RESULTS AND DISCUSSION

¹⁵N enrichment in seawater suspended particles, collected from control bottles without corals or with corals but without ¹⁵N addition, was insignificantly different from the natural levels (ANOVA, P=0.098). Following 24 h of incubation, particles collected from ¹⁵N-enriched bottles, containing coral nubbins, were significantly enriched with ¹⁵N (ANOVA, P=0.001). ¹⁵N enrichment ranged from 0.006% to 0.0241% depending on the coral species incubated (Fig. 1A). These values correspond to 1.6–22 ng N l⁻¹ day⁻¹ (i.e. 0.12–1.6 nmol N l⁻¹ day⁻¹) or to 0.08–0.32 ng N cm⁻² day⁻¹ when normalized to the surface area of the coral nubbins (Fig. 1B). As seawater was filtered at the beginning of the incubation, and coral nubbins had a very small fraction of apparent skeleton, this result suggests that microorganisms contained in the freshly released mucus are able to fix N₂. Our measurements are in the same range as N₂ fixation rates measured for other oligotrophic waters (from 0.1 to 4 nmol N l⁻¹ day⁻¹) (reviewed in Ridame et al., 2013), suggesting that there is significant N₂ fixation, and that the enrichment technique used in this study allows an accurate measurement of this N₂ fixation. The %¹⁵ N atom excess values were significantly higher in particles released by S. pistillata from deep water and C. gracilis than in samples containing S. pistillata from surface waters and Porites sp. (ANOVA, P<0.01). This could be due to species-specific populations of microbes, which remain to be further studied in a larger experiment including determination of the microbial communities involved. The main finding of this study is the lack of significant nitrogen enrichment in the coral compartments, despite high rates of N₂ fixation in the incubation medium. Indeed, the %¹⁵N atom excess in the coral tissue and in the symbionts was very low (between 0.0002% and 0.0015% in both compartments, Fig. 1A) and in the same range as the standard deviation of the natural samples (0.0010%). Thus, no reliable uptake could be established for corals. This observation suggests that, in our experiment, corals did not benefit from the nitrogen fixed by diazotrophs contained in their mucus layer and released into the surrounding medium, at least within 24 h. Indeed, colonies of S. pistillata, maintained for 3 days in ¹⁵N₂-enriched medium, presented a ¹⁵N excess enrichment as high as 5% (data not shown). However, such a long incubation in a closed system may entail the death of bacterial particles and the release of ammonium and/or dissolved organic nitrogen, and abnormal release of mucus and bacteria by corals, which precludes any reliable estimations on the processes occurring on natural reefs. This study contradicts that by Lesser et al. (Lesser et al., 2007), who found some evidence that dinoflagellates in symbiosis with the coral Montastrea cavernosa were able to acquire nitrogen from N₂ fixation by diazotroph symbionts. The nature and location of these symbionts within the coral is, however, subject to debate (Oswald et al., 2007). Interpretations/conclusions from ARA experiments should be taken with care, because ARA measures total N₂ fixation without tracing the fate of the new nitrogen into the organic matter. Nutrients can sometimes be acquired from skeletal endoliths (Fine and Loya, 2002). Taken together, these observations suggest that the microbial community in coral tissues and mucus is highly dynamic and can change rapidly (Lema et al., 2012), which in turn may alter its capacity to transfer nutrients to the host.

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Fig. 1.

¹⁵N enrichment and N₂ fixation rates in Red Sea corals. (A) ¹⁵N enrichment in coral tissue [light grey bars (left)], in symbionts [white bars (middle)] and in seawater particles [dark grey bars (right)] of the three coral species: Cladopsammia gracilis, Porites sp. and Stylophora pistillata [collected at 5 m depth (shallow) and 40 m depth (deep)]. Values were multiplied by 100 for clarity. (B) N₂ fixation rates by seawater particles.

The use of ¹⁵N₂ tracer for the first time with corals has clearly revealed that diazotrophs released with the mucus of scleractinian tropical corals can present high rates of N₂ fixation. These rates are equivalent to those previously measured in other oligotrophic environments. This study, however, clearly demonstrates the lack of rapid uptake of new nitrogen by the coral host and its symbionts, although N₂ fixation occurs in their immediate surroundings. This observation thus contradicts previous interpretation from ARA experiments on corals.