Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis

Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis craig wheelock, Marina Botana, Robert Lewis, Alessandro Quaranta, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5690019/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Oxylipin signaling has been suggested as a potential mechanism for the inter-partner recognition and homeostasis regulation of cnidarian-dinoflagellate symbiosis, which maintains the ecological viability of coral reefs. Here we assessed the effects of symbiosis and symbiont identity on a model cnidarian, the sea anemone Exaiptasia diaphana , using mass spectrometry to quantify octadecanoid oxylipins ( i.e. , 18-carbon-derived oxygenated fatty acids). A total of 84 octadecanoids were reported, and distinct stereospecificity was observed for the synthesis of R- and S -enantiomers for symbiont-free anemones and free-living cultured dinoflagellate symbionts, respectively. Symbiont-derived 13( S )-hydroxy-octadecatetraenoic acid (13( S )-HOTE) linked to a 13( S )-lipoxygenase was translocated to the host anemone with a 32-fold increase, suggesting it as a biomarker of symbiosis and as a potential agonist of host receptors that regulate inflammatory transcription. Only symbiosis with the native symbiont Breviolum 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 Durusdinium trenchii was marked by higher abundance of autoxidation-derived octadecanoids, corroborating previous evidence for cellular stress in this association. The putative octadecanoid signaling pathways reported here suggest foundational knowledge gaps that can support the bioengineering and selective breeding of more optimal host-symbiont pairings to enhance resilience and survival of coral reefs. cell signaling coral reef Aiptasia Exaiptasia diaphana Symbiodiniaceae zooxanthellae lipoxygenase octadecanoid mass spectrometry supercritical fluid chromatography Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ecological success of coral reefs stems from the symbiotic relationship between the cnidarian host ( e.g. , corals, sea anemones) and its dinoflagellate algal endosymbionts of the family Symbiodiniaceae. Evolution of this symbiosis over space and time has resulted in a high diversity of host and symbiont genotypes, as well as a high specificity of host-symbiont pairings and therefore, the occupation of distinct ecological niches by the holobiont ( i.e. , the whole symbiosis) 1 – 3 . The symbiotic dinoflagellates supply their host with products from photosynthesis, including sugars, lipids and amino acids 4 – 6 and receive inorganic substrates and shelter from the host 7 , 8 . As in other symbiotic systems ( e.g. , rhizobia-leguminous plants, human gut-microbiota), host-symbiont exchange not only involves nutritional compounds, but also cell signaling molecules that regulate inter-partner recognition and symbiosis function 9 , 10 . These signaling molecules might cross the symbiosome membrane, the host-derived vacuole membrane that encloses the dinoflagellate symbionts within the cnidarian host’s gastrodermal cells, and which acts as the primary host-symbiont interface 11 . Glycans, small peptides, inositols, polar lipids, oxylipins and non-coding RNA have all been proposed as candidate signaling molecules that may interact with specific transmembrane transporters, enzyme-coupled receptors, and voltage-gated ion channels ( e.g. , G-protein coupled receptors, lectins, peroxisome proliferator-activated receptors) for mediation of symbiosis establishment and cellular homeostasis; however, the underlying regulatory mechanisms involved in these processes remain poorly understood 12 . Oxylipins are a group of metabolites derived from oxygenated fatty acids that have gained recent attention in the cnidarian-dinoflagellate symbiosis 12 , 13 , while also being reported as important bioactive mediators of cellular function and fate in many biological systems 14 . Oxylipins can be synthesized by the oxidation of mono- and polyunsaturated fatty acids (MUFA, PUFA) either following release from the cell membrane by lipase hydrolysis ( e.g. , phospholipase A 2 , PLA 2 ) 15 or by direct oxidation of the membrane lipids. They can also be formed by autoxidation processes ( e.g ., via free radicals), which are not stereoselective 16 (Fig. 1 a). Enzymatic formation of oxylipins is generally selective for carbon and double bond positions within the precursor fatty acid, so that only a specific regioisomer ( e.g. , 9- or 13-hydroxy-octadecadienoic acid (HODE)) is synthesized by a given enzyme 17 . Biosynthesis of oxylipins is also often stereoselective 18 , meaning that the geometrical configuration of the oxylipin product has a specific spatial 3D structure – for instance, either right-handed (the ( R ) enantiomer) or left-handed (the left ( S ) enantiomer) for molecules with only one chiral center 17 (Fig. 1 b). As such, determining the chirality of an oxylipin can indicate the synthetic source of the compound ( i.e. , enzymatic or autoxidation). Determination of oxylipin biological function and regulation is aided by knowledge of the regio- and stereo-isomer configuration because the propagation of signaling cascades often relies on their interaction with stereoselective membrane receptors 19 , 20 (Fig. 1 c). To date, only oxylipins derived from oxidation of 20-carbon fatty acids ( i.e. , eicosanoids), particularly products of arachidonic acid (ARA, 20:4, n-6), have been suggested to exert key functions for host-symbiont communication in the cnidarian-dinoflagellate symbiosis 21 – 23 . Nevertheless, the high diversity of PUFA precursors, from both the host and symbiont, suggests that this symbiosis a promising model for exploration of novel oxylipins and the pathways responsible for their synthesis 13 , 24 , 25 . PUFA oxidation is mediated by the availability of both the substrate ( i.e. , fatty acid) and initiator ( i.e. , an oxygenating agent – enzymes, free radicals or singlet oxygen), and by the scavenging capacity of the antioxidant machinery present in the biological system being studied. It is therefore necessary to explore the distribution of intact PUFAs and their modifying enzymes to better predict oxylipin profiles and their synthetic routes 26 , 27 . Stearidonic acid (SDA; 18:4, n-3) and octadecapentaenoic acid (ODPA; 18:5, n-3) are the most abundant PUFAs integral to the chloroplast membranes of the symbionts 13 , 28 . Oxylipins derived from 18-carbon fatty acids are termed octadecanoids 29 and have been primarily studied in plants, with the classical octadecanoid pathway critical for the formation of the phytohormone jasmonic acid (derived from alpha-linolenic acid, ALA 18:3, n-3) 30 – 32 . These ALA-derived metabolites are compounds of interest in the cnidarian-dinoflagellate symbiosis 33 , 34 . However, octadecanoids originating from SDA and ODPA are less studied and have only been reported to occur in cultures of Symbiodiniaceae 13 ; their role in the cnidarian-dinoflagellate symbiosis remains unexplored. Here we assessed the effects of symbiotic state and symbiont identity in the cnidarian-dinoflagellate symbiosis using a chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) method to perform metabolic profiling of octadecanoids 35 . We used the sea anemone Exaiptasia diaphana (commonly called ‘Aiptasia’) as a host model, in symbiosis with either its native (homologous) symbiont Breviolum minutum or the non-native (heterologous) symbiont Durusdinium trenchii . This latter symbiont was chosen as it is a widely studied opportunist that provides less nutritional benefit to the host and induces cellular stress when it populates Aiptasia under experimental conditions 22 , 36 . Symbiont-free anemones ( i.e. , aposymbiotic) and cultured symbionts were also analyzed to determine species specificity for the biosynthesis of specific oxylipin stereoisomers before addressing the impact of symbiotic state. Our comprehensive phenotypic analyses combined with top-down bioinformatics for determination of candidate biosynthetic enzymes of 18-carbon fatty acids enabled us to further describe the signaling cascades and pathways present in the cnidarian-dinoflagellate symbiosis and provide evidence for the inter-partner exchange of octadecanoids and their potential roles as lipid mediators. Results Distinct biosynthesis patterns in aposymbiotic anemones vs. cultured dinoflagellates By chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) (SFC-MS/MS), we were able to quantify 84 octadecanoids across all host and symbiont samples, including compounds derived from oleic acid (OA) (18:1, n-9), linoleic acid (LA) (18:2, n-6), alpha-linolenic acid (ALA) (18:3, n-3), gamma-linolenic acid (GLA) (18:3, n-6) and stearidonic acid (SDA) (18:4, n-3). There was significant structural diversity in the detected octadecanoids; however, the overall concentrations of the different functional groups were in the order of mono-hydroxy > epoxy > oxo > trihydroxy > dihydroxy (Fig. 3 ; see Fig. 2 for octadecanoid nomenclature). Diols derived from OA and ALA, as well as triols from ALA were absent from all sample groups (a detailed list of octadecanoids and their concentrations are provided in Table S1 ). Aposymbiotic anemones and cultured dinoflagellates were first assessed for species-specificity with respect to the biosynthesis and potential origins of octadecanoids before evaluating the effects of symbiotic state. Differential stereochemistry specificity was observed (Fig. 3 a), with aposymbiotic anemones predominantly containing the ( R ) enantiomer of mono-hydroxy octadecanoids and cultured dinoflagellates the ( S ) enantiomer (Fig. 3 b and c ). Aposymbiotic anemones primarily produced ( R ) mono-hydroxylated forms of all regioisomers of SDA ( i.e. , HOTEs), GLA ( i.e. , HOTrEs-γ) and ALA ( i.e. , HOTrEs). The observed stereoselectivity was determined using the % enantiomeric excess (ee), with values ranging from 64–100% ( Table S2 ). Cultured B. minutum and D. trenchii exclusively contained the SDA-derived monohydroxy 13( S )-HOTE ( ee = 100%), consistent with enzymatic biosynthesis. In both dinoflagellate species, ee values also suggested the biosynthesis of the 13( S )-HOTrE enantiomer ( ee = 100% and 94% in Breviolum minutum and Durusdinium trenchii , respectively) ( Table S2 ). The only octadecanoid that exhibited the same stereospecificity for both aposymbiotic anemones and cultured dinoflagellates was 9( R ),10( S ),13( R )-TriHOME ( ee = 100% in all cases for position C-9) ( Table S2 ). Alterations in octadecanoid pathways in response to symbiotic state Symbiont population densities in symbiotic anemones were not significantly different between the two dinoflagellate species (0.60 ± 0.16 and 0.57 ± 0.12 cells/ng host protein for B. minutum and D. trenchii , respectively). Any differences in the octadecanoid profile were therefore likely to be symbiont species-specific rather than a symbiont density effect. Profiling oxylipins with regio- and stereo-specificity enabled us to investigate alterations in distinct oxygenation pathways associated with symbiotic state and symbiont identity. There was a greater shift in the octadecanoid profile associated with symbiotic state ( i.e ., symbiosis vs . aposymbiosis) than with symbiont identity (Fig. 4 ). Phenotypic remodeling was broadly manifested as upregulation of octadecanoids in both the host and symbiont when in symbiosis. Additionally, both partners showed lower ee values for multiple octadecanoid enantiomers when in symbiosis than in their isolated states ( Table S2 ), with S enantiomers upregulated in the host and R enantiomers upregulated in the symbiont. Specifically, in symbiosis, the host exhibited an increased abundance of the 9( R ),10( S ),13( R )-TriHOME diastereoisomer and the ( S ) stereoisomers of HOTEs and HOTrEs-γ, with the biggest increases observed in D. trenchii -colonized anemones (p < 0.05 with FDR) (Fig. 5 a; Table S3 ). For EpODEs, EpOMEs and epoxy-HOMEs, an increase of up to 30-fold only occurred in anemones containing D. trenchii ( Tables S4 ). In contrast, in B. minutum -colonized anemones, the level of 9( R )-HODE decreased 3-fold relative to aposymbiotic anemones ( Fig. 5 a). In the symbionts, the abundance of all octadecanoids except for 13( S )-HOTE was upregulated in symbiosis vs. culture, with most increases being more marked in D. trenchii than in B. minutum (Fig. 5 b; Table S5 ). Increased enzymatic oxidation of ALA by 9-lipoxygenase (9-LOX) was evident for both dinoflagellate species. The abundance of HOTrEs from ALA was also elevated for both species. The abundance of epoxy-HOMEs and EpODEs increased in both species, but in D. trenchii this increase was 3-fold greater than in B. minutum (Fig. 5 b; Table S5 ). Evidence for inter-partner exchange of octadecanoids Aposymbiotic anemones did not contain 13( S )-HOTE, although this compound appeared in the host when colonized by either symbiont species, with the concentration 4-fold greater in anemones containing D. trenchii than those containing B. minutum (Fig. 5 a; p < 0.05, with FDR). The ( S ) ee was 22% and 29% for anemones symbiotic with B. minutum and D. trenchii , respectively (Fig. 5 ). In comparison, the abundance of 13( S )-HOTE was significantly diminished in the symbiont when in symbiosis, in contrast to the pattern observed for all other octadecanoids (Fig. 5 b; Table S5 ). The ( S ) ee values decreased from 100–10% in B. minutum and from 100–23% in D. trenchii in the symbiotic state ( Table S2 ). These patterns suggest potential translocation from symbiont to host when in symbiosis. Novel 9( S )- and 13 ( S )-lipoxygenases in Symbiodiniaceae One B. minutum (MSTRG. 