Using Transcript Levels of Nitrate Transporter 2 as Molecular Indicators to Estimate the Potentials of Nitrate Transport in Symbiodinium, Cladocopium, and Durusdinium of the Fluted Giant Clam, Tridacna squamosa

Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of phototrophic Symbiodiniaceae dinoflagellates of mainly genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid dinoflagellates donate photosynthate and amino acids to the clam host, which in return needs to supply inorganic carbon and nitrogen to them. The host can conduct light-enhanced absorption of nitrate (NO3–), which can only be metabolized by the symbionts. This study aimed to clone nitrate transporter 2 (NRT2) from the symbionts of the fluted giant clam, Tridacna squamosa. Here, we report three major sequences of NRT2 derived from Symbiodinium (Symb-NRT2), Cladocopium (Clad-NRT2) and Durusdinium (Duru-NRT2). Phenogramic analysis and molecular characterization confirmed that these three sequences were NRT2s derived from dinoflagellates. Immunofluorescence microscopy localized NRT2 at the plasma membrane and cytoplasmic vesicles of the symbiotic dinoflagellates, indicating that it could partake in the uptake and transport of NO3–. Therefore, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 could be used as molecular indicators to estimate the potential of NO3– transport in five organs of 13 T. squamosa individuals. The transcript levels of form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcII) of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII) and Durusdinium (Duru-rbcII) were also determined in order to calculate the transcript ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII. These ratios expressed the potentials of NO3– transport with reference to the phototrophic potentials in a certain genus of coccoid dinoflagellate independent of its quantity. Results obtained indicate that Symbiodinium generally had a higher potential of NO3– transport than Cladocopium and Durusdinium at the genus level. Furthermore, some phylotypes (species) of Symbiodinium, particularly those in the colorful outer mantle, had very high Symb-NRT2/Symb-rbcII ratio (7–13), indicating that they specialized in NO3– uptake and nitrogen metabolism. Overall, our results indicate for the first time that different phylotypes of Symbiodiniaceae dinoflagellates could have dissimilar abilities to absorb and assimilate NO3–, alluding to their functional diversity at the genus and species levels.

Stress Resistance in an Extreme Environment: Lessons Learnt from a Temperate Symbiotic Sea Anemone

<p>Coral bleaching, the loss of symbiotic dinoflagellates (zooxanthellae) or their photosynthetic pigments in response to environmental stress, is of huge global concern. In contrast to tropical corals, which are highly sensitive to fluctuations in environmental parameters such as temperature, light and salinity, zooxanthellate invertebrates in temperate waters rarely bleach despite highly variable conditions. In this study, we tested the effects of salinity with combined effects of light and temperature stress on the photophysiology and stability of the temperate symbiotic sea anemone, Anthopleura aureoradiata, through chlorophyll fluorescence. In the field it was demonstrated that A. aureoradiata was resilient to abiotic fluctuations of considerable magnitude in the intertidal zone. Salinity was revealed to range naturally between a winter low of 30 and summer high of 40 ppt in an elevated tide pool with no measurable effects on the photophysiology of A. aureoradiata residing within. In a controlled environment, only extreme high and low salinities had an effect on the zooxanthellar photosystem, with a wide range of tolerance between 15-50 ppt dependent on the levels of temperature and light. Both high and low light, and temperature, also impacted upon photophysiology. Moreover, each of these variables independently, as well as combined, exacerbated the impact of salinity stress. In addition, the duration of exposure played an important role in the survival of this symbiosis, with only 48-96 h exposure to the extreme salinities of 5, 10, 55 and 60 ppt inducing irreversible photosynthetic failure, bleaching and death. Thus, the data supports the idea that this anemone-zooxanthellar symbiosis is highly resilient to considerable amounts of abiotic stress, a likely a function of the robust photophysiology of its zooxanthellae. This resilience to bleaching suggests that A. aureoradiata and its zooxanthallae have evolved a combination of powerful defensive mechanisms to help aid against the heterogenous environment from which they come. I will present an overview of these osmoregulatory mechanisms, photoacclimatory strategies and behaviours that this symbiosis likely deploys in order to combat environmentally realistic ranges in abiotic factors. Further studies would be necessary to deduce whether it is the host or zooxanthellae which are responsible for the breakdown of this symbiosis.</p>

Stress Resistance in an Extreme Environment: Lessons Learnt from a Temperate Symbiotic Sea Anemone

