Coupled carbon and nitrogen cycling regulates the cnidarian–algal symbiosis

Domain: earth_science
Published: 2023-11-01
Editorial outcome: minor_revision

Abstract
Efficient nutrient recycling underpins the ecological success of cnidarian-algal symbioses in oligotrophic waters. In these symbioses, nitrogen limitation restricts the growth of algal endosymbionts in hospite and stimulates their release of photosynthates to the cnidarian host. However, the mechanisms controlling nitrogen availability and their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Combining 13C and 15N stable isotope labeling experiments with physiological analyses and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by labile carbon availability. Light regimes optimal for algal photosynthesis increase carbon availability in the holobiont and stimulate nitrogen assimilation in the host metabolism. Consequently, algal symbiont densities are lowest under optimal environmental conditions and increase toward the lower and upper light tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. Yet, the dependence on resource competition may favor parasitic interactions, explaining the instability of the cnidarian-algal symbiosis as environmental conditions in the Anthropocene shift towards its tolerance limits.

Introduction
Photosymbioses between heterotrophic hosts and phototrophic symbionts are diverse and widespread in the aquatic environment1. The efficient recycling of organic and inorganic nutrients in these associations provides a critical advantage under oligotrophic conditions and has enabled their repeated evolutionary formation2,3. Coral reefs, with their immense biodiversity and productivity, are testimony to this ecological success and the fundamental role of photosymbioses in the marine environment4,5. Yet, the dependence of these ecosystems on the cnidarian-algal symbiosis may also prove their Achilles heel in times of global change. Marine heatwaves, among other environmental disturbances, now repeatedly cause the disruption of this symbiosis in so-called mass bleaching events that result in widespread reef degradation6. Thus, understanding the processes that maintain the stable cnidarian-algal symbiosis could elucidate the evolutionary origins of photosymbioses and help understand their apparent susceptibility to the accelerating environmental change of the Anthropocene.

The photosynthetic activity of algal symbionts implies that the functioning of the cnidarian-algal symbiosis is intimately linked to light availability7. Latitudinal, seasonal, and tidal fluctuations in light intensity, attenuation with depth, shading, and turbidity differences create a complex mosaic of light conditions in aquatic environments8–10. Such variability poses a challenge to photosynthetic organisms as low light levels may limit photosynthetic carbon fixation, while high light levels may result in excessive photooxidative damage7,11. Yet, cnidarian-algal symbioses can be found across a wide range of light regimes ranging from intertidal to mesophotic environments12,13. The key to this broad ecological tolerance lies in the efficient photo-acclimation of both symbiotic partners. Specifically, changes in symbiont densities, host pigments, morphology, and behavior (e.g., locomotion) may modulate light microenvironments for algal symbionts within the host14–18. Likewise, changes in photosynthetic pigments and antioxidant levels of the algae enable optimal light harvesting while avoiding excessive photodamage18–20. However, holobiont responses to changes in light availability are highly species- and context-dependent and are often confounded by changes in other environmental parameters (e.g., along depths gradients)15,21. Our understanding of the general regulatory processes shaping the ecological niche of cnidarian-algal symbiosis is thus limited.

The regulation of the cnidarian-algal symbiosis is directly linked to the nutrient exchange between the host and its symbionts22. In this symbiosis, the release of excess photosynthates by the algae supports the metabolic energy demands of the host16. Host respiration enhances CO2 availability for algal photosynthesis, forming an efficient recycling loop that supports high gross productivity23. Constant nutrient limitation is required to initiate and maintain carbon translocation and recycling in the symbiosis24,25. In a stable state, low nitrogen availability limits algal symbiont growth and ensures the translocation of excess carbon to the host26–29. Consequently, the onset of nitrogen limitation has been proposed to play a vital role in the establishment of the cnidarian-algal symbiosis as it promotes life stage transition and controls the population density of the algal endosymbionts30–32. Likewise, failure to maintain nitrogen-limited conditions has been linked to the breakdown of the symbiosis during heat stress, i.e., bleaching33–35.

However, while the importance of nitrogen cycling in the cnidarian-algal symbiosis is widely accepted, the factors regulating nitrogen availability in hospite remain poorly understood36,37. Previous studies propose that algal symbiont nitrogen limitation arises from symbiont-symbiont as well as host-symbiont competition for inorganic nitrogen30,34,38–43. Deciphering the factors regulating this interplay of intra- and interspecific nitrogen competition could be key to understanding the functional regulation of the symbiosis. Here, we thus investigated the nutritional and environmental controls of nutrient cycling in the cnidarian-algal symbiosis to elucidate the processes shaping its ecological niche and tolerance limits. Using the photosymbiotic model organism Aiptasia (Exaiptasia diaphana clonal line CC7 harboring Symbiodinium linucheae strain SSA01 endosymbionts)44,45, we conducted two complementary experiments to elucidate the role of nitrogen competition in shaping symbiotic interactions. First, we investigated the effects of labile carbon availability on host ammonium assimilation and its consequences on nitrogen availability for algal symbionts. Secondly, we tested how light intensities affect symbiotic nitrogen competition and the resulting eco-evolutionary dynamics in photosymbioses.

We show that symbiotic nutrient cycling in the cnidarian-algal symbiosis is regulated by resource competition. Increases in labile carbon availability enhance host ammonium assimilation thereby reducing nitrogen availability for its algal symbionts. Increases in algal photosynthesis and associated labile carbon availability thus stimulate nitrogen competition in the symbiosis. Consequently, algal symbiont densities are lowest under conditions optimal for algal photosynthesis and increase toward the environmental tolerance limits of the symbiosis.

Results
Labile carbon availability limits ammonium assimilation in the cnidarian host metabolism
In the cnidarian-algal symbiosis, the host and its algal symbionts have the cellular machinery to use carbon backbones from the tricarboxylic acid (TCA) cycle for amino acid synthesis by assimilating ammonium (NH4+) via the glutamate metabolism (Fig. 1A)31,46,47. Carbon translocation by algal symbionts could thus enhance nitrogen assimilation by their host24,39,43. However, the metabolic controls of host ammonium assimilation and its consequences for algal symbiont nitrogen uptake remain largely speculative28,36,37,42.

Here, we investigated the effects of 10 mM pyruvate addition on ammonium assimilation in aposymbiotic (algal symbiont-free; Fig. 1B) and symbiotic (algal symbiont-bearing; Fig. 1F) Aiptasia. Previous studies suggest that algal symbionts may lack the cellular machinery to utilize pyruvate48,49. Indeed, bulk isotope analysis confirmed that host tissues of both aposymbiotic and symbiotic Aiptasia efficiently assimilated [2,3−13C]-pyruvate, while 13C enrichments of algal symbionts were two orders of magnitude lower (Fig. S1A). Pyruvate addition hence increased labile carbon availability for the host but not the algal symbiont, allowing us to study how the availability of labile carbon in the host metabolism affects ammonium assimilation by symbiotic partners. Consistent with previous observations50, incubations with 10 µM ammonium revealed that aposymbiotic Aiptasia showed net release of ammonium during 6 h incubations. In contrast, symbiotic Aiptasia showed net uptake of ammonium from the seawater (Tukey HSD, p < 0.001). Pyruvate addition, however, increased ammonium uptake by both aposymbiotic and symbiotic Aiptasia (ANOVA, F = 181.8, p < 0.001), resulting in a depletion of ammonium in the seawater to the limit of detection within 3 h of incubation (Fig. S1B–D). The retention and uptake of ammonium by the Aiptasia holobiont thus appear to be limited by labile carbon availability. Algal photosynthesis or environmental carbon sources (here pyruvate) thus enhance metabolic nitrogen demand in the holobiont, likely resulting in nitrogen limitation for symbiotic partners under oligotrophic conditions.

To disentangle the contribution of symbiotic partners to these changes in holobiont ammonium uptake, we quantified the assimilation of ammonium-15N in the host tissue layers and algal symbiont cells following 6 h incubations. NanoSIMS imaging revealed that aposymbiotic Aiptasia showed significantly less ammonium assimilation in their tissues than their symbiotic counterparts (ANOVA, F = 94.0, p < 0.001; Fig. 1B–I). However, pyruvate addition caused a pronounced increase in aposymbiotic ammonium assimilation (Tukey HSD, p < 0.001 for epidermis and gastrodermis, respectively) to 15N enrichment levels resembling those of symbiotic Aiptasia. Likewise, pyruvate addition also enhanced ammonium assimilation in symbiotic Aiptasia. However, this effect was restricted to the gastrodermal tissue, i.e., the tissue hosting the algal symbionts (Tukey HSD, p = 0.979 for the epidermis and p < 0.001 for the gastrodermis), potentially reflecting the elevated expression and localization of bi-directional ammonium transporters reported for gastrodermal cells in symbiotic Aiptasia43.

Importantly, algal symbiont ammonium assimilation showed a reversed pattern with significantly reduced 15N-enrichment following pyruvate addition (Tukey HSD, p < 0.001). These contrasting effects of pyruvate addition on the host and algal symbiont ammonium assimilation likely reflect their contrasting carbon utilization. Our results show that the uptake (and recycling) of ammonium by the host is limited by their access to labile carbon; here pyruvate. While this limitation appears to be most pronounced in aposymbiotic animals, even symbiotic hosts were able to utilize the increased labile carbon availability to stimulate their amino acid synthesis42. Consequently, under our experimental conditions (i.e., after 1 week without heterotrophic nutrient sources) algal symbiont photosynthate translocation appears insufficient to fully saturate the metabolic carbon requirements of their host in a stable symbiosis in Aiptasia. The observed reduction in ammonium assimilation by algal symbionts during pyruvate addition is thus likely a direct consequence of increased host anabolic activity. These results suggest that symbiotic partners compete for ammonium and that the outcome of this competition is directly linked to carbon availability.

These findings imply that a positive feedback loop regulates nutrient cycling in the symbiosis: translocation of algal photosynthates stimulates anabolic ammonium assimilation by their host. Reduced nitrogen availability for algal symbionts limit their growth and further enhances photosynthate translocation to the host. Environmental conditions affecting algal photosynthetic performance and/or organic nutrient pollution could thus directly alter nitrogen availability for algal symbionts because of changes in carbon availability in the host metabolism.

Algal photosynthesis alters symbiotic nitrogen availability
To test this hypothesis, we performed a long-term experiment on the effects of light availability on symbiotic Aiptasia. Over 6 months, Aiptasia were gradually acclimated to seven different light levels spanning an exponential gradient from near dark (photosynthetically active radiation (PAR < 6.25 µE s−1 m−2) to high light intensity (PAR = 400 µE s−1 m−2), and animals were left at their final treatment levels for 1 month without supplemental feeding. The lowest and highest light treatment levels exceeded the tolerance limits of a stable symbiosis. At near-dark conditions, Aiptasia exhibited a near-complete loss of algal symbionts (bleaching) while the host remained viable. At the highest light intensity, Aiptasia still hosted algal symbionts but suffered from severe host mortality (63%). These symbiotic tolerance limits, i.e., PAR from 12.5 to 200.0 µE s−1 m−2, likely reflect the trade-offs between increasing light limitation toward the lower end of the light gradient and increasing photooxidative stress toward the higher end of the light gradient, respectively11,21,51,52.

