Differential responses of hard coral Montipora digitata and soft coral Xenia umbellata to nutrient stoichiometry under heat stress

Pulsation rate measurements

Pulsation rates of X. umbellata colonies were monitored on days 0, 14, and 35. Each measurement day, we randomly selected three polyps from a marked colony in each experimental tank and measured pulsation according to the method described by Vollstedt et al. (2020). Measurements were always conducted in the morning to minimize the influence of circadian rhythms. Pulsations were counted over 30 s and then converted to a standard unit of beats per minute (beats min⁻¹). Only complete tentacle contractions (open–fully closed–open) were considered. Average pulsation rates were calculated per colony, providing four independent replicates per treatment.

Symbiodiniaceae density and Chlorophyll a concentration measurements

Coral fragments were sampled on days 0, 14, and 35 with one random colony chosen per species and tank, removed from their plug, rinsed with demineralized water, and frozen at −20 °C until further analysis. Surface area (cm²) was calculated for the normalization of both Symbiodiniaceae density and chlorophyll a concentration measurements. For X. umbellata, the number of polyps in a colony was counted 3 times using tweezers, and the mean number of polyps was then multiplied by an internal surface area lab standard (0.4988 cm² per polyp), following the method developed by Bednarz et al. (2012). For M. digitata the surface area was determined using the advanced geometry method by Naumann et al. (2009), measuring the length, as well as the minimum and maximum diameter in the center of the respective fragment. If there were several branches in a sample, all were measured individually, and the surface areas were added together. Surface area was then calculated using the following formulas:

(1)${\displaystyle r=\frac{1}{2}\ast d}$ (2)${\displaystyle A=2\ast \pi \ast h\ast r}$

with d = diameter, A = surface area, h = height and r = radius.

Coral tissue for further analysis was obtained by homogenization in demineralized water using a high-speed homogenizer (VEVOR, FSH-2A) for X. umbellata, and airbrushing of coral fragments with demineralized water (DM) and subsequent homogenization for M. digitata fragments. After homogenisation, two subsamples of two mL each were transferred into Eppendorf cups and centrifuged for 10 min to separate coral tissue from algal cells. Supernatants were discarded and the pellets resuspended in two mL DM and centrifuged for another 10 min. Supernatants were discarded and the pellets frozen until further analysis.

Symbiodiniaceae density was analysed using the haemocytometer counting method (LeGresley & McDermott, 2010). We resuspended the pellets of the subsamples in two mL DM and mixed them thoroughly using a pipette and a vortex. Afterwards, 10 µL were transferred into both chambers of a glass haemocytometer (Improved Neubauer counting chamber, depth 0.1 mm) for two replicate counts of each sample. Counts were then adjusted to the initial sample volume and surface area to determine Symbiodiniaceae density of the colony.

All extraction steps for chlorophyll a (Chl a) analysis were conducted under dark conditions. We resuspended the pellets of the samples in 90% acetone by using a pipette and a vortex to extract the chlorophyll from the algal cells. Samples were then incubated at 4 °C under dark conditions for 24 h. Subsequently we centrifuged samples for 5 minutes and then transferred one mL each into glass cuvettes, allowing for two replicate readings of each sample. Afterwards, we measured Chl a concentrations using a photometer (Turner Design Trilogy Laboratory Fluorometer; excitation: 436 nm; emission: 685 nm) with a respective module for extracted Chl a (non-acidic). Concentrations were standardised to the surface area of the colony and subsequently to the Symbiodiniaceae density to calculate the concentration per algal cell.

Survival analysis

Survival rates were determined daily by counting the number of dead and alive fragments of each species in each tank. Fragments of M. digitata were considered dead when no coral tissue remained on the skeleton. Colonies of X. umbellata were considered dead if no pulsation and tissue necrosis was observed. Dead fragments were subsequently removed from the experiment.

Nutrient enrichment alone did not cause mortality in M. digitata and X. umbellata

Regardless of N:P ratio, none of the hard coral fragments died during the experiment. This aligns with earlier studies that reported no significant mortality under nutrient enrichment alone (Fabricius et al., 2013; Kline et al., 2006; Kuntz et al., 2005). While some studies reported mortality in hard corals exposed to nutrient enrichment, they did not report whether these rates were significantly different from controls (Renegar & Riegl, 2005; Samlansin, Chawakitchareon & Rungsupa, 2020). For X. umbellata, initial mortality occurred after two weeks of nutrient enrichment in both N:P treatments, though it was not significant. Similar results were obtained by Thobor et al. (2022), who reported initial mortality in X. umbellata exposed to 37 µM nitrate for three weeks, without significant differences from controls.

