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1 Bermuda Biological Station for Research, Inc., Ferry Reach GE-01, Bermuda
2 Environment Agency, Westbury-on-Trym, Bristol BS106BF, UK
3 Department of Environmental Sciences, University of Technology, Sydney, New South Wales 2065, Australia
4 Department of Oceanography, University of Hawaii, Honolulu, Hawaii 96822
5 Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, Nishihara, Okinawa, 903-0213 Japan
6 Department of Biology, Evergreen State University, Olympia, Washington 98505
* To whom correspondence should be addressed, at School of Ocean and Earth Science and Technology, University of Hawaii, Honolulu 96822. E-mail: hank{at}bbsr.edu
Coral bleaching, the breakdown of the symbiotic relationship between host corals and their photosynthetic dinoflagellate endosymbionts, is a phenomenon of major ecological significance. The cellular and molecular mechanisms underlying bleaching are poorly understood. Here we report substantial increases in nitric oxide synthase (NOS) activity in symbionts associated with bleaching corals. Nitric oxide (NO), a product of NOS activity, is a highly reactive and membrane-permeable molecule that has the potential to exert cytotoxic effects in host cells either directly or in combination with molecular oxygen or superoxide radicals. This potential allows us to suggest that coral bleaching may be a stress response initiated by the symbiont, rather than by the host.
Coral reefs are biodiverse ecosystems of enormous ecological and economic importance (1, 2). The corals that constitute the foundations of these ecosystems are symbiotic assemblages consisting of the host coral and symbiotic dinoflagellate algae of the genus Symbiodinium, known as zooxanthellae, which provide the host organism with photosynthetically derived nutrients and promote calcification. Environmental stressors can induce the phenomenon of bleaching, which refers to the loss of symbiotic algae or, less commonly, the degradation of algal pigments (3). Severely bleached corals can suffer depressed growth rates, increased susceptibility to disease and, at the extreme, mortality (4, 5). The cellular and molecular mechanisms that underlie the breakdown of the symbiotic partnership are still poorly understood. The study of these mechanisms is an active research area, in which a consensus is developing that a primary step in the bleaching process occurs in the zooxanthellae (3, 6).
Many investigations into the molecular mechanisms of bleaching have used experimentally bleached symbioses—that is, assemblages exposed to acute temperature stress 2–5 °C above or below ambient for 12–72 h (4). However, bleaching in the field is not usually associated with such abrupt, acute changes in temperature and usually occurs after a more gradual onset of temperature stress that may persist for several weeks (7). The extent to which the mechanisms of experimental bleaching relate to the observed declines in symbiont densities in the field is uncertain, unless these mechanisms are investigated under both experimental and field conditions. Results from our laboratory indicate that nitric oxide (NO), generated by algal nitric oxide synthase (NOS), is closely associated with coral bleaching both in artificially induced bleaching in the laboratory setting and during naturally occurring bleaching episodes in the field.
Nitric oxide synthases convert arginine to citrulline and NO (8). Zooxanthellae that have been freshly isolated from a non-stressed symbiotic relationship with a host cnidarian have a free amino acid pool that is dominated by arginine (9). In addition, these symbionts possess a low basal level of NOS activity (10). We examined the effects of elevated or depressed water temperatures that induce the breakdown of cnidarian-dinoflagellate symbioses on symbiont NOS activity, which was measured by monitoring the conversion of radiolabeled arginine to citrulline (8, 10).
We examined the effect of elevated temperature on zooxanthellar NOS activity (amol citrulline/zooxanthella/min) isolated from the branching scleractinian coral Madracis mirabilis (sensu Wells, 1973). Corals, from the coastal waters of Bermuda collected by scuba diving, were kept in seawater collected at the site and returned to the laboratory within 1 h. For temperature treatments in the laboratory, corals were maintained at ambient temperature in continuously flowing seawater for at least 48 h before treatment. Corals were subjected to temperature stress by placing them in beakers of artificial seawater (ASW; 423 mM NaCl, 26 mM MgSO4·7H2O, 23 mM MgCl2·6H2O, 9 mM KCl, 12.6 mM CaCl2·2H2O, 0.2 mM NaHCO3, 10 mM HEPES; pH 7.8). The beakers were then placed into incubators set at the desired temperatures and remained there at constant temperature and irradiance for 24 h. Algal NOS activity was determined for those zooxanthellae retained within the coral host tissue (i.e., in hospite) over the course of the incubation and for those zooxanthellae that had left the host as a result of temperature stress. For in hospite zooxanthellae, corals were removed from the incubation at selected time points; zooxanthellae were then separated from the host tissue, as described by Owen et al. (11). For determination of algal NOS activity for zooxanthellae that had left the host, algae were quantitatively collected from beakers by centrifugation. In all cases zooxanthellar numbers were determined by counting with a hemacytometer; cells were then rinsed with homogenization buffer (HB; 50 mM HEPES, 1 mM EDTA; pH 7.4, ambient temperature) and homogenized in HB, in a total volume of 1 ml, by vortexing with sterile borosilicate glass beads (a single 5-mm bead, 0.4 g 710–1180-µm beads, and 0.2 g 15–212-µm beads). Homogenates were centrifuged at 16,000 x g for 10 min, after which NOS activity in the supernatant fraction was determined by measuring conversion of the NOS substrate 3H-arginine (New England Nuclear, specific activity adjusted to 3.22 mCi/mmol) to 3H-citrulline as previously described (8, 10, 12). Results are presented as the amount of citrulline generated per cell per unit time.