<p>Coral bleaching, the loss of symbiotic dinoflagellates (zooxanthellae) or their photosynthetic pigments in response to environmental stress, is of huge global concern. In contrast to tropical corals, which are highly sensitive to fluctuations in environmental parameters such as temperature, light and salinity, zooxanthellate invertebrates in temperate waters rarely bleach despite highly variable conditions. In this study, we tested the effects of salinity with combined effects of light and temperature stress on the photophysiology and stability of the temperate symbiotic sea anemone, Anthopleura aureoradiata, through chlorophyll fluorescence. In the field it was demonstrated that A. aureoradiata was resilient to abiotic fluctuations of considerable magnitude in the intertidal zone. Salinity was revealed to range naturally between a winter low of 30 and summer high of 40 ppt in an elevated tide pool with no measurable effects on the photophysiology of A. aureoradiata residing within. In a controlled environment, only extreme high and low salinities had an effect on the zooxanthellar photosystem, with a wide range of tolerance between 15-50 ppt dependent on the levels of temperature and light. Both high and low light, and temperature, also impacted upon photophysiology. Moreover, each of these variables independently, as well as combined, exacerbated the impact of salinity stress. In addition, the duration of exposure played an important role in the survival of this symbiosis, with only 48-96 h exposure to the extreme salinities of 5, 10, 55 and 60 ppt inducing irreversible photosynthetic failure, bleaching and death. Thus, the data supports the idea that this anemone-zooxanthellar symbiosis is highly resilient to considerable amounts of abiotic stress, a likely a function of the robust photophysiology of its zooxanthellae. This resilience to bleaching suggests that A. aureoradiata and its zooxanthallae have evolved a combination of powerful defensive mechanisms to help aid against the heterogenous environment from which they come. I will present an overview of these osmoregulatory mechanisms, photoacclimatory strategies and behaviours that this symbiosis likely deploys in order to combat environmentally realistic ranges in abiotic factors. Further studies would be necessary to deduce whether it is the host or zooxanthellae which are responsible for the breakdown of this symbiosis.</p>

Detecting reactive oxygen species in free-living symbiotic dinoflagellates exposed to nanoparticles v1

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a popular fluorescent probe for the detection of oxidative stress in cells. Since the probe can be prone to auto-oxidation, carboxy-2',7'-dichlorodihydrofluorescein-diacetate (carboxy-H2DCF-DA) which is more stable and easily penetrates cell membranes is often reported in the literature instead. Upon crossing the cell membrane, esterases hydrolyse DCFH-DA to DCFH, which remains trapped within cells. The oxidation of DCFH yields DCF, a fluorescent compound which can be measured using excitation/emission wavelengths of 485-495/520-530 nm. Due to several cases of interference when used in cellular systems, DCFH-DA is a general marker of the cellular oxidative stress rather than a specific indicator H2O2 formation or other ROS.

Detecting nitric oxide in free-living symbiotic dinoflagellates exposed to nanoparticles v1

Diaminofuorescein-2 diacetate (DAF-2 DA) is a fluorescent indicator of nitric oxide (NO). The DAF reacts with the nitric anhydride (N2O3) which formed by oxidation of NO. Upon crossing the cell membrane, esterases hydrolyse DAF-2 DA to DAF-2, which remains trapped within cells. The DAF-2 reacts to the oxidation of intracellular NO (or more accurately the N2O3) to produce the highly fluorescent triazolofluorescein (DAF-2T) by nitrosation and dehydration (Kojima et al., 1999). Protocol adapted from Bouchard & Yamasaki (2009) and Kojima et al. (1999)

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian–dinoflagellate symbiosis

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan–lectin interactions in host–symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular β-D-galactose) residues in the establishment of the cnidarian–dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.

Impacts of Seagrass on Benthic Microalgae and Phytoplankton Communities in an Experimentally Warmed Coral Reef Mesocosm

The effects of seagrass on microalgal assemblages under experimentally elevated temperatures (28°C) and CO2 partial pressures (pCO2; 800 μatm) were examined using coral reef mesocosms. Concentrations of nitrate, ammonium, and benthic microalgal chlorophyll a (chl-a) were significantly higher in seagrass mesocosms, whereas phytoplankton chl-a concentrations were similar between seagrass and seagrass-free control mesocosms. In the seagrass group, fewer parasitic dinoflagellate OTUs (e.g., Syndiniales) were found in the benthic microalgal community though more symbiotic dinoflagellates (e.g., Cladocopium spp.) were quantified in the phytoplankton community. Our results suggest that, under ocean acidification conditions, the presence of seagrass nearby coral reefs may (1) enhance benthic primary productivity, (2) decrease parasitic dinoflagellate abundance, and (3) possibly increase the presence of symbiotic dinoflagellates.