Over the years, the acetylene reduction assay (ARA) has been widely used to quantify N₂ fixation of coral holobiont due to its easy operation and low cost (Crossland and Barnes, 1976; Williams et al., 1987; Shashar et al., 1994a; Shashar et al., 1994b; Davey et al., 2007; Lesser et al., 2007; Rädecker et al., 2014; Bednarz et al., 2015; Cardini et al., 2015; Cardini et al., 2016a; Cardini et al., 2016b; Bednarz et al., 2018; Tilstra et al., 2019a; El-Khaled et al., 2020b). However, ARA cannot provide DDN (Diazotroph-Derived Nitrogen) measurement because its measurement object is C₂H₄ gas derived from the whole coral holobiont in the incubator. Grover et al. were the first to use the ¹⁵N tracer method (the bubble method (Montoya et al., 1996)) to measure the symbiotic N₂ fixation rates of coral holobionts (Grover et al., 2014). The ¹⁵N tracer method not only provides net N₂ fixation rates but also enables to trace the fate and assimilation of DDN within different coral compartments. Currently, there are three protocols to measure N₂ fixation using ¹⁵N₂ tracer: the bubble method, dissolution method, and the modified bubble method (reviewed in (Wilson et al., 2012; White et al., 2020)). However, the bubble method underestimates the N₂ fixation rates seriously due to the slow N₂ dissolution. While the modified bubble method is less preferred since it requires shaking incubator, which may interfere coral activity. Therefore, the most popular method is the dissolution method. Inevitably, the dissolution method has the potential to introduce trace metal contamination and alter pH and alkalinity, which can bias the results (White et al., 2020).

N₂ fixation of coral holobionts have temporal and spatial variation. From temporal aspect, diazotrophs (Lema et al., 2014; Zhang et al., 2016; Cai et al., 2018) and N₂ fixation rates (Bednarz et al., 2015; Cardini et al., 2015; Cardini et al., 2016a; Bednarz et al., 2018) in coral holobiont show significant seasonal variation regulated by environmental factors. For example, in summer season with lower DIN supply and stronger light relative to winter, the rates of N₂ fixation in coral holobionts are higher correspondingly (Bednarz et al., 2015; Cardini et al., 2015; Cardini et al., 2016a; Bednarz et al., 2018; Cai et al., 2018; Tong et al., 2020). An opposite case presented by Bednarz (Bednarz et al., 2021) showed that N₂ fixation is detectable in winter under opposite environmental conditions, but not detected in summer, suggesting extra factors, such as food availability may also influence coral holobionts’ N₂ fixation.

Besides seasonality, diel light cycle also modulates N₂ fixation of coral holobionts. Most research results show higher N₂ fixation rates in the light compared with the dark (Crossland and Barnes, 1976; Williams et al., 1987; Shashar et al., 1994a; Rädecker et al., 2014; Cardini et al., 2016b; Zhang et al., 2016), yet, the light stimulation is likely indirect. More specifically, light provide energy for photosynthesis in algae, and the supply of photosynthate promotes N₂ fixation of heterotrophic bacterial diazotrophs (HBDs) subsequently (Shashar et al., 1994a). This statement is supported by molecular biology showing HBDs such as α and γ-proteobacteria are the main diazotrophs in many coral holobionts (Olson et al., 2009; Lema et al., 2014; Santos et al., 2014; Bednarz et al., 2019; Bednarz et al., 2021; Glaze et al., 2022; Sheng et al., 2023) and by manipulation experiments, which showed N₂ fixation activity was enhanced after adding DOC (Pogoreutz et al., 2017a). Couple researches showed that there was no significant difference of N₂ fixation rates between the day and night (Cardini et al., 2016b; Lesser et al., 2018; Glaze et al., 2022), such phenomenon can be explained by sufficient carbon provision to HBDs in coral holobionts in the night. Interestingly, Lesser’s experiments not only show a higher rate at night but also the second peak of N₂ fixation between 6 a.m. and 8 a.m. (Lesser et al., 2007). That may be explained by the effect of oxygen, an important factor controlling N₂ fixation, although it has become clear that different diazotrophs could adopt different strategies to combat oxygen damage (Gallon, 1981; Zehr and Capone, 2020). For oceanic N₂ fixation, there are still amounts of unknowns about HBDs themselves’ physiological information and their relative roles and contribution (Zehr and Capone, 2020), however, their wide-distributed and high-diverse characteristics suggest HBDs definitely play an important role (Delmont et al., 2018; Salazar et al., 2019; Zehr and Capone, 2020). The existing limited evidences show that HBDs are largely associated with organic-rich and low-DO environment (Pedersen et al., 2018; Farnelid et al., 2019). Actually, that is exactly consistent with the microenvironment that coral holobionts provide. The specific mechanism of the diel variation of N₂ fixation in coral holobiont is still not clear, but the inconsistencies suggests that the mechanism is far more complex than we imagined. In deeper waters where environment is relatively stable, N₂ fixation showed less seasonality (Bednarz et al., 2018), and reasonably less diel variation.

