Molecular Signalling in the Cnidarian-Dinoflagellate Symbiosis

The cnidarian-dinoflagellate endosymbiosis enables the success of coral reefs, though there are still major knowledge gaps concerning the molecular and cellular biology that underpins this symbiosis, limiting our capacity to understand coral reef function, and its potential responses and adaptability to climate change. One significant knowledge gap concerns the molecular signalling pathways that regulate the onset, persistence and dysfunction of the symbiosis, and determine the success of particular host-symbiont pairings. This thesis therefore aimed to elucidate mechanisms of molecular signalling in the cnidarian-dinoflagellate symbiosis. The specific objectives were: (1) to characterise a potentially key molecular signalling pathway, the phosphatidylinositol (PI) signalling pathway, through the application of bioinformatics, and determine putative functionality of its downstream effects; (2) to characterise how symbiosis and symbiont identity impacts the phosphoproteome of the model cnidarian Exaiptasia diaphana (‘Aiptasia’); (3) to characterise the phosphoproteome of Aiptasia during colonisation with both homologous (native) and heterologous (non-native) symbionts; and (4) to determine whether the symbiotic dinoflagellates release extracellular vesicles to their surrounding culture medium, and if so characterise their proteome, to elucidate their potential role in inter-partner communication. In Chapter 2, bioinformatic evidence is provided for a functionally complete PI pathway in both anthozoans and symbiotic dinoflagellates, through the identification of PI-kinases and PI-phosphatases using publicly available transcriptomes and genomes. The pathways identified in anthozoans were conserved relative to other multicellular model organisms, while the pathway identified in Symbiodiniaceae shared a high level of conservation with the parasitic Apicomplexa. The downstream function of phosphoinositide (PIP)-binding proteins was further elucidated, revealing a diverse array of roles associated to the likes of vesicle formation, MAPK-, Notch-, Wnt- and NF-κB signalling, and the JAK-STAT pathway. The diverse range of potential cellular impacts revealed in Chapter 2 led to the decision to analyse cellular signalling events more widely in the host, via the application of phosphoproteomics (Chapter 3). Indeed, the activation and activity of the PI pathway and many aspects of the downstream effects of PIP-binding proteins are controlled by phosphorylation, a powerful post-translational modification. Aiptasia in long-term symbiosis (> 1 year) with the homologous Breviolum minutum and heterologous Durusdinium trenchii was analysed alongside aposymbiotic (i.e., symbiont-free) Aiptasia. Many of the same signalling pathways identified in Chapter 2 were again identified here, such as MAPK signalling, NF-κB signalling, and the JAK-STAT pathway, while interactions with Ca2+ and cytokines were also apparent. Symbiont identity impacted the phosphoproteomic response, however, with the homologous symbionts dampening host immunity, as well as evidence of the regulation of cell adhesion and cell signalling pathways. Conversely, the heterologous symbiont induces an abundance of phosphoproteins associated with nutritional and oxidative stresses, and an increased host inflammatory response. Phosphoproteomics analysis was then applied (Chapter 4) to examine the signalling events that occur during the establishment of the symbiosis, from onset to the fully symbiotic state. Once again, two symbiont species were compared: the homologous B. minutum vs. the heterologous Symbiodinium microadriaticum. This heterologous species was used to provide a marked contrast with the homologous symbiont as it is known to struggle to form a symbiosis with this culture of Aiptasia (‘NZ1’), thereby aiding differentiation of phosphorylation events underpinning the establishment of a successful symbiosis. Pathways such as MAPK-, Wnt- and NF-κB signalling were again identified in the symbiotic state, plus modifications in cytokine and Ca2+ activity. Symbiosis onset was characterised by an abundance of phosphoproteins associated with inflammation, mRNA decay, cell proliferation/apoptosis, and oxidative and nutritional stress, irrespective of symbiont species. However, as colonisation progressed, these latter stress related-phosphopeptides declined in abundance and presence as the homologous symbiont successfully proliferated, but remained at elevated levels in the presence of the heterologous symbiont, which persisted at much lower densities. The late stages of symbiosis establishment were characterised by an abundance of phosphopeptides related to cellular homeostasis, with putative roles such as cell cycle and mRNA/DNA regulation, Ca2+ signalling, and vesicle trafficking, though interestingly, many of these phosphoproteins exhibited elevated abundances irrespective of symbiont identity or density. Finally, Chapter 5 provided evidence for the release of extracellular vesicles (EVs) by B. minutum and the free-living Symbiodiniaceae species Effrenium voratum, and successfully characterised their proteome. Putative functions for proteins that were significantly enriched in the EVs relative to the dinoflagellate cell, or indeed were EV-specific, were explored to elucidate possible roles that EVs might play in communication and molecular exchange between cnidarians and dinoflagellates. Consistent with the results of Chapters 2, 3 and 4, proteins were identified with putative functionality in MAPK signalling, cytokine interactions, Ca2+ activity, and more broadly in immunity, cell adhesion and host cell modulation. The proteome of EVs from B. minutum was characterised by a higher proportion of proteins with putative roles in cell adhesion, whereas E. voratum EVs were characterised by a higher proportion of immune suppressing and anti-inflammatory-related proteins. In both EV proteomes there were proteins identified with putative roles in host cell invasion and symbiosis maintenance. In summary, this study employed a range of powerful bioinformatic and omics methods to provide insight into the molecular signalling events that underlie the successful establishment and persistent of the cnidarian-dinoflagellate symbiosis. As discussed further in Chapter 6, it has identified candidate molecules and pathways for future study. It has thereby provided valuable targets for the engineering of more thermally-resistant, novel host-symbiont partnerships for helping to conserve, or at lease slow the decline of the world’s critically endangered coral reefs.