Lipid mediators and a new HOPE in the cnidarian-dinoflagellate symbiosis

Oxylipin lipid signalling could be a potential mechanism for inter-partner recognition and homeostasis regulation in the cnidarian–dinoflagellate symbiosis, which forms the ecological basis of coral reefs. Oxylipins have also been proposed as mediators of coral symbiosis breakdown under elevated seawater temperatures, which are continuing to increase due to anthropogenic climate change. Until now, however, these hypotheses lacked analytical evidence to elucidate the synthesis, metabolism, and signalling pathways of oxylipins and other lipid mediators. In this thesis, I explore lipid signalling pathways in the cnidarian-dinoflagellate symbiosis by combining different analytical techniques to characterize and quantify lipids and oxylipins in both the host cnidarian and dinoflagellate symbiont in response to symbiotic state and elevated temperature. This approach enabled the building of a conceptual model that describes the universal role of specific oxylipin stereoisomers as agonists and antagonists of receptors that might regulate symbiosis homeostasis and dysbiosis via the activation or deactivation of the cnidarian host immune system and inflammation transcription factors. My specific objectives were to: 1) characterize and monitor lipids and photosynthetic pigments in the cnidarian-dinoflagellate symbiosis using the Aiptasia-Breviolum minutum model system; 2) determine how octadecanoid profiles and their potential regulatory signalling pathways influence symbiosis homeostasis, using the same cnidarian host colonized with the native B. minutum and the non-native Durusdinium trenchii; and 3) to determine how elevated temperature can lead to dysbiosis and thermal bleaching via alterations in octadecanoids, eicosanoids and docosanoids synthesis and lipid signalling pathways, in both Aiptasia and B. minutum.

Chapter 2 describes the development of a semi-quantitative untargeted analytical platform for the characterization and monitoring of the lipidome and photosynthetic pigments in this model system to establish a baseline for the proceeding chapters. Important analytical aspects for compound annotation and method reproducibility and sensitivity are discussed with suggestions for good practices for generating high quality datasets and for allowing comparison between different studies. The platform allowed the monitoring of 107 analytes with coefficients of variation below 30% across distinct sample batches. The anemone host tissue uniquely presented plasmalogen phospholipids and ceramide aminoethylphosphonate sphingolipids. Glycolipids characteristic of chloroplast membranes and betaine lipids with diacyl chains were exclusively found in the symbiont. Symbiosis increased omega-3 fatty acids abundance in Aiptasia tissues, mainly in the free-form, but also in the acyl chains of membrane and storage lipids. In B. minutum, symbiosis was marked by increased abundance of free fatty acids and storage lipids and an up to 40-fold increase of betaine lipids. Significantly, because oxylipin synthesis is universally regulated by fatty acid substrate availability, these findings explored different complex membrane lipids as substrates for oxylipins synthesis that were monitored in Chapters 3 and 4.

The chloroplast membranes of the symbiont were enriched with C-18 fatty acids, which in Chapter 3 were explored as precursors of 84 different octadecanoid stereoisomers, which were reported in the cnidarian-dinoflagellate symbiosis for the first time. Distinct stereochemistry specificity for the synthesis of R and S octadecanoids enantiomers was seen in aposymbiotic anemones (i.e., symbiont-free) and both free-living cultured dinoflagellate symbiont species (i.e., the native B. minutum and the non-native D. trenchii), respectively. These data showed that the symbiont derived 13(S)-hydroxy-octadecatetraenoic acid (13(S)- HOTE) serves as a potential agonist of host nuclear receptors that downregulates inflammatory transcription. Only symbiosis with the native symbiont B. minutum decreased the abundance of pro-inflammatory 9(R)-hydroxy-octadecadienoic acid (9(R)-HODE) in the host. In contrast, symbiosis with the non-native symbiont D. trenchii was marked by higher abundance of autoxidation-derived octadecanoids, corroborating previous evidence for cellular stress in this association.

The lipidome and octadecanoids profiling were subsequently integrated with the monitoring of eicosanoids and docosanoids in Chapter 4 to determine which oxylipin stereoisomers and potential signalling pathways were altered by elevated temperature in the Aiptasia-B. minutum model. A total of 244 oxylipins were monitored in combination with 107 lipids. This study reported stereoisomers of hydroxy-octadecapentaenoic acid (HOPE) for the first time in any biological system, with the 13(S)-HOPE enantiomer being suggested to have the same bioactivity as the 13(S)-HOTE reported previously. Both were symbiosis biomarkers that could potentially contribute to symbiosis homeostasis via binding with nuclear receptors that might trigger the suppression of the host’s immune system. No signs of dysbiosis were reported for the host, but abrupt changes marked by an up to 92-fold drop in the abundance of epoxides and diols from cytochrome P450-derived enzymes were reported for the symbiont, which also exhibited a decrease in chlorophyll-a content and photosynthetic efficiency. This indicates that the symbiont might be the initiator of the thermal bleaching cascade, consistent with previous hypotheses. Prostaglandin 1 series might have their fatty acid substrate oxylipin precursor i.e., dihomo-gamma linolenic acid (DGLA, 20:3 n-6) translocated to the host from the symbiont. The symbiont derived downstream DGLA product i.e., PGE1 might compete at the substrate level with the host derived PGE2 for the binding with symbiosome membrane receptors and consequently inhibit the activation of the host immune system. This chapter provided strong evidence that symbiosis homeostasis, including under elevated temperature, might be mediated by the fine spatial and temporal tuning of oxylipins.

Overall, this study provides foundational knowledge of regulatory lipid signalling in the cnidarian-dinoflagellate symbiosis, highlighting potential links with immuno-regulation in particular. Moreover, it reveals potential target pathways for supporting the development of conservation and restoration tools, which have been widely proposed for enhancing the survival of coral reefs in warmer future oceans. Structural and comprehensive phenotypic screening might be combined with reverse genetics in future studies, to validate and further describe the enzymatic candidates involved in the signalling pathways suggested here. There should also be increased effort to understand oxylipin formation, endogenous levels, and host-symbiont signalling, as such information could be hugely valuable for informing the bioengineering and selective breeding of more optimal and thermally resilient host-symbiont pairings.