Here, we compiled data of global N₂ fixation rates in coral holobionts ( Figure 1 ). It is worth mentioning that the N₂ fixation rates reported in “¹⁵N based studies” only reflect the rates in bulk tissue, which includes both algal symbiont and coral host tissue. However, N₂ fixation in coral skeleton may also play a non-negligible role for the coral holobiont (Bednarz et al., 2019; Bednarz et al., 2021; Moynihan et al., 2022). Some studies have found diazotrophs in coral skeletons (Yang et al., 2016; Yang et al., 2019) and measured nitrogen fixation rates (Shashar et al., 1994a; Bednarz et al., 2019; Bednarz et al., 2021; Moynihan et al., 2022). Existing literature indicates that the nitrogen fixation rate in coral skeletons can be 20% to 300% of the rate in bulk tissues (Bednarz et al., 2019; Bednarz et al., 2021; Moynihan et al., 2022). However, due to the limited research on this topic, we will not delve into it further here. Nonetheless, we call on coral scientists to pay attention to the role of coral skeletons. Additionally, while it is unclear whether coral-released mucus benefits the coral holobiont itself, it does stimulate N₂ fixation process in the mucus and water column (Rädecker et al., 2014).

Spatially, there are significant differences in N₂ fixation rates in coral holobionts within and between regions ( Figure 1 ), which can be attributed to coral heterogeneity (Pogoreutz et al., 2017b), environmental condition such as light (Lesser et al., 2018), and food availability (Benavides et al., 2016; Pupier et al., 2019; El-Khaled et al., 2020b). In Figure 1 , the median of 4 boxes (ARA based studies: Red sea; ¹⁵N based studies: Red sea, the Great Barrier Reef and New Caledonian) analyzed are 13.2, 10.5, 225.6, 2.4 μmol N m^-2d^-1 respectively. For ¹⁵N based studies, N₂ fixation rates in the Great Barrier Reef were relatively higher than those in Red Sea and New Caledonian (P< 0.05), probably indicating that the corals in the Great Barrier Reef are more autotrophic and require more nitrogen compensation. There is also a significant difference in the amount of data available for N₂ fixation rates between different regions ( Figure 1 ), which is inseparable from the convenience of research in certain areas and the level of attention paid to coral-related research. The Coral Triangle and the coastal areas of East Asia have high coral abundance and diversity (Veron et al., 2015), but there is a lack of sufficient research, especially the Coral Triangle, which is significantly impacted by human activities (Andrello et al., 2022). Therefore, in addition to emphasizing conservation efforts, it is important to increase research in these regions.