Indeed, the role of photodamage was reflected in the maximum photosynthetic efficiency of algal symbionts declining by more than 20% at the upper tolerance limit (PAR 200.0 µE s−1 m−2) compared to the lower tolerance limit of the symbiosis (PAR 12.5 µE s−1 m−2; LM, F = 62.9, p < 0.001; Fig. 2A). This trade-off between light limitation and excess light stress was evident in the gross photosynthetic activity of individual algal cells showing an optimum response with the highest activities at intermediate light levels, i.e., PAR 50–100 µE s−1 m−2 (LM, F = 3.5, p = 0.048; Fig. 2B). Bulk stable isotope measurements of Aiptasia incubated with 13C-bicarbonate confirmed this pattern as algal cells contributed the most photosynthetically fixed carbon to the holobiont at intermediate light levels (LM, F = 11.9, p < 0.001; Fig. 2C). In line with this, atomic carbon to nitrogen ratios (C:N ratios) of holobionts followed a similar pattern along the light gradient (LM, F = 5.1, p = 0.016; Fig. 2D). Given that environmental nitrogen availability, i.e., dissolved seawater nutrients and heterotrophic feeding, was identical for all light treatments, these changes in the C:N ratio likely reflect the direct changes in carbon availability in the holobiont as a function of gross photosynthetic activity. Consequently, our results suggest that labile carbon availability in the symbiosis is highest under intermediate light levels and decreases toward the tolerance limits of the symbiosis at lower and higher light intensities.

The link between carbon availability and symbiotic nitrogen cycling identified above implies that algal photosynthesis should alter nitrogen assimilation in the symbiosis. Indeed, the optimum in photosynthetic carbon fixation and C:N ratios were consistent with a similar pattern of bulk stable isotope measurements of 15N-ammonium labeled Aiptasia showing the highest ammonium assimilation in the holobiont at intermediate light levels (LM, F = 4.6, p = 0.022; Fig. S2A). To further disentangle the respective contribution of symbiotic partners to holobiont ammonium assimilation, we used NanoSIMS imaging. This revealed that the optimum in holobiont ammonium assimilation was the composite result of the asymmetric responses of the host and algal symbionts, respectively. On average, algal symbionts showed an approximately ten-fold higher 15N enrichment than the surrounding host tissue (Fig. 2E–G). However, algal ammonium assimilation peaked at low light availability, i.e., PAR 25–50 µE s−1 m−2, while host ammonium assimilation in the gastrodermis and epidermis peaked at intermediate light levels, 50–100 µE s−1 m−2 (Figs. 2E–G and S2B; algal symbionts: LM, F = 10.6, p < 0.001; host gastrodermis: LM, F = 9.9, p < 0.001; host epidermis: LM, F = 4.6, p = 0.008). While 15N enrichments only reflect the uptake of environmental nitrogen in the symbiosis, the recycling of ammonium from the host catabolism is likely regulated by the same processes and follow the same patterns. High 15N enrichments thus indicate an increased nitrogen demand in the symbiosis resulting in reduced production of catabolic ammonium by the host. In this light, the combined C:N ratio and 15N enrichment patterns (Figs. 2D, E and S2) imply that symbiotic competition for nitrogen is highest under intermediate light levels and decreases toward the tolerance limits of the symbiosis. Hence, our data suggest that increases in photosynthesis and associated labile carbon availability effectively enhance nitrogen limitation for algal symbionts.

Nitrogen availability shapes the symbiotic phenotype in Aiptasia
Given that nitrogen limitation controls algal growth in the cnidarian-algal symbiosis30,31,36,37, the here-observed light-dependent changes in symbiotic nitrogen availability should directly affect the regulation of the symbiosis. Indeed, light availability affected the phenotype of photosymbiotic Aiptasia. Pigmentation and chlorophyll autofluorescence were at their highest levels in Aiptasia at the low and high end of the light tolerance range of the symbiosis (Fig. 3A). However, chlorophyll a content of individual algal cells remained unaffected by light availability suggesting that long-term photo acclimation was driven by other holobiont responses, e.g., changes in host pigmentation (LM, F = 0.0, p = 0.995; Fig. S3A). Nonetheless, chlorophyll a content in relation to host protein content increased by more than two-fold toward the symbiosis tolerance limits compared to intermediate light levels (LM, F = 7.4, p = 0.004; Fig. S3B). This response was driven by changes in algal symbiont densities, which were lowest at 50 µE s−1 m−2 and increased by more than three-fold toward the upper and lower light tolerance limit of the symbiosis (LM, F = 8.9, p = 0.002, Fig. 3B). Consequently, symbiont density was lowest when their gross photosynthetic performance was highest. Indeed, algal symbiont densities showed a pronounced negative correlation with gross photosynthetic activity along the experimental light gradient (Pearson’s r = −0.578, p = 0.002) and holobiont C:N ratios (Pearson’s r = −0.489, p = 0.001) suggesting that the algal symbiont population is regulated by nitrogen availability (as a consequence of resource competition). Under optimal environmental conditions, high photosynthetic carbon availability ensures efficient recycling of catabolic nitrogen in the host metabolism and limits nitrogen availability for algal symbionts. Under suboptimal environmental conditions, reduced carbon availability increases the relative availability of nitrogen in the holobiont, thereby supporting higher symbiont densities, consistent with the feedback loop described above.

Nutrient cycling controls the eco-evolutionary dynamics of the cnidarian-algal symbiosis
Our results provide direct experimental support for the role of metabolic interactions in the passive regulation of the cnidarian-algal symbiosis. Specifically, these findings lend support to the bioenergetic models of symbiosis regulation by Cunning et al.25 and Cui et al.39 by highlighting the role of carbon and nitrogen cycling in the eco-evolutionary dynamics of photosymbiotic cnidarians. We found that nitrogen competition between the host and its algal symbionts effectively limits nitrogen availability for algal symbionts under optimal environmental conditions. Toward the environmental tolerance limits of the symbiosis, however, this competition is gradually replaced by nitrogen competition between algal symbionts as their population density increases38. This dynamic interplay between inter- and intraspecific nitrogen competition extends the symbiosis’ tolerance range by maintaining nitrogen-limited conditions across a wide range of environmental conditions. Indeed, previous reports found that corals with a broad depth distribution harbored the highest symbiont densities at their upper and lower distribution limits, with the lowest symbiont densities occurring toward the center of their distribution range15,53. Our results thus suggest that the passive metabolic regulation of symbiotic interaction through resource competition facilitates the ecological success and stability of the cnidarian-algal symbiosis across a wide range of environmental conditions.

Finally, the processes described here improve our understanding of the maintenance of the symbiosis in a changing environment. These mechanisms are unlikely limited to the light-dependent regulation of the symbiosis but likely control other acclimation and stress responses as well. Specifically, our findings may contribute to deciphering the processes leading to the breakdown of cnidarian-algal symbiosis during heat stress54. During mass bleaching events, severe and prolonged heat stress causes a dramatic loss of algal symbionts in photosymbiotic Cnidaria, thereby promoting host starvation and reef degradation55,56. Yet, algal symbiont densities have been shown to increase prior to bleaching during early and moderate warming, and corals with higher symbiont densities show increased susceptibility to bleaching34,57–59. Here, we show that algal symbiont densities may increase toward the symbiosis tolerance limits following reduced carbon availability for the host. Hence, an initial proliferation of algal symbionts during early heat stress is unlikely to be a beneficial response. Instead, increasing symbiont densities in the early phases of environmental stress should be considered a sign of destabilization of symbiotic nutrient cycling, which might ultimately contribute to the breakdown of the symbiosis during heat stress34,40,60.

Taken together, we conclude that the cnidarian-algal symbiosis is passively controlled by coupling carbon and nitrogen cycling in the symbiosis. Resource competition stabilizes the symbiosis under a wide range of environmental conditions. At the same time, the resulting negative relationship between host and symbiont performance (zero sum) may promote the evolution of parasitic behavior and destabilize the symbiosis in times of rapid environmental change.

Methods
Animal culture maintenance
All experiments were performed with Aiptasia clonal lineage CC7 (sensu Exaiptasia diaphana)61. Prior to the experiments, the animals were reared in illuminated growth chambers (Algaetron 230, Photo System Instruments, Czech Republic) at a constant temperature of 20°C in 2L clear food containers (Rotho, Switzerland) filled with artificial seawater (35 PSU, Pro-Reef, Tropic Marin, Switzerland). Each week, Aiptasia anemones were fed with freshly hatched Artemia nauplii (Sanders GSLA, USA), thoroughly cleaned, and the seawater was exchanged. Photosymbiotic animals harboring their native algal symbiont community dominated by Symbiodinium linucheae (subclade A4, strain SSA01) were reared in a 12h:12h light-dark cycle with photosynthetic active radiation (PAR) of 50 µE m−2 s−1. Further, aposymbiotic animal cultures deprived of algal symbionts were generated following established cold shock bleaching protocols62. Briefly, photosymbiotic animals were cold-shocked at 4°C for 4 h followed by 8 weeks of culturing in artificial seawater containing 50 µM DCMU at constant irradiance of 50 µE m−2 s−1. The absence of algal symbionts was confirmed using fluorescence microscopy and aposymbiotic cultures were maintained at continuous darkness thereafter.

Experiment 1: effect of pyruvate on nitrogen cycling
To study the effect of labile carbon availability on symbiotic nitrogen cycling, we assessed how pyruvate affects ammonium (NH4+) assimilation in aposymbiotic and symbiotic Aiptasia. Following 1 week of starvation, 26 aposymbiotic and 26 photosymbiotic animals were transferred into individual 50 ml glass vials for isotope labeling. For the incubations, a minimal artificial seawater medium (35 PSU, pH 8.1, 355.6 mM NaCl, 46.2 mM MgCl2, 10.8 mM Na2SO4, 9.0 mM CaCl2, 7.9 mM, KCl, 2.5 mM NaHCO3) containing 10 µM 15NH4Cl (≥98 atom% 15N) was used following Harrison et al.63. While half of the animals (13 aposymbiotic and 13 photosymbiotic) were incubated in minimal artificial seawater medium, the other half was incubated in minimal artificial seawater medium spiked with 10 mM [2,3-13C]-pyruvate (99 atom% 13C). Incubations were performed using the culturing conditions outlined above, with aposymbiotic animals being kept in the dark and photosymbiotic animals being kept in constant light. After 3 h, the incubations were terminated for each combination of treatment and symbiotic state and the incubation water was collected to quantify ammonium concentrations (see below). After 6 h, the remaining incubations were terminated. For each combination of treatment and symbiotic state, the water was collected from 5 incubations for ammonium measurements, and Aiptasia were sampled for either bulk isotope measurements (5 animals) of 13C assimilation or NanoSIMS analysis (3 animals) of 15N assimilation (see below).

