appeared differentially susceptible to NO donated by pharmacological agents. Taken together, these differences in NO synthesis and tolerance could potentially influence the varying bleaching responses seen among hosts harboring different Symbiodinium types.

Partitioning of Respiration in an Animal-Algal Symbiosis: Implications for Different Aerobic Capacity between Symbiodinium spp.

Cnidarian-dinoflagellate symbioses are ecologically important and the subject of much investigation. However, our understanding of critical aspects of symbiosis physiology, such as the partitioning of total respiration between the host and symbiont, remains incomplete. Specifically, we know little about how the relationship between host and symbiont respiration varies between different holobionts (host-symbiont combinations). We applied molecular and biochemical techniques to investigate aerobic respiratory capacity in naturally symbiotic Exaiptasia pallida sea anemones, alongside animals infected with either homologous ITS2-type A4 Symbiodinium or a heterologous isolate of Symbiodinium minutum (ITS2-type B1). In naturally symbiotic anemones, host, symbiont, and total holobiont mitochondrial citrate synthase (CS) enzyme activity, but not host mitochondrial copy number, were reliable predictors of holobiont respiration. There was a positive association between symbiont density and host CS specific activity (mg protein(-1)), and a negative correlation between host- and symbiont CS specific activities. Notably, partitioning of total CS activity between host and symbiont in this natural E. pallida population was significantly different to the host/symbiont biomass ratio. In re-infected anemones, we found significant between-holobiont differences in the CS specific activity of the algal symbionts. Furthermore, the relationship between the partitioning of total CS activity and the host/symbiont biomass ratio differed between holobionts. These data have broad implications for our understanding of cnidarian-algal symbiosis. Specifically, the long-held assumption of equivalency between symbiont/host biomass and respiration ratios can result in significant overestimation of symbiont respiration and potentially erroneous conclusions regarding the percentage of carbon translocated to the host. The interspecific variability in symbiont aerobic capacity provides further evidence for distinct physiological differences that should be accounted for when studying diverse host-symbiont combinations.