59407) and two D. trenchii (MSTRG.68745/7968) novel LOX-domain encoding mRNA sequences were proposed as novel LOX enzymes for Symbiodiniaceae (Fig. 6 ). The sequence for B. minutum was incomplete, but the alignment with AtLOX3 showed a combination of alanine and phenylalanine residues at the active site ( Fig. S2 ), which revealed it as a 13( S )-LOX 41 . MSTRG.59407 had no blast hits with identity percentages above 79.9% in any of the searched public databases ( e.g. , GeneBank, Reef Genomics and UNIPROT). The sequences for D. trenchii were 99.9% identical to two other sequences published in GenBank (CAK8999115.1 and CAK9070246.1), which have been described as “unnamed proteins”. The alanine alignment and valine alignments with the active site residues of AtLOX 1 and 5 revealed that MSTRG.68745/7968 were 9( S )-LOXes ( Fig. S2 ). All transcripts demonstrated active constitutive expression across both cultured and symbiotic Symbiodiniaceae RNA-seq datasets. Homology comparisons grouped the new Symbiodiniaceae LOXes within their own clade in the tree of life, being evolutionarily divergent from green algal ( i.e., Chlamydomonas reinhardtii and Lobosphera incisa ) and vascular plant LOXes, but rather closely related to a fungal Mn-LOX (Fig. 6 ). Discussion Interest in oxylipins as potential lipid mediators of inter-partner signaling in the cnidarian-dinoflagellate symbiosis has increased recently 12 , 13 . Here, octadecanoid biosynthesis by aposymbiotic anemones was found to be restricted to ( R) enantiomers (as indicated by ee values of 100% for most HOTEs, HOTrEs and HOTrEs-γ). This trend was also reported for eicosanoid biosynthesis in various soft corals 37 – 40 and the stony corals Acropora sp. and Galaxea fascicularis 24 ; however, the stereospecificity of octadecanoid formation in cnidarians was previously unknown. In contrast, octadecanoid biosynthesis in both the cultured homologous symbiont Breviolum minutum and the heterologous Durusdinium trenchii was restricted to ( S ) enantiomers, with 13( S )-HOTE and 13( S )-HOTrE especially prominent ( ee values from 94–100%). Both compounds are likely formed via a 13( S )-lipoxygenase (LOX), which is enantioselective for the synthesis of ( S ) octadecanoids in the jasmonic acid pathway 41 . In microalgae, studies of the LOX pathways have primarily focused on the synthesis of eicosanoids and docosanoids (derived from 22-carbon fatty acids) 42 – 44 , and there is a paucity of investigations of octadecanoid biosynthesis 45 , 46 . To our knowledge, this is the first study of LOX enzymes in Symbiodiniaceae and applied top-down bioinformatics to elucidate biosynthetic pathways in the cnidarian-dinoflagellate symbiosis. Symbiosis resulted in increased levels of octadecanoids in both host and symbionts; however, there was a commensurate up to 3-fold decrease in their ee values. These findings suggest a potential dynamic inter-partner exchange of compounds and/or alterations to symbiosis homeostasis marked by a higher autoxidative state 47 , 48 . Specifically, D. trenchii promoted greater abundance of octadecanoids in the host, consistent with a stress response, which was further indicated by an up to 30-fold increase in epoxides ( i.e. , EpODEs, EpOMEs and epoxy-HOMEs). In contrast, symbiosis with the homologous B. minutum resulted in only a slight increase in octadecanoid abundance, which was more consistent with a homeostatic adjustment. 9( R )-HODE has been linked to inflammation in mammals 49 – 51 and was exclusively downregulated in B. minutum -colonized anemones, perhaps serving to dampen cellular stress and so contribute to the persistence of this host-symbiont pairing 2 , 22 . A putative regulatory role can be attributed to 9( R ),10( S ),13( R )-TriHOME (ee = 80% and 64% for position C-9, for anemones with B. minutum and D. trenchii , respectively), for which the abundance increased in symbiotic host tissues, although the associated pathway is unknown. In plants and humans, TriHOMEs have been linked to lipoxygenases with species and tissue stereospecificity 52 – 55 . In plants, TriHOMEs may also be linked to epoxyalcohol synthases (EAS), which together with allene oxide synthase (AOS), are part of CYP74 in the cytochrome P450 family 41 , 56 . In cnidarians, AOS and 8( R )-LOX genes have been linked to eicosanoid synthesis 39 , 57 , 58 ; but whether they are also involved in the synthesis of TriHOMEs and other octadecanoids remains to be investigated, as does the potential synthesis role by EAS. Symbiosis also led to a general increase in the abundance of octadecanoids in the symbiont, with the greatest increase in D. trenchii . For both symbiont species, symbiosis caused changes in the ALA oxidation pathway, possibly via one of the linoleate-LOXes described here. This pathway occurs in plants 41 , where synthesis of mono-hydroxy forms and a wide diversity of ALA downstream products ( e.g. , jasmonic acid and jasmonates) occurs in response to wounding and necrotrophic fungal infection and recognition 59 , as well as during network communication in mycorrhiza 10 , 60 . The involvement of ALA oxidation products in host cell invasion and/or host-symbiont communication in the cnidarian-dinoflagellate symbiosis is therefore conceivable. However, the upregulation of LA and ALA oxidation pathways could also be linked to oxidative stress, with the increased abundance of EpOMEs and EpODEs in D. trenchii suggesting that this non-native symbiont species could be under greater cellular stress than the native B. minutum . Such stress could have arisen due to the greater levels of oxidative stress in the host’s tissues discussed above, or perhaps other aspects of physiological dysfunction associated with the poorer degree of host-symbiont integration that is typical of this symbiotic pairing 2 , 22 , 61 , 62 . Octadecanoids as putative mediators of lipid signaling Regulatory signaling involves both intracellular and intercellular cascades, and the combination of both is crucial for endosymbioses 9 , 60 , 63 , 64 . In the cnidarian-dinoflagellate symbiosis, intercellular signaling between the partners may occur via G protein-coupled receptors associated with the symbiosome membrane 12 , 65 , ultimately leading to alterations in host nuclear transcription factors 66 , 67 ; however, the molecular pathways involved are poorly understood 47 , 68 . In the case of intracellular signaling, a given molecule may bind to nuclear peroxisome proliferator-activated receptors (PPAR) that also regulate transcriptional factors, such as nuclear factor kappa-B (NF-kB) and tumor necrosis factors (TNF), mediators of inflammatory and immune responses 64 , 69 . This latter mechanism has been widely studied in mammals but not in the cnidarian-dinoflagellate symbiosis. Nevertheless, the expression of both NF-kB and TNF has been observed to change in the host in response to symbiosis, leading to the suggestion that they help regulate symbiosis stability 47 , 68 . The role of octadecanoids as PPAR ligands and controllers of transcriptional factors has received considerable attention in the biomedical field, and they have been extensively targeted for the development of metabolic regulatory drugs 70 . 13( S )-HODE and 9( S )-HODE are PPAR-γ ligands and controllers of the immune response in mammals, showing that different oxylipins can bind to the same receptor, although binding affinity is variable 70 , 71 . The SDA-derived octadecanoid 13( S )-HOTE, extracted from cultures of the green microalga Chlamydomonas debaryana , has also been shown to be a strong PPAR-γ ligand and to promote downregulation of NF-kB and other transcription factors in cell cultures and in a murine recurrent colitis model 72 – 74 . In our experimental system, the abundance of the symbiont-derived 13( S )-HOTE decreased in the symbiont fraction and increased in the host fraction while in symbiosis, supporting evidence for inter-partner communication via translocation of this compound. While confirmation is needed, our findings support this octadecanoid-receptor binding as a potential mechanism of host immune modulation. Transport of oxylipins through membranes often occurs through lipoproteins and we have previously reported these proteins to be 20-fold more abundant when Aiptasia is colonized with B. minutum 75 . Ultimately, oxylipin-receptor binding and translocation are promising themes for future research on the cnidarian-dinoflagellate symbiosis, with considerable potential to draw parallels with the biomedical field given that the cnidarian immune system is similar to that of higher organisms, including mammals 76 . Novel lipoxygenases in Symbiodiniaceae We also studied the enzymatic candidates for the biosynthesis of 13( S ) octadecanoids, which included a novel 13( S )-LOX for B. minutum and two 9( S )-LOXes for D. trenchii . In vascular plants, most 9-LOXes described to date are cytosolic and only free fatty acids serve as substrate, whereas 13-LOXes are either chloroplast-derived or cytosolic; and both esterified membranes or free fatty acids can serve as substrates 41 . 13-LOX activity forms the 13-positional mono-hydroperoxy fatty acids ( e.g. , 13( S )-HpOTE and 13( S )-HpOTrE), whereas 9-LOXes synthesize the 9-positional forms ( e.g. , 9( S )-HpOTE and 9( S )-HpOTrE) 77 . These fatty acid hydroperoxides can serve either as substrates for additional metabolic transformation ( e.g. , AOS or EAS), or are reduced to form the corresponding mono-hydroxys ( e.g. , HOTrEs or HOTEs). In cyanobacteria LOX, both positional mono-hydroxy forms can be synthesized by the same enzyme, because the insertion of molecular oxygen into the fatty acid chain is guided by steric shielding of the radical intermediate rather than by the positional orientation of the fatty acid when entering the enzyme active site 78 . We do not know which mechanism occurs in the Symbiodiniaceae, but the novel LOX identified in B. minutum is a candidate for the synthesis of symbiont-derived 13( S ) octadecanoids. The active constitutive expression of both 13( S ) - and 9( S )-LOX candidates across both cultured and symbiotic Symbiodiniaceae RNA-seq datasets also suggests that they might not only be real sequences, but also of paramount importance for symbiont metabolism. In vascular plants, chloroplast-derived 13( S )-LOX has the highest substrate oxygenation kinetics with ALA 41 , 79 , which is also true for LiLOX in the green alga Lobosphera incisa 80 and the cyanobacterium Cyanothece sp. 78 . In plants, this initial oxygenation step is part of the jasmonic acid pathway, where jasmonic acid and other jasmonates help regulate the immune response, growth and development 31 , 81 . In Symbiodiniaceae, SDA and ODPA are more abundant than ALA 13 , 28 , suggesting that their as yet unidentified downstream products might be key metabolic mediators and perhaps play a role in host-symbiont recognition. This hypothesis needs further investigation; however, it suggests that future studies should focus on ODPA-derived octadecanoids as well as oxylipins from 20-carbon and 22-carbon