Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Abstract

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.

Fig. 1. Characterization of coral-associated diazotroph communities after 10 days of heat stress.

A Relative diazotroph community composition of individual colonies under control (left) and heat stress (right) conditions based on nifH amplicon sequencing. Notably, no archaeal diazotrophs could be detected in the present study. B Change in the relative abundance of dominant diazotroph orders during heat stress relative to control conditions (mean ± SE). Asterisks indicate a significant change (p < 0.05) under heat stress compared to control corals.

Fig. 2. Nitrogen fixation activity and assimilation in the coral holobiont after 10 days of heat stress.

A Nitrogen fixation activity of coral holobionts (n = 5 per treatment) was quantified via the acetylene reduction assay. B Net assimilation of diazotroph-derived nitrogen in the coral holobiont (n = 5 per treatment) quantified by ¹⁵N₂ isotope labeling and bulk stable isotope analysis. C Nitrogen fixation activity and assimilation of diazotroph-derived nitrogen show a positive correlation across colonies (symbols) under control (blue) and heat stress conditions (red) with corresponding confidence intervals (gray). Pearson’s correlation coefficients (r) correspond to correlations among each condition of the corresponding color. Samples from both control and heat stress conditions of the colony with the highest enrichment (ellipse) were imaged in detail using the NanoSIMS ion microprobe. Bars and error bars indicate mean ± SE. Asterisks indicate significant differences between treatments (p < 0.05).

Fig. 3. NanoSIMS imaging of ¹⁵N₂ assimilation in the coral holobiont after 10 days of heat stress.

A Mosaic of hue saturation images of ¹⁵N¹²C⁻/¹⁴N¹²C⁻ ratios illustrating the spatial distribution of ¹⁵N enrichment in the coral tissue under control conditions. B, C Enlarged ¹⁴N¹²C⁻ NanoSIMS images of epithelial ¹⁵N₂ hotspots highlighted in A. Epithelial hotspots were primarily characterized by two distinct morphologies: oval compartments/cells with 2–3 µm length (yellow circles) and clusters of smaller rod-shaped compartments/cells with 1–3 µm in length (white circles). D, E Hue saturation images of ¹⁵N¹²C⁻/¹⁴N¹²C⁻ ratios illustrating ¹⁵N assimilation in the coral host and algal symbionts under control (D) and heat stress (E) conditions. F ¹⁵N₂ assimilation in the coral tissue and algal symbionts under control (blue) and heat stress (red) conditions based on NanoSIMS images (n = 15 per treatment). Differing letters above the boxplot indicate significant differences between groups (p < 0.05). All scale bars are 10 µm.

Fig. 4. Conceptual view of the effect of heat stress on nitrogen assimilation in the coral holobiont.

A In a stable state, low environmental nitrogen availability limits nitrogen uptake in the coral holobiont. Under these conditions, diazotroph-derived nitrogen provides an important nitrogen source for the coral-algal symbiosis. B During heat stress, the release of excess nitrogen waste from the host metabolism increases nitrogen availability in the coral holobiont. Under these conditions, the coral-algal symbiosis does not depend on diazotroph-derived nitrogen. The color of arrows indicates the proportion of diazotroph-derived nitrogen in the nitrogen flux/pool: continuous scale ranging from “low contribution of diazotroph-derived nitrogen (dark blue)” on one end of the spectrum to “high contribution of diazotroph-derived nitrogen (pink)” on the other end of the spectrum. The line width of arrows in the heat stress (B) indicates their proportional increase relative to control conditions (A).