On the vertical scale, the research results show consistency basically. The relative nifH abundance associated with coral holobionts decreased with the water depth (Tilstra et al., 2019b; Bednarz et al., 2021), but the assimilation of DDN increased (Grover et al., 2014; Bednarz et al., 2017; Bednarz et al., 2021), especially in winter (Bednarz et al., 2018). That is explained by the shift of trophic strategy for coral holobionts as depth increases (Bednarz et al., 2017; Tilstra et al., 2019b). Mesophotic coral holobionts generally rely on heterotrophy, which may result more DDN assimilation from heterotrophic path. However, the profile of N₂ fixation in coral holobionts is affected by multiple factors, thus, in some cases N₂ fixation rates show no significant effect of depth (Lesser et al., 2018) even may decrease with depth in summer due to the enhancement of N₂ fixation of coral holobionts in the upper layer (Bednarz et al., 2018).

Recently, the impact of environmental stresses on the N₂ fixation of coral holobionts has garnered significant attention. For the heat stress, studies have shown an increase in the abundances and diversities of diazotrophs and the total N₂ fixation activity (from ARA) in coral holobiont, especially during the daytime (Santos et al., 2014; Cardini et al., 2016b; Radecker et al., 2022). However, the net N₂ fixation rates were found to decrease in parallel experiments using ¹⁵N₂, indicating reduced utilization efficiency of DDN by coral holobionts (Radecker et al., 2022). For acidification, which mainly focused on pCO₂ elevation, inconsistent results were documented. Some studies demonstrate a decrease in N₂ fixation rates as pCO₂ increased, explained by the reallocation of energy for calcification (Rädecker et al., 2014; Zheng et al., 2021). However, some field studies showed that N₂ fixation rates and diazotrophs in coral holobionts were higher in high pCO₂ sites than low pCO₂ sites (Biagi et al., 2020; Meunier et al., 2021b; Prada et al., 2023), such enhancement of N₂ fixation in space was attributable to the higher N requirements induced by the enhanced photosynthesis under high pCO₂ condition (Biagi et al., 2020). These contrasting findings were not surprising, since the effects of acidification, including pCO2 and pH effect, on N₂ fixation is a complicated issue (Hutchins et al., 2015; Hong et al., 2017). For nutrient stress, elevated nutrient levels may stimulate N₂ fixation (Bednarz et al., 2017; El-Khaled et al., 2020b) after coral holobionts’ reorganizing in-and-out nitrogen pathways. The above results indicate that diazotrophs in coral holobionts could take different strategies to counter various adverse circumstances.

Oceanic environment changes, such as warming (Schoepf et al., 2015), acidification (Anthony et al., 2008), eutrophication (Hall et al., 2018) and deoxygenation (Johnson et al., 2021; Alderdice et al., 2022), all may lead to coral bleaching after transgressing a certain threshold eventually. Researches have shown that after bleaching coral holobionts’ N₂ fixation increases dozens of times due to occupation of some pioneer communities on coral substrate (Larkum, 1988; Davey et al., 2007; Bednarz et al., 2019; Meunier et al., 2019; Roth et al., 2020), and such enhancement of N₂ fixation is crucial for coral recovery and coral reef succession. Obviously, the sustainability and resilience of coral system depends not only to understand and predict the persistence of coral reefs. Further studies are necessary to subdivide the effects of different nutrients, such as NO 3 − and phosphorus, to avoid coordinated effect. Furthermore, investigating the relationship between diazotrophs and other coral compartments’ response to environmental change is essential.

Nitrification is crucial process for coral holobionts, involving two steps of ammonia oxidation and nitrite oxidation. Ammonia oxidation converts NH₃/ NH 4 + to NO 2 − by either ammonia oxidation archaea (AOA) or ammonia oxidation bacteria (AOB). Nitrite oxidation then further converts NO 2 − to NO 3 − by nitrite oxidation bacteria (NOB). This process helps to reduce their internal NH 4 + levels to maintain the growth of symbiotic algae stable, meanwhile, retain the dissolved inorganic nitrogen in oxidized state (Radecker et al., 2015).