PUFAs to provide a broader understanding of the lipid signaling pathways present in the cnidarian-dinoflagellate symbiosis. Conclusion This work presents the first detailed quantification of octadecanoids in cnidarians and their associated symbionts. Determination of the octadecanoid stereochemistry provided insight into their biosynthetic route of formation. Our findings indicate a dynamic exchange of octadecanoids between symbiont and host and suggest that specific oxylipin stereoisomers act as putative lipid signaling mediators of this symbiosis. Understanding the molecular processes that underpin host-symbiont communication and symbiosis stability is of importance across symbiotic systems and is key in determining how to stabilize or strengthen host-symbiont interactions. For this purpose, the combination of lipidomics and reverse genetics is a powerful tool for further elucidation and discovery of new molecular pathways, which can contribute to our improved understanding of coral reef function. Accordingly, increased efforts should be made in understanding octadecanoid formation, endogenous levels, and signaling in relation to host-symbiont communication. This approach has the potential to improve the development of restoration and conservation tools involving bioengineering and selective breeding of more optimal host-symbiont pairings to enhance coral reef survival in the face of environmental stressors. Material and methods Maintenance of animal and algal cultures Symbiodiniaceae cultures ( Breviolum minutum , culture ID CCMP830; Durusdinium trenchii , culture ID D1A001 (see Table S6 for species confirmation) were grown for eight years in f/2-enriched 0.22 µm filtered seawater (FSW) 82 . Culture flasks were maintained inside an incubator set to 25°C (± 0.5°C), under cool fluorescent lights (Osram Dulux 36/W890) at 90–110 µmol photons m − 2 s − 1 on a 12:12 h light/dark cycle. Long-term clonal stocks of the sea anemone Exaiptasia diaphana (Aiptasia, culture ID: NZ1) were rendered aposymbiotic using a menthol protocol 83 . The anemones were confirmed to be aposymbiotic based on the absence of chlorophyll autofluorescence under confocal microscopy (Olympus Provis AX70; 100x magnification) and the absence of symbiont DNA amplification through polymerase chain reaction using ITS2_F and ITS_R primers 84 . Two thirds of the aposymbiotic stock (n = 150 anemones) were inoculated with either homologous B. minutum or heterologous D. trenchii and maintained in symbiosis for 15 months as described by Wuerz et al. (2023) 85 . All anemones were maintained in FSW and fed once a week with freshly hatched Artemia sp. nauplii. Stocks were kept at 25°C and 90–110 µmol photons m − 2 s − 1 on a 12:12 h light/ dark cycle using Philips 6500K bulbs. Experimental setup Symbiodiniaceae cultures (n = 3 per species) were grown in 500 mL Erlenmeyer flasks under the same conditions for long-term maintenance. Initial cell densities in flasks were 1000 cells per mL and culture growth was monitored using the confocal imaging system IN Cell analyzer 6500 Hs, where cell density was quantified with Image J processing software (ten counts per replicate). Cultures were monitored over time and sampling for octadecanoid analysis occurred after 16 days, while the cultures were still in exponential growth ( Fig. S1 ), using sterile pipettes (150 mL per replicate). Samples were centrifuged at 4000 g for 5 min and the resulting algal pellets flash-frozen and freeze-dried before storage at -80°C in sterile 1.5 mL Eppendorf tubes. On the day of sampling, cultures were dark acclimated for 15 min within 4 h of initiating the light cycle, before measurements of ‘photosynthetic health’ (F v /F m ) using a Diving Pulse Amplitude Modulated Fluorometer (Diving-PAM, Walz, Effeltrich, Germany; settings: measuring light = 4, saturation intensity = 8, saturation width = 0.8 s, gain = 3, and damping = 3) ( Fig. S1 ). Anemones for each experimental treatment ( i.e. , aposymbiotic; symbiotic with B. minutum ; and symbiotic with D. trenchii ) were split across 3 x 400 mL jars per treatment (n = 15 anemones per jar) that contained FSW. These jars were then transferred to water baths set at 25°C (± 0.5°C) and under the same light conditions as described for the anemone stocks. All anemones were fed with Artemia sp. nauplii at the same time of day and the old water was changed with fresh FSW 8 h after feeding. Anemones were maintained under these conditions for 16 days prior to sampling and analyses. On the sampling day, F v /F m of symbiotic anemones was measured ( Fig. S1 ) as described above for the Symbiodiniaceae cultures. Anemones (N = 10 per jar) were then pooled, washed, and homogenized with 1 mL of ice-cold 10 mM sodium phosphate buffer (pH 7.4) containing 100 µM of the Fe chelator deferoxamine mesylate, using a glass tissue grinder. Aposymbiotic anemone homogenates were flash-frozen, freeze-dried and stored at -80°C in 1.5 mL sterile Eppendorf tubes. Homogenates from pooled symbiotic anemones were transferred to sterile Eppendorf tubes and centrifuged at 500 g for 5 min at 4°C, to separate host and symbiont fractions. Host tissue supernatant was transferred to a separate sterile tube and the pelleted symbiont dinoflagellate fraction was washed free of residual host material by resuspension and centrifugation using the same buffer solution. A 20 µL aliquot of each host supernatant was analyzed for protein content using a fluorometric Qubit Protein Assay Kit. After resuspension for a second time, the symbiont fraction was aliquoted equally for cell counts, conducted using the confocal imaging system IN Cell analyzer 6500 Hs (as described above), and then normalized to host protein content (see Results). All steps were performed on ice and final fractions were flash-frozen, freeze-dried and stored at -80°C until further analysis. Sample preparation and extraction Octadecanoids were extracted from freeze-dried biomass by adding 1.5 mL of methanol (MeOH) and 10 µL of internal standard (IS) mix ( Table S7 ). All samples were thoroughly mixed by vortexing and subsequently sonicated in an ice bath for 30 min. Following centrifugation for 10 min at 15,000 x g at 4°C, the supernatant was transferred to a glass tube and evaporated to dryness under nitrogen. To maximize compound recovery, this methanolic extraction was repeated a second time and combined with the first extract. The dried extracts were then reconstituted with 1 mL of a solution of 0.2 M Na 2 HPO 4 and 0.1 M citric acid (pH 5.6). To concentrate the extract, solid phase extraction (SPE) was performed as described in Quaranta et al. (2022), using an Extrahera automated sample preparation system (Biotage, Uppsala, Sweden). Briefly, the 1 mL reconstituted extract was loaded onto a preconditioned 3 mL (3 cc/60 mg) Waters Oasis HLB cartridge (Milford, MA, USA). Samples were washed three times with 3 mL of HPLC-grade water and a fourth time with 3 mL MeOH:H 2 O (1:9). Octadecanoids were eluted with 2.5 mL of MeOH and the extract was further evaporated to dryness under a nitrogen stream. Reconstitution was performed with 80 µL MeOH, and the samples were then filtered through a 0.1 µm polyvinyl-i-dene fluoride membrane spin-filter (Amicon, Merck Millipore Cooperation, Billerica, MA, USA) before being transferred to liquid chromatography vials for analysis. Blanks comprised of FSW only, and culture medium was added to ensure that oxylipin phenotypic profiles in samples were not related to possible contamination from the experimental environment. Octadecanoid profiling and analysis A chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) platform was used to perform quantitative metabolic profiling of octadecanoids as previously reported 35 . The published method was expanded by the addition of 27 novel custom synthesized standards derived from oleic acid (OA), alpha-linolenic acid (ALA, n-3), gamma-linolenic acid (GLA, n-6) and SDA ( Table S7 ). The discrimination of R and S enantiomers for the monohydroxy octadecanoids was based on the elution order in the chiral column following the same patterns as observed for the 18 enantiopure standards detailed in Quaranta et al . (2022) 35 . Extracts were first analyzed using the SFC method on a Waters UPC 2 system coupled to a Waters Xevo TQ-XS mass spectrometer. Chiral separation was performed on a polysaccharide Waters Trefoil AMY1 column (3.0 x 150 mm, 2.5 µm) set at 35°C and an injection volume of 2 µL. In addition to supercritical CO 2 as the main eluent (phase A), a co-modifier consisting of MeOH:EtOH (8:2 by vol.) and CH 3 COOH 0.1% v/v was used as phase B. The gradient started with 5% B maintained until 1 min, before increasing it linearly to 25% B at 11 min and 30% B at 12.3 min. The column was then washed with 50% B for 2–5 min and re-equilibrated under the initial conditions for 2.2 min. Flow rate was 2.0 mL/min during separation and equilibration but was decreased to 1.5 mL/min during washing. The active backpressure regulator (ABPR) was set to 2000 psi and the make-up solvent consisting of MeOH and CH 3 COONH 4 (5mM) was flow rate-based on the co-modifier to avoid excess organic solvent at the source. It started at a flow rate of 0.2 mL/min before linearly decreasing to 0 mL/min after 6 min. The chromatographic system had an MS source operating in negative-ion ESI mode with a capillary voltage of 1.9 kV, the source temperature at 150°C, the desolvation temperature at 600°C, the source offset at 30.0 V, the cone gas flow at 150 L h − 1 , the desolvation gas flow at 1000 L h − 1 and the nebulizer gas pressure at 7.0 bar. Negative multiple reaction monitoring transitions along with the collision energy, cone voltage and dwell time were manually optimized for each octadecanoid, and one transition per analyte was selected based upon sensitivity and selectivity. Calibration was performed using an 11-point linear calibration model, applying a 1/x weighted least-squared regression. Retrieved octadecanoid quantity was normalized using the dry biomass, such that concentrations are reported in ng/g. MassLynx software version 4.2 was used for data acquisition and TargetLynx for data processing. Stereochemistry of the octadecanoids was evaluated using the enantiomeric excess ( ee ) (see IUPAC compendium for chemical terminology). For each characterized regioisomer with chiral configuration, the ee was calculated using the equation: % of ( R ) or ( S ) enantiomer = 100 x total amounts of ( R ) or ( S )/ ( R + S ) De novo transcriptome assembly and mRNA expression quantification To describe the transcriptomic responses of both cultured and symbiotic B. minutum and D. trenchii , a splice-aware transcriptome was assembled from previously sequenced RNA libraries (Maor-Landaw et al. , 2019 86 , BioProject: PRJNA544863 and Bellantuono et al. , 2019 87 , BioProject PRJNA508937). Using Fastp 88 , the raw paired-end RNA sequence files were trimmed and filtered to remove sequencing adaptors and bases with Phred quality scores below 30. Reads with more than 30% low-quality bases, containing more than 5 N bases, and reads shorter than 50 bases after trimming were discarded. Using the RNA-Seq aligner Spliced Transcripts Alignment to a Reference (STAR v2.7.10b 89 ), all filtered sample reads were first mapped without guidance to the Aiptasia genome (assembly v1.1; GCF_001417965.1 90 ) to filter anemone RNA reads and double mapping sequences for downstream expression quantitation. Un-mapped RNA reads were collected and mapped without guidance to the respective symbiont genomes of B. minutum (strain: Mf 1.05b.01; GenBank assembly: GCA_000507305.1 91 ) and D. trenchii (strain: CCMP2556; GenBank assembly: GCA_963970005.1 92 ). Splice-junction aware alignment outputs were assembled into sample transcriptomes using StringTie2 v2.0.3 93 . Sample transcriptomes were then merged into species RNA transcriptomes with a minimum transcript coverage of one mapped read per base pair. All assembled RNA transcript open reading frames (ORFs) greater than 100 amino acids were predicted using Transdecoder (TransDecoder.Predict v5.7, see Github in references), to predict the most likely protein coding sequences (CDs regions) and reduce the ORF false discovery rate. The ORFs were then filtered to retain only transcripts with either Blastp protein sequence homology (e-value < 1e − 5 ) to any described or hypothetical protein