Experiment 2: effect of light on nitrogen cycling
To study the effect of light on symbiotic nitrogen cycling, photosymbiotic animals were kept in an exponential series of light levels in a long-term experiment. All seven light treatments followed a 12h:12h light-dark cycle and used the same culturing conditions outlined above. Over the course of 6 months, 30 animals per treatment were gradually acclimated in monthly steps to the following PAR levels: <6.25, 12.50, 25.00, 50.00, 100.00, 200.00, 400.00 µE m−2 s−1. Following acclimation, animals were starved for 1 month to minimize confounding feeding responses on symbiotic nitrogen cycling. After this, the rate of survival of animals was recorded (any asexual offspring were removed during the weekly cleaning routines throughout the experiment). Aiptasia showed severe bleaching at low light levels while hosts suffered from high mortality at high light levels, respectively. Hence, only host mortality and symbiont densities were quantified for all light treatments. All other parameters were only recorded within the tolerance range of the stable symbiosis, i.e., PAR 12.5–200.0 µE m−2 s−1. To quantify bicarbonate and ammonium assimilation in the symbiosis, five animals per treatment were incubated in 50 ml minimal artificial seawater medium containing 2.5 mM NaH13CO3 (≥98 atom% 13C) and 10 µM 15NH4Cl (≥98 atom% 15N) for 24 h at their respective treatment conditions. Following the incubation, tentacles from two animals were dissected and fixed for NanoSIMS analysis, and all animals were immediately collected for bulk elemental and isotope analysis (see below). Further, the dark-adapted photosynthetic efficiency and oxygen fluxes were recorded for six and five animals per treatment, respectively (see below).

Ammonium uptake
Collected seawater samples were immediately filtered (PES, 0.22 µm), transferred into sterile 15 ml centrifuge tubes, and stored at −20°C for subsequent analysis. Within 1 week of sampling, samples were defrosted, and ammonium concentrations were immediately analyzed using a Smartchem 450 wet chemistry analyzer (AMS Alliance, Italy). Changes in ammonium concentrations were corrected for volume and duration of incubations, and fluxes were normalized to the dry weight of animals (freeze-dried after carefully removing any excess water using filter paper).

Fixation, embedding, and NanoSIMS imaging
Collected animals (experiment 1) and individual tentacles (experiment 2) were rinsed in artificial seawater without isotope tracers and immediately transferred into a fixative solution (2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M Sorensen’s phosphate buffer) and incubated for 1 h at room temperature followed by 24 h at 4°C. Samples were transferred into a storage solution (1% paraformaldehyde in 0.1 M Sorensen’s phosphate buffer) and processed for dehydration and resin infiltration within 4 days of collection. Samples were incubated for 1 h in 1% OsO4, rinsed, and dehydrated in a series of increasing ethanol concentrations (30% for 10 min, 50% for 10 min, 2×70% for 10 min, 3×90% for 10 min, and 3×100% for 10 min). For resin infiltration, samples were transferred to 100% acetone and gradually infiltrated with Spurr resin (Electron Microscopy Sciences, USA) at increasing concentrations (25% for 30 min, 50% for 30 min, 75% for 1 h, and 100% overnight), and resins were cured for 48 h at 65°C. Embedded samples were cut into 200 nm thin sections with an Ultracut E ultra-microtome (Leica, Germany), transferred onto glow-discharged silicon wafers, and coated with a 12 nm gold layer.

The surface isotopic composition of sample sections on silicon wafers was analyzed using a NanoSIMS 50L (Cameca, France)64. Following pre-sputtering for 5 min with a primary beam of ca. 6 pA to remove the metal coating, samples were bombarded with a 16 keV primary ion beam of ca. 2 pA Cs+ focused to a spot size of about 150 nm on the sample surface. Secondary molecular cyanide ions 12C14N− and 12C15N− were simultaneously collected in electron multipliers at a mass resolution of about 9000 (Cameca definition), sufficient to resolve the 12C15N− ions from potentially problematic interferences. Eight to nine sample areas were analyzed for each of the samples by rastering the primary beam across a 40×40 µm sample surface with a 256×256 pixels resolution and a pixel dwell time of 5 ms for five consecutive image layers. The resulting isotope images were processed using the ImageJ plug-in OpenMIMS (https://github.com/BWHCNI/OpenMIMS/wiki). Mass images were drift and dead-time corrected, the individual planes were summed, and the 12C15N−/12C14N− ratio images were expressed as a hue-saturation-intensity image, where the color scale represents the 15N/14N isotope ratio. 15N assimilation was quantified by drawing regions of interest (ROIs) of host epidermis, host gastrodermis, and algal symbionts based on 12C14N− images, respectively (Fig. S4). As individual host cells were not clearly distinguishable in the NanoSIMS images, all epidermal and gastrodermal tissue areas within one image (excluding algal symbionts and symbiosomal contents) were recorded as one ROI per tissue layer, respectively (Fig. S4). For experiment 1, this yielded 8 epidermal and gastrodermal ROIs per Aiptasia respectively. For experiment 2, this yielded 4 epidermal and 8 gastrodermal ROIs per Aiptasia. Algal symbionts ROIs were drawn based on individual algal cells and only the largest ROIs (40 ROIs per Aiptasia in experiment 1 and 16 ROIs per Aiptasia in experiment 2) were included in the analysis to minimize potential measurement variability due to lower signal-to-noise ratio of smaller ROIs. Algal symbiont ROI size thus had no significant effect on 15N enrichments (LM, F = 1.5, p = 0.224 for experiment 1, F = 0.1, p = 0.720 for experiment 2; Fig. S5). For each ROI, 15N enrichment was expressed as atom% express (APE) relative to unlabeled controls. Notably, these enrichment values are likely an underestimation of the actual enrichment levels for these organisms as fixation, dehydration, and resin embedding during sample preparation extract and dilute soluble compounds from the sample matrix. However, any methodological bias arising from this is consistent across samples, NanoSIMS images hence allow for a robust assessment of relative enrichment values. In light of the clonal nature of Aiptasia and the identical environmental conditions of animals within the same treatment, individual ROIs were considered as independent measurements at the microscale level regardless of the animal replicate for the purpose of the analysis.

Bulk elemental and isotope analysis
Anemones were collected following stable isotope labeling, rinsed in artificial seawater without isotope tracers, and immediately homogenized in 1 ml MilliQ water using a Polytron PT1200E immersion dispenser (Kinematica, Switzerland). For experiment 1, host and algal symbiont fractions were separated by centrifugation (1000×g for 5 min, sufficient to pellet >95% of algal symbionts from the sample), the algal pellet was rinsed twice by resuspension and centrifugation, and both fractions were snap-frozen in liquid nitrogen and kept at −80°C until further analysis. For experiment 2, homogenized samples were immediately snap-frozen and kept at −80°C without separating host and algal symbiont fractions. All samples were freeze-dried, and the dry mass was recorded. The atomic carbon (C) to nitrogen (N) ratios were quantified using a Carlo Erba 1108 elemental analyzer (Fisons Instruments, Italy). This was coupled via a Conflo III interface to a Delta V Plus isotope ratio mass spectrometer (Thermo Fisher Scientific, Germany) to determine the carbon (experiments 1 and 2) and nitrogen (experiment 2) stable isotope composition. 13C/12C and 15N/14N ratios were calibrated with six in-house urea standards with defined isotope ratios as described in Spangenberg and Zufferey65 and normalized against the international Vienna Pee Dee Belemnite limestone (VPDB) and Air-N2 scale, respectively. To assess the isotope enrichment of samples, the isotope ratios were converted to atom% and converted to APE by subtracting isotopic values measured in unlabeled control animals. For experiment 2, absolute isotope tracer assimilations of Aiptasia holobionts were normalized to their algal symbiont content to account for potential differences in symbiont densities between samples.

Photosynthetic efficiency and gross photosynthesis
To assess the effect of light availability on the photosynthetic efficiency of algal symbionts, pulse amplitude modulated (PAM) fluorometry was used. Anemones were dark-acclimated for 1 h before the maximum quantum yield (Fv/Fm) was recorded using the blue light version of the Mini-PAM-II (Walz, Germany). For each specimen, the initial fluorescence (Fo) was recorded, followed by a saturating pulse, and maximum fluorescence (Fm) was measured directly afterward. The maximum quantum yield was calculated as the ratio between the variable fluorescence (Fv = Fm − Fo) and the maximum fluorescence.

To quantify gross photosynthetic rates of the anemones, respiration, and net photosynthesis were measured in separate consecutive incubations. For this, anemones were transferred into 12.3 ml borosilicate vials filled with artificial seawater and equipped with an OXSP5 oxygen sensor spot (Pyroscience, Germany). Following the attachment of the animals at the vial bottom, a 6 mm magnetic stirrer was added, and each vial was sealed bubble-free, inverted, and transferred to a water bath. Vials were continuously stirred at 240 rpm using a magnetic stirring plate, and oxygen concentrations were continuously recorded with an FSO2-4 oxygen meter (PyroScience) connected via a SPFIB-BARE optical fiber (PyroScience). All incubations were performed at 20°C for ~2 h each. Respiratory oxygen consumption was quantified during dark incubations. Subsequently, net photosynthetic activity was recorded during light incubations with light levels according to the respective treatment conditions of the animals. Oxygen fluxes were corrected for artificial seawater control incubations and normalized to the incubation duration and the animals’ algal symbiont content (see below). Gross photosynthesis was calculated as the sum of the net photosynthesis rate and the absolute value of the respiration rate of each animal. Notably, this method does not account for potential increases in respiration rates during the light66. The here presented gross photosynthesis rates thus likely represent an underestimation of the actual rates. However, this methodological bias is consistent across treatments and does not impair the conclusions presented here.

