The Assimilation of Diazotroph-Derived Nitrogen by Scleractinian Corals Depends on Their Metabolic Status

N₂ fixation by suspended particles.N₂ fixation by suspended particles quantified in unfiltered seawater samples (containing the natural planktonic diazotroph community) averages 9.80 ± 1.90 ng N liter⁻¹ h⁻¹. This rate is higher than those previously measured in other ¹⁵N₂-labeling studies (<1.1 ng N liter⁻¹ h⁻¹) for seawater of the northern Gulf of Aqaba (32, 33) but is in the range of those measured (10.8 ± 0.6 ng N liter⁻¹ h⁻¹) in coastal waters of New Caledonia (34). N₂ fixation rates vary significantly with the abundance and activity of diazotrophs, themselves depending on environmental factors such as temperature, depth, light, and nutrient and trace metal availability (35). Here we show that N₂ fixation rates in coral mucus samples are 12 times as high as those in unfiltered natural seawater (without mucus). This is likely due to a high diazotroph abundance in the mucus, which can be up to 400-fold higher than that in the surrounding seawater (31). Coral mucus is rich in dissolved organic C and inorganic phosphate, which is required for bacterial growth (36, 37). Once released into seawater, it stimulates the activity of both mucus- and seawater-associated bacteria by providing a suitable energy and nutrient source (37–39). Thus, the increased N₂ fixation observed in our mucus incubations could be the result of both the net release of diazotrophs into seawater and the stimulation of their activity. Our results are more pronounced than those of Camps et al. (31), who found only an insignificant 2-fold increase in N₂ fixation when coral mucus was added to seawater samples. This may result from more active diazotrophs associated with the coral mucus in the present study. Those authors also measured up to 70-fold lower planktonic N₂ fixation rates than previous measurements in the same area and season (34), also indicating that planktonic diazotrophs were less active.

During the coral incubations, suspended particles particularly released by the shallow corals showed up to 2-fold higher N₂ fixation rates than suspended particles during the mucus-alone incubations (without corals). Corals continuously release approximately half of their photosynthetically acquired C as organic matter into the surrounding seawater (40, 41). This continuous supply of fresh mucus during coral incubations may have further increased the abundance of diazotrophs and stimulated their activity, while the availability of C was likely much reduced during the 24-h incubation with mucus alone. Mucus is indeed rapidly degraded (7 to 10% h⁻¹) by bacteria (42), which, in particular, draw down its content in essential molecules such as phosphate and N (43). Our results are in agreement with the previous findings of Grover et al. (22), who also detected significant rates of N₂ fixation by suspended particles released after the incubation of coral colonies, but the rates were much lower than those in the present study. The discrepancies between the two studies might be explained by the use of the ¹⁵N₂ bubble rather than the ¹⁵N₂-enriched seawater addition method, but also by seasonal variations (end of summer versus autumn) in diazotroph composition/activity, in the seawater nutrient concentrations, or in the N needs of the coral colonies, respectively. N₂ fixation stimulated by corals is indeed highly variable on a seasonal scale, with the highest rates measured during the nutrient-depleted warm summer season (18, 19). We found the highest rates of N₂ fixation by suspended particles after the incubation of S. pistillata (bleached). This may result from a higher abundance/activity of mucus-associated diazotrophs and may be linked to an increased release of labile mucus compounds by the coral (44). Interestingly, N₂ fixation by suspended particles did not increase when a deep-water coral colony was incubated. Deep corals show reduced photosynthesis rates as a result of decreased light availability (45, 46). This may be accompanied by less C translocation from the symbiotic dinoflagellates to the host and reduced rates of organic matter release, as previously demonstrated in shallow-water corals exposed to reduced light levels (47, 48). Together, these processes may have affected the activity of diazotrophs, leading to decreased N₂ fixation rates by suspended particles. In addition, deep corals rely more on heterotrophy because of reduced photosynthesis (46). Increased heterotrophic feeding on organic matter and on diazotrophs in the mucus may all have lowered the amount of fixed N in suspended particles. Also, we detected in deep corals a higher ¹⁵N enrichment in both the animal tissue and the dinoflagellates compared to shallow corals (see discussion below). This further supports the idea that deep corals may have assimilated more suspended particles (containing diazotrophs and fixed N) from seawater as a result of increased heterotrophic feeding that, in turn, lowered the amount of fixed N in the suspended particles. Overall, it was suggested that mucus-associated diazotrophs, once released into seawater, provide an additional source of N to the pelagic and benthic reef compartments (31). In addition, our results indicate that this input of N to the coral reef may strongly differ between shallow and deep reef habitats, in relation to the autotrophic or heterotrophic status of corals.