in a collection of Stramenopile and Alveolate genomes ( Table S8 ) and/or HMMER (hmmscan v3.4) Pfam domain homology. Internal unique transcripts were used as training data for the species-specific Markov Model-derived coding potential of the ORFs and to revise start codon choice where statistically appropriate. After prediction, the best ORF candidates with revised starts were propagated to each genome and written into new genomic protein-coding transcript annotations. To quantify transcript abundance and test for differential expression, un-normalized read counts summarized at the gene (meta-feature) level for the homologous protein-coding transcript annotations were performed on the RNA sequence STAR genomic alignments for each sample, using FeatureCounts (Rsubread v2.16.1). All counted genes were tested for differential expression among experimental conditions from the original raw datasets using DeSeq2 94 , with default internal gene-filtering procedures. Identification of Symbiodiniaceae LOX candidates and phylogeny To identify lipoxygenase (LOX) sequences in the protein-coding transcriptomes, a collection of previously annotated LOX protein sequences, from both prokaryotic and eukaryotic species was curated ( Table S9 ). All hypothetical Symbiodiniaceae protein sequences were scanned for sequence homology to the LOX references, using a Blastp cutoff of e-value = 1e − 4 . Presence of Pfam LOX domains within the collection of Blastp candidates was confirmed with InterProScan (interproscan.sh v5.68-101.0) 95 . Protein sequence phylogeny of the 13( S )-LOX candidates was constructed from MUSCLE multiple sequence alignments, together with the other LOX references 96 . The resulting distance matrix provided the information to generate a phylogenetic tree using the neighbor-joining clustering method, with branch lengths optimized via a phylogenetic maximum likelihood criterion 97 , with Le and Gascuel model parameters 98 . The fitted tree was then bootstrapped 100 times. Statistical analysis Before evaluating the alterations caused by symbiotic state in both the host and symbiont, oxylipin profiles of both aposymbiotic anemones and cultured dinoflagellates were characterized, and symbiont species-specific compounds and the potential origins of octadecanoid enantiomer biosynthesis patterns ( i.e., R and/or S ) were assessed by ee ( Table S2 ). Principal component analysis (PCA) was used to identify the main differences in the octadecanoid profiles associated with symbiotic state and symbiont identity, in the host and symbiont separately. A two-way crossed ANOVA using symbiotic state (Symb; two levels in the host: aposymbiotic and symbiotic; two levels in the symbiont: cultured and symbiotic) orthogonal to the dinoflagellate species (Symb; two levels: B. minutum and D. trenchii ) was further applied for univariate validation of octadecanoids highlighted in the PCA. Additionally, pairwise comparisons were analyzed with Tukey's HSD test, and the significance threshold set at p < 0.05 with false discovery rate corrections. A volcano plot analysis that considered pairwise fold-change differences of greater than two was also applied to the whole octadecanoid matrix, to investigate whether oxidation pathways were altered due to the symbiotic state in the host and symbiont. MetaboAnalyst version 6.0 99 was used for all statistical analyses. Declarations Acknowledgements We thank Prof. Miguel Mies (University of Sao Paulo, Brazil), Dr. Matthias Kellerman (Carl-von-Ossietzky University, Germany), Dr. Matthew Nitschke, Dr. Gerhard Hagn and Prof. Robert Keyzers for reviewing the manuscript. We also thank Prof. Blair Paul (Marine Biological Laboratory, Woods Hole, USA) for the assistance provided with the bioinformatics pipeline and Dr. Ellen Hornung (Georg-August-University, Germany) for the assistance with the LOX analysis. This manuscript is in memory of Professor Raymond Valentine (University of California, Davis, USA), may his incessant curiosity in understanding life always remains with us. Funding This research was supported by the Marsden Fund of the Royal Society Te Apārangi, grant number 19VUW086, awarded to S.K.D., C.A.O., A.R.G., D.J.S. and V.M.W., including a postgraduate scholarship awarded to M.T.B. I.F. acknowledges funding from the German Research Foundation (DFG) grant numbers GRK 1422 and GRK 2172. C.E.W. acknowledges support from the Swedish Research Council (2022-00796) and the Cayman Biochemical Research Institute (CABRI). Contributions M.T.B. and S.K.D. conceptualization; M.T.B. experimental design and sample processing, data analysis and manuscript writing; R.E.L. bioinformatics and data analysis; A.Q. and O.S. analytical sample runs; J.R.C. standards synthesis, M.H. standards synthesis and pathway analysis supervision; I.F. LOX analysis and pathway analysis supervision; I.F., C.A.O., A.R.G., D.J.S., V.M.W. manuscript editing; C.E.W. and S.K.D. funding, supervision and manuscript editing. Corresponding authors Craig Wheelock: [email protected] Simon Davy: [email protected] Ethics declarations Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Baker, A. C. 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11:56:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOctadecanoid oxylipins can be formed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e multiple synthetic routes\u003c/strong\u003e. (\u003cstrong\u003ea)\u003c/strong\u003e Autoxidation in which free radicals (\u003cem\u003ee.g.,\u003c/em\u003e hydroxyl radical (\u003csup\u003e●\u003c/sup\u003eOH and superoxide (\u003csup\u003e●\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e)) or singlet oxygen oxidize the fatty acid substrate. Notably, this process results in a racemic mixture of the resulting mono-hydroxyl species. Here we only represent the products for the 9-regioisomer, but the same process occures for the 9-, 10-, 12- and 15- regioisomers (\u003cstrong\u003eb)\u003c/strong\u003e Enzymatic formation can result in stereo-selective formation of mono-hydroxy species (\u003cem\u003ei.e.,\u003c/em\u003e \u003cem\u003eR\u003c/em\u003e \u003cem\u003evs\u003c/em\u003e \u003cem\u003eS\u003c/em\u003e enantiomer). \u003cstrong\u003e(c) \u003c/strong\u003ePropagation of signaling cascades can rely on binding with stereoisomer-specific receptors. ALA = alpha\u003cu\u003e-\u003c/u\u003elinolenic acid; HOTrE = hydroxy-octadecatrienoic acid; COX =\u003cu\u003e \u003c/u\u003ecyclooxygenase; 13-LOX =\u003cu\u003e \u003c/u\u003e13-lipoxygenase.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/76f1292d40d00ca1b24e84b7.png"},{"id":73666429,"identity":"1cc17482-b310-4a1a-a5bf-87cee8e3573e","added_by":"auto","created_at":"2025-01-13 11:56:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":184350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbbreviated nomenclature system for octadecanoids\u003c/strong\u003e. Compound names start with the positioning of the oxygenated moiety on the carbon backbone. The number(s) is followed by the abbreviation of the moiety (‘Hp' for hydroperoxides, ‘H’ for mono-hydroxy, ‘DiH' for diols, ‘TriH' for triols, ‘oxo' for ketones, and ‘Ep' for epoxide) and by the alkyl chain length (for octadecanoids, a capital ‘O') and number of unsaturations (‘M': mono-unsaturated; ‘D': di-unsaturated; ‘Tr': tri-unsaturated; ‘T': tetra-unsaturated). The last letter indicates whether the compound is unsaturated (‘E' for enoic, unsaturated; ‘DA' for decanoic, saturated).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/f7d2dc9202824122b8b907b7.png"},{"id":73667016,"identity":"a1bda8ad-3320-4042-b9db-7658d491f57d","added_by":"auto","created_at":"2025-01-13 12:04:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of all quantified octadecanoids in the: a) aposymbiotic Aiptasia host; and cultured b) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBreviolum minutum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and c) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDurusdinium trenchii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e symbionts.\u003c/strong\u003eOctadecanoids are grouped as: mono-hydroxy octadecanoids (MONO=42 compounds), epoxides (EPOXY=26 compounds), ketones (OXO=9 compounds), and triols (TRIOLS=5 compounds). Percentages of the abundance of the \u003cem\u003eR\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003eenantiomers were calculated for mono-hydroxy octadecanoids to highlight the distinct stereochemistry specificity between aposymbiotic anemones and dinoflagellate symbionts. Octadecanoid levels are presented as ng of octadecanoid per gram of dry biomass (ng/g). The abundance of diols (n=2) was \u0026lt;100 ng/g for all cases and data for these are not presented in the graphs. Abbreviations: OA (oleic acid, 18;1, n-9), LA (linoleic acid, 18:2, n-6), ALA (alpha-linolenic acid, 18:3, n-3), GLA (gamma-linolenic acid, 18:3, n-6), SDA (stearidonic acid, 18:4, n-3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/eaa229ee3a8594bc355d8b87.png"},{"id":73666430,"identity":"04487cc5-8ec2-42eb-9691-bec59db4e6e1","added_by":"auto","created_at":"2025-01-13 11:56:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrincipal component analysis of changes to the octadecanoid profile of (a) host and (b) symbiont caused by symbiotic state\u003c/strong\u003e. The sea anemone Aiptasia was either aposymbiotic or colonized by the native symbiont \u003cem\u003eBreviolum minutum\u003c/em\u003e or non-native symbiont \u003cem\u003eDurusdinium trenchii. \u003c/em\u003eBlue circles represent individual octadecanoids and vector length is proportional to the individual contribution of each octadecanoid to the spatial distribution of sample replicates and groups. Compounds of interest are highlighted on the vectors and were further validated with a univariate test (\u003cstrong\u003eTable S3\u003c/strong\u003e; \u003cstrong\u003eFig. 5\u003c/strong\u003e). 13(\u003cem\u003eS\u003c/em\u003e)-HOTE=13(\u003cem\u003eS\u003c/em\u003e)-hydroxy-octadecatetraenoic acid; 9(\u003cem\u003eR\u003c/em\u003e)-HODE=9(\u003cem\u003eR\u003c/em\u003e)-hydroxy-octadecadienoic acid.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/32a6ca312f2025f4e275a2ec.png"},{"id":73668384,"identity":"e5a7171e-80a7-4fe2-b7ac-2f979ff67c90","added_by":"auto","created_at":"2025-01-13 12:20:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations to octadecanoid pathways in response to symbiosis: (a) in the host (Aiptasia) when symbiotic with the dinoflagellate symbionts \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBreviolum minutum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDurusdinium trenchii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (Symbiotic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Aposymbiotic); and (b) in the symbionts (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIn hospite\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Cultured).\u003c/strong\u003e Venn diagrams indicate the number of octadecanoids significantly altered when in symbiosis, including compounds shared by both species with pairwise fold-change differences \u0026gt;2; p \u0026lt;0.05 with FDR (see \u003cstrong\u003eTables S4\u003c/strong\u003e and \u003cstrong\u003eS5\u003c/strong\u003e for details). Species-specific changes are color-coded. The enantiomeric excess values (ee %) for the \u003cem\u003eS\u003c/em\u003e and \u003cem\u003eR\u003c/em\u003e enantiomers of 13-HOTE and 9-HODE are shown in each graph.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/bfc171014102231458f2011a.png"},{"id":73667017,"identity":"b106c9a0-e024-4233-ad78-22a376cdf8ed","added_by":"auto","created_at":"2025-01-13 12:04:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":476922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of lipoxygenases (LOXes) from different kingdoms and the novel LOXes in Symbiodiniaceae based on protein sequence homology.\u003c/strong\u003e Accession numbers were obtained from GenBank and are described in the Materials \u0026amp; Methods section.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/4023e6b67b99a5b50c76a2f1.png"},{"id":95179738,"identity":"31599694-8446-48e5-a9d1-a95169d7fe12","added_by":"auto","created_at":"2025-11-05 08:09:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3181478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/36eddfcd-5ef4-4f60-87a7-5f948e40821f.pdf"},{"id":73666427,"identity":"e3ec4783-8422-4659-997e-e2fee478517d","added_by":"auto","created_at":"2025-01-13 11:56:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":351065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Botanaetal21122024supplemental.