Algal symbiont densities, chlorophyll a content, and protein content
Following oxygen flux incubations, anemones were immediately homogenized in 0.5 ml 2x PBS using a Polytron PT1200E immersion dispenser (Kinematica), and host and symbiont fractions were separated by centrifugation (1000×g for 5 min, sufficient to pellet >95% of algal symbionts). The host supernatant was stored at −20°C until further analysis. The algal symbiont pellet was resuspended in 1 ml 2x PBS and divided into two equal aliquots. The first aliquot was used to quantify the concentration of algal symbiont cells in six replicate measurements using a Countess cell counter Countess II FL Automated Cell Counter (Thermo Fisher Scientific, USA) with a Cy5 light cube. Algal symbiont cells could be clearly identified by the cell counter based on their size, roundness, and chlorophyll fluorescence. The second aliquot was used for chlorophyll a content quantification. For this, cells were pelleted (5000×g for 5 min), resuspended in 90% ethanol, and incubated at 4°C for 24 h in the dark under constant agitation. After this, cell debris was pelleted (5000×g for 5 min), and two 200 µl supernatant per sample were transferred into a transparent flat-bottom 96-well plate. Sample absorbances were immediately recorded at 630, 664, and 750 nm using a CLARIOstar Plus microplate reader (BMG Labtech, Germany), and chlorophyll content was calculated according to Jeffrey and Humphrey67:

Chlorophyll a [µg mL−1] = 11.43 × (OD664 − OD750) − 0.644 × OD630 ( − OD750)

Symbiont densities and chlorophyll a content were normalized against host tissue protein content as a proxy of host biomass. Protein content in the defrosted host supernatant was quantified according to Bradford68. Three technical 5 µl replicates were analyzed for each sample using the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific, US) according to the manufacturer’s instruction, and the absorbance of samples, as well as BSA calibration standards, was recorded at 595 nm using a CLARIOstar Plus microplate reader (BMG Labtech).

Statistical analysis
For the first experiment, NanoSIMS data from the host and symbiont compartments were square root transformed to meet model assumptions analyzed separately. Bulk isotope, ammonium flux, and NanoSIMS measurements were analyzed with an analysis of variance (ANOVA) based on symbiotic state (aposymbiotic vs. photosymbiotic) and treatment (control vs. pyruvate addition) followed by Tukey’s honest significance test. Changes in Ammonium concentrations over time were analyzed using an analysis of variance (ANOVA) based on symbiotic state (aposymbiotic vs. photosymbiotic) and treatment (control vs. pyruvate addition) and time point (3 h vs. 6 h) followed by Tukey’s honest significance test.

For the second experiment, only measurements within the defined tolerance range of the stable symbiosis, i.e., PAR 12.5–200.0 µE m−2 s−1, were used for the statistical analysis. All data were analyzed using linear regression models (LM) using light treatment levels as an explanatory variable. Where necessary, a second-degree (symbiont density, chlorophyll a content normalized by host protein, gross photosynthesis, C:N ratio, bulk 13C and 15N assimilation) or third-degree (NanoSIMS measurements for host tissues and symbiont cells) polynomial transformation was applied to the data to meet model assumptions. Further, Pearson correlations were used to test the linear relationship between gross photosynthesis rates and symbiont densities.