Low N:P ratio significantly increased Symbiodiniaceae density in M. digitata, while high N:P ratio did not affect algal symbionts

While no mortality was expected, we expected nutrient enrichment to increase Symbiodiniaceae densities and Chl a concentrations, as previous studies observed such responses at moderate to high dissolved inorganic nitrogen (DIN) levels (Nalley et al., 2023). However, in our study, only the low N:P ratio (5:1) significantly increased Symbiodiniaceae density in M. digitata, while the high N:P ratio (19:1) showed no notable effect on either Symbiodiniaceae or Chl a levels after two weeks. Studies using similar low N:P ratios (between 3−7.5:1) report mixed outcomes regarding their impact on Symbiodiniaceae and Chl a (Béraud et al., 2013; Blanckaert et al., 2023; Ezzat et al., 2015; Ezzat et al., 2016b; Stambler et al., 1991), indicating that a low N:P ratios alone may not be a strong driver of this coral physiological response. Conversely, research using high N:P ratios (16–22:1) often observed increased Symbiodiniaceae density and Chl a concentrations (Hall et al., 2018; Rosset et al., 2017; Tanaka et al., 2014; Wiedenmann et al., 2013), but these studies typically involved longer exposure periods (4–6 months). This discrepancy suggests that short-term experiments, like ours, may be insufficient for detecting such changes.

Besides looking at the N:P ratio, the nitrogen source itself can have significantly different effects. Fernandes de Barros Marangoni et al. (2020) observed no significant effect on Symbiodiniaceae density or Chl a at a 17:1 N:P ratio using nitrate for 5 weeks, though ammonium enrichment in the same ratio caused substantial increases. Generally, studies testing ammonium consistently reported a stronger response than nitrate, increasing the amount of Symbiodiniaceae and overall Chl a concentrations (Béraud et al., 2013; Ezzat et al., 2015; Fernandes de Barros Marangoni et al., 2020; Muller-Parker et al., 1994; Muscatine et al., 1989; Stambler et al., 1991). This highlights the importance of the nitrogen source, as ammonium is more bioavailable and directly assimilated by both corals and Symbiodiniaceae, enhancing photosynthesis and growth (Nalley et al., 2023; Shantz & Burkepile, 2014). Nitrate, in contrast, must first be reduced to ammonium, an energy-intensive process that may limit its physiological impact (Dagenais-Bellefeuille & Morse, 2013; Patterson et al., 2010).

Another important factor to look at, in addition to N:P ratio and nitrogen source, is the actual nutrient concentration in the water. Regarding nitrate concentrations, studies with low nitrate concentrations (3–7 µM) typically report no significant changes in Symbiodiniaceae density or Chl a (Blanckaert et al., 2023; Dobson et al., 2021; Fernandes de Barros Marangoni et al., 2020; Rosset et al., 2017). Higher concentrations (>20 µM) can induce increases, though this is not consistent across studies (Marubini & Davies, 1996; Schlöder & D’Croz, 2004). Importantly, nutrient uptake rates appear highest when DIN is paired with dissolved inorganic phosphorus (DIP) (Blanckaert et al., 2023). This could explain the increased Symbiodiniaceae density observed under the low N:P ratio in our study, where phosphate was less limiting compared to the high N:P ratio, allowing for higher nutrient uptake rates of the corals in the 5:1 ratio treatment.

The duration of exposure may also play a role. Longer experiments (e.g., 30–40 days) with comparably high nitrate concentrations have shown stronger responses (Marubini & Davies, 1996; Schlöder & D’Croz, 2004). Nevertheless, a meta-analysis by Nalley et al. (2023) found that exposure duration was not a significant predictor for most coral response parameters, except for growth and survival, suggesting our two-week period should have been sufficient.

Overall, the limited response of M. digitata to nutrient enrichment in our study likely reflects a combination of N:P ratio, nitrogen source, concentration, and exposure duration. These findings underscore the need for further research to clarify the interplay of these factors in coral nutrient dynamics on hard corals.