Actually, molecular biological evidences shows that ammonia oxidation occurs in coral tissues (Wafar et al., 1990; Yang et al., 2013), mucus (Siboni et al., 2008; Kimes et al., 2010) and skeletons (Risk and Muller, 1983). Furthermore, different coral holobionts in various areas may contain varying species and abundance of ammonia-oxidizing organisms, e.g., solely AOA (Beman et al., 2007; Siboni et al., 2008; Siboni et al., 2012), solely AOB (Yang et al., 2013), AOA coexisted with AOB (Zhang et al., 2015; Zhang et al., 2021) and none (Beman et al., 2007), although the survival strategies of AOA and AOB are completely different. It remains unclear what determines the habitat to host AOA or/and AOB in coral holobionts, which requires further studied.

Molecular biology evidences suggest that coral holobionts have the potential to conduct nitrification, but it remains uncertain whether they actually do so. However, there have been limited quantitative studies on the topic. In 1990, Wafar et al. were the first to quantify the gross nitrification rate in coral holobionts after holding in the dark in running seawater for 1 or 2 d to minimize dark uptake of NH 4 + by the symbiotic algae, which was found to be on average of 9. 4 ± 6. 0 nmol (mg coral tissue N) ^-1 h^-1, accounting for even up to 17% of the total NH 4 + consumption (Wafar et al., 1990). This suggests a potential competitive relationship between nitrification and the uptake process of symbiotic algae. However, until 2021, there have been other quantitative studies on the nitrification rate in coral holobionts. Babbin and Glaze measured the net nitrification in coral holobiont using ¹⁵N tracer method and their results were less than 2 μmol N m^-2 d^-1 and an average 2. 70 µmol N m^-2 d^-1 respectively (Babbin et al., 2021; Glaze et al., 2022). However, their studies did not report the uptake rate, so the percentage of nitrification to total NH 4 + consumption remains unknown.

Furthermore, Glaze’s experiment demonstrated that the rate of dark incubation (4. 75 µmol N m^-2 d^-1) was significantly greater than the rate observed in light (0. 65 µmol N m^-2 d^-1) (Glaze et al., 2022). This finding is in line with our current understanding of the photoinhibition on the ammonia oxidation process (Schoen and Engel, 1962; Merbt et al., 2012; Horak et al., 2018; Glaze et al., 2022). However, in comparison to other processes such as the well-studied N₂ fixation, these rates are relatively low. Thus, upcoming research efforts should concentrate on examining the allocation and condition of the NH 4 + consumption pathway to verify the role of nitrification.

Denitrification is a complex process that involves a sequence of four steps, whereby the NO 3 − is reduced to NO 2 − , NO, N₂O and finally N₂, with each step corresponding to different reductase genes (nas/narG/napA/euk-nr, nir, norB, nosZ). The significance of denitrification for coral holobionts may lie in the fact that, during the night, when the nitrification is more intense (Glaze et al., 2022), corals convert excess NH 4 + to NO 2 − and NO 3 − through nitrification, while anoxic microenvironment causes denitrifying bacteria to expel nitrogen from the coral holobiont. By coupling of nitrification and denitrification, corals can not only survive when the nutrient level increases, but also avoid the excessive growth of symbiotic algae in coral holobionts (Siboni et al., 2008; Radecker et al., 2015). Research on denitrification in coral holobionts began relatively late compared to nitrification. With the advent of molecular biology, it was gradually discovered that there are denitrifying microorganisms present in coral holobionts (Siboni et al., 2008), including in tissues and mucus (Kimes et al., 2010). Further studies have shown that different corals or even different color morphs of the same coral, have different types and numbers of denitrifying-related genes (Yang et al., 2013; Lesser et al., 2019; Tilstra et al., 2019a). It is now widely accepted that coral holobionts have the potential to perform denitrification.