For both experiments, the effects of algal symbiont ROI size on algal 15N enrichments were analyzed using LM using the number of pixels per algal ROI as the explanatory variable.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review: Coupled carbon and nitrogen cycling regulates cnidarian-algal symbiosis\nRädecker et al. present a two experiment study focused on a model sea anemone (Aptasia) and its symbiotic algal partners (Symbiodiniuum linuchae str SSA01). Two experiments were performed using stable isotope labeling of 15NH4 and NanoSIMS imaging for measuring the ammonium uptake in both partners. The overarching goal was to improve the understanding of the regulation between the partners as it relates to nitrogen limitation and carbon availability. One experiment assessed the effect of labile carbon by addition of pyruvate, and in the second experiment, the light regime was altered from low to high intensities. The key finding of the first experiment was that the partners are competing for N, while in the second experiment, the competition between the partners persists under optimal conditions, and switches to only among the symbionts at the extremes (e.g. high and low light). The authors argue the switch in competition from inter to intra allows a more stable mutualism across a broader range of conditions.\nThe experiments were performed well, and analyses of the results are conclusive. One weakness in the experimental design was to not utilize a 13C labelled pyruvate, however, the authors likely have an explanation.\nThe significance of the work is that it provides an important explanation for stabilizing a model representative of cnidarian-algal symbioses. These populations in the wild play an important role in the coral reef ecosystems, and hence the work contributes new information. It seems the authors have a convincing dataset with multiple measurements that should be worthy input (photosynthesis, biosynthesis, respiration, etc.) for a hypothetical flux model. Moreover, this flux model could be used to test different future climate conditions expected for reefs. Perhaps outside the scope or intention of the manuscript, it could be something to consider. A few suggestions and comments are provided with the intention of improving the presentation. The article could also benefit from the help of a native speaker to edit or review the text.\nAbstract\n-It seems appropriate to mention a bit more details on which form of nitrogen e.g. e.g.15NH4\nLine 34, add “stable” before the isotope\nGeneral comment. The first sentence of the abstract is difficult to comprehend, consider to read aloud and re-write. .. Efficient nutrient cycling underpins the ecological success of cnidarian-algal symbioses which thrive in oligotrophic waters and represent the foundation of coral reef ecosystems.\nIntroduction.\nGeneral comment. The introduction provides the details necessary for the role of nutrient exchange in the symbiotic system, but lacks an equal attention on light intensity. Given the second experiment focuses on light, perhaps the introduction should provide some information on the current state of knowledge for light intensity on the symbiotic system.\nL54- phrase “shine a light” could be replaced with “could elucidate the evolutionary…”\nGeneral comment. Can the end of the introduction strengthen the rationale and motivation for the general audience; for example, besides the fact that the factors are poorly understood for nutrient regulation in cnidarian-algal symbioses, is there a larger motivation/impact for this …e.g. its relevance to the environment and/or future expected conditions.\nResults & Discussion\nL 87 -why have the authors not considered a multi label experiment of 13C-pyruvate and 15NH4? The nanoSIMS 50L can measure both up to 6 masses simultaneously (e.g. 13C-, 12C-, 12C14N-, 12C15N-). It seems like a shortcoming in the experimental design, but perhaps there is an explanation?\nL90. The use of “and/or” which is it-the algal symbionts have limited access and lack machinery OR have limited access or lack machinery? This detail seems rather crucial to the experimental design.\nL92. What is the label percent of 15NH4+? This is important for the SIMS measures, and should be described here or in the materials and methods.\nL107- Can the authors clarify how it is known that the photosynthetic hosts can utilize the additional carbon for their amino acid synthesis. Is this from previous works or the experiment described in the study?\nFig 1 explain what APE is in the caption, add in the number of ROIs or cells analyzed-this detail should also be added to the materials and methods.\nL138-140. Seems like this speculation/interpretation could use a reference? Or a comparison to another study?\nGeneral comment: With the data acquired in cell abundances and chla content, is there any indication that the cells are altering their chla content relative to their cell size? e.g. under high/low light algal cells can alter the pigment content for energy conservation or dissipation.\nMaterials and methods\nL 277-278. Rephrase, seems like host mortality is not necessary in the first part of the sentence.\nGeneral comment. Fixation procedures could lead to a dilution of the enrichment, the authors could consider making a statement in the methods section to explain this. Several papers have reported this for bacteria, perhaps similar response is valid for the host and algal cells.\nL309. It is probably better to state that the 15N/14N ratios were based on the 12C15N/12C14N.\nL312. I think “ion images” is a better descriptor than “maps.”\nL329-330. Have the authors also tried to normalize to time in order to report rate?\nFigures\nGeneral comment. Authors should consider to move and combine Fig S1and Fig S2 to replace Fig. 3. These two supplementary figures are quite clear overview of the results from Exp. 2.\nSuggestion/Question on analyses. Given that nanoSIMS is an amazing MSI technique with high lateral resolution, have the authors tried to draw and compare regions of high enrichment in the individual Symbiodinium. In Fig S2, a striking observation is the variation in the enrichment within the subpopulations of Symbiodinium in the sections-one can see clusters of cells of high enrichment co-occurring with cells of less enrichment. Futhermore, within a Symbiodinium smaller areas of extreme enrichment e.g., are these representative of storage granules? Is this variation due to variable physiology amongst the Symbiodinum OR related to the depth of the sectioning into the Symbiodinium? This is an important technical caveat in SIMS imaging on populations of cells embedded (or in colonies, etc), as they are not all in the same orientation, and hence one should comment/explain this variation. One could measure the diameter of the Symbiodinium, and plot the enrichment as a function of the cell diameter. This figure also makes me wonder how the ROIs for the host tissue were made? Perhaps the authors can show this as a Suppl. figure as well.", "reviewer_2": "The cnidarian-phototroph symbiosis in principle relies on the transfer of photosynthetically-derived carbon from the symbiont to the host, while the host provides ideal conditions for the symbiont to thrive. It has been shown previously that increased nitrogen availability can lead to increased symbiont growth and subsequent decreases in the carbon transfer to the host (e.g. O’Neil and Capone, 2008) suggesting a tight coupling and/or host regulation of the symbiotic metabolism under stable environmental conditions. Here, Rädecker et al. present single-cell ammonium uptake by an anemone host (Aiptasia) and their photosynthetic symbionts under different carbon availabilities, accomplished by either additions of pyruvate or changes in light (and resulting changes in photosynthesis by the symbionts). The key finding is that when pyruvate is added to incubations of symbiotic Aiptasia, the ammonium uptake of the host increases while the ammonium uptake of the symbiont decreases (Fig. 1G). The authors argue that under these increased external (organic) carbon availabilities (i.e. the pyruvate addition), the host competes for ammonium with its symbionts, leading to the decreased ammonium uptake by the symbionts. This conclusion would indicate that the host could, to some extent, control the availability of nitrogen to the symbionts and the subsequent transfer of carbon to themselves.\nI see one major issue with the authors’ argumentation. While I do not necessarily disagree that the host may have the capacity to control symbiont growth (via coupling of carbon and nitrogen cycling), the authors do not provide enough evidence of the ‘resource competition’ between host and symbiont or that nitrogen availability was different for host and symbiont. In more detail, the authors used only one concentration of ammonium in their experiments, i.e. ten micromolar ammonium, and do not show that i) this concentration was limiting during the incubation or ii) that ammonium was used up during the incubation. Generally, competition for a resource, such as nitrogen, should only occur if substrate availability is limiting. However, the authors do not provide evidence that this is the case here. In contrast, the ten micromolar ammonium concentration is substantially more than typical in situ concentrations in the oligotrophic conditions of coral reef ecosystems. In addition, incubations using 13C-labeled compounds could have possibly provided the means to constrain the coupled carbon-nitrogen cycling but such data is not provided here. These types of incubations/approaches could have provided additional information on what type of carbon and nitrogen cycling was occurring, and how strongly carbon and nitrogen cycling were coupled. In summary, this manuscript does not provide enough evidence for the authors’ conclusions.\nAdditional comments:\nl 37: Please clarify what is meant by ‘enhanced symbiotic competition’\nl 39: Please clarify what is meant by ‘tolerance limits of the symbiosis’. While this is explained later in the manuscript, I think it is essential that the reader already understands in the abstract what is meant.\nl 83/86: I don’t think that ammonium is ‘fixed’; rather it is taken up and assimilated or incorporated. Using the term ‘fixing’ or ‘fixation’ may mislead the reader to think that N2 fixation plays a role here.\nl 107: How do you know whether the pyruvate was used for amino acid synthesis and not for, for example, lipid biosynthesis?\nl 176: Doesn’t the light mediate photosynthesis and therefore carbon rather than N availability?\nFig. 1: It would be good to have some microscopic context to the nanoSIMS images (and/or outlines of tissues and so on).\nFig. 1: The panels H and I in Fig. 1 do not seem to show substantial differences in the 15N-enrichment in the symbionts between the control and +pyruvate treatment. They do not seem to reflect the findings shown in panel G.\nFig. 1: In panel C, I would rather show the ‘Epi’ and ‘Gas’ data on top of the ‘Epi’ and ‘Gas’ data in label G, highlighting that there are no symbionts in the aposymbiotic Aiptasia.\nReferences:\nO’Neil, Judith M., and Douglas G. Capone. \"Nitrogen cycling in coral reef environments.\" Nitrogen in the marine environment (2008): 949-989.", "reviewer_3": "In this manuscript, Raedecker and colleagues investigate how organic C availability shapes competition for nitrogen and regulates symbiont abundance in the model photosymbiotic system Aiptasia (Exaiptasia diaphana) and its phototrophic Symbiodinium symbionts. The authors compare 15N-ammonium assimilation under labile C-replete vs. C-deplete conditions, in symbiotic and aposymbiotic Aiptasia on single cell level using nanoSIMS. Furthermore, they track symbiont abundance and activity under varying light regimes, leading to differing availabilities of organic C from photosynthates for the host. This manuscript sheds light on the mechanism sustaining (and destabilizing) photosymbiotic systems, and adds to the growing body of literature highlighting nitrogen availability as key factor for this type of symbiosis. While I am not an expert in photosymbiosis, I very much enjoyed reading this interesting and well written manuscript and particularly liked the elegant experiments and combination of methods from single cell to bulk activity measurements. I do have some comments on the manuscript, mainly pertaining to the methodological setup (e.g. the rather high concentration of organic C used in this study), as detailed below.\nMain points:\n- I would like to hear the authors’ opinion on the transferability of the labile C experiments to in situ conditions. The used labile C concentration (10 mM pyruvate) seems rather extreme compared to the natural conditions these animals live in (typical surface seawater DOC concentration is 100-500 µM). I wonder whether the highly elevated pyruvate concentrations used here might distort the host (and symbiont) responses in 15N-assimilation reported in the manuscript. Was the response to lower (more natural) pyruvate additions tested?\n- To me it is unclear how many replicates were measured on the nanoSIMS. Please state this in the figure legend (fig 1) and in the main text. This is important to know, as it can otherwise not be assessed how much of the observed differences between +/- pyruvate is due to biological heterogeneity of individual animals, or response to the treatment.\n- Likewise, I would suggest to show data from individual Aiptasia as separate points in Fig 2, 3, and the supplementary figures, to show variability between individual animals.\nMinor comments:\nIntroduction:\n- I suggest to briefly introduce the photosymbiotic system (species) used here, and especially mention the Symbiodiniaceae symbionts by name.\nResults & Discussion:\n- L100: Do the authors have an explanation for the lack of significant response to pyruvate addition in the gastrodermal tissue of the photosymbiotic animals? Higher general labile C-availability due to the physical proximity to the symbionts and thus lack of response to elevated external C?\n- Can the authors make a rough calculation on how much of the added 15N-ammonium was consumed by the holobiont in the 6h (pyruvate experiment) and 24h (light levels experiment)? Was there residual ammonium available?\n- Figure 2: In the legend, descriptions of panel A and B are swapped. Please explain the abbreviation APE in the legend (atom percent excess), this is also missing in the other figures depicting nanoSIMS or isotope enrichment data. Please double check the data sheet for the nanoSIMS data of experiment 2 – AP and APE columns contain identical values.\nMethods:\n- L313-316: I don’t understand what is meant by this statement. Please specify how ROIs of the host were drawn. The data sheet suggests that the areas of host ROIs were much larger than the symbiont ROIs – I find this surprising, as I would have intuitively drawn ROIs around individual host cells, which should not be that much larger than the symbionts, to depict the variability in host tissue enrichment.