DDN assimilation by shallow-water corals.All corals assimilated DDN into the animal tissue and the dinoflagellates, except for Alveopora species (shallow) and S. pistillata (shallow). This confirms the first observations of Grover et al. (22) on tropical corals that, under some conditions, corals do not assimilate fixed N, although this additional N source would be particularly beneficial for shallow corals. Shallow corals receive photosynthetically derived C in excess and are thus N rather than C limited. To cope with this N deficiency, shallow corals have an efficient uptake of dissolved inorganic N from seawater that is energetically fueled by high light availability (49; L. Ezzat and C. Ferrier-Pagès, unpublished data). Although we observed no DDN assimilation by shallow corals after 24 h, S. pistillata (72 h) assimilated approximately 30% of the total fixed N into the animal tissue (0.97 ± 0.14 ng N cm⁻² h⁻¹) and dinoflagellates (0.39 ± 0.08 ng N cm⁻² h⁻¹) after an incubation period of 72 h. This suggests that assimilation of DDN by shallow corals is a slow process and that N₂ fixation is associated with the released coral mucus rather than with the corals, at least in shallow reef environments. A recent study using the ¹⁵N₂-enriched seawater addition method on shallow S. pistillata corals from New Caledonia detected DDN assimilation exclusively in the dinoflagellates (~330 ng N cm⁻² h⁻¹) with assimilation rates up to 3 orders of magnitude higher than in the present study (23). Although both studies used the same incubation technique, different ¹⁵N₂ enrichment percentages (10% versus 20%) at the start of incubation and different incubation times (4 h versus 24 h) may have affected both the diazotrophic activity and the transfer of DDN into the different coral compartments. Dinoflagellates assimilate ammonium (the end product of N₂ fixation) much faster than the coral host (50). Also, Kopp et al. (51) observed a time lag of 6 h between N assimilation by the dinoflagellates and subsequent N translocation to the coral host, suggesting that incubation periods of >4 h are necessary to detect DDN assimilation in the animal tissue. Also, S. pistillata from the Red Sea may be associated with a diazotroph community that is different from or less active than that of corals from New Caledonia. Last, N₂ fixation, as well as the assimilation of dissolved inorganic N from the surrounding seawater, can strongly vary with the current environmental conditions (19, 49, 52) and can be a reason for the observed differences. In the following, we provide new insights into DDN assimilation by shallow-water corals exposed to increased phosphate levels in seawater, as well as after bleaching.

(i) Effect of phosphate availability.Phosphate availability in seawater can positively affect the N₂ fixation activity of planktonic diazotrophs (53). The high phosphorus demand for N₂ fixation can be explained by the high ATP requirements for nitrogenase function, as previously demonstrated on plants (54, 55). This phosphorus constraint can occur either because fixation uniquely requires phosphorus-rich metabolites (56–58) or because fixation allows fixers to grow and thus creates phosphorus demand (59). In addition, it was previously shown for S. pistillata that ammonium uptake from seawater increases up to 2.5-fold in the presence of 0.5 or 3.0 μM phosphate because one nutrient is limiting the use of the other (52). Since ammonium is the final product of N₂ fixation, it may explain the up to 5-fold higher DDN assimilation by the coral holobiont when it is exposed to pulses of 3 μM phosphate. Also, N₂ fixation can be significantly reduced under low-phosphate conditions (60), and background phosphate levels in the Gulf of Eilat are very low (<0.1 μM) throughout the year (32, 61). This low availability of phosphate in seawater may also explain the significant effect we found on N₂ fixation rates upon exposure to 3 μM phosphate. We cannot calculate the total N₂ fixation under increased phosphate availability because of the lack of quantification of ¹⁵N enrichment in the suspended particles released by the coral. However, total N₂ fixation by S. pistillata (phosphate) likely increased since the amount of DDN assimilated in the animal tissue and dinoflagellate compartment of S. pistillata (phosphate) already exceeded the total N₂ fixation by S. pistillata (shallow).