Nutrient enrichment alone did not affect algal symbionts of X. umbellata

Compared to studies on hard corals, few experimental studies have explored the effects of nutrient enrichment on soft corals, and even fewer report specific N:P ratios (Table S1). Blanckaert et al. (2023) investigated a low N:P ratio (3:1) using nitrate in Sarcophyton glaucum and found no significant effects on Symbiodiniaceae density or Chl a concentration after 5 weeks, aligning with our findings for X. umbellata at a 5:1 ratio. Similarly, experiments on X. umbellata with isolated nitrate (Thobor et al., 2022) or phosphate enrichment (Klinke et al., 2022) also reported no significant changes in algal symbionts, despite vastly different N:P ratios (200–1200:1 and 0.06–0.5:1, respectively). These results suggest that the N:P ratio alone may not strongly influence algal symbionts in X. umbellata under nutrient enrichment.

Soft corals exhibit lower nitrogen and phosphorus uptake rates compared to scleractinian corals, even under high nutrient concentrations (Blanckaert et al., 2023). This limited uptake likely minimizes nutrient accumulation in soft coral tissue, preventing significant shifts in host-symbiont interactions or elemental stoichiometry (Blanckaert et al., 2023). Consequently, soft corals may be less sensitive to reef nutrification than hard corals (Baum et al., 2016; Schleyer & Celliers, 2003). However, studies using ammonium as the nitrogen source (Bednarz et al., 2012; McCauley & Goulet, 2019) reported increases in Symbiodiniaceae density and Chl a concentration in soft corals after 1–4 weeks at 10–20 µM NH₄. This suggests a nitrogen source-dependent effect, with the higher bioavailability of ammonium facilitating more efficient uptake by algal symbionts and stimulating growth (Nalley et al., 2023; Shantz & Burkepile, 2014). In contrast, the lack of response in nitrate-based studies may reflect slower assimilation or energy constraints associated with nitrate reduction (Dagenais-Bellefeuille & Morse, 2013; Patterson et al., 2010).

Thermal stress alone caused bleaching in M. digitata but not in X. umbellata

Temperature alone led to a significant reduction in both Symbiodiniaceae density and cellular Chl a concentration in the hard coral M. digitata. This is consistent with the common bleaching response of hard corals to temperature stress (Douglas, 2003; Fitt et al., 2000), characterized by the loss of color due to the partial elimination of Symbiodiniaceae and the degradation of their algal pigments. In contrast, for X. umbellata, the addition of warming did not significantly influence Symbiodiniaceae density or cellular Chl a concentration. This indicates that the photophysiology of X. umbellata was not affected by thermal stress, which may be due to the origin of the colonies from the Red Sea, a region known for corals exhibiting high thermal tolerance (Fine, Gildor & Genin, 2013). Additionally, the ability to pulsate allows soft corals of the family Xeniidae to enhance water mixing across the coral-water boundary layer (Kremien et al., 2013), potentially helping to mitigate increases in water temperature near the coral in a warming scenario.

Combined factors significantly decrease survival in M. digitata regardless of N:P ratio

In our study, both N:P ratios combined with elevated temperature stress significantly reduced survival in M. digitata. In both eutrophication treatments we observed increased algal growth, a potential additional stress factor facilitating coral mortality (Smith et al., 2006). Only two studies have explicitly reported hard coral mortality under combined nutrient enrichment and temperature stress. Most studies without mortality data likely observed no coral death during their experiments. Of the two available studies, Fabricius et al. (2013) reported no effect of single or combined stressors on coral mortality. However, their experiments featured prolonged exposure to lower nitrate levels (four µM), suggesting that nutrient concentration plays a crucial role in survival outcomes. Similarly, most studies reporting no mortality often used nitrogen concentration below six µM. Conversely, Wiedenmann et al. (2013) observed 100% mortality of coral fragments exposed to a 42:1 ratio under combined stress, compared to no mortality at a 21:1 ratio, both using 6.5 µM nitrate. This underscores the additional critical role of N:P ratios in coral survival. Surprisingly, in our study, M. digitata experienced complete mortality under the 5:1 ratio and nearly complete mortality under the 19:1 ratio, indicating no significant differences between these ratios. While oligotrophic reefs typically exhibit natural N:P ratios between 4.3:1 and 7.2:1 (Rosset et al., 2017), ambient nutrient concentrations in such systems are extremely low, often in the sub-micromolar range (Lesser, 2021). The higher nutrient concentrations in our study likely induced nitrogen toxicity, causing coral collapse regardless of the N:P ratio. These findings emphasize that not only the N:P ratio but also absolute nutrient concentrations must be considered when evaluating coral responses to combined stressors.

