Dinoflagellate Symbiosis Research Articles

A journey into the wild of the cnidarian model system Aiptasia and its symbionts

The existence of coral reef ecosystems relies critically on the mutualistic relationship between calcifying cnidarians and photosynthetic, dinoflagellate endosymbionts in the genus Symbiodinium. Reef-corals have declined globally due to anthropogenic stressors, for example, rising sea-surface temperatures and pollution that often disrupt these symbiotic relationships (known as coral bleaching), exacerbating mass mortality and the spread of disease. This threatens one of the most biodiverse marine ecosystems providing habitats to millions of species and supporting an estimated 500 million people globally (Hoegh-Guldberg et al. 2007). Our understanding of cnidarian–dinoflagellate symbioses has improved notably with the recent application of genomic and transcriptomic tools (e.g. Voolstra et al. 2009; Bayer et al. 2012; Davy et al. 2012), but a model system that allows for easy manipulation in a laboratory environment is needed to decipher underlying cellular mechanisms important to the functioning of these symbioses. To this end, the sea anemone Aiptasia, otherwise known as a ‘pest’ to aquarium hobbyists, is emerging as such a model system (Schoenberg & Trench 1980; Sunagawa et al. 2009; Lehnert et al. 2012). Aiptasia is easy to grow in culture and, in contrast to its stony relatives, can be maintained aposymbiotically (i.e. dinoflagellate free) with regular feeding. However, we lack basic information on the natural distribution and genetic diversity of these anemones and their endosymbiotic dinoflagellates. These data are essential for placing the significance of this model system into an ecological context. In this issue of Molecular Ecology, Thornhill et al. (2013) are the first to present genetic evidence on the global distribution, diversity and population structure of Aiptasia and its associated Symbiodinium spp. By integrating analyses of the host and symbiont, this research concludes that the current Aitpasia taxonomy probably needs revision and that two distinct Aiptasia lineages are prevalent that have probably been spread through human activity. One lineage engages in a specific symbiosis with Symbiodinium minutum throughout the tropics, whereas a second, local Aiptasia sp. population in Florida appears more flexible in partnering with more than one symbiont. The existence of symbiont-specific and symbiont-flexible Aiptasia lineages can greatly complement laboratory-based experiments looking into mechanisms of symbiont selectivity. In a broader context, the study by Thornhill et al. (2013) should inspire more studies to target the natural environment of model systems in a global context targeting all participating member species when establishing ecological and genetic baselines.

Photosynthate translocation increases in response to low seawater pH in a coral–dinoflagellate symbiosis

Abstract. This study has examined the effect of low seawater pH values (induced by an increased CO2 partial pressure) on the rates of photosynthesis, as well as on the carbon budget and carbon translocation in the scleractinian coral species Stylophora pistillata, using a new model based on 13C labelling of the photosynthetic products. Symbiont photosynthesis contributes to a large part of the carbon acquisition in tropical coral species, and it is thus important to know how environmental changes affect this carbon acquisition and allocation. For this purpose, nubbins of S. pistillata were maintained for six months at two pHTs (8.1 and 7.2, by bubbling seawater with CO2). The lowest pH value was used to tackle how seawater pH impacts the carbon budget of a scleractinian coral. Rates of photosynthesis and respiration of the symbiotic association and of isolated symbionts were assessed at each pH. The fate of 13C photosynthates was then followed in the symbionts and the coral host for 48 h. Nubbins maintained at pHT 7.2 presented a lower areal symbiont concentration, and lower areal rates of gross photosynthesis and carbon incorporation compared to nubbins maintained at pHT 8.1. The total carbon acquisition was thus lower under low pH. However, the total percentage of carbon translocated to the host as well as the amount of carbon translocated per symbiont cell were significantly higher under pHT 7.2 than under pHT 8.1 (70% at pHT 7.2 vs. 60% at pHT 8.1), such that the total amount of photosynthetic carbon received by the coral host was equivalent under both pHs (5.5 to 6.1 μg C cm−2 h−1). Although the carbon budget of the host was unchanged, symbionts acquired less carbon for their own needs (0.6 compared to 1.8 μg C cm−2 h−1), explaining the overall decrease in symbiont concentration at low pH. In the long term, such decrease in symbiont concentration might severely affect the carbon budget of the symbiotic association.