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/fd80ab969685574b51d1ea7c.pdf"},{"id":73666426,"identity":"c232f7ab-6336-48d2-be9b-cbd6ee1934a8","added_by":"auto","created_at":"2025-01-13 11:56:39","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":60352,"visible":true,"origin":"","legend":"","description":"","filename":"BotanaetalSuplementarytables141224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5690019/v1/fa1f64c592d6e11a42760367.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEcological success of coral reefs stems from the symbiotic relationship between the cnidarian host (\u003cem\u003ee.g.\u003c/em\u003e, corals, sea anemones) and its dinoflagellate algal endosymbionts of the family Symbiodiniaceae. Evolution of this symbiosis over space and time has resulted in a high diversity of host and symbiont genotypes, as well as a high specificity of host-symbiont pairings and therefore, the occupation of distinct ecological niches by the holobiont (\u003cem\u003ei.e.\u003c/em\u003e, the whole symbiosis)\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The symbiotic dinoflagellates supply their host with products from photosynthesis, including sugars, lipids and amino acids\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and receive inorganic substrates and shelter from the host\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. As in other symbiotic systems (\u003cem\u003ee.g.\u003c/em\u003e, rhizobia-leguminous plants, human gut-microbiota), host-symbiont exchange not only involves nutritional compounds, but also cell signaling molecules that regulate inter-partner recognition and symbiosis function\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These signaling molecules might cross the symbiosome membrane, the host-derived vacuole membrane that encloses the dinoflagellate symbionts within the cnidarian host\u0026rsquo;s gastrodermal cells, and which acts as the primary host-symbiont interface\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Glycans, small peptides, inositols, polar lipids, oxylipins and non-coding RNA have all been proposed as candidate signaling molecules that may interact with specific transmembrane transporters, enzyme-coupled receptors, and voltage-gated ion channels (\u003cem\u003ee.g.\u003c/em\u003e, G-protein coupled receptors, lectins, peroxisome proliferator-activated receptors) for mediation of symbiosis establishment and cellular homeostasis; however, the underlying regulatory mechanisms involved in these processes remain poorly understood\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOxylipins are a group of metabolites derived from oxygenated fatty acids that have gained recent attention in the cnidarian-dinoflagellate symbiosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, while also being reported as important bioactive mediators of cellular function and fate in many biological systems\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Oxylipins can be synthesized by the oxidation of mono- and polyunsaturated fatty acids (MUFA, PUFA) either following release from the cell membrane by lipase hydrolysis (\u003cem\u003ee.g.\u003c/em\u003e, phospholipase A\u003csub\u003e2\u003c/sub\u003e, PLA\u003csub\u003e2\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e or by direct oxidation of the membrane lipids. They can also be formed by autoxidation processes (\u003cem\u003ee.g\u003c/em\u003e., \u003cem\u003evia\u003c/em\u003e free radicals), which are not stereoselective\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Enzymatic formation of oxylipins is generally selective for carbon and double bond positions within the precursor fatty acid, so that only a specific regioisomer (\u003cem\u003ee.g.\u003c/em\u003e, 9- or 13-hydroxy-octadecadienoic acid (HODE)) is synthesized by a given enzyme\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Biosynthesis of oxylipins is also often stereoselective\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, meaning that the geometrical configuration of the oxylipin product has a specific spatial 3D structure \u0026ndash; for instance, either right-handed (the (\u003cem\u003eR\u003c/em\u003e) enantiomer) or left-handed (the left (\u003cem\u003eS\u003c/em\u003e) enantiomer) for molecules with only one chiral center\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). As such, determining the chirality of an oxylipin can indicate the synthetic source of the compound (\u003cem\u003ei.e.\u003c/em\u003e, enzymatic or autoxidation). Determination of oxylipin biological function and regulation is aided by knowledge of the regio- and stereo-isomer configuration because the propagation of signaling cascades often relies on their interaction with stereoselective membrane receptors\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo date, only oxylipins derived from oxidation of 20-carbon fatty acids (\u003cem\u003ei.e.\u003c/em\u003e, eicosanoids), particularly products of arachidonic acid (ARA, 20:4, n-6), have been suggested to exert key functions for host-symbiont communication in the cnidarian-dinoflagellate symbiosis\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the high diversity of PUFA precursors, from both the host and symbiont, suggests that this symbiosis a promising model for exploration of novel oxylipins and the pathways responsible for their synthesis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. PUFA oxidation is mediated by the availability of both the substrate (\u003cem\u003ei.e.\u003c/em\u003e, fatty acid) and initiator (\u003cem\u003ei.e.\u003c/em\u003e, an oxygenating agent \u0026ndash; enzymes, free radicals or singlet oxygen), and by the scavenging capacity of the antioxidant machinery present in the biological system being studied. It is therefore necessary to explore the distribution of intact PUFAs and their modifying enzymes to better predict oxylipin profiles and their synthetic routes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Stearidonic acid (SDA; 18:4, n-3) and octadecapentaenoic acid (ODPA; 18:5, n-3) are the most abundant PUFAs integral to the chloroplast membranes of the symbionts\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Oxylipins derived from 18-carbon fatty acids are termed octadecanoids\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and have been primarily studied in plants, with the classical octadecanoid pathway critical for the formation of the phytohormone jasmonic acid (derived from alpha-linolenic acid, ALA 18:3, n-3)\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These ALA-derived metabolites are compounds of interest in the cnidarian-dinoflagellate symbiosis\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, octadecanoids originating from SDA and ODPA are less studied and have only been reported to occur in cultures of Symbiodiniaceae\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; their role in the cnidarian-dinoflagellate symbiosis remains unexplored.\u003c/p\u003e \u003cp\u003eHere we assessed the effects of symbiotic state and symbiont identity in the cnidarian-dinoflagellate symbiosis using a chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) method to perform metabolic profiling of octadecanoids\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We used the sea anemone \u003cem\u003eExaiptasia diaphana\u003c/em\u003e (commonly called \u0026lsquo;Aiptasia\u0026rsquo;) as a host model, in symbiosis with either its native (homologous) symbiont \u003cem\u003eBreviolum minutum\u003c/em\u003e or the non-native (heterologous) symbiont \u003cem\u003eDurusdinium trenchii\u003c/em\u003e. This latter symbiont was chosen as it is a widely studied opportunist that provides less nutritional benefit to the host and induces cellular stress when it populates Aiptasia under experimental conditions\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Symbiont-free anemones (\u003cem\u003ei.e.\u003c/em\u003e, aposymbiotic) and cultured symbionts were also analyzed to determine species specificity for the biosynthesis of specific oxylipin stereoisomers before addressing the impact of symbiotic state. Our comprehensive phenotypic analyses combined with top-down bioinformatics for determination of candidate biosynthetic enzymes of 18-carbon fatty acids enabled us to further describe the signaling cascades and pathways present in the cnidarian-dinoflagellate symbiosis and provide evidence for the inter-partner exchange of octadecanoids and their potential roles as lipid mediators.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDistinct biosynthesis patterns in aposymbiotic anemones\u003c/b\u003e \u003cb\u003evs.\u003c/b\u003e \u003cb\u003ecultured dinoflagellates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBy chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) (SFC-MS/MS), we were able to quantify 84 octadecanoids across all host and symbiont samples, including compounds derived from oleic acid (OA) (18:1, n-9), linoleic acid (LA) (18:2, n-6), alpha-linolenic acid (ALA) (18:3, n-3), gamma-linolenic acid (GLA) (18:3, n-6) and stearidonic acid (SDA) (18:4, n-3). There was significant structural diversity in the detected octadecanoids; however, the overall concentrations of the different functional groups were in the order of mono-hydroxy\u0026thinsp;\u0026gt;\u0026thinsp;epoxy\u0026thinsp;\u0026gt;\u0026thinsp;oxo\u0026thinsp;\u0026gt;\u0026thinsp;trihydroxy\u0026thinsp;\u0026gt;\u0026thinsp;dihydroxy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for octadecanoid nomenclature). Diols derived from OA and ALA, as well as triols from ALA were absent from all sample groups (a detailed list of octadecanoids and their concentrations are provided in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAposymbiotic anemones and cultured dinoflagellates were first assessed for species-specificity with respect to the biosynthesis and potential origins of octadecanoids before evaluating the effects of symbiotic state. Differential stereochemistry specificity was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), with aposymbiotic anemones predominantly containing the (\u003cem\u003eR\u003c/em\u003e) enantiomer of mono-hydroxy octadecanoids and cultured dinoflagellates the (\u003cem\u003eS\u003c/em\u003e) enantiomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb \u003cb\u003eand c\u003c/b\u003e). Aposymbiotic anemones primarily produced (\u003cem\u003eR\u003c/em\u003e) mono-hydroxylated forms of all regioisomers of SDA (\u003cem\u003ei.e.\u003c/em\u003e, HOTEs), GLA (\u003cem\u003ei.e.\u003c/em\u003e, HOTrEs-γ) and ALA (\u003cem\u003ei.e.