The Role of Nitrogen Metabolism in the Stability and Dysfunction of the Cnidarian-Dinoflagellate Symbiosis

<p><strong>Coral reefs are able to thrive in oligotrophic waters due to their symbiosis with dinoflagellate algae (family Symbiodiniaceae), which enables efficient nitrogen recycling and conservation within the holobiont. However, increased seawater temperatures can destabilise this partnership, causing bleaching, which can result in coral mortality. Thermally-tolerant symbionts can withstand higher temperatures, but they may provide the host with less carbon while assimilating more of the host’s nitrogen. The extent of bleaching can also be mitigated or exacerbated depending on the nitrogen form and concentration in the seawater. To elucidate nitrogen’s role in coral bleaching, this thesis aimed to determine how nitrogen assimilation of the host and symbiont varies with temperature and whether this response is influenced by symbiont identity.</strong></p><p>In Chapter 2, the tropical scleractinian coral Stylophora pistillata was grown at different temperatures (26°C, 30°C and 34°C), before being placed in nutrient-replete or -depleted seawater for 24 h. The corals were then incubated with 13C-labelled sodium bicarbonate and different 15N-labelled nitrogen forms (ammonium, urea, and dissolved free amino acids) to determine their assimilation rates. I found that nutrient depletion inhibited the assimilation of all nitrogen sources studied and that heat stress reduced the assimilation of ammonium and dissolved free amino acids. However, the host assimilated over 3-fold more urea at 30°C relative to 26°C. Overall, both moderate heat stress (30°C) and nutrient depletion individually decreased the total nitrogen assimilated by the symbiont by 66%, and combined, they decreased assimilation by 79%. This led to the symbiotic algae becoming nitrogen starved, with the C:N ratio increasing by over 3-fold at 34°C, potentially exacerbating the impacts of coral bleaching.</p><p>In Chapter 3, the model anemone Exaiptasia diaphana (“Aiptasia”), colonised with either relatively heat-resistant or -susceptible symbiont species, was maintained at ambient or elevated temperatures and fed with one of four 15N-labelled nitrogen sources (ammonium, nitrate, dissolved free amino acids, and brine shrimp) and simultaneously incubated with 13C-labelled bicarbonate. This allowed quantification of nitrogen and carbon assimilation in host tissues vs. algal symbionts. Despite lower in hospite densities, the non-native symbiont Durusdinium trenchii translocated similar amounts of fixed carbon to the host as the native symbiont Breviolum minutum, contrasting with previous reports of reduced carbon translocation by this non-native symbiont, which may indicate improved inter-partner integration with time spent in symbiosis. The increased temperatures in this study, prior to visible bleaching, also led to increased photosynthetic carbon fixation and translocation to the host and increases in the proportion of heterotrophic nitrogen assimilated by the host. This raises the possibility of the symbionts becoming nitrogen limited at an early stage of bleaching.</p><p>In Chapter 4, cultures of the thermally-susceptible B. minutum and the thermally-tolerant Breviolum psygmophilum were maintained under nitrogen-depleted conditions to mimic the nitrogen environment inside their cnidarian host. Cultures were then subjected to one of three temperature treatments (cold stress, ambient control, or heat stress) and incubated with 15N-labelled ammonium, nitrate, or urea to track nitrogen assimilation. To assess how ammonium inhibition of nitrate assimilation differs across temperatures, I also incubated samples with ammonium and nitrate simultaneously. In parallel,13C-labelled bicarbonate incubations quantified carbon fixation, allowing carbon and nitrogen fluxes to be linked. I found that temperature effects on ammonium assimilation and carbon fixation increased with temperature in both species, but nitrate assimilation only increased with temperature in B. minutum, and was strongly modulated by the availability of ammonium.</p><p>In Chapter 5, Aiptasia colonised with the native symbiont B. minutum or non-native D. trenchii was incubated with 13C-labelled bicarbonate and either 15N-labelled ammonium or DFAA for 1 h, and then chased for 120 h. At multiple intervals, I determined the quantity of 13C and 15N in both host tissue and symbiont cells to assess species-specific translocation patterns and nutrient fate. Both symbionts assimilated similar amounts of ammonium per symbiont cell, but D. trenchii more efficiently incorporated host-derived nitrogen released via DFAA metabolism, indicating a competitive advantage in scavenging waste nitrogen. Contrary to expectations based on previous, shorter-term experiments, D. trenchii translocated photosynthate to the host at rates comparable to B. minutum, providing further evidence that inter-partner integration may improve with time spent in symbiosis.</p><p>This thesis provides a mechanistic link between photosynthesis, nitrogen conservation, and bleaching, and helps to explain, at least in part, why corals with dense symbiont populations bleach more readily under heat stress, and why elevated ammonium can mitigate coral bleaching. This understanding can allow us to develop tools to prevent coral bleaching from occurring at a local scale.</p>