Specimens of M. mirabilis that are subjected to acute heat shock in the laboratory undergo visible bleaching. After 16 h of exposure to a water temperature of 32 °C (4 degrees above the ambient temperature of 28 °C), in hospite zooxanthellae isolated from corals subjected to such treatment had higher levels of NOS activity (6.09 ± 0.60 x 10–18 mol citrulline/zooxanthella/min, n = 3) than did zooxanthellae isolated from non-heat-stressed control corals (0.13 ± 0.04 x 10–18 moles citrulline/zooxanthella/min, n = 3); this difference was significant (Student’s t = 17.31, df = 4, P < 0.0001). Time-course thermal stress experiments also showed that in hospite zooxanthellae within the coral, as well as those that had vacated the symbiosis, showed increases in algal NOS activity associated with thermal stress and the bleaching response (Fig. 1).
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Morrall and colleagues have shown that the host fractions of symbiotic cnidarians exhibit NOS activity, but that, unlike the zooxanthellae, their NOS activity decreases when they are subjected to either heat (33 °C) or cold (17 °C) shock (12). In light of our current studies, their results can be interpreted as a compensatory response of the host to the higher, possibly cytotoxic levels of symbiont-generated NO within the coral endodermal cells.
Cytotoxic reactions resulting from elevated amounts of NO can be direct and indirect. Direct reactions of NO may cause cytotoxicity by inactivating key metabolic pathways (14). The reaction of NO with photosynthetically generated molecular oxygen or superoxide also has the potential to form reactive nitrogen oxide species (RNOS), notably N2O3, the major NO autoxidation intermediate in aqueous media, and peroxynitrite (15). RNOS are generally thought to cause deleterious effects such as chemical damage to proteins and DNA and irreversible inhibition of mitochondrial respiration (15, 16, 17) by modifying important biomacromolecules. Notable key reactions of RNOS are nitration reactions (particularly of tyrosine), nitrosation of sulfhydryl-containing moieties (e.g., glutathione and cysteine), and oxidation of various substrates (17); proteins containing reduced thiol-rich environments are particularly susceptible, forming stable and biologically active protein S-nitrosothiols.
Since NO and O2 are more than 20 times more soluble in the lipid layers of cells than in aqueous media (13, 16), cell membranes are predicted to be a primary site of RNOS formation. Cell-membrane-associated proteins would be vulnerable to nitrosation and nitration reactions, forming species such as S-nitrosothiols (themselves NO-donors), nitrosamines, and nitrotyrosine. Histological studies have shown that, in addition to degradation of zooxanthellae in situ and loss of algal cells (18), loss of intact host endodermal cells containing algae can be a feature of the bleaching process; the shedding of these cells is suggested to result from cell adhesion dysfunction (19). It is worth noting that the most abundant, symbiosis-enhanced protein in the symbiotic anemone Anthopleura elegantissima has been identified as a protein that shares significant homology with the fasciclin I (Fas I) family of cysteine-containing cell adhesion proteins (20), which are themselves significant targets for RNOS nitrosative reactions.
Algal stress is a common initiating feature of the bleaching response, with reports of bleaching-associated destabilization of photosystem II D1 reaction centers and photosynthetic dysfunction (21, 22), and photosynthetic inhibition by Vibrio toxins (23). We hypothesize that upregulation of NOS activity is an important component of the zooxanthellar response to such stress. Such upregulation might be analogous to the process of induction of NOS by a number of cell types (e.g., activated macrophages) as a cytotoxic defense response against microbial infection (15). This may provide the zooxanthellae with the means to vacate the now stressful conditions of the symbiosis by induction of host cell necrosis and bleaching; the algal cells can emerge from the symbiosis in photosynthetically active, and thus presumably healthy and viable, condition (24). It is interesting to speculate that bleaching may be an adaptive response of the zooxanthellae rather than the coral host, as one aspect of a multiphased dinoflagellate life cycle.
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This article has been cited by other articles:
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  J. N. Bouchard and H. Yamasaki Heat Stress Stimulates Nitric Oxide Production in Symbiodinium microadriaticum: A Possible Linkage between Nitric Oxide and the Coral Bleaching Phenomenon Plant Cell Physiol., April 1, 2008; 49(4): 641 - 652. [Abstract] [Full Text] [PDF]
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 S. Perez and V. Weis Nitric oxide and cnidarian bleaching: an eviction notice mediates breakdown of a symbiosis J. Exp. Biol., July 15, 2006; 209(14): 2804 - 2810. [Abstract] [Full Text] [PDF]
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) and zooxanthellae released (
) from host corals were determined as described in the text. Activities are represented as means and standard deviations, in attomoles (amol; 10–18 moles) of citrulline generated per cell per minute of triplicate incubations; when no error bars are depicted, the bars lie within the graphical representation of the mean.
, 12-Mar-02, 17.8 (18.2); 
, 18-Mar-02, 20.0 (18.6); 
, 22-Aug-02, 28.4 (28.7); 
, 28-Aug-02, 29.8 (28.9); 
, 19-Sep-02, 26.9 (29.4); 
, 10-Dec-02, 20.6 (20.8); •, 12-Dec-02, 20.8 (20.8);