As for quantitative studies, currently, there are the two methods used to measure denitrification in coral holobionts, ¹⁵N tracer method and COBRA (a combination of acetylene blockage analysis and acetylene reduction assay) (El-Khaled et al., 2020a). Acetylene blockage analysis uses the property of acetylene to inhibit the reduction of N₂O, which leads to the accumulation of N₂O in the incubation system. The denitrification rate can be calculated by measuring the change in N₂O content using gas chromatography (Balderston et al., 1976; Yoshinari and Knowles, 1976). Recent studies have measured the denitrification rate of coral holobionts (Middelburg et al., 2015; El-Khaled et al., 2020a; El-Khaled et al., 2021a; El-Khaled et al., 2021b; Zhang et al., 2021; Glaze et al., 2022), and almost all samples have detected denitrification, indicating that this process is widespread in coral holobionts (Tilstra et al., 2019a; El-Khaled et al., 2020b; Babbin et al., 2021; El-Khaled et al., 2021b; Glaze et al., 2022). The results showed higher rates of denitrification at night, which may be closely related to lower oxygen concentrations, although not all corals exhibit this characteristic (Babbin et al., 2021; Glaze et al., 2022). Interestingly, Glaze’s results indicate NO 3 − stimulates the production of the combined gases of NO, N₂O and N₂ less than NH 4 + (Glaze et al., 2022). From a symbiotic perspective, NH 4 + can stimulate nitrogen-related gas production to reduce the nitrogen content in the holobiont, which may be an important part of coral internal regulation to maintain the density of symbiotic algae and homeostasis.

Figure 2 presents the denitrification and nitrification rates of coral holobionts. The median rates for the 4 boxes (Denitrification: Red sea, the Great Barrier Reef and Caribbean Sea and Bahamas; Nitrification: the Great Barrier Reef) are 1.8, 13.3, 0.4 and 3.4 μmol N m^-2 d^-1 respectively. Although the range of these rates is smaller than that of N₂ fixation, they still show differ by orders of magnitude, possibly due to coral heterogeneity, regional differences and variations in denitrification rate measurement protocols ( Table S2 ).

The relationship of denitrification and N₂ fixation is also discussed these years. Previous studies have shown a positive correlation between denitrification and N₂ fixation rates in coral holobiont, suggesting the balance between denitrification and N₂ fixation process in coral holobionts (Tilstra et al., 2019a). Furthermore, Tilstra et al. (El-Khaled et al., 2021b) found that nirS/nifH of different coral species was positively correlated with the concentration of NO 3 − in the environment, suggesting the availability of environmental NO 3 − could drive the changes of denitrification and N₂ fixation in coral holobionts. Further experiment has confirmed that denitrification of coral holobionts is enhanced with the increase of nutrient concentration (El-Khaled et al., 2020b). Additionally, Redfield ratio of coral holobionts may be an important index presenting the metabolic balance of coral, which is also used to explain the balance of denitrification and N₂ fixation (Tilstra et al., 2019a; El-Khaled et al., 2021b).

Furthermore, Xiang et al. found when the external environment had an increased DOC concentration, the C/N ratio of corals increased, while the abundance of denitrifying organisms in coral holobionts decreased by an order of magnitude (Xiang et al., 2022). Hypotheses for this phenomenon were presented by Xiang et al.: (1) The abundance of denitrifying organisms is mainly controlled by the availability of nitrogen, so they grow normally under nitrogen restriction even if DOC content is increased. (2) Different species of denitrifying organisms prefer different carbon sources, and using glucose as the only carbon source in the experiment may affect the experimental results (Xiang et al., 2022). Together, these funding suggested that changes in the external environment may cause Redfield ratio changes in coral holobionts, thereby promoting changes in the denitrification and N₂ fixation to maintain the original ratio.

