Nutritional Interactions in the Cnidarian-Dinoflagellate Symbiosis and their Role in Symbiosis Establishment
Authors:   , Sproles Ashley 
Published: August 2022

<p><b>Mass bleaching events induced by climate change are threatening coral reef ecosystems worldwide. Elevated seawater temperatures cause breakdown of the coral’s endosymbiotic relationship with dinoflagellate algae from the genus Symbiodinium. Corals rely on these symbionts for the majority of their metabolic carbon (provided by photosynthetic products) and for efficient nitrogen cycling in the reef ecosystem. Researchers have predicted that corals may potentially adapt to higher ocean temperatures according to the Adaptive Bleaching Hypothesis (ABH), which states that bleaching can facilitate a change in symbiont communities, allowing more thermally tolerant symbionts to become dominant. Hosting thermally tolerant symbionts thus grants the coral a higher resistance to bleaching. However, confidence in this adaptation method has wavered due to the nutritional impairments of hosts harbouring thermally tolerant symbionts under normal environmental conditions. It is also unknown if coral species that do not currently associate with thermally tolerant symbionts would be able to successfully switch their symbiont communities. Here, I explore the mechanisms driving nutritional exchange in the symbiosis and determine the effect of establishing a non-native (heterologous) association with a thermally tolerant symbiont. A combined approach of bioinformatic analysis with proteomic and isotopic labelling experiments is used to uncover a link between host-symbiont cellular integration, the potential for nutrient exchange, and the success of establishing a symbiosis.</b></p>
<p>In Chapter 2, I characterized membrane protein sequences discovered in publicly available cnidarian and Symbiodinium transcriptomes and genomes to identify potential transporters of sugars into cnidarian cells and nitrogen products into Symbiodinium cells. I examined the facilitated glucose transporters (GLUT), sodium/glucose cotransporters (SGLT), and aquaporin (AQP) channels in the cnidarian host as mechanisms for sugar uptake, and the ammonium and high-affinity nitrate transporters (AMT and NRT2, respectively) in the algal symbionts as mechanisms for nitrogen uptake. Homologous protein sequences were used for phylogenetic analysis and tertiary structure deductions. In cnidarians, I identified putative glucose transporters of the GLUT family and glycerol transporting AQP proteins, as well as sodium monocarboxylate transporters and sodium myo-inositol cotransporters homologous to SGLT proteins. I predict that cnidarians use GLUT proteins as the primary mechanism for glucose uptake, while glycerol moves into cells by passive diffusion. I also identified putative AMT proteins in several Symbiodinium clades and putative NRT2 proteins only in a single clade. I further observed a high expression of putative AMT proteins in Symbiodinium, which may have resulted from adaptations to conditions experienced inside the host cell. This study is the first to identify transporter sequences from a diversity of cnidarian species and Symbiodinium clades.</p>
<p>The phylogenetic patterns seen in chapter 2 led to the hypothesis that symbiont types may have different influences on host-symbiont cellular integration. In chapter 3, I explored this notion using the model cnidarian Aiptasia. A population of anemones was rendered aposymbiotic using a menthol-bleaching method developed by my colleagues and I, and anemones then experimentally infected with either the native (homologous) symbiont (Symbiodinium minutum, clade B1) or a thermally tolerant heterologous symbiont (Symbiodinium trenchii, clade D1a). The response of the host proteome to these associations was examined by analysing the extracted host proteins with liquid chromatography-nano-electrospray-tandem mass spectrometry (LC-nano-ESI-MS/MS), and identifying resulting peptides against a cnidarian database. Proteins were compared between B1-colonised anemones, D1a-colonised anemones, and aposymbiotic anemones to determine which proteins were affected by the different treatments. Overall, I found that the response of D1a-colonised anemones mimicked that of aposymbiotic anemones to some degree, and showed signs of less efficient carbon and nitrogen pathways. Additionally, I discovered that the symbiosome protein NPC2 was upregulated only in B1-colonised anemones and could therefore be an important determinant of symbiont success.</p>
<p>I further investigated the apparent inadequacy of carbon and nitrogen pathways in hosts harbouring Symbiodinium D in chapter 4, using isotope labelling and sub-cellular imaging. Carbon and nitrogen fluxes between experimentally infected Aiptasia with both homologous (Symbiodinium B1) and heterologous (Symbiodinium D1a) symbionts were compared over multiple time points during symbiosis establishment. This was accomplished by incubating anemones in 13C- and 15N-labelled seawater to measure metabolite transfer to the host, and anemones were also fed 13C- and 15N-labelled Artemia to measure reverse translocation to the symbiont. Carbon and nitrogen enrichment in host tissues and symbiont cells were imaged using nanoscale secondary ion mass spectrometry (nanoSIMS). In both the earlier stages of symbiosis establishment (2 days post-inoculation) and later stages of symbiont proliferation (14 days post-inoculation), Symbiodinium B1 reached significantly higher population densities (> 300%) than Symbiodinium D1a. Differences in symbiont nitrogen uptake from host tissues were detectable, however they were likely due to nitrogen limitation imposed by the comparatively high densities of the homologous symbiont. While Symbiodinium B1 cells maintained a high degree of carbon fixation throughout symbiosis establishment, less than 50% of Symbiodinium D1a cells were found to be fixing carbon in the later stages of establishment, despite still receiving substantial amounts of host-derived nitrogen. These findings support my previous prediction that Symbiodinium D1a affects the host’s carbon and nitrogen pathways, acting as a less mutually beneficial symbiont.</p>
<p>Overall, my results provide new insights into the underlying factors determining the success of symbiotic interactions. The results indicate that cnidarian hosts and their dinoflagellate symbionts have adapted to the symbiotic life-style by cellular integration, thereby making compatibility of certain biological processes a key factor in determining symbiosis success. Furthermore, the findings presented here show how the potential for nutritional exchange may be linked to host-symbiont compatibility and hence specificity. This information is relevant when making predictions about which corals may successfully adapt to climate change via the ABH.</p>