\u003c/em\u003e, HOTrEs). The observed stereoselectivity was determined using the % enantiomeric excess (ee), with values ranging from 64\u0026ndash;100% (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Cultured \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e exclusively contained the SDA-derived monohydroxy 13(\u003cem\u003eS\u003c/em\u003e)-HOTE (\u003cb\u003eee\u003c/b\u003e\u0026thinsp;=\u0026thinsp;100%), consistent with enzymatic biosynthesis. In both dinoflagellate species, \u003cb\u003eee\u003c/b\u003e values also suggested the biosynthesis of the 13(\u003cem\u003eS\u003c/em\u003e)-HOTrE enantiomer (\u003cb\u003eee\u003c/b\u003e\u0026thinsp;=\u0026thinsp;100% and 94% in \u003cem\u003eBreviolum minutum\u003c/em\u003e and \u003cem\u003eDurusdinium trenchii\u003c/em\u003e, respectively) (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). The only octadecanoid that exhibited the same stereospecificity for both aposymbiotic anemones and cultured dinoflagellates was 9(\u003cem\u003eR\u003c/em\u003e),10(\u003cem\u003eS\u003c/em\u003e),13(\u003cem\u003eR\u003c/em\u003e)-TriHOME (\u003cb\u003eee\u003c/b\u003e\u0026thinsp;=\u0026thinsp;100% in all cases for position C-9) (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAlterations in octadecanoid pathways in response to symbiotic state\u003c/h2\u003e \u003cp\u003eSymbiont population densities in symbiotic anemones were not significantly different between the two dinoflagellate species (0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 and 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 cells/ng host protein for \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e, respectively). Any differences in the octadecanoid profile were therefore likely to be symbiont species-specific rather than a symbiont density effect. Profiling oxylipins with regio- and stereo-specificity enabled us to investigate alterations in distinct oxygenation pathways associated with symbiotic state and symbiont identity. There was a greater shift in the octadecanoid profile associated with symbiotic state (\u003cem\u003ei.e\u003c/em\u003e., symbiosis \u003cem\u003evs\u003c/em\u003e. aposymbiosis) than with symbiont identity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Phenotypic remodeling was broadly manifested as upregulation of octadecanoids in both the host and symbiont when in symbiosis. Additionally, both partners showed lower ee values for multiple octadecanoid enantiomers when in symbiosis than in their isolated states (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), with \u003cem\u003eS\u003c/em\u003e enantiomers upregulated in the host and \u003cem\u003eR\u003c/em\u003e enantiomers upregulated in the symbiont. Specifically, in symbiosis, the host exhibited an increased abundance of the 9(\u003cem\u003eR\u003c/em\u003e),10(\u003cem\u003eS\u003c/em\u003e),13(\u003cem\u003eR\u003c/em\u003e)-TriHOME diastereoisomer and the (\u003cem\u003eS\u003c/em\u003e) stereoisomers of HOTEs and HOTrEs-γ, with the biggest increases observed in \u003cem\u003eD. trenchii\u003c/em\u003e-colonized anemones (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with FDR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; \u003cb\u003eTable S3\u003c/b\u003e). For EpODEs, EpOMEs and epoxy-HOMEs, an increase of up to 30-fold only occurred in anemones containing \u003cem\u003eD. trenchii\u003c/em\u003e (\u003cb\u003eTables S4\u003c/b\u003e). In contrast, in \u003cem\u003eB. minutum\u003c/em\u003e-colonized anemones, the level of 9(\u003cem\u003eR\u003c/em\u003e)-HODE decreased 3-fold relative to aposymbiotic anemones \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In the symbionts, the abundance of all octadecanoids except for 13(\u003cem\u003eS\u003c/em\u003e)-HOTE was upregulated in symbiosis \u003cem\u003evs.\u003c/em\u003e culture, with most increases being more marked in \u003cem\u003eD. trenchii\u003c/em\u003e than in \u003cem\u003eB. minutum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; \u003cb\u003eTable S5\u003c/b\u003e). Increased enzymatic oxidation of ALA by 9-lipoxygenase (9-LOX) was evident for both dinoflagellate species. The abundance of HOTrEs from ALA was also elevated for both species. The abundance of epoxy-HOMEs and EpODEs increased in both species, but in \u003cem\u003eD. trenchii\u003c/em\u003e this increase was 3-fold greater than in \u003cem\u003eB. minutum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; \u003cb\u003eTable S5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvidence for inter-partner exchange of octadecanoids\u003c/h3\u003e\n\u003cp\u003eAposymbiotic anemones did not contain 13(\u003cem\u003eS\u003c/em\u003e)-HOTE, although this compound appeared in the host when colonized by either symbiont species, with the concentration 4-fold greater in anemones containing \u003cem\u003eD. trenchii\u003c/em\u003e than those containing \u003cem\u003eB. minutum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with FDR). The (\u003cem\u003eS\u003c/em\u003e) \u003cb\u003eee\u003c/b\u003e was 22% and 29% for anemones symbiotic with \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In comparison, the abundance of 13(\u003cem\u003eS\u003c/em\u003e)-HOTE was significantly diminished in the symbiont when in symbiosis, in contrast to the pattern observed for all other octadecanoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; \u003cb\u003eTable S5\u003c/b\u003e). The (\u003cem\u003eS\u003c/em\u003e) \u003cb\u003eee\u003c/b\u003e values decreased from 100\u0026ndash;10% in \u003cem\u003eB. minutum\u003c/em\u003e and from 100\u0026ndash;23% in \u003cem\u003eD. trenchii\u003c/em\u003e in the symbiotic state (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). These patterns suggest potential translocation from symbiont to host when in symbiosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNovel 9(\u003c/b\u003e \u003cb\u003eS\u003c/b\u003e \u003cb\u003e)- and 13\u003c/b\u003e(\u003cb\u003eS\u003c/b\u003e\u003cb\u003e)-lipoxygenases in Symbiodiniaceae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne \u003cem\u003eB. minutum\u003c/em\u003e (MSTRG. 59407) and two \u003cem\u003eD. trenchii\u003c/em\u003e (MSTRG.68745/7968) novel LOX-domain encoding mRNA sequences were proposed as novel LOX enzymes for Symbiodiniaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The sequence for \u003cem\u003eB. minutum\u003c/em\u003e was incomplete, but the alignment with AtLOX3 showed a combination of alanine and phenylalanine residues at the active site (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), which revealed it as a 13(\u003cem\u003eS\u003c/em\u003e)-LOX\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. MSTRG.59407 had no blast hits with identity percentages above 79.9% in any of the searched public databases (\u003cem\u003ee.g.\u003c/em\u003e, GeneBank, Reef Genomics and UNIPROT). The sequences for \u003cem\u003eD. trenchii\u003c/em\u003e were 99.9% identical to two other sequences published in GenBank (CAK8999115.1 and CAK9070246.1), which have been described as \u0026ldquo;unnamed proteins\u0026rdquo;. The alanine alignment and valine alignments with the active site residues of AtLOX 1 and 5 revealed that MSTRG.68745/7968 were 9(\u003cem\u003eS\u003c/em\u003e)-LOXes (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). All transcripts demonstrated active constitutive expression across both cultured and symbiotic Symbiodiniaceae RNA-seq datasets. Homology comparisons grouped the new Symbiodiniaceae LOXes within their own clade in the tree of life, being evolutionarily divergent from green algal (\u003cem\u003ei.e., Chlamydomonas reinhardtii\u003c/em\u003e and \u003cem\u003eLobosphera incisa\u003c/em\u003e) and vascular plant LOXes, but rather closely related to a fungal Mn-LOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInterest in oxylipins as potential lipid mediators of inter-partner signaling in the cnidarian-dinoflagellate symbiosis has increased recently\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Here, octadecanoid biosynthesis by aposymbiotic anemones was found to be restricted to (\u003cem\u003eR)\u003c/em\u003e enantiomers (as indicated by \u003cb\u003eee\u003c/b\u003e values of 100% for most HOTEs, HOTrEs and HOTrEs-γ). This trend was also reported for eicosanoid biosynthesis in various soft corals\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and the stony corals \u003cem\u003eAcropora\u003c/em\u003e sp. and \u003cem\u003eGalaxea fascicularis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e; however, the stereospecificity of octadecanoid formation in cnidarians was previously unknown. In contrast, octadecanoid biosynthesis in both the cultured homologous symbiont \u003cem\u003eBreviolum minutum\u003c/em\u003e and the heterologous \u003cem\u003eDurusdinium trenchii\u003c/em\u003e was restricted to (\u003cem\u003eS\u003c/em\u003e) enantiomers, with 13(\u003cem\u003eS\u003c/em\u003e)-HOTE and 13(\u003cem\u003eS\u003c/em\u003e)-HOTrE especially prominent (\u003cb\u003eee\u003c/b\u003e values from 94\u0026ndash;100%). Both compounds are likely formed \u003cem\u003evia\u003c/em\u003e a 13(\u003cem\u003eS\u003c/em\u003e)-lipoxygenase (LOX), which is enantioselective for the synthesis of (\u003cem\u003eS\u003c/em\u003e) octadecanoids in the jasmonic acid pathway\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In microalgae, studies of the LOX pathways have primarily focused on the synthesis of eicosanoids and docosanoids (derived from 22-carbon fatty acids)\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and there is a paucity of investigations of octadecanoid biosynthesis\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To our knowledge, this is the first study of LOX enzymes in Symbiodiniaceae and applied top-down bioinformatics to elucidate biosynthetic pathways in the cnidarian-dinoflagellate symbiosis.\u003c/p\u003e \u003cp\u003eSymbiosis resulted in increased levels of octadecanoids in both host and symbionts; however, there was a commensurate up to 3-fold decrease in their \u003cb\u003eee\u003c/b\u003e values. These findings suggest a potential dynamic inter-partner exchange of compounds and/or alterations to symbiosis homeostasis marked by a higher autoxidative state\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Specifically, \u003cem\u003eD. trenchii\u003c/em\u003e promoted greater abundance of octadecanoids in the host, consistent with a stress response, which was further indicated by an up to 30-fold increase in epoxides (\u003cem\u003ei.e.\u003c/em\u003e, EpODEs, EpOMEs and epoxy-HOMEs). In contrast, symbiosis with the homologous \u003cem\u003eB. minutum\u003c/em\u003e resulted in only a slight increase in octadecanoid abundance, which was more consistent with a homeostatic adjustment. 9(\u003cem\u003eR\u003c/em\u003e)-HODE has been linked to inflammation in mammals\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and was exclusively downregulated in \u003cem\u003eB. minutum\u003c/em\u003e-colonized anemones, perhaps serving to dampen cellular stress and so contribute to the persistence of this host-symbiont pairing\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. A putative regulatory role can be attributed to 9(\u003cem\u003eR\u003c/em\u003e),10(\u003cem\u003eS\u003c/em\u003e),13(\u003cem\u003eR\u003c/em\u003e)-TriHOME (ee\u0026thinsp;=\u0026thinsp;80% and 64% for position C-9, for anemones with \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e, respectively), for which the abundance increased in symbiotic host tissues, although the associated pathway is unknown. In plants and humans, TriHOMEs have been linked to lipoxygenases with species and tissue stereospecificity\u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In plants, TriHOMEs may also be linked to epoxyalcohol synthases (EAS), which together with allene oxide synthase (AOS), are part of CYP74 in the cytochrome P450 family\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In cnidarians, AOS and 8(\u003cem\u003eR\u003c/em\u003e)-LOX genes have been linked to eicosanoid synthesis\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e; but whether they are also involved in the synthesis of TriHOMEs and other octadecanoids remains to be investigated, as does the potential synthesis role by EAS.\u003c/p\u003e \u003cp\u003eSymbiosis also led to a general increase in the abundance of octadecanoids in the symbiont, with the greatest increase in \u003cem\u003eD. trenchii\u003c/em\u003e. For both symbiont species, symbiosis caused changes in the ALA oxidation pathway, possibly \u003cem\u003evia\u003c/em\u003e one of the linoleate-LOXes described here. This pathway occurs in plants\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, where synthesis of mono-hydroxy forms and a wide diversity of ALA downstream products (\u003cem\u003ee.g.