It is possible that denitrifying organisms within coral holobiont may be opportunistic rather than dominant. As ocean nutrient levels continue to rise, denitrification is likely to play a more significant role in regulating the carbon and nitrogen balance of coral holobionts. It is important to consider whether corals can actively promote changes in denitrification and N₂ fixation to maintain homeostasis under varying environmental conditions, and this should be discussed in future studies.

DNRA (dissimilatory nitrate reduction to ammonium) is a process reducing NO 3 − to NH 4 + (Burgin and Hamilton, 2007). This pathway has the potential to enhance the efficiency of internal nitrogen circulation within coral holobionts, resulting in the retention of dissolved inorganic nitrogen in the form of NH 4 + . When coupled with nitrification, this bidirectional process helps to maintain a stable NH 4 + level, promoting healthy coral growth. However, direct measurement of DNRA in coral holobionts using ¹⁵N tracer method is challenging, and therefore, there are limited studies on this process. Molecular evidences showed that DNRA-associated organisms such as fungi and vibrios, are common in coral holobionts, but their prevalence varies with coral species, regions and depths (Wegley et al., 2007; Zhang et al., 2015; Babbin et al., 2021; Bednarz et al., 2021; Zhang et al., 2021; Glaze et al., 2022). Some researches showed that the DNRA-associated genes can account for up to ~12% of the nitrogen-associated genes (Glaze et al., 2022), underscoring the importance of DNRA. Indirect calculation using NO 3 − mass balance models has shown that the DNRA rates obtained at night could reach 460–600 µmol N m^-2 d^-1, assuming nitrification is less than 1% of ammonia assimilation (Glaze et al., 2022). However, due to the considerable variation of nitrogen processes rates within coral holobionts, the indirect rate obtained was with great uncertainty.

ANAMMOX (anaerobic ammonium oxidation) is a reaction between NO 2 − and NH 4 + that produces N₂, which is currently thought to occur only in a strictly anoxic environment (Dalsgaard et al., 2005). ANAMMOX has been hypothesized as a possible means of nitrogen removal for coral holobionts (Radecker et al., 2015). However, only a few studies have been conducted on this topic, and they have found that ANAMMOX-associated genes exist in small amounts in coral holobionts in South China Sea. These studies also found a weak positive correlation between ANAMMOX-associated genes and NH 4 + concentration (Zhang et al., 2015; Zhang et al., 2021). In contrast, other studies have not found any evidences of ANAMMOX in coral holobionts (Babbin et al., 2021; Glaze et al., 2022). These findings suggest that ANAMMOX may not play an important role in coral holobionts.

Mixotrophy, the primary mechanism by which coral holobionts acquire energy, has been extensively studied for its role in coral physiology. However, there is still much to be learned about nitrogen fixation, which has only recently begun to be explored in depth. Current investigations have been limited to different regions, seasons, and responses to environmental stress. Quantitative research on nitrification and denitrification processes has just begun, while DNRA and other processes have yet to receive sufficient attention. Based on the number of studies published on various nitrogen processes related to corals (as shown in Figure 3 ), we can gain some insight into the above-mentioned issue. The amount of researches on assimilation and N₂ fixation related to coral is the largest, which is closely related to their long research history and their important role in balance nutrition, while the studies about nitrification and other processes is less significantly due to technical difficulties. We still need to strengthen further nitrogen research of coral holobionts in the future. So that we can know more about the influence of the different processes on coral health, which is crucial for understanding how coral holobionts build and collapse.

Coral holobionts are complex ecosystems consisting of the coral host and a diverse range of microorganisms. Below we listed some of the critical factors to consider when designing and conducting experiments in order to obtain accurate and reliable data for studying this ecosystem.

Recent experiment designs have been closer to real in situ conditions, such as pulsed nutrient addition (Van Der Zande et al., 2021), fluctuation of pCO₂ control (Jiang et al., 2018), and short-term acute heat stress (Guan et al., 2020). These experiments have produced results significantly different from those maintaining constant external conditions, providing a more accurate assessment of the impact of the environment on corals. Hence, researchers should focus more on these types of experiments.