Intracellular pH of symbiotic dinoflagellates

Intracellular pH (pHi) is likely to play a key role in maintaining the functional success of cnidarian–dinoflagellate symbiosis, yet until now the pHi of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pHi over a daily light/dark cycle. The pHi of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHi 7.21–7.39 in the light and 7.06–7.14 in the dark. Of note, there was a significant genotypic difference in pHi, irrespective of irradiance.

Host autophagic degradation and associated symbiont loss in response to heat stress in the symbiotic anemone, Aiptasia pallida

AbstractCoral bleaching involves the loss of essential photosynthetic dinoflagellates (Symbiodinium sp.) from host gastrodermal cells in response to temperature or light stress. Although numerous potential cellular bleaching mechanisms have been proposed, there remains much uncertainty regarding which cellular events occur during early breakdown of the host–dinoflagellate symbiosis. In this study, transmission electron microscopy was used to conduct a detailed examination of symbiotic tissues of the tropical anemone Aiptasia pallida during early stages of host stress. Bleaching was induced by exposing specimens to a stress treatment of 32.5±0.5°C at 140±7 μ mol photons m−2 s−1 light intensity for 12 h, followed by 12 h at 24±1°C in darkness, repeated over a 48 h period. Ultrastructural examinations revealed numerous dense autophagic structures and associated cellular degradation in tentacle tissues after ~12 h of the stress treatment. Anemones treated with rapamycin, a known autophagy inducer, exhibited the same ultrastructural characteristics as heat‐stressed tissues, confirming that the structures observed during heat stress treatment were autophagic. In addition, symbionts appeared to be expelled from host cells via an apocrine‐like detachment mechanism from the apical ends of autophagic gastrodermal cells. This study provides the first ultrastructural evidence of host autophagic degradation during thermal stress in a cnidarian system and also supports earlier suggestions that autophagy is an active cellular mechanism during early stages of bleaching.

Ecological and biogeographic implications of Siderastrea symbiotic relationship with Symbiodinium sp. C46 in Sal Island (Cape Verde, East Atlantic Ocean)

The relative abundance of the genus Siderastrea and its relationship with temperature and irradiance was assessed around Sal Island (Cape Verde). In some of the surveyed sites, these corals accounted for 80–90 % of the living cover, making it a biotope-dominant organism. Unlike Siderastrea corals from West Atlantic and Caribbean locations, genetic analyses of the dinoflagellate symbiotic partner revealed high specificity between Siderastrea sp. in Cape Verde and the Symbiodinium type C46. Biotope restriction of the ecological success of Siderastrea in Cape Verde may be explained in part by this host–symbiont partnership, resulting locally in a small optimum ecological niche with specific light intensity regimes. Distinctively, West Atlantic and Caribbean Siderastrea associates with a much broader range of Symbiodinium diversity, suggesting that these symbioses exhibit some flexibility under differing environmental conditions where these corals occupy a wider range of ecological niches. Geographic isolation and/or long-standing environmental conditions are probably responsible for such adaptions and coral–dinoflagellate symbioses. Additional genetic analyses on Clade C Symbiodinium associated with Siderastrea were conducted with the hyper-variable plastid psbA minicircle to resolve phylogeographic patterns that indicate the relative connectivity and/or isolation of these symbionts throughout the tropical Atlantic.