\u003c/em\u003e, jasmonic acid and jasmonates) occurs in response to wounding and necrotrophic fungal infection and recognition\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, as well as during network communication in mycorrhiza\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The involvement of ALA oxidation products in host cell invasion and/or host-symbiont communication in the cnidarian-dinoflagellate symbiosis is therefore conceivable. However, the upregulation of LA and ALA oxidation pathways could also be linked to oxidative stress, with the increased abundance of EpOMEs and EpODEs in \u003cem\u003eD. trenchii\u003c/em\u003e suggesting that this non-native symbiont species could be under greater cellular stress than the native \u003cem\u003eB. minutum\u003c/em\u003e. Such stress could have arisen due to the greater levels of oxidative stress in the host\u0026rsquo;s tissues discussed above, or perhaps other aspects of physiological dysfunction associated with the poorer degree of host-symbiont integration that is typical of this symbiotic pairing\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eOctadecanoids as putative mediators of lipid signaling\u003c/h3\u003e\n\u003cp\u003eRegulatory signaling involves both intracellular and intercellular cascades, and the combination of both is crucial for endosymbioses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. In the cnidarian-dinoflagellate symbiosis, intercellular signaling between the partners may occur \u003cem\u003evia\u003c/em\u003e G protein-coupled receptors associated with the symbiosome membrane\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, ultimately leading to alterations in host nuclear transcription factors\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e; however, the molecular pathways involved are poorly understood\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In the case of intracellular signaling, a given molecule may bind to nuclear peroxisome proliferator-activated receptors (PPAR) that also regulate transcriptional factors, such as nuclear factor kappa-B (NF-kB) and tumor necrosis factors (TNF), mediators of inflammatory and immune responses\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. This latter mechanism has been widely studied in mammals but not in the cnidarian-dinoflagellate symbiosis. Nevertheless, the expression of both NF-kB and TNF has been observed to change in the host in response to symbiosis, leading to the suggestion that they help regulate symbiosis stability\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe role of octadecanoids as PPAR ligands and controllers of transcriptional factors has received considerable attention in the biomedical field, and they have been extensively targeted for the development of metabolic regulatory drugs\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. 13(\u003cem\u003eS\u003c/em\u003e)-HODE and 9(\u003cem\u003eS\u003c/em\u003e)-HODE are PPAR-γ ligands and controllers of the immune response in mammals, showing that different oxylipins can bind to the same receptor, although binding affinity is variable\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. The SDA-derived octadecanoid 13(\u003cem\u003eS\u003c/em\u003e)-HOTE, extracted from cultures of the green microalga \u003cem\u003eChlamydomonas debaryana\u003c/em\u003e, has also been shown to be a strong PPAR-γ ligand and to promote downregulation of NF-kB and other transcription factors in cell cultures and in a murine recurrent colitis model\u003csup\u003e\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. In our experimental system, the abundance of the symbiont-derived 13(\u003cem\u003eS\u003c/em\u003e)-HOTE decreased in the symbiont fraction and increased in the host fraction while in symbiosis, supporting evidence for inter-partner communication \u003cem\u003evia\u003c/em\u003e translocation of this compound. While confirmation is needed, our findings support this octadecanoid-receptor binding as a potential mechanism of host immune modulation. Transport of oxylipins through membranes often occurs through lipoproteins and we have previously reported these proteins to be 20-fold more abundant when Aiptasia is colonized with \u003cem\u003eB. minutum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Ultimately, oxylipin-receptor binding and translocation are promising themes for future research on the cnidarian-dinoflagellate symbiosis, with considerable potential to draw parallels with the biomedical field given that the cnidarian immune system is similar to that of higher organisms, including mammals\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eNovel lipoxygenases in Symbiodiniaceae\u003c/h3\u003e\n\u003cp\u003eWe also studied the enzymatic candidates for the biosynthesis of 13(\u003cem\u003eS\u003c/em\u003e) octadecanoids, which included a novel 13(\u003cem\u003eS\u003c/em\u003e)-LOX for \u003cem\u003eB. minutum\u003c/em\u003e and two 9(\u003cem\u003eS\u003c/em\u003e)-LOXes for \u003cem\u003eD. trenchii\u003c/em\u003e. In vascular plants, most 9-LOXes described to date are cytosolic and only free fatty acids serve as substrate, whereas 13-LOXes are either chloroplast-derived or cytosolic; and both esterified membranes or free fatty acids can serve as substrates\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. 13-LOX activity forms the 13-positional mono-hydroperoxy fatty acids (\u003cem\u003ee.g.\u003c/em\u003e, 13(\u003cem\u003eS\u003c/em\u003e)-HpOTE and 13(\u003cem\u003eS\u003c/em\u003e)-HpOTrE), whereas 9-LOXes synthesize the 9-positional forms (\u003cem\u003ee.g.\u003c/em\u003e, 9(\u003cem\u003eS\u003c/em\u003e)-HpOTE and 9(\u003cem\u003eS\u003c/em\u003e)-HpOTrE)\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. These fatty acid hydroperoxides can serve either as substrates for additional metabolic transformation (\u003cem\u003ee.g.\u003c/em\u003e, AOS or EAS), or are reduced to form the corresponding mono-hydroxys (\u003cem\u003ee.g.\u003c/em\u003e, HOTrEs or HOTEs). In cyanobacteria LOX, both positional mono-hydroxy forms can be synthesized by the same enzyme, because the insertion of molecular oxygen into the fatty acid chain is guided by steric shielding of the radical intermediate rather than by the positional orientation of the fatty acid when entering the enzyme active site\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. We do not know which mechanism occurs in the Symbiodiniaceae, but the novel LOX identified in \u003cem\u003eB. minutum\u003c/em\u003e is a candidate for the synthesis of symbiont-derived 13(\u003cem\u003eS\u003c/em\u003e) octadecanoids. The active constitutive expression of both 13(\u003cem\u003eS\u003c/em\u003e)\u003cem\u003e-\u003c/em\u003e and 9(\u003cem\u003eS\u003c/em\u003e)-LOX candidates across both cultured and symbiotic Symbiodiniaceae RNA-seq datasets also suggests that they might not only be real sequences, but also of paramount importance for symbiont metabolism.\u003c/p\u003e \u003cp\u003eIn vascular plants, chloroplast-derived 13(\u003cem\u003eS\u003c/em\u003e)-LOX has the highest substrate oxygenation kinetics with ALA\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, which is also true for LiLOX in the green alga \u003cem\u003eLobosphera incisa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e and the cyanobacterium \u003cem\u003eCyanothece\u003c/em\u003e sp.\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. In plants, this initial oxygenation step is part of the jasmonic acid pathway, where jasmonic acid and other jasmonates help regulate the immune response, growth and development\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. In Symbiodiniaceae, SDA and ODPA are more abundant than ALA\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, suggesting that their as yet unidentified downstream products might be key metabolic mediators and perhaps play a role in host-symbiont recognition. This hypothesis needs further investigation; however, it suggests that future studies should focus on ODPA-derived octadecanoids as well as oxylipins from 20-carbon and 22-carbon PUFAs to provide a broader understanding of the lipid signaling pathways present in the cnidarian-dinoflagellate symbiosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work presents the first detailed quantification of octadecanoids in cnidarians and their associated symbionts. Determination of the octadecanoid stereochemistry provided insight into their biosynthetic route of formation. Our findings indicate a dynamic exchange of octadecanoids between symbiont and host and suggest that specific oxylipin stereoisomers act as putative lipid signaling mediators of this symbiosis. Understanding the molecular processes that underpin host-symbiont communication and symbiosis stability is of importance across symbiotic systems and is key in determining how to stabilize or strengthen host-symbiont interactions. For this purpose, the combination of lipidomics and reverse genetics is a powerful tool for further elucidation and discovery of new molecular pathways, which can contribute to our improved understanding of coral reef function. Accordingly, increased efforts should be made in understanding octadecanoid formation, endogenous levels, and signaling in relation to host-symbiont communication. This approach has the potential to improve the development of restoration and conservation tools involving bioengineering and selective breeding of more optimal host-symbiont pairings to enhance coral reef survival in the face of environmental stressors.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of animal and algal cultures\u003c/h2\u003e \u003cp\u003eSymbiodiniaceae cultures (\u003cem\u003eBreviolum minutum\u003c/em\u003e, culture ID CCMP830; \u003cem\u003eDurusdinium trenchii\u003c/em\u003e, culture ID D1A001 (see \u003cb\u003eTable S6\u003c/b\u003e for species confirmation) were grown for eight years in f/2-enriched 0.22 \u0026micro;m filtered seawater (FSW)\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Culture flasks were maintained inside an incubator set to 25\u0026deg;C (\u0026plusmn;\u0026thinsp;0.5\u0026deg;C), under cool fluorescent lights (Osram Dulux 36/W890) at 90\u0026ndash;110 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on a 12:12 h light/dark cycle.\u003c/p\u003e \u003cp\u003eLong-term clonal stocks of the sea anemone \u003cem\u003eExaiptasia diaphana\u003c/em\u003e (Aiptasia, culture ID: NZ1) were rendered aposymbiotic using a menthol protocol\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. The anemones were confirmed to be aposymbiotic based on the absence of chlorophyll autofluorescence under confocal microscopy (Olympus Provis AX70; 100x magnification) and the absence of symbiont DNA amplification through polymerase chain reaction using ITS2_F and ITS_R primers\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Two thirds of the aposymbiotic stock (n\u0026thinsp;=\u0026thinsp;150 anemones) were inoculated with either homologous \u003cem\u003eB. minutum\u003c/em\u003e or heterologous \u003cem\u003eD. trenchii\u003c/em\u003e and maintained in symbiosis for 15 months as described by Wuerz et al. (2023)\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. All anemones were maintained in FSW and fed once a week with freshly hatched \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii. Stocks were kept at 25\u0026deg;C and 90\u0026ndash;110 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on a 12:12 h light/ dark cycle using Philips 6500K bulbs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental setup\u003c/h2\u003e \u003cp\u003eSymbiodiniaceae cultures (n\u0026thinsp;=\u0026thinsp;3 \u003cem\u003eper\u003c/em\u003e species) were grown in 500 mL Erlenmeyer flasks under the same conditions for long-term maintenance. Initial cell densities in flasks were 1000 cells per mL and culture growth was monitored using the confocal imaging system IN Cell analyzer 6500 Hs, where cell density was quantified with Image J processing software (ten counts \u003cem\u003eper\u003c/em\u003e replicate). Cultures were monitored over time and sampling for octadecanoid analysis occurred after 16 days, while the cultures were still in exponential growth (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), using sterile pipettes (150 mL \u003cem\u003eper\u003c/em\u003e replicate). Samples were centrifuged at 4000 \u003cem\u003eg\u003c/em\u003e for 5 min and the resulting algal pellets flash-frozen and freeze-dried before storage at -80\u0026deg;C in sterile 1.5 mL Eppendorf tubes. On the day of sampling, cultures were dark acclimated for 15 min within 4 h of initiating the light cycle, before measurements of \u0026lsquo;photosynthetic health\u0026rsquo; (F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e) using a Diving Pulse Amplitude Modulated Fluorometer (Diving-PAM, Walz, Effeltrich, Germany; settings: measuring light\u0026thinsp;=\u0026thinsp;4, saturation intensity\u0026thinsp;=\u0026thinsp;8, saturation width\u0026thinsp;=\u0026thinsp;0.8 s, gain\u0026thinsp;=\u0026thinsp;3, and damping\u0026thinsp;=\u0026thinsp;3) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAnemones for each experimental treatment (\u003cem\u003ei.e.