(ii) Effect of bleaching.In the present study, incubations of bleached coral colonies revealed the highest total N₂ fixation. The N₂ fixation by suspended particles in this experiment was twice as high as the N₂ fixation by suspended particles in the S. pistillata (shallow) incubation. Also, the DDN assimilation into tissue and dinoflagellates was greatly increased by S. pistillata (bleached) similarly to what was found for S. pistillata (phosphate). This enhanced activity of total (net) N₂ fixation is similar to what was recently found for overall (gross) N₂ fixation activities in bleached corals with the acetylene reduction assay (30). An increased seawater temperature also increases the abundance and diversity of coral-associated diazotrophs, shifting toward a more heat-resistant diazotroph community (62). Also, the bacterial community associated with the mucus undergoes drastic changes when exposed to heat stress (63). In the present study, ¹⁵N₂ incubations with S. pistillata (bleached) were conducted after the heat stress period under normal temperature conditions. Thus, increased activity of the nitrogenase enzyme due to higher temperature can be excluded, but the corals most likely experienced a shift in the diazotrophic community that could be one reason for the increased N₂ fixation rates seen (62). Furthermore, decreased photosynthesis rates in bleached corals lead to reduced O₂ levels in the animal tissue and mucus layer. This, in turn, favors the activity of the O₂-sensitive nitrogenase enzyme and can result in increased N₂ fixation rates (64). Decreased photosynthesis reduces not only the O₂ levels around the corals but also the autotrophic C supply to the host. This makes the coral more dependent on heterotrophic food sources, although the heterotrophic capacity (i.e., grazing) in bleached corals was also found to be impaired. A shift from catching larger high-energy prey items (i.e., nanoflagellates) to smaller low-energy prey (i.e., bacteria and picoflagellates) was observed in bleached S. pistillata because of the reduced energy required for catching nonmotile bacteria (65). Consequently, increased grazing on bacteria from the surrounding water may explain the higher DDN assimilation into the tissue of bleached corals observed here. Moreover, net organic matter fluxes in S. pistillata (bleached) changed from net uptake toward net release likely to reduce the effects of photoinhibition or cell membrane disruption (65–67). Increased release of organic matter can provide additional energy to seawater and mucus-associated diazotrophs, subsequently stimulating their abundance and/or N₂ fixation activity in the surrounding water. Overall, the results show not only that total N₂ fixation increases in incubations with bleached versus nonbleached corals but also that bleached corals assimilate more DDN. To what extent this additional N is beneficial for the recovery of the coral host after a bleaching event needs further investigation.

DDN assimilation by deep-water corals.Overall, the total N₂ fixation was ~2-fold higher in incubations of S. pistillata (deep) than in incubations of S. pistillata (shallow or 72 h). One explanation for the increased N₂ fixation activity with depth may be reduced light availability (350 versus 20 μmol m⁻² s⁻¹) leading to reduced O₂ production via photosynthesis in corals (46). On a diurnal scale, coral-associated N₂ fixation was found to be highest during twilight, when O₂ production is not too high to inhibit nitrogenase, the enzyme responsible for N₂ fixation (68, 69), but high enough for corals to deliver photosynthetic energy to the diazotrophs (15). Thus, the diurnal window with optimal O₂ levels for N₂ fixation may be greatly increased with increasing water depth, leading to higher N₂ fixation rates. Previously, the abundance of scleractinian corals (Montastraea cavernosa, Bahamas) associated with diazotrophs was found to correlate positively with increasing water depth (15). This suggests that the diazotrophic community composition associated with corals may vary with depth and that the assimilation of DDN by corals may increase with depth as well. Also, the natural δ¹⁵N isotopic signature of scleractinian corals tends to be depleted as depth increases (70, 71). This can result from the assimilation of isotopically depleted DDN or from the uptake of isotopically depleted nitrate derived from atmospheric deposition, as for the Gulf of Aqaba/Eilat (72, 73). Here we show not only that the total N₂ fixation increased in the incubations with deep versus shallow corals but also that deep corals assimilated 80% (8.65 ng N cm⁻² h⁻¹) of the total fixed N into the animal tissue or dinoflagellates. In contrast, shallow corals assimilated only 0% or 30% (1.36 ng N cm⁻² h⁻¹) of the total fixed N when incubated for 24 or 72 h, respectively. The higher assimilation by deep corals may result from a shift in the heterotrophic status of the coral. Corals in mesophotic reef environments generally rely more on other trophic strategies (i.e., heterotrophy) than their shallow counterparts in order to meet their metabolic demand and to compensate for the reduced photosynthetic C supply from their symbiotic dinoflagellates (45, 74). We found that the DDN assimilation by shallow and deep corals is inversely correlated with the amount of fixed N quantified in suspended particles in the coral incubation. Furthermore, the amount of fixed N in suspended particles was much smaller after the incubation of deep coral colonies than after the incubation alone of mucus extracted from deep coral colonies. This indicates that the high DDN assimilation into the tissue and dinoflagellates of deep corals is derived mainly via increased heterotrophic feeding on suspended particles rich in fixed N and diazotrophs that have been associated with the mucus. Although deep corals are considered to be less N limited by receiving both C and N from heterotrophic sources, they assimilate more DDN than their shallow counterparts. This could be explained by the fact that deep corals are light limited not only for photosynthesis but also for the uptake of dissolved inorganic N (49; Ezzat and Ferrier-Pagès, unpublished). Therefore, the increased DDN assimilation by deep corals may help to compensate for the reduced inorganic N uptake and may be particularly beneficial during periods when organic particles in seawater are insufficient to fully cover their N demand. The ingestion of planktonic free-living diazotrophs can also provide a substantial amount of N to the coral host, even exceeding the N input from coral-associated diazotrophs (23). Thus, it will be interesting to also investigate the importance of planktonic free-living diazotrophs for the N budget of mesophotic corals.