A single-cell view of ammonium assimilation in coral–dinoflagellate symbiosis

Assimilation of inorganic nitrogen from nutrient-poor tropical seas is an essential challenge for the endosymbiosis between reef-building corals and dinoflagellates. Despite the clear evidence that reef-building corals can use ammonium as inorganic nitrogen source, the dynamics and precise roles of host and symbionts in this fundamental process remain unclear. Here, we combine high spatial resolution ion microprobe imaging (NanoSIMS) and pulse-chase isotopic labeling in order to track the dynamics of ammonium incorporation within the intact symbiosis between the reef-building coral Acropora aspera and its dinoflagellate symbionts. We demonstrate that both dinoflagellate and animal cells have the capacity to rapidly fix nitrogen from seawater enriched in ammonium (in less than one hour). Further, by establishing the relative strengths of the capability to assimilate nitrogen for each cell compartment, we infer that dinoflagellate symbionts can fix 14 to 23 times more nitrogen than their coral host cells in response to a sudden pulse of ammonium-enriched seawater. Given the importance of nitrogen in cell maintenance, growth and functioning, the capability to fix ammonium from seawater into the symbiotic system may be a key component of coral nutrition. Interestingly, this metabolic response appears to be triggered rapidly by episodic nitrogen availability. The methods and results presented in this study open up for the exploration of dynamics and spatial patterns associated with metabolic activities and nutritional interactions in a multitude of organisms that live in symbiotic relationships.

Host–symbiont recombination versus natural selection in the response of coral–dinoflagellate symbioses to environmental disturbance

Mutualisms between reef-building corals and endosymbiotic dinoflagellates are particularly sensitive to environmental stress, yet the ecosystems they construct have endured major oscillations in global climate. During the winter of 2008, an extreme cold-water event occurred in the Gulf of California that bleached corals in the genus Pocillopora harbouring a thermally 'sensitive' symbiont, designated Symbiodinium C1b-c, while colonies possessing Symbiodinium D1 were mostly unaffected. Certain bleached colonies recovered quickly while others suffered partial or complete mortality. In most colonies, no appreciable change was observed in the identity of the original symbiont, indicating that these partnerships are stable. During the initial phases of recovery, a third species of symbiont B1(Aiptasia), genetically identical to that harboured by the invasive anemone, Aiptasia sp., grew opportunistically and was visible as light-yellow patches on the branch tips of several colonies. However, this symbiont did not persist and was displaced in all cases by C1b-c several months later. Colonies with D1 were abundant at inshore habitats along the continental eastern Pacific, where seasonal turbidity is high relative to offshore islands. Environmental conditions of the central and southern coasts of Mexico were not sufficient to explain the exclusivity of D1 Pocillopora in these regions. It is possible that mass mortalities associated with major thermal disturbances during the 1997-1998 El Niño Southern Oscillation eliminated C1b-c holobionts from these locations. The differential loss of Pocillopora holobionts in response to thermal stress suggests that natural selection on existing variation can cause rapid and significant shifts in the frequency of particular coral-algal partnerships. However, coral populations may take decades to recover following episodes of severe selection, thereby raising considerable uncertainty about the long-term viability of these communities.

Immunocytochemical evidence that symbiotic algae secrete potential recognition signal molecules in hospite

Cnidarian–dinoflagellate symbioses are not well understood at the molecular level. Observed specificity between partners during initiation, establishment, and maintenance of the relationship strongly implies a role for chemical signaling. This report presents biochemical and immunocytochemical evidence for potential signaling molecules, as large molecular weight glycoproteins, secreted by Symbiodinium dinoflagellates both in culture and in symbiosis. Polyclonal antibodies directed against recovered exudate from S. microadriaticum, the natural endosymbiont of Cassiopea xamachana, the upside–down jellyfish, were highly specific in recognizing exudates from Symbiodinium species that can successfully induce developmental metamorphosis in the host but did not recognize exudates from Symbiodinium species that do not. Immunoblot analyses showed S. microadriaticum exudate to be protease sensitive. Release of antigenic material by symbiotic S. microadriaticum was demonstrated through light and electron microscopy using immunogold-labeled anti-S. microadriaticum (anti-Sm-XuLg) antibodies as probes. These secreted, symbiont-derived glycoconjugates may be candidates for interspecific molecular signals.

Symbiosis-dependent gene expression in coral-dinoflagellate association: cloning and characterization of a P-type H+-ATPase gene.