\u003c/em\u003e, aposymbiotic; symbiotic with \u003cem\u003eB. minutum\u003c/em\u003e; and symbiotic with \u003cem\u003eD. trenchii\u003c/em\u003e) were split across 3 x 400 mL jars \u003cem\u003eper\u003c/em\u003e treatment (n\u0026thinsp;=\u0026thinsp;15 anemones \u003cem\u003eper\u003c/em\u003e jar) that contained FSW. These jars were then transferred to water baths set at 25\u0026deg;C (\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) and under the same light conditions as described for the anemone stocks. All anemones were fed with \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii at the same time of day and the old water was changed with fresh FSW 8 h after feeding. Anemones were maintained under these conditions for 16 days prior to sampling and analyses. On the sampling day, F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e of symbiotic anemones was measured (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) as described above for the Symbiodiniaceae cultures. Anemones (N\u0026thinsp;=\u0026thinsp;10 \u003cem\u003eper\u003c/em\u003e jar) were then pooled, washed, and homogenized with 1 mL of ice-cold 10 mM sodium phosphate buffer (pH 7.4) containing 100 \u0026micro;M of the Fe chelator deferoxamine mesylate, using a glass tissue grinder. Aposymbiotic anemone homogenates were flash-frozen, freeze-dried and stored at -80\u0026deg;C in 1.5 mL sterile Eppendorf tubes. Homogenates from pooled symbiotic anemones were transferred to sterile Eppendorf tubes and centrifuged at 500 \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C, to separate host and symbiont fractions. Host tissue supernatant was transferred to a separate sterile tube and the pelleted symbiont dinoflagellate fraction was washed free of residual host material by resuspension and centrifugation using the same buffer solution. A 20 \u0026micro;L aliquot of each host supernatant was analyzed for protein content using a fluorometric Qubit Protein Assay Kit. After resuspension for a second time, the symbiont fraction was aliquoted equally for cell counts, conducted using the confocal imaging system IN Cell analyzer 6500 Hs (as described above), and then normalized to host protein content (see Results). All steps were performed on ice and final fractions were flash-frozen, freeze-dried and stored at -80\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation and extraction\u003c/h2\u003e \u003cp\u003eOctadecanoids were extracted from freeze-dried biomass by adding 1.5 mL of methanol (MeOH) and 10 \u0026micro;L of internal standard (IS) mix (\u003cb\u003eTable S7\u003c/b\u003e). All samples were thoroughly mixed by vortexing and subsequently sonicated in an ice bath for 30 min. Following centrifugation for 10 min at 15,000 x \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C, the supernatant was transferred to a glass tube and evaporated to dryness under nitrogen. To maximize compound recovery, this methanolic extraction was repeated a second time and combined with the first extract. The dried extracts were then reconstituted with 1 mL of a solution of 0.2 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 0.1 M citric acid (pH 5.6). To concentrate the extract, solid phase extraction (SPE) was performed as described in Quaranta et al. (2022), using an Extrahera automated sample preparation system (Biotage, Uppsala, Sweden). Briefly, the 1 mL reconstituted extract was loaded onto a preconditioned 3 mL (3 cc/60 mg) Waters Oasis HLB cartridge (Milford, MA, USA). Samples were washed three times with 3 mL of HPLC-grade water and a fourth time with 3 mL MeOH:H\u003csub\u003e2\u003c/sub\u003eO (1:9). Octadecanoids were eluted with 2.5 mL of MeOH and the extract was further evaporated to dryness under a nitrogen stream. Reconstitution was performed with 80 \u0026micro;L MeOH, and the samples were then filtered through a 0.1 \u0026micro;m polyvinyl-i-dene fluoride membrane spin-filter (Amicon, Merck Millipore Cooperation, Billerica, MA, USA) before being transferred to liquid chromatography vials for analysis. Blanks comprised of FSW only, and culture medium was added to ensure that oxylipin phenotypic profiles in samples were not related to possible contamination from the experimental environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOctadecanoid profiling and analysis\u003c/h2\u003e \u003cp\u003eA chiral supercritical fluid chromatography (SFC) coupled to tandem mass spectrometry (MS/MS) platform was used to perform quantitative metabolic profiling of octadecanoids as previously reported\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The published method was expanded by the addition of 27 novel custom synthesized standards derived from oleic acid (OA), alpha-linolenic acid (ALA, n-3), gamma-linolenic acid (GLA, n-6) and SDA (\u003cb\u003eTable S7\u003c/b\u003e). The discrimination of \u003cem\u003eR\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e enantiomers for the monohydroxy octadecanoids was based on the elution order in the chiral column following the same patterns as observed for the 18 enantiopure standards detailed in Quaranta \u003cem\u003eet al\u003c/em\u003e. (2022)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExtracts were first analyzed using the SFC method on a Waters UPC\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e system coupled to a Waters Xevo TQ-XS mass spectrometer. Chiral separation was performed on a polysaccharide Waters Trefoil AMY1 column (3.0 x 150 mm, 2.5 \u0026micro;m) set at 35\u0026deg;C and an injection volume of 2 \u0026micro;L. In addition to supercritical CO\u003csub\u003e2\u003c/sub\u003e as the main eluent (phase A), a co-modifier consisting of MeOH:EtOH (8:2 by vol.) and CH\u003csub\u003e3\u003c/sub\u003eCOOH 0.1% v/v was used as phase B. The gradient started with 5% B maintained until 1 min, before increasing it linearly to 25% B at 11 min and 30% B at 12.3 min. The column was then washed with 50% B for 2\u0026ndash;5 min and re-equilibrated under the initial conditions for 2.2 min. Flow rate was 2.0 mL/min during separation and equilibration but was decreased to 1.5 mL/min during washing. The active backpressure regulator (ABPR) was set to 2000 psi and the make-up solvent consisting of MeOH and CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e (5mM) was flow rate-based on the co-modifier to avoid excess organic solvent at the source. It started at a flow rate of 0.2 mL/min before linearly decreasing to 0 mL/min after 6 min.\u003c/p\u003e \u003cp\u003eThe chromatographic system had an MS source operating in negative-ion ESI mode with a capillary voltage of 1.9 kV, the source temperature at 150\u0026deg;C, the desolvation temperature at 600\u0026deg;C, the source offset at 30.0 V, the cone gas flow at 150 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the desolvation gas flow at 1000 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the nebulizer gas pressure at 7.0 bar. Negative multiple reaction monitoring transitions along with the collision energy, cone voltage and dwell time were manually optimized for each octadecanoid, and one transition \u003cem\u003eper\u003c/em\u003e analyte was selected based upon sensitivity and selectivity. Calibration was performed using an 11-point linear calibration model, applying a 1/x weighted least-squared regression. Retrieved octadecanoid quantity was normalized using the dry biomass, such that concentrations are reported in ng/g. MassLynx software version 4.2 was used for data acquisition and TargetLynx for data processing. Stereochemistry of the octadecanoids was evaluated using the enantiomeric excess (\u003cb\u003eee\u003c/b\u003e) (see IUPAC compendium for chemical terminology). For each characterized regioisomer with chiral configuration, the \u003cb\u003eee\u003c/b\u003e was calculated using the equation:\u003c/p\u003e \u003cp\u003e% of (\u003cem\u003eR\u003c/em\u003e) or (\u003cem\u003eS\u003c/em\u003e) enantiomer\u0026thinsp;=\u0026thinsp;100 x total amounts of (\u003cem\u003eR\u003c/em\u003e) or (\u003cem\u003eS\u003c/em\u003e)/ (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eS\u003c/em\u003e)\u003c/p\u003e \u003cp\u003e \u003cb\u003eDe novo\u003c/b\u003e \u003cb\u003etranscriptome assembly and mRNA expression quantification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo describe the transcriptomic responses of both cultured and symbiotic \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e, a splice-aware transcriptome was assembled from previously sequenced RNA libraries (Maor-Landaw \u003cem\u003eet al.\u003c/em\u003e, 2019\u003csup\u003e86\u003c/sup\u003e, BioProject: PRJNA544863 and Bellantuono \u003cem\u003eet al.\u003c/em\u003e, 2019\u003csup\u003e87\u003c/sup\u003e, BioProject PRJNA508937). Using Fastp\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, the raw paired-end RNA sequence files were trimmed and filtered to remove sequencing adaptors and bases with Phred quality scores below 30. Reads with more than 30% low-quality bases, containing more than 5 N bases, and reads shorter than 50 bases after trimming were discarded.\u003c/p\u003e \u003cp\u003eUsing the RNA-Seq aligner Spliced Transcripts Alignment to a Reference (STAR v2.7.10b\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e), all filtered sample reads were first mapped without guidance to the Aiptasia genome (assembly v1.1; GCF_001417965.1\u003csup\u003e90\u003c/sup\u003e) to filter anemone RNA reads and double mapping sequences for downstream expression quantitation. Un-mapped RNA reads were collected and mapped without guidance to the respective symbiont genomes of \u003cem\u003eB. minutum\u003c/em\u003e (strain: Mf 1.05b.01; GenBank assembly: GCA_000507305.1\u003csup\u003e91\u003c/sup\u003e) and \u003cem\u003eD. trenchii\u003c/em\u003e (strain: CCMP2556;\u003c/p\u003e \u003cp\u003eGenBank assembly: GCA_963970005.1\u003csup\u003e92\u003c/sup\u003e). Splice-junction aware alignment outputs were assembled into sample transcriptomes using StringTie2 v2.0.3\u003csup\u003e93\u003c/sup\u003e. Sample transcriptomes were then merged into species RNA transcriptomes with a minimum transcript coverage of one mapped read \u003cem\u003eper\u003c/em\u003e base pair. All assembled RNA transcript open reading frames (ORFs) greater than 100 amino acids were predicted using Transdecoder (TransDecoder.Predict v5.7, see Github in references), to predict the most likely protein coding sequences (CDs regions) and reduce the ORF false discovery rate. The ORFs were then filtered to retain only transcripts with either Blastp protein sequence homology (e-value\u0026thinsp;\u0026lt;\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) to any described or hypothetical protein in a collection of Stramenopile and Alveolate genomes (\u003cb\u003eTable S8\u003c/b\u003e) and/or HMMER (hmmscan v3.4) Pfam domain homology. Internal unique transcripts were used as training data for the species-specific Markov Model-derived coding potential of the ORFs and to revise start codon choice where statistically appropriate. After prediction, the best ORF candidates with revised starts were propagated to each genome and written into new genomic protein-coding transcript annotations.\u003c/p\u003e \u003cp\u003eTo quantify transcript abundance and test for differential expression, un-normalized read counts summarized at the gene (meta-feature) level for the homologous protein-coding transcript annotations were performed on the RNA sequence STAR genomic alignments for each sample, using FeatureCounts (Rsubread v2.16.1). All counted genes were tested for differential expression among experimental conditions from the original raw datasets using DeSeq2\u003csup\u003e94\u003c/sup\u003e, with default internal gene-filtering procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Symbiodiniaceae LOX candidates and phylogeny\u003c/h2\u003e \u003cp\u003eTo identify lipoxygenase (LOX) sequences in the protein-coding transcriptomes, a collection of previously annotated LOX protein sequences, from both prokaryotic and eukaryotic species was curated (\u003cb\u003eTable S9\u003c/b\u003e). All hypothetical Symbiodiniaceae protein sequences were scanned for sequence homology to the LOX references, using a Blastp cutoff of e-value\u0026thinsp;=\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e. Presence of Pfam LOX domains within the collection of Blastp candidates was confirmed with InterProScan (interproscan.sh v5.68-101.0)\u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. Protein sequence phylogeny of the 13(\u003cem\u003