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. 2023 Nov 1;14(1):6949.
doi: 10.1038/s41467-023-42582-y.

A carbon-nitrogen negative feedback loop underlies the repeated evolution of cnidarian-Symbiodiniaceae symbioses

Guoxin Cui #  1 , Jianing Mi #  2   3 , Alessandro Moret #  4 , Jessica Menzies  4 , Huawen Zhong  4 , Angus Li  4 , Shiou-Han Hung  4 , Salim Al-Babili  2   5 , Manuel Aranda  6
Affiliations

A carbon-nitrogen negative feedback loop underlies the repeated evolution of cnidarian-Symbiodiniaceae symbioses

Guoxin Cui et al. Nat Commun. .

Abstract

Symbiotic associations with Symbiodiniaceae have evolved independently across a diverse range of cnidarian taxa including reef-building corals, sea anemones, and jellyfish, yet the molecular mechanisms underlying their regulation and repeated evolution are still elusive. Here, we show that despite their independent evolution, cnidarian hosts use the same carbon-nitrogen negative feedback loop to control symbiont proliferation. Symbiont-derived photosynthates are used to assimilate nitrogenous waste via glutamine synthetase-glutamate synthase-mediated amino acid biosynthesis in a carbon-dependent manner, which regulates the availability of nitrogen to the symbionts. Using nutrient supplementation experiments, we show that the provision of additional carbohydrates significantly reduces symbiont density while ammonium promotes symbiont proliferation. High-resolution metabolic analysis confirmed that all hosts co-incorporated glucose-derived 13C and ammonium-derived 15N via glutamine synthetase-glutamate synthase-mediated amino acid biosynthesis. Our results reveal a general carbon-nitrogen negative feedback loop underlying these symbioses and provide a parsimonious explanation for their repeated evolution.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Nutrient-flux-based negative feedback mechanism underlying symbiont population control.
a Aposymbiotic hosts are limited by the availability of energy-rich carbohydrates. They take up organic carbon from food and release nitrogenous waste to the surrounding environment as they do not have excess carbon backbones to assimilate the nitrogenous waste. b During the initial stages of symbiosis, a few colonizing symbionts have access to all host nitrogenous waste. This results in high nitrogen availability per symbiont that promotes symbiont cell proliferation. However, with increasing symbiont density, the competition for nitrogen increases until nitrogen becomes limiting and photosynthates are produced in excess and translocated to the host. In response, the host experiences an increasing provision of energy-rich photosynthates from the symbionts while symbiont proliferation slows down gradually. c In fully symbiotic hosts, symbiont-provided glucose now provides an excess of carbon backbones that allow the host to assimilate a substantial amount of its nitrogenous waste. This further reduces nitrogen availability to the symbionts and decreases symbiont proliferation rates to eventually reach a balance between symbiont proliferation and symbiont decay. Adapted from Cui, et al. .
Fig. 2
Fig. 2. Symbiont cell density changes induced by the availability of glucose and ammonium.
a–g The effects of 10 mM glucose, 250 μM ammonium, or both on symbiont density, represented by symbionts per μg host protein, were assessed in four cnidarian species: the coral S. pistillata (a, d), the sea anemone E. diaphana (b, e), the jellyfish C. andromeda (c, f), and the coral A. hemprichii (g). h Symbiont density changes in E. diaphana induced in response to different concentrations of glucose. i Symbiont density changes in E. diaphana induced in response to different concentrations of ammonium. For all plots, error bars represent the standard error of the mean. p-values were calculated using two-sided Welch’s t-tests, comparing each condition to its respective control within each experiment. The n value indicates the number of biologically independent animals used in each experiment.
Fig. 3
Fig. 3. Amino acid biosynthesis in response to glucose supplementation.
a, b Biological process GO terms enriched for DEGs identified from the comparisons between symbiotic anemones incubated with or without 10 mM glucose (a) and aposymbiotic anemones incubated with or without 10 mM glucose (b). GO terms were clustered based on their semantic similarity. Word cloud summarizes the features with keywords in each GO cluster, with different font sizes representing the level of the enrichment. c Overrepresented pathways enriched in glucose-regulated DEGs in aposymbiotic and symbiotic E. diaphana. The enrichment score (ES) was calculated by dividing the actual by the expected number of DEGs associated with the corresponding pathway. The p-values were calculated with overrepresentation analysis implemented in the R package clusterProfiler. d Expression changes of hub genes involved in amino acid biosynthesis in response to symbiosis and glucose supplementation.
Fig. 4
Fig. 4. Identification of isotope-labeled metabolites using UHPLC-HR-MS.
a Extracted ion chromatograms (EIC, Left) and the isotopic distributions (Right) of glutamine from E. diaphana incubated with 13C6-glucose and 15N-ammonium (Top) and the corresponding glutamine standard (Bottom). The inset corresponds to a zoom of the area in which different isotopologue compositions of glutamine (dashed box) were identified using HR-MS. The gray ball and square indicate 12C atom and 14N atom, respectively; the red ball indicates 13C atom, the blue square indicates 15N, and the number of carbon and nitrogen atoms are inserted in the corresponding shapes. b Metabolic footprinting of stable isotopes in the three selected cnidarian species. The proposed 13C and 15N isotope labeling is indicated as red dots or written in blue color in the structural formulas. Heatmap color indicates the relative abundance of isotope-labeled metabolites (specifically summarized as 13C14N, 12C15N, and 13C15N) normalized to their non-labeled counterparts (12C14N). The isotopic forms of each compound shown in the heatmaps follow the same order as in the figure legend. The numeric values for heatmaps are included in Supplementary Data 5. Sym, symbiotic state; Apo, aposymbiotic state; *, undetectable metabolite.
Fig. 5
Fig. 5. The incorporation of 13C across metabolites of GS–GOGAT-mediated amino acid synthesis.
a, b 13C incorporation in response to supplementation with 13C6-glcuose (a) or 13C6-glucose + 15N-ammonium (b). The relative abundance of 13C was calculated for each intermediate metabolite associated with GS–GOGAT-mediated amino acid biosynthesis. Error bars represent the standard error of the average 13C percentage. n indicates the number of biologically independent animals used in each experiment. p-values were calculated using two-sided Welch’s t-tests, comparing each condition to its respective control within each experiment.
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