Symbiodinaceae density and Chl a response of M. digitata is driven by temperature stress

In M. digitata fragments surviving the combined nutrient enrichment and heat stress, Symbiodiniaceae density and Chl a concentrations significantly decreased, yet the nutrient enrichment did not exacerbate the temperature-induced effects. This aligns with several laboratory studies, which also found temperature-driven changes in these parameters without exacerbation or mitigation by nutrient enrichment (Béraud et al., 2013; Higuchi, Yuyama & Nakamura, 2015; Hoadley et al., 2016; Schlöder & D’Croz, 2004; Tanaka et al., 2014). The absence of a synergistic effect with nutrient enrichment suggests that nutrient levels alone do not significantly influence temperature-driven physiological or photosynthetic stress in Symbiodiniaceae and that temperature stress appears to be the primary driver of changes in these parameters. Furthermore, several studies report no differences between nutrient enrichment alone and combined nutrient enrichment with heat stress in response parameters (Ezzat et al., 2016b; Hadjioannou et al., 2019; Hall et al., 2018). This further supports the idea that nutrient enrichment does not directly exacerbate physiological stress in corals under combined stress conditions.

Combined factors significantly decrease survival X. umbellata, but less under high N:P ratios

Compared to hard corals, only three studies have investigated the combined effects of inorganic nutrient enrichment under temperature stress on soft corals (Klinke et al., 2022; Mezger et al., 2022; Thobor et al., 2022). Our study found significantly increased mortality of X. umbellata fragments under both N:P ratios tested. This aligns with previous findings where temperature alone did not cause mortality in X. umbellata (Klinke et al., 2022; Thobor et al., 2022), but combined nitrate enrichment reduced survival to 74% of the fragments (Thobor et al., 2022). In our experiment, survival probability was even lower, at 67% in the 19:1 ratio and only 13% in the 5:1 ratio treatment. These differences in survival may be explained by varying N:P ratios across studies. While nutrient uptake rates increase with higher seawater concentrations (Aubriot & Bonilla, 2018; Lubsch & Timmermans, 2020; Perini & Bracken, 2014), the limitation of one nutrient restricts the assimilation of the other, as seen with combined DIN and DIP enrichment (Blanckaert et al., 2023). Therefore, the corals in Thobor et al. (2022), using the same nitrate concentration but in a highly imbalanced N:P ratio (200-1200:1), experienced the lowest mortality, followed by the 19:1 ratio in our study, and the 5:1 ratio being the most detrimental. This suggests that higher, more imbalanced N:P ratios at similar nitrate concentrations could reduce the stress caused by nutrient enrichment.

Combined stress significantly reduces pulsation rates of X. umbellata under high N:P ratio

Under combined stressors, we did not observe any changes in Symbiodiniaceae density or Chl a concentration for X. umbellata, consistent with the results of Thobor et al. (2022). Pulsation rates were reduced to zero in the combined treatment, similar to the findings of Thobor et al. (2022), regardless of the N:P ratio. However, it is noteworthy that in our study, pulsation under the 5:1 ratio was already reduced by 100% under pure nutrient enrichment, meaning the addition of temperature stress could not exacerbate this effect. This early collapse of pulsation under the 5:1 ratio could serve as an indicator of the higher stress levels in X. umbellata exposed to this treatment compared to the 19:1 ratio, as corals in the 5:1 ratio showed significantly lower survival probability under combined stressors. It has been suggested that pulsation rate can be used as a non-invasive, easily detectable, and inexpensive early warning indicator for changes in water quality and ocean warming (Vollstedt et al., 2020), and our study further supports this parameter as a valuable early bioindicator. In contrast, under the 19:1 ratio, nutrient enrichment alone reduced pulsation, but not significantly; however, under combined stress, pulsation was significantly reduced by 100%. This may be explained by potentially different nitrate uptake rates, resulting in lower stress in the high N:P ratio treatment alone (Blanckaert et al., 2023), which, when combined with temperature stress, led to a collapse of pulsation by the end of our experiment.