We report the molecular cloning of a H(+)-ATPase in the symbiotic dinoflagellate, Symbiodinium sp. previously suggested by pharmacological studies to be involved in carbon-concentrating mechanism used by zooxanthellae when they are in symbiosis with corals. This gene encodes a protein of 975 amino acids with a calculated mass of about 105 kDa. The structure of the protein shows a typical P-type H(+)-ATPase structure (type IIIa plasma membrane H(+)-ATPases) and phylogenetic analyses show that this new proton pump groups with diatoms in the Chromoalveolates group. This Symbiodinium H(+)-ATPase is specifically expressed when zooxanthellae are engaged in a symbiotic relationship with the coral partner but not in free-living dinoflagellates. This proton pump, therefore, could be involved in the acidification of the perisymbiotic space leading to bicarbonate dehydration by carbonic anhydrase activity in order to supply inorganic carbon for photosynthesis as suggested by earlier studies. To our knowledge, this work provides the first example of a symbiosis-dependent gene in zooxanthellae and confirms the importance of H(+)-ATPase in coral-dinoflagellate symbiosis.

Diazotrophic bacteria associated with Hawaiian Montipora corals: Diversity and abundance in correlation with symbiotic dinoflagellates

The primary productivity of coral–dinoflagellate symbiosis is classically viewed as nitrogen (N)-limited. However, the recent discovery of N-fixing bacteria residing within coral tissues challenges this view. This study investigated the diversity and abundance of N-fixing bacteria associated with Hawaiian corals of the genus Montipora. Phylogenetic analysis of a partial sequence of the nitrogenase gene ( nifH), revealed a diverse group of N-fixing bacteria in association with Montipora capitata and Montipora flabellata. These nifH sequences were closely affiliated with known taxa in α-, β-, γ-, and δ-proteobacteria, as well as cyanobacteria. Gamma-proteobacteria were the dominant bacterial class represented, and bacteria closely related to the genus Vibrio were the prevailing group within this class. Quantitative PCR was used to evaluate the abundance of the dominant class of N-fixing bacteria from different parts of four M. capitata colonies. The abundance of the dominant γ-proteobacteria was positively correlated ( p = 0.001) with the abundance of symbiotic dinoflagellates, suggesting a physiological relationship between the two coral symbionts. The evidence obtained in this study suggests that N-limitation may not be a major regulator of the productivity of dinoflagellate symbionts of corals, but provides impetus for renewed investigation into N-cycling in this multi-partner symbiosis.

Size-related stable isotope changes in Late Cretaceous planktic foraminifera: Implications for paleoecology and photosymbiosis

The paleoecology of Cretaceous planktic foraminifera during the Late Cenomanian to Coniacian period (∼ 95–86 Ma) remains controversial since much of the tropical marine record is preserved as chalk and limestone with uncertain geochemical overprints. Here we present δ 13C and δ 18O data from sieve size fractions of monospecific samples of exceptionally well preserved planktic foraminifera recovered during Ocean Drilling Program Leg 207 (Demerara Rise, western tropical Atlantic). Our results suggest that all species studied ( Hedbergella delrioensis, Heterohelix globulosa, Marginotruncana sinuosa, Whiteinella baltica) grew primarily in surface waters and did not change their depth habitat substantially during their life cycle. Comparison of size-related ontogenetic trends in δ 13C in Cretaceous and modern foraminifera further suggests that detection of dinoflagellate photosymbiosis using δ 13C is confounded by physiological effects during the early stages of foraminifer growth, raising doubts about previous interpretations of photosymbiosis in small foraminifera species. We propose that obligate photosymbiosis involving dinoflagellates may not have evolved until the Campanian or Maastrichtian since our survey of Cenomanian–Coniacian species does not find the δ 18O and δ 13C size-related trends observed in modern foraminifer–dinoflagellate symbioses.

Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression

BackgroundCnidarian – dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian – algal symbiosis.ResultsWe detected statistically significant differences in host gene expression profiles between sea anemones (Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian – dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress.ConclusionOur data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian – dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

OXYGEN MICROSENSOR STUDIES ON ZOOXANTHELLATE CLIONAID SPONGES FROM THE COSTA BRAVA, MEDITERRANEAN SEA1

Photosynthetic properties of two symbiotic demosponges were compared using Clark‐type oxygen microsensors. The putatively distinct sponge species, Cliona viridis (Schmidt, 1862) and Cliona nigricans (Schmidt, 1862) were discriminated by their mean megasclere lengths of 296 and 387 μm, respectively. Photosynthetic behavior was used to generate additional taxonomic information. Sponge–dinoflagellate symbioses were well adapted to low light due to the hosts' endolithic lifestyle. Both sponges reached light compensation and saturation at similar light levels with means close to 10 and 30 μmol photons·m−2·s−1, respectively. The gross photosynthetic activity was closely related to symbiont cell density in the sponge surface tissue. Mean symbiont densities, chl a content, and gross photosynthesis were about six times higher in C. viridis than in C. nigricans, with respective values of 3000 and 440 symbiont·mm−2, 1.3 and 0.2 μg chl a·g−1, and 5.4 and 1.0 μmol O2·cm−3·s−1 gross photosynthesis. Net photosynthesis and respiration could not be calculated accurately from the oxygen gradients, because significant gas exchange occurs through the pumping activity. Thus, assumptions of diffusional oxygen exchange via the surface do not hold for sponges. Combined data of this study indicate that the metabolic activity of C. viridis depends on photosynthetic activity of its symbionts, whereas C. nigricans appears to have a higher pumping intensity and is more actively filter feeding. The difference in photosynthetic activities is not caused by different light adaptations but provides new evidence against the conspecifity of C. viridis and C. nigricans.

Dinoflagellate symbioses: strategies and adaptations for the acquisition and fixation of inorganic carbon.

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3-and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.

Development of host- and symbiont-specific monoclonal antibodies and confirmation of the origin of the symbiosome membrane in a cnidarian-dinoflagellate symbiosis.

The "symbiosome membrane" as defined by Roth et al. (1988) is a single, host-derived membrane that surrounds an endosymbiotic organism, separating it from the cytoplasm of the host cell. However, in the case of cnidarian-dinoflagellate endosymbioses, clear identification of the symbiosome membrane is complicated by the fact that each algal symbiont is surrounded by multiple layers of apparent membrane. The origin and molecular nature of these membranes has been the subject of considerable debate in the literature. Here we report the development of host-specific (G12) and symbiont-specific (PC3) monoclonal antibodies that allow separation of the host and symbiont components of these multiple membranes. Using immunocytochemistry at both the light and the electron microscopic level, we present data supporting the conclusion that the definitive symbiosome membrane is a single, host-derived membrane, whereas the remainder of the underlying apparent membranes surrounding the algal cell are symbiont-derived. The potential for macromolecules associated with these membranes to act as cellular signals critical to recruiting symbionts and maintaining established symbioses is discussed.

The Physiological Mechanisms of Acclimatization in Tropical Reef Corals

SYNOPSIS. The ability of scleractinian corals to survive changes that are predicted in the global environment over the next century will lie in their physiological mechanisms of acclimatization. Corals display rapid modifications in behavior, morphology and physiology enabling them to photoacclimate to changing light conditions, a scenario that demonstrates considerable biological flexibility. Here we argue that the acclimatization mechanisms in corals are fundamentally similar to those exhibited by other invertebrate taxa. We discuss protein metabolism as a mechanism underlying acclimatization responses in reef corals, and explore the relationship between protein turnover, metabolic rate, growth rate, and acclimatization capacity. Our preliminary analyses suggest that corals with low growth rates ([micro]Ca/mgN/h) and high metabolic rates ([micro][O.sub.2]/cm2/hr), such as the massive species, acclimatize more effectively than those with high growth rates and low metabolic rates, a feature that is characteristic of branching species. We conclude that studies of protein turnover, combined with temporally relevant investigations into the dynamic aspects of coral dinoflagellate symbioses will provide considerable insight into why corals exhibit such a high level of variation in response to the same environmental challenge. Furthermore, a more detailed understanding of acclimatization mechanisms is essential if we are to predict how a coral assemblage will respond to present and future environmental challenges.