Contribution of DDN to the coral N budget.Tropical corals are generally well adapted to flourish in nutrient-poor waters by efficiently recycling ammonium between the animal host and symbiotic dinoflagellates, by heterotrophic feeding on organic N sources, and by actively scavenging dissolved inorganic N from seawater even at very low dissolved N concentrations. In order to estimate the importance of DDN in relation to other inorganic N sources, we compared the present results with data on rates of inorganic N uptake by corals. The rate of ammonium uptake by S. pistillata at nearly natural ambient concentrations (0.2 μM) was ca. 30 ng N cm⁻² h⁻¹, taking into account the animal and dinoflagellate assimilation rates, except for highly fed corals, for which the uptake was lower (49). In addition, the total rate of nitrate uptake by S. pistillata at the same low concentrations was equal to 1.5 ng N cm⁻² h⁻¹ (75). Overall, dissolved inorganic N contributes for a mean of 30 to 32 ng N cm⁻² h⁻¹, although these rates are an approximation, as they vary with light or temperature levels, as well as with other physicochemical and biological factors. If we assume similar inorganic N uptake rates for all of the corals in the present study, DDN can contribute up to ~1.4 ng N cm⁻² h⁻¹ in shallow corals or ca. 5% of the inorganic N uptake. This contribution increases up to ~6 ng N cm⁻² h⁻¹ in bleached or phosphate-enriched corals and up to ~9.5 ng N cm⁻² h⁻¹ in deep corals, representing ca. 20 and 30% of the inorganic N uptake, respectively. In deep corals, with respect to their reduced inorganic N uptake capacity, this contribution is likely to be higher, underlining the importance of DDN for the N requirements of deep corals in relation to other inorganic N sources. As the ambient inorganic N concentrations on tropical coral reefs are often too low to sustain net growth of the coral holobiont (49), the association with diazotrophs likely serves as additional adaptation of corals to overcome N limitation in oligotrophic reef environments. In order to calculate the contribution of DDN to cover the N demand of deep and shallow corals, we used gross photosynthesis rates, C/N ratios, and assimilation rates of photosynthetically acquired C from previous studies with S. pistillata corals from the Red Sea exposed to high- and low-light conditions (45, 48, 70). This shows that DDN contributes only ~1% of the total N demand of shallow corals, while it can increase up to ~15% in deep corals (Fig. 3). Furthermore, in deep corals, DDN satisfies the N demand of dinoflagellates (~26%) to a greater extent than the N demand of the animal host (~9%), likely because of more efficient DDN assimilation by the former. Dinoflagellates can use N₂ fixation products in the form of ammonium much faster than the coral host, as previously demonstrated for ammonium uptake from seawater (50). Instead, the coral host assimilates DDN after the synthesis of host macromolecules deriving from prey digestion or after translocation of recycled DDN by dinoflagellates (23). The higher contribution in deep corals is likely a product of both their higher DDN assimilation and their lower N demand, indicating that deep corals are likely less N limited than shallow corals (76). Overall, the present study shows that DDN can contribute importantly to the overall N budget of mesophotic corals, but whether this additional N is beneficial for the resistance and recovery of deep corals from environmental disturbances requires further investigation.

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FIG 3 

Overview of the percent contribution of DDN assimilation to the N demand (ND) of the animal host (CAND), the dinoflagellates (CDND), and the total symbiotic association (CTND) from shallow and deep S. pistillata corals. The ND was calculated from gross photosynthesis (P_gross) measurements (45), from values of C incorporation into the animal host and the dinoflagellates (48), and from C/N ratios (70) of S. pistillata corals at 5- and 50-m water depths in the Red Sea.