Nitrogen processes in coral holobiont are dynamic, and different phenomena may be observed in a longer incubation time, just like sheng et al.’s (Sheng et al., 2023) experiment results show. Therefore, new ideas and methods need to be developed to explore more detailed processes in the future. For example, future experiments exploring nitrogen processes in coral holobionts should set more time points in the sequential experiment to estimate nitrogen migration and transformation rates. And as various processes occur simultaneously in coral holobiont, this brings some difficulties in analyzing a single path. Researches can also use isotope matrix methods, which proposed by Xu et al., in 2017 for the quantification of simultaneous multi-path nitrogen flow (Xu et al., 2017), to study coral holobionts and clarify the respective contributions of each path under different environmental stresses.

Maintaining the appropriate balance of environmental factors in incubation experiments is crucial. Some experiments require long incubation periods in a gas-tight incubator, which can cause a sharp decrease in dissolved oxygen levels. This low level of dissolved oxygen can affect the redox-sensitive processes like nitrification (Bristow et al., 2016) and denitrification. Therefore, it is important to pay attention to the synergistic control of environmental factors in the incubation experiments, for example, maintaining a suitable balance between oxygen consumption rate and incubator volume.

Enhancing comparability across experiments and data is a critical challenge in coral research, given that the coral holobiont is a meta-organism with multiple dimensions. This complexity creates numerous design and operational choices for coral experiments compared to other marine investigations. For instance, heat-stress experiments require careful consideration of several factors, such as exposure duration, light conditions, feeding regimes, collection, preservation, and laboratory operation as highlighted by existing reviews (Mclachlan et al., 2020; Grottoli et al., 2021; Mclachlan et al., 2021; Vega Thurber et al., 2022). Furthermore, quantitative normalization is fundamental for data analysis. Different studies have used various normalization units, like cross-section area, dry weight, nitrogen content of coral tissue, and coral surface area. The mainstream method is to normalize tothe surface area, representing the habitat of coral microbes. At present, several methods are available for measuring surface area, including simple geometry, advanced geometry, aluminum foil, methylene blue dye, secondary wax drops, primary wax drops, CT, and laser scanning. Therefore, it is recommended to measure the coral surface area at least as a constant quantitative indicator. This approach would help standardize the results across different studies and avoid the confusion that may arise from using different normalization units.

Research on nitrogen processes, such as nitrification, denitrification, and DNRA, in coral holobionts is still limited and requires futher efforts. In addition, there is a lack of research that measures all nitrogen processes simultaneously in coral holobionts, even for model species. The heterogeneity of coral holobionts creates great uncertainty in what we have learned about nitrogen fluxes. Therefore, future work on fundamental nitrogen flux of coral holobiont still need to be strengthened.

Understanding the short-term responses of corals to turbulent environments on diurnal and tidal timescales, as well as regulation of internal nitrogen cycle, is essential to explain the inconsistencies in previous studies. Invitigations into the effects of long-term environmental changes, such as warming (Radecker et al., 2021), acidification (Hoegh-Guldberg et al., 2017), eutrophication (Zhao et al., 2021), hypoxia (Altieri et al., 2021; Hughes et al., 2022), and synergistic effects of those factors (Donovan et al., 2020; Zhang et al., 2023), are also critical for predicting future of coral reefs. However, these investigations should be treated differently and their differences and connections should be clarifed by purposeful experiments in future.

Given the dynamic nature of coral holobionts including symbiont transfer and symbiosis evolution, it is currently challenging to quantify relative contributions of different factors to coral holobiont function, making it difficult to predict future impacts accurately. Thus, researchers must remain attentive to new technologies, and the development and utilization of new tools and methods may represent important breakthroughs in furthering our understanding of coral holobionts.