\n- Bulk elemental analysis: Please specify how Aiptasia individuals were washed before elemental analysis (I assume they were washed, else, 15N ammonium likely sticks to the biomass and biases the measurement)\n- L347-352: Is the respiration rate of the host identical in light and dark conditions? This would need to be the case to be able to calculate gross photosynthesis the way it was done. This could be at least roughly assessed by measuring oxygen consumption by aposymbiotic Aiptasia in light vs. dark conditions.\n- L354-374: Can the authors confirm that no (or very few) symbiont cells are retained in the supernatant after pelleting?"}, "author_response": "Response to reviewer comments\nCoupled carbon and nitrogen cycling regulates the cnidarian-algal symbiosis\nComments by the reviewers are indicated in black.\nResponses by the authors are indicated in blue.\nNew and revised manuscript sections are indicated in green.\nReviewer #1\nR1: Rädecker et al. present a two experiment study focused on a model sea anemone (Aptasia) and its symbiotic algal partners (Symbiodiniuum linuchae str SSA01). Two experiments were performed using stable isotope labeling of 15NH4 and NanoSIMS imaging for measuring the ammonium uptake in both partners. The overarching goal was to improve the understanding of the regulation between the partners as it relates to nitrogen limitation and carbon availability. One experiment assessed the effect of labile carbon by addition of pyruvate, and in the second experiment, the light regime was altered from low to high intensities. The key finding of the first experiment was that the partners are competing for N, while in the second experiment, the competition between the partners persists under optimal conditions, and switches to only among the symbionts at the extremes (e.g. high and low light). The authors argue the switch in competition from inter to intra allows a more stable mutualism across a broader range of conditions.\nA: Excellent summary! We are delighted to see that we were able to convey the key messages. Thank you for taking the time to review the work and for your excellent suggestions.\nR1: The experiments were performed well, and analyses of the results are conclusive. One weakness in the experimental design was to not utilize a 13C labelled pyruvate, however, the authors likely have an explanation.\nA: Thank you for raising this valid point. We have now included additional bulk isotope measurements of 13C assimilation for both experiments. These additional analyses confirm that algal symbionts do not assimilate pyruvate, thereby supporting our interpretation of the results (please refer to Figure S1, as well as the revised results/discussion which now includes:\nL107: “Previous studies suggest that algal symbionts may lack the cellular machinery to utilize pyruvate (48, 49). Indeed, bulk isotope analysis confirmed that host tissues of aposymbiotic and symbiotic Aiptasia efficiently assimilated [2,3-13C]-pyruvate, while 13C enrichments of algal symbionts were two orders of magnitude lower (Fig. S1A). Pyruvate addition hence increased labile carbon availability for the host but not the algal symbiont, allowing us to study how the availability of labile carbon in the host metabolism affects ammonium assimilation by symbiotic partners.”\nR1: The significance of the work is that it provides an important explanation for stabilizing a model representative of cnidarian-algal symbioses. These populations in the wild play an important role in the coral reef ecosystems, and hence the work contributes new information. It seems the authors have a convincing dataset with multiple measurements that should be worthy input (photosynthesis, biosynthesis, respiration, etc.) for a hypothetical flux model. Moreover, this flux model could be used to test different future climate conditions expected for reefs. Perhaps outside the scope or intention of the manuscript, it could be something to consider. A few suggestions and comments are provided with the intention of improving the presentation. The article could also benefit from the help of a native speaker to edit or review the text.\nA: Good point. We agree that modeling fluxes in the symbioses would be the best way to transfer the findings of this work to other settings. Unfortunately, NanoSIMS analyses are an excellent tool to quantify relative changes in assimilation but do not provide a reliable assessment of absolute fluxes. For the time being, we thus point the reader to the previous excellent work of Cunning et al. 2017 J Theo Biol and hope to develop robust flux models for future applications.\nThe manuscript now states:\nL251: “Specifically, these findings lend support to the bioenergetic models of symbiosis regulation by Cunning et al. (25) and Cui et al. (39)…”\nAbstract\nR1: -It seems appropriate to mention a bit more details on which form of nitrogen e.g. e.g.15NH4\nA: The abstract now clearly states that we used 13C and 15N labeling to study the link between ammonium uptake and labile carbon availability.\nR1: Line 34, add “stable” before the isotope\nA: Done.\nR1: General comment. The first sentence of the abstract is difficult to comprehend, consider to read aloud and re-write. .. Efficient nutrient cycling underpins the ecological success of cnidarian-algal symbioses which thrive in oligotrophic waters and represent the foundation of coral reef ecosystems.\nA: Agreed. We have removed the reference to coral reef ecosystems as the ecological success of these symbioses is not limited to coral reefs.\nIntroduction.\nR1: General comment. The introduction provides the details necessary for the role of nutrient exchange in the symbiotic system, but lacks an equal attention on light intensity. Given the second experiment focuses on light, perhaps the introduction should provide some information on the current state of knowledge for light intensity on the symbiotic system.\nA: Great suggestion. The introduction has been extended to include the following paragraph:\nL57: “The photosynthetic activity of algal symbionts implies that the functioning of the cnidarian-algal symbiosis is intimately linked to light availability (7). Latitudinal, seasonal and tidal fluctuations in light intensity, attenuation with depth, shading, and turbidity differences create a complex mosaic of light conditions in aquatic environments (8–10). Such variability poses a challenge to photosynthetic organisms as low light levels may limit photosynthetic carbon fixation while high light levels may result in excessive photooxidative damage (7, 11). Yet, cnidarian-algal symbioses can be found across a wide range of light regimes ranging from intertidal to mesophotic environments (12, 13). The key to this broad ecological tolerance lies in the efficient photo-acclimation of both symbiotic partners. Specifically, changes in symbiont densities, host pigments, morphology, and behavior (e.g., locomotion) may modulate light microenvironments for algal symbionts within the host (14–18). Likewise, changes in photosynthetic pigments and antioxidant levels of the algae enable optimal light harvesting while avoiding excessive photodamage (18–20). However, holobiont responses to changes in light availability are highly species- and context-dependent and are often confounded by changes in other environmental parameters (e.g., along depths gradients) (15, 21). Our understanding of the regulatory processes shaping the ecological niche of cnidarian-algal symbioses is thus limited.”\nR1: L54- phrase “shine a light” could be replaced with “could elucidate the evolutionary…”\nA: Implemented as suggested.\nR1: General comment. Can the end of the introduction strengthen the rationale and motivation for the general audience; for example, besides the fact that the factors are poorly understood for nutrient regulation in cnidarian-algal symbioses, is there a larger motivation/impact for this …e.g. its relevance to the environment and/or future expected conditions.\nA: Thanks for raising this point. We have now elaborated on the justification for the study.\nL85: “However, while the importance of nitrogen cycling in the cnidarian-algal symbiosis is widely accepted, the factors regulating nitrogen availability in hospite remain poorly understood (36, 37). Previous studies propose that algal symbiont nitrogen limitation arises from symbiont-symbiont as well as host-symbiont competition for inorganic nitrogen (30, 34, 38–43). Deciphering the factors regulating this interplay of intra- and interspecific nitrogen competition could be key to understanding the functional regulation of the symbiosis. Here, we thus investigated the nutritional and environmental controls of nutrient cycling in the cnidarian-algal symbiosis to elucidate the processes shaping its ecological niche and tolerance limits in the Anthropocene.”\nResults & Discussion\nR1: L 87 -why have the authors not considered a multi label experiment of 13C-pyruvate and 15NH4? The nanoSIMS 50L can measure both up to 6 masses simultaneously (e.g. 13C-, 12C-, 12C14N-, 12C15N-). It seems like a shortcoming in the experimental design, but perhaps there is an explanation?\nA: Please refer to the response above. 13C measurements have now been included.\nR1: L90. The use of “and/or” which is it-the algal symbionts have limited access and lack machinery OR have limited access or lack machinery? This detail seems rather crucial to the experimental design.\nA: This was rephrased to clarify that Symbiodinicaeae lost the molecular machinery for pyruvate utilization.\nR1: L92. What is the label percent of 15NH4+? This is important for the SIMS measures, and should be described here or in the materials and methods.\nA: Notably, we used artificial seawater to exclude any background nutrient “contamination”. The purity of used isotope labels (≥ 98 atom %) has now been included for all isotopes in the Material and Methods.\nR1: L107- Can the authors clarify how it is known that the photosynthetic hosts can utilize the additional carbon for their amino acid synthesis. Is this from previous works or the experiment described in the study?\nA: While there are older works on this, we recommend the preprint by Cui et al. on this topic showing that labile carbon (in their study glucose) is converted into carbon backbones for amino acid synthesis: https://doi.org/10.1101/2022.06.30.498212\nThis study was cited throughout this manuscript and the reference is now included in this sentence as well.\nR1: Fig 1 explain what APE is in the caption, add in the number of ROIs or cells analyzed-this detail should also be added to the materials and methods.\nA: We apologize for the oversight. Where relevant, figure legends now mention the number of replicates involved and define what APE means.\nR1: L138-140. Seems like this speculation/interpretation could use a reference? Or a comparison to another study?\nA: Valid point. References for bleaching and/or lack of algal proliferation in the dark (Rouan et al. 2022 Mol Ecol, Jinkerson et al. 2022 Curr Biol) and photooxidative stress at high light (Richier et al. 2008 JEMBE, Levy et al. 2016 Roy Soc B) have now been added.\nR1: General comment: With the data acquired in cell abundances and chla content, is there any indication that the cells are altering their chla content relative to their cell size? e.g. under high/low light algal cells can alter the pigment content for energy conservation or dissipation.\nA: Great point. Indeed, these responses have been observed for some species, but the patterns strongly depend on species and environmental context. We have now included a new normalization of chlorophyll content per algal cell (see Figure S3A). As you can see, the chlorophyll a content of algal cells was not affected by light availability suggesting that other mechanisms of photoacclimation were in place. This is now also highlighted in the Results & Discussion:\nL232: “However, chlorophyll a content of individual algal cells remained unaffected by light availability suggesting that long-term photo acclimation was driven by other holobiont responses, e.g., changes in host pigmentation (LM, F = 0.0, p = 0.995; Fig. S3A). Nonetheless, chlorophyll a content in relation to host protein content increased by more than two-fold towards the symbiosis tolerance limits compared to intermediate light levels (LM, F = 7.4, p = 0.004; Fig. S3B).”\nMaterials and methods\nR1: L 277-278. Rephrase, seems like host mortality is not necessary in the first part of the sentence.\nA: The sentence was split for clarity.\nR1: General comment. Fixation procedures could lead to a dilution of the enrichment, the authors could consider making a statement in the methods section to explain this. Several papers have reported this for bacteria, perhaps similar response is valid for the host and algal cells.\nA: Point well taken. The following has been included in the methods section:\nL386: “Notably, these enrichment values are likely an underestimation of the actual enrichment levels for these organisms as fixation, dehydration, and resin embedding during sample preparation extract and dilute soluble compounds from the sample matrix. However, any methodological bias arising from this is consistent across samples, NanoSIMS images hence allow for a robust assessment of relative enrichment values.”\nR1: L309. It is probably better to state that the 15N/14N ratios were based on the 12C15N/12C14N.\nA: Implemented as suggested.\nR1: L312. I think “ion images” is a better descriptor than “maps.”\nA: Replaced as suggested.\nR1: L329-330. Have the authors also tried to normalize to time in order to report rate?\nA: We have now normalized bulk isotope measurements for experiment 2 per day and symbiont sell to allow easy comparison with oxygen measurements.\nFigures\nR1: General comment. Authors should consider to move and combine Fig S1and Fig S2 to replace Fig. 3. These two supplementary figures are quite clear overview of the results from Exp. 2.\nA: Great suggestion. Former Figures S1 and S2 have been moved to the main text to replace the previous figure.\nR1: Suggestion/Question on analyses. Given that nanoSIMS is an amazing MSI technique with high lateral resolution, have the authors tried to draw and compare regions of high enrichment in the individual Symbiodinium. In Fig S2, a striking observation is the variation in the enrichment within the subpopulations of Symbiodinium in the sections-one can see clusters of cells of high enrichment co-occurring with cells of less enrichment. Futhermore, within a Symbiodinium smaller areas of extreme enrichment e.g., are these representative of storage granules? Is this variation due to variable physiology amongst the Symbiodinum OR related to the depth of the sectioning into the Symbiodinium? This is an important technical caveat in SIMS imaging on populations of cells embedded (or in colonies, etc), as they are not all in the same orientation, and hence one should comment/explain this variation. One could measure the diameter of the Symbiodinium, and plot the enrichment as a function of the cell diameter. This figure also makes me wonder how the ROIs for the host tissue were made? Perhaps the authors can show this as a Suppl. figure as well.\nA: Indeed, NanoSIMS is an excellent tool to illustrate and analyze single-cell variability. However, in the present datasets, algal cell ROI size had no effect on 15N enrichments beyond introducing high variability in very small ROIs (which were hence excluded from the analysis where possible). We have now updated the methods accordingly and included a new Figure S5 showing that algal ROI size did not affect 15N enrichment.