Characterisation of Oxylipin Receptors in the Cnidarian-Dinoflagellate Symbiosis

<p dir="ltr"><b>The success of scleractinian corals relies on their ability to establish a symbiotic relationship with dinoflagellate algae from the family Symbiodiniaceae. These dinoflagellate symbionts reside within the host's gastrodermal cells, enclosed in a host-derived membrane known as the 'symbiosome membrane'. The symbionts can communicate with the host via molecular signalling, dampening its immune system and facilitating symbiosis establishment and homeostasis. Oxylipins, short-lived signalling molecules formed through the oxidation of polyunsaturated fatty acids, are important inflammatory regulators and may play an important signalling role in the cnidarian-dinoflagellate symbiosis through the action of specific receptors, though this has yet to be confirmed. This thesis therefore aimed to localise and quantify specific oxylipin receptors within the different tissue layers of a model cnidarian – the sea anemone ‘Aiptasia’ (</b><b><i>Exaiptasia diaphana</i></b><b>) - and the reef coral </b><b><i>Acropora tenuis</i></b><b> in response to symbiotic state, symbiont identity, thermal stress and/or life-history stage. The potential functional role of a specific oxylipin receptor in Aiptasia was then characterised further, through exposure to a pharmacological inhibitor, and the application of metabolomic and lipidomic analyses.</b></p><p dir="ltr">In Chapter 2, specific antibodies were developed, and immunohistochemistry (IHC) and immunocytochemistry (ICC) used to localise and quantify four receptors: Transient Receptor Potential Cation Channel, Subfamily A, Member 1 (TRPA1); Prostaglandin E2 Receptors 2 and 4 (EP2 and EP4); and Glutamate Ionotropic Receptor Kainate Type Subunit 2 (GRIK2), in aposymbiotic anemones and symbiotic anemones hosting either the native symbiont <i>Breviolum minutum</i> or the non-native symbiont <i>Durusdinium trenchii</i>. TRPA1, EP2 and EP4 receptors are important for the signalling of the oxylipins hydroxyoctadecadienoic acid (HODEs) and prostaglandin E2 (PGE2), while GRIK2 is part of the ionotropic glutamate receptor (iGluRs) family, which help sense chemicals and pathogens in invertebrates. The receptors were found in both the gastrodermis and epidermis, and of particular interest, were further localised to the symbiosome membrane, suggesting a direct role in inter-partner signalling. However, TRPA1, EP2 and EP4 were all less abundant in the gastrodermis when it housed the native symbiont relative to the other symbiotic states, suggesting that the associated pathways are downregulated in a compatible symbiosis. GRIK2, in contrast, was upregulated in the gastrodermis and the epidermis of anemones hosting the non-native symbiont. This observationis consistent with previous suggestions that this receptor may play a role in the detection of potentially harmful microbes, especially given the reputation of <i>D. trenchii</i> as a nutritionally sub-optimal opportunist.</p><p dir="ltr">In Chapter 3, IHC was used to observe how these receptors respond to thermal stress (i.e., ‘bleaching’), again using Aiptasia containing its native symbiont <i>B. minutum</i>. Whereas EP4 abundance did not change in response to elevated temperature, TRPA1 and EP2 increased in abundance in both the epidermis and gastrodermis, consistent with increased inflammation, symbiosis dysfunction and a potential role in the cellular bleaching cascade. GRIK2 once again behaved differently, exhibiting an initial increase in the gastrodermis and then in the whole animal in response to elevated temperature. One interpretation is that high temperature caused the native symbiont to shift from a beneficial to less beneficial state, triggering the GRIK2- mediated pathway to initiate its removal.</p><p dir="ltr">In Chapter 4, IHC was used to localise and quantify EP2 and EP4 in the tissues of A. tenuis larvae and polyps, when in the aposymbiotic state or when hosting one of their two native dinoflagellate symbionts: <i>Cladocopium goreaui</i> and <i>Durusdinium trenchii</i>. Corals were inoculated with these symbionts and sampled at 3 and 30 days post-inoculation. EP2 and EP4 were consistently present in both the gastrodermis and epidermis. However, EP2 and EP4 abundance changed with symbiotic state, symbiont identity, coral life-stage, and time in symbiosis. <i>D. trenchii</i>, but not <i>C. goreaui</i>, was found to decrease EP2 levels in both coral larvae and polyps, as well as EP4 levels specifically in coral polyps. Conversely, <i>C. goreaui</i>, but not <i>D. trenchii</i>, reduced EP4 levels in coral larvae. These findings suggest that <i>D. trenchii</i> has a greater ability to modulate host immune pathways, particularly in the polyp stage. This may explain why <i>D. trenchii</i> frequently dominates <i>A. tenuis</i> polyps in the wild. Additionally, the study revealed that various coral life stages activate distinct pathways while forming symbiosis, regardless of the symbiont type.</p><p dir="ltr">In Chapter 5, to elucidate the function of the EP4 receptor in the cnidarian-dinoflagellate symbiosis, the lipidomic and metabolomic profiles of Aiptasia and its native symbiont <i>B. minutum</i> were analysed before and after chemical inhibition. The host was the most affected, with several metabolites and lipids upregulated in the absence of EP4 function. Many of these upregulated molecules had possible roles in immune pathway regulation and membrane dynamics. In contrast, the symbiont fraction was only marginally affected, with some lipids involved in membrane dynamics again being upregulated. These patterns are consistent with greater cellular stress in the host than the symbiont in response to the inhibition of EP4- mediated signalling, which ultimately induces an immune response, potentially linking this pathway to homeostatic maintenance of the symbiosis.</p><p dir="ltr">This thesis provides insights suggesting that oxylipin receptors and oxylipin signalling may contribute to inter-partner communication and homeostatic regulation of the cnidarian- dinoflagellate symbiosis, though further studies are necessary to validate these roles. Furthermore, a new pathway by which the host can identify potential pathogens and/or sub- optimal symbionts is suggested. My study augments our knowledge of the fundamental cellular mechanisms that underlie a successful cnidarian-dinoflagellate symbiosis, which is essential for developing effective tools, such as <i>via</i> the engineering of thermally-resistant corals (as discussed in Chapter 6), to mitigate climate change.</p>