\nFurther, we have included an additional Figure S4 to illustrate how ROIs correspond to SEM and NanoSIMS images and have extended the methods to include the following:\nL375: “15N assimilation was quantified by drawing regions of interest (ROIs) of host epidermis, host gastrodermis, and algal symbionts based on 12C14N− images, respectively (Fig. S4). As individual host cells were not clearly distinguishable in the NanoSIMS images, all epidermal and gastrodermal tissue areas within one image (excluding algal symbionts and symbiosomal contents) were recorded as one ROI per tissue layer, respectively (Fig. S4). For experiment 1, this yielded 8 epidermal and gastrodermal ROIs per Aiptasia respectively. For experiment 2, this yielded 4 epidermal and 8 gastrodermal ROIs per Aiptasia. Algal symbionts ROIs were drawn based on individual algal cells and only the largest ROIs (40 ROIs per Aiptasia in experiment 1 and 16 ROIs per Aiptasia in experiment 2) were included in the analysis to minimize potential measurement variability due to lower signal-to-noise ratio of smaller ROIs. Algal symbiont ROI size thus had no significant effect on 15N enrichments (LM, F = 1.5, p = 0.224 for experiment 1, F = 0.1, p = 0.720 for experiment 2; Fig. S5).”\nReviewer #2\nR2: The cnidarian-phototroph symbiosis in principle relies on the transfer of photosynthetically-derived carbon from the symbiont to the host, while the host provides ideal conditions for the symbiont to thrive. It has been shown previously that increased nitrogen availability can lead to increased symbiont growth and subsequent decreases in the carbon transfer to the host (e.g. O’Neil and Capone, 2008) suggesting a tight coupling and/or host regulation of the symbiotic metabolism under stable environmental conditions. Here, Rädecker et al. present single-cell ammonium uptake by an anemone host (Aiptasia) and their photosynthetic symbionts under different carbon availabilities, accomplished by either additions of pyruvate or changes in light (and resulting changes in photosynthesis by the symbionts). The key finding is that when pyruvate is added to incubations of symbiotic Aiptasia, the ammonium uptake of the host increases while the ammonium uptake of the symbiont decreases (Fig. 1G). The authors argue that under these increased external (organic) carbon availabilities (i.e. the pyruvate addition), the host competes for ammonium with its symbionts, leading to the decreased ammonium uptake by the symbionts. This conclusion would indicate that the host could, to some extent, control the availability of nitrogen to the symbionts and the subsequent transfer of carbon to themselves.\nI see one major issue with the authors’ argumentation. While I do not necessarily disagree that the host may have the capacity to control symbiont growth (via coupling of carbon and nitrogen cycling), the authors do not provide enough evidence of the ‘resource competition’ between host and symbiont or that nitrogen availability was different for host and symbiont. In more detail, the authors used only one concentration of ammonium in their experiments, i.e. ten micromolar ammonium, and do not show that i) this concentration was limiting during the incubation or ii) that ammonium was used up during the incubation. Generally, competition for a resource, such as nitrogen, should only occur if substrate availability is limiting. However, the authors do not provide evidence that this is the case here. In contrast, the ten micromolar ammonium concentration is substantially more than typical in situ concentrations in the oligotrophic conditions of coral reef ecosystems. In addition, incubations using 13C-labeled compounds could have possibly provided the means to constrain the coupled carbon-nitrogen cycling but such data is not provided here. These types of incubations/approaches could have provided additional information on what type of carbon and nitrogen cycling was occurring, and how strongly carbon and nitrogen cycling were coupled. In summary, this manuscript does not provide enough evidence for the authors’ conclusions.\nA: Thank you for this very constructive feedback and the interest in our work.\nIndeed, ammonium concentrations used here exceed concentrations found on most reefs. Yet, these concentrations are necessary to ensure sufficient signal for a robust quantification using NanoSIMS imaging and to account for the depletion of ammonium over time. As suggested, we have now included additional measurements to show that the uptake of ammonium from the seawater is limited by carbon (pyruvate) availability and that ammonium can be depleted to the limit of detection within 3 hours of incubation (Figure S1). Hence, nitrogen availability was indeed limited for the holobiont supporting the interpretation that host and symbionts compete for this valuable resource.\nFurther, we have now included additional 13C stable isotope measurements (based on bulk analysis), which show that 1) only the host assimilates pyruvate effectively and 2) photosynthetic carbon assimilation is highest at intermediate light levels within the cnidarian-algal symbiosis.\nWe are thus confident that the reviewer’s suggestion to include these additional measurements substantiated our previous interpretations and has strengthened the narrative of the manuscript.\nAmong other sections, the results & discussion now include the following paragraph:\nL107: “Previous studies suggest that algal symbionts may lack the cellular machinery to utilize pyruvate (48, 49). Indeed, bulk isotope analysis confirmed that host tissues of aposymbiotic and symbiotic Aiptasia efficiently assimilated [2,3-13C]-pyruvate, while 13C enrichments of algal symbionts were two orders of magnitude lower (Fig. S1A). Pyruvate addition hence increased labile carbon availability for the host but not the algal symbiont, allowing us to study how the availability of labile carbon in the host metabolism affects ammonium assimilation by symbiotic partners. Consistent with previous observations (50), incubations with 10 µM ammonium revealed that aposymbiotic Aiptasia showed net release of ammonium during 6 h incubations. In contrast, symbiotic Aiptasia showed net uptake of ammonium from the seawater (Tukey HSD, p < 0.001). Pyruvate addition, however, increased ammonium uptake by aposymbiotic and symbiotic Aiptasia (ANOVA, F = 181.8, p < 0.001), resulting in a depletion of ammonium in the seawater to the limit of detection within 3 h of incubation (Fig. S1B-D). The retention and uptake of ammonium by the Aiptasia holobiont thus appear to be limited by labile carbon availability. Algal photosynthesis or environmental carbon sources (here pyruvate) thus enhance metabolic nitrogen demand in the holobiont, likely resulting in nitrogen limitation for symbiotic partners under oligotrophic conditions.”\nR2: 37: Please clarify what is meant by ‘enhanced symbiotic competition’\nA: Rephrased to clarify that increased host nitrogen assimilation is limiting nitrogen availability for the algae.\nR2: 39: Please clarify what is meant by ‘tolerance limits of the symbiosis’. While this is explained later in the manuscript, I think it is essential that the reader already understands in the abstract what is meant.\nA: The abstract now states that this refers to the lower and upper light tolerance limits of the stable symbiosis.\nR2: 83/86: I don’t think that ammonium is ‘fixed’; rather it is taken up and assimilated or incorporated. Using the term ‘fixing’ or ‘fixation’ may mislead the reader to think that N2 fixation plays a role here.\nA: Changed to “assimilation” throughout the manuscript.\nR2: 107: How do you know whether the pyruvate was used for amino acid synthesis and not for, for example, lipid biosynthesis?\nA: Importantly, we do not claim that pyruvate is exclusively used for amino acid synthesis and nothing else. Indeed, it is very likely, that excess carbon will also be converted into lipids and other forms of carbon storage when nitrogen is limited. However, our data clearly show that increased labile carbon availability stimulates ammonium assimilation in the animal as a consequence of amino acid synthesis. The sentence in question has been rephrased for clarity:\nL139: “…even symbiotic hosts were able to utilize the increased labile carbon availability to stimulate their amino acid synthesis…”\nR2: 176: Doesn’t the light mediate photosynthesis and therefore carbon rather than N availability?\nA: Indeed. The link between photosynthetic carbon and nitrogen availability is highlighted in the previous sentence. The sentence in question has been reworded to clarify that this statement refers to the previous results:\nL228: “Given that nitrogen limitation controls algal growth in the cnidarian-algal symbiosis (30, 31, 36, 37), the here-observed light-dependent changes in symbiotic nitrogen availability should directly affect the regulation of symbiosis.”\nR2: Fig. 1: It would be good to have some microscopic context to the nanoSIMS images (and/or outlines of tissues and so on).\nA: Great suggestion. We have now included a correlated SEM & NanoSIMS image as part of Figure S4 to help the reader understand the NanoSIMS images and have extended Figure 1 to include a label indicating the position of the mesoglea.\nR2: Fig. 1: The panels H and I in Fig. 1 do not seem to show substantial differences in the 15N-enrichment in the symbionts between the control and +pyruvate treatment. They do not seem to reflect the findings shown in panel G.\nA: Given that host and symbiont enrichment differ by nearly one order of magnitude, the selected color scale reflects a compromise between illustrating changes in the host and the symbiont tissue. Even in this color scale, algae in panel H are homogenously pink (maximum enrichment) while algae in panel I show larger areas of blue (no enrichment) or yellow (intermediate enrichment).\nR2: Fig. 1: In panel C, I would rather show the ‘Epi’ and ‘Gas’ data on top of the ‘Epi’ and ‘Gas’ data in label G, highlighting that there are no symbionts in the aposymbiotic Aiptasia.\nA: Implemented as suggested.\nReferences:\nR2: O’Neil, Judith M., and Douglas G. Capone. \"Nitrogen cycling in coral reef environments.\" Nitrogen in the marine environment (2008): 949-989.\nA: This study is now cited in the manuscript.\nReviewer #3\nR3: In this manuscript, Raedecker and colleagues investigate how organic C availability shapes competition for nitrogen and regulates symbiont abundance in the model photosymbiotic system Aiptasia (Exaiptasia diaphana) and its phototrophic Symbiodinium symbionts. The authors compare 15N-ammonium assimilation under labile C-replete vs. C-deplete conditions, in symbiotic and aposymbiotic Aiptasia on single cell level using nanoSIMS. Furthermore, they track symbiont abundance and activity under varying light regimes, leading to differing availabilities of organic C from photosynthates for the host. This manuscript sheds light on the mechanism sustaining (and destabilizing) photosymbiotic systems, and adds to the growing body of literature highlighting nitrogen availability as key factor for this type of symbiosis. While I am not an expert in photosymbiosis, I very much enjoyed reading this interesting and well written manuscript and particularly liked the elegant experiments and combination of methods from single cell to bulk activity measurements. I do have some comments on the manuscript, mainly pertaining to the methodological setup (e.g. the rather high concentration of organic C used in this study), as detailed below.\nA: Thank you for the encouraging feedback. We appreciate your interest in our work. Please see below for a detailed response to your comments.\nMain points:\nR3: - I would like to hear the authors’ opinion on the transferability of the labile C experiments to in situ conditions. The used labile C concentration (10 mM pyruvate) seems rather extreme compared to the natural conditions these animals live in (typical surface seawater DOC concentration is 100-500 µM). I wonder whether the highly elevated pyruvate concentrations used here might distort the host (and symbiont) responses in 15N-assimilation reported in the manuscript. Was the response to lower (more natural) pyruvate additions tested?\nA: Importantly, the purpose of the pyruvate treatment was not to mimic natural conditions. Rather, we wanted to use this carbon source to selectively enhance carbon availability for the host but not the algal symbiont. As such, we chose a high concentration to ensure sufficient uptake by the host. Even at the used concentrations, 13C signature of pyruvate-labeled animals were still below those of bicarbonate-labeled animals from experiment 2 (even when considering the longer incubation time in experiment 2). Hence, we are confident that pyruvate addition is a powerful experiment to test the effect of photosynthate translocation on the host metabolism and its consequences for symbiotic nitrogen competition.\nWe now elaborate further on the reasoning behind choosing pyruvate labeling in the results & discussion:\nL107: “Previous studies suggest that algal symbionts may lack the cellular machinery to utilize pyruvate (48, 49). Indeed, bulk isotope analysis confirmed that host tissues of aposymbiotic and symbiotic Aiptasia efficiently assimilated [2,3-13C]-pyruvate, while 13C enrichments of algal symbionts were two orders of magnitude lower (Fig. S1A). Pyruvate addition hence increased labile carbon availability for the host but not the algal symbiont, allowing us to study how the availability of labile carbon in the host metabolism affects ammonium assimilation by symbiotic partners.”\nR3: To me it is unclear how many replicates were measured on the nanoSIMS. Please state this in the figure legend (fig 1) and in the main text. This is important to know, as it can otherwise not be assessed how much of the observed differences between +/- pyruvate is due to biological heterogeneity of individual animals, or response to the treatment.\nA: Thanks for this suggestion. All figure legends now state the number of replicates used in the respective analysis.\nR3: Likewise, I would suggest to show data from individual Aiptasia as separate points in Fig 2, 3, and the supplementary figures, to show variability between individual animals.\nA: We agree that it would be desirable to plot all raw data where possible. We have now included plots showing all raw data associated with both experiments as part of the Zenodo submission.\nFor experiment 1, we have included the raw data in the individual figures of the manuscript. For experiment 2, however, we have chosen to stick with plotting mean ± se in the main manuscript for two reasons: 1) Some of the figures appear too crowded when all individual responses are included. 2) The spread of the raw data results in an extended y-axis making the shape of the response curve less visible to the reader in some cases.\nMinor comments:\nIntroduction:\nR3: I suggest to briefly introduce the photosymbiotic system (species) used here, and especially mention the Symbiodiniaceae symbionts by name.\nA: The introduction has been extended to include:\nL91: “Exaiptasia diaphana clonal line CC7 harboring Symbiodinium linucheae strain SSA01 endosymbionts”\nResults & Discussion:\nR3: L100: Do the authors have an explanation for the lack of significant response to pyruvate addition in the gastrodermal tissue of the photosymbiotic animals? Higher general labile C-availability due to the physical proximity to the symbionts and thus lack of response to elevated external C?\nA: We believe there is a misunderstanding here, L100 stated that the gastrodermis showed a significant increase (note the log scale in the figure, this reflects an 80 % increase between treatments). L99 however, states that no significant increase was observed for the epidermis. Unfortunately, we can only speculate regarding the absence of a response in the epidermis. Potentially, pyruvate is more efficiently assimilated in the gastrodermal tissue of symbiotic animals or the lower abundance of ammonium transporters throughout the epidermis limits higher ammonium assimilation. The latter hypothesis is now mentioned in the manuscript:\nL129: “However, this effect was restricted to the gastrodermal tissue, i.e., the tissue hosting the algal symbionts (Tukey HSD, p = 0.979 for the epidermis and p < 0.001 for the gastrodermis), potentially reflecting the elevated expression and localization of bi-directional ammonium transporters reported for gastrodermal cells in symbiotic Aiptasia (43).”\nR3: Can the authors make a rough calculation on how much of the added 15N-ammonium was consumed by the holobiont in the 6h (pyruvate experiment) and 24h (light levels experiment)? Was there residual ammonium available?\nA: Great suggestion. In line with the other reviewers’ feedback, we have now included additional ammonium uptake experiments to show that pyruvate-supplemented animals are able to deplete ammonium in the incubation vial within 3 h of incubation. Even symbiotic animals were able to deplete ammonium concentrations by about 70 % within 6 h of incubation suggesting that 24 h incubations are long enough for animals to deplete ammonium concentrations under optimal light conditions. Hence, both experiments presented here allow us to study the partitioning of ammonium under nitrogen-limited conditions. These additional measurements are now presented in Figure S3 and the Results & Discussion now include the following:\nL112: “Consistent with previous observations (50), incubations with 10 µM ammonium revealed that aposymbiotic Aiptasia showed net release of ammonium during 6 h incubations. In contrast, symbiotic Aiptasia showed net uptake of ammonium from the seawater (Tukey HSD, p < 0.001). Pyruvate addition, however, increased ammonium uptake by aposymbiotic and symbiotic Aiptasia (ANOVA, F = 181.8, p < 0.001), resulting in a depletion of ammonium in the seawater to the limit of detection within 3 h of incubation (Fig. S1B-D). The retention and uptake of ammonium by the Aiptasia holobiont thus appear to be limited by labile carbon availability. Algal photosynthesis or environmental carbon sources (here pyruvate) thus enhance metabolic nitrogen demand in the holobiont, likely resulting in nitrogen limitation for symbiotic partners under oligotrophic conditions.”\nR3: Figure 2: In the legend, descriptions of panel A and B are swapped. Please explain the abbreviation APE in the legend (atom percent excess), this is also missing in the other figures depicting nanoSIMS or isotope enrichment data. Please double check the data sheet for the nanoSIMS data of experiment 2 – AP and APE columns contain identical values.\nA: Thanks for spotting this important point. All figure legends have been corrected and now include number of replicates as well as definitions of abbreviations. Further, the supplementary data files have been updated to now include the correct AP and APE values of 15N assimilation.\nMethods:\nR3: L313-316: I don’t understand what is meant by this statement. Please specify how ROIs of the host were drawn. The data sheet suggests that the areas of host ROIs were much larger than the symbiont ROIs – I find this surprising, as I would have intuitively drawn ROIs around individual host cells, which should not be that much larger than the symbionts, to depict the variability in host tissue enrichment.\nA: We agree that it would be ideal to analyze individual host cells as separate ROIs. However, neither SEM nor NanoSIMS images provided sufficient information to allow identifying individual host cells. As any segmentation of host tissues would thus be arbitrary, we decided to group host tissue area in only one host epidermal and gastrodermal ROI per image. To clarify, how ROIs were drawn we have now included supplementary Figure S1. Further the methods now state:\nL376: “As individual host cells were not clearly distinguishable in the NanoSIMS images, all epidermal and gastrodermal tissue areas within one image (excluding algal symbionts and symbiosomal contents) were recorded as one ROI per tissue layer, respectively (Fig. S4). For experiment 1, this yielded 8 epidermal and gastrodermal ROIs per Aiptasia respectively. For experiment 2, this yielded 4 epidermal and 8 gastrodermal ROIs per Aiptasia. Algal symbionts ROIs were drawn based on individual algal cells and only the largest ROIs (40 ROIs per Aiptasia in experiment 1 and 16 ROIs per Aiptasia in experiment 2) were included in the analysis to minimize potential measurement variability due to lower signal-to-noise ratio of smaller ROIs.”\nR3: Bulk elemental analysis: Please specify how Aiptasia individuals were washed before elemental analysis (I assume they were washed, else, 15N ammonium likely sticks to the biomass and biases the measurement)\nA: Thanks for spotting this. The methods now clarify that all samples were rinsed in artificial seawater without isotope tracers before the samples were prepared for analysis.\nR3: L347-352: Is the respiration rate of the host identical in light and dark conditions? This would need to be the case to be able to calculate gross photosynthesis the way it was done. This could be at least roughly assessed by measuring oxygen consumption by aposymbiotic Aiptasia in light vs. dark conditions.\nA: The here-used method is the most commonly employed technique to measure gross photosynthesis in cnidarians (e.g., Ferrier-Pagès et al. 2000 Coral Reefs, Cardini et al. 2015 Roy Soc B, Carbonne et al. 2021 Limnol Ocean). Nonetheless, we acknowledge that this method cannot account for potential increases in light respiration rates due to enhanced carbon availability. As such, the reported gross photosynthesis may underestimate actual rates. However, any methodological bias arising from this is consistent across treatments and thus doesn’t affect the conclusions presented here.\nThis methodological bias is now acknowledged in the methods:\nL434: “Notably, this method does not account for potential increases in respiration rates during the light (65). The here presented gross photosynthesis rates thus likely represent an underestimation of the actual rates. However, this methodological bias is consistent across treatments and does not impair the conclusions presented here.”\nR3: L354-374: Can the authors confirm that no (or very few) symbiont cells are retained in the supernatant after pelleting?\nA: Indeed, we quantified the symbiont content in the host supernatant as well to confirm that symbionts were efficiently pelleted by centrifugation in all samples. This information is not included in the manuscript.\nL397: “…host and algal symbiont fractions were separated by centrifugation (1000 g for 5 min, sufficient to pellet > 95 % of algal symbionts from the sample)…”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of my considerations and suggestions were properly and thoroughly addressed. I appreciate the detailed and thoughtful responses to all my comments. The additional figure (S4) and details in the methods and nanoSIMS analyses are important improvements and well implemented. THANK you for the opportunity to review this important and timely work.", "reviewer_2": "Review of revised manuscript:\nI am very happy to see that the authors were able to provide additional data to support their conclusions. Particularly, the addition of the ammonium concentrations is very valuable to show that indeed N limitation sets in after some hours and that therefore organisms are likely competing for it. All other comments have also been resolved, and the manuscript is really well written and structured. I have no further comments.", "reviewer_3": "Thank you for addressing my previous comments on the manuscript.\nThe additional data on 13C-pyruvate uptake by host and symbionts, and the inclusion of the ammonium concentration data have further strengthened the manuscript.\nI have only few remaining suggestions/comments:\nL89-90: Ecological niche and tolerance limits of this symbiosis were probably the same/similar in their recent evolutionary history also before entering the Anthropocene - please rephrase.\nL116: maybe better \"...ammonium uptake by *both* aposymbiotic and symbiotic...\" for clarity\nL134-135: Is it possible that the 10mM pyruvate addition has an inhibitory/detrimental effect on the symbionts' activity? Has this been tested in symbiont pure culture?\nL141, L310: please clarify what starvation refers to here - I assume no feeding with Artemia, but it could also mean no light.\nL206-209: Can higher 15N enrichment by the symbionts at lower light intensities not simply be due to less excess released organic C from the symbionts due to lower light stress, and thus less available organic C for the host (and thus less host N-assimilation), and a more tightly coupled C fixation : N assimilation ratio by the symbionts?\nL278: While the experiments conducted look at light intensities, the discussion about environmental conditions is about heat stress - it might be useful for readers to link this more explicitly in the discussion.\nL323: The methods section states that 3 animals were sampled for nanoSIMS, the nanoSIMS figure legends state that 2 were analyzed. Please mention in the methods section that two replicates were analyzed by nanoSIMS."}, "author_response": "NCOMMS-22-51840A\nResponse to reviewer comments\nCoupled carbon and nitrogen cycling regulates the cnidarian-algal symbiosis\nComments by the reviewers are indicated in black.\nResponses by the authors are indicated in blue.\nNew and revised manuscript sections are indicated in green.\nReviewer 1:\nR1: All of my considerations and suggestions were properly and thoroughly addressed. I appreciate the detailed and thoughtful responses to all my comments. The additional figure (S4) and details in the methods and nanoSIMS analyses are important improvements and well implemented. THANK you for the opportunity to review this important and timely work.\nA: We thank the reviewer for their constructive feedback and support.\nReviewer 2:\nR2: Review of revised manuscript:\nI am very happy to see that the authors were able to provide additional data to support their conclusions. Particularly, the addition of the ammonium concentrations is very valuable to show that indeed N limitation sets in after some hours and that therefore organisms are likely competing for it. All other comments have also been resolved, and the manuscript is really well written and structured. I have no further comments.\nA: We thank the reviewer for their constructive feedback and support.\nReviewer 3:\nR3: Thank you for addressing my previous comments on the manuscript.\nThe additional data on 13C-pyruvate uptake by host and symbionts, and the inclusion of the ammonium concentration data have further strengthened the manuscript.\nA: We thank the reviewer for their constructive feedback and support. Please see below for detailed responses.\nR3: I have only few remaining suggestions/comments:\nL89-90: Ecological niche and tolerance limits of this symbiosis were probably the same/similar in their recent evolutionary history also before entering the Anthropocene - please rephrase.\nA: Agreed. The reference to the Anthropocene was removed from the sentence accordingly.\nR3: L116: maybe better \"...ammonium uptake by *both* aposymbiotic and symbiotic...\" for clarity\nA: Implemented as suggested.\nR3: L134-135: Is it possible that the 10mM pyruvate addition has an inhibitory/detrimental effect on the symbionts' activity? Has this been tested in symbiont pure culture?\nA: While we did not test the effects of pyruvate on algal cultures, we did test prolonged pyruvate exposure in Aiptasia. Over the course of two-week experiments, no decline in algal photosynthetic efficiency or pigmentation was observed. Hence, it is unlikely that the used pyruvate concentrations had severe negative effects on algal symbionts or their host.\nR3: L141, L310: please clarify what starvation refers to here - I assume no feeding with Artemia, but it could also mean no light.\nA: Indeed. We now clarify: “…without heterotrophic nutrient sources…”\nR3: L206-209: Can higher 15N enrichment by the symbionts at lower light intensities not simply be due to less excess released organic C from the symbionts due to lower light stress, and thus less available organic C for the host (and thus less host N-assimilation), and a more tightly coupled C fixation : N assimilation ratio by the symbionts?\nA: Light stress is unlikely a major driver of carbon translocation in the symbiosis. If high algal 15N enrichment would indicate reduced competition by the host, host and algae should have inversed 15N enrichment patterns. Yet, both symbiotic partners show their highest 15N enrichment at intermediate light levels. Hence, we propose that increased nitrogen competition reduces host catabolic nitrogen release and increases the relative proportion of environmental ammonium in the nitrogen pool of either symbiotic partner. For clarification, we have rephrased this section:\n“While 15N enrichments only reflect the uptake of environmental nitrogen in the symbiosis, the recycling of ammonium from the host catabolism is likely regulated by the same processes and follows the same patterns. High 15N enrichments thus indicate an increased nitrogen demand in the symbiosis resulting in reduced production of catabolic ammonium by the host.”\nR3: L278: While the experiments conducted look at light intensities, the discussion about environmental conditions is about heat stress - it might be useful for readers to link this more explicitly in the discussion.\nA: Great suggestion. The introduction of the paragraph has been rephrased for clarity:\n“Finally, the processes described here improve our understanding of the maintenance of the symbiosis in a changing environment. These processes are unlikely limited to the light-dependent regulation of the symbiosis but likely control other acclimation and stress responses as well. Specifically, our findings may contribute to deciphering the processes leading to the breakdown of cnidarian-algal symbiosis during heat stress (54).”\nR3: L323: The methods section states that 3 animals were sampled for nanoSIMS, the nanoSIMS figure legends state that 2 were analyzed. Please mention in the methods section that two replicates were analyzed by nanoSIMS.\nA: Thanks for spotting! In this case, the figure legend was wrong. Indeed, 3 animals were analyzed per treatment for experiment 1. The figure legend for Fig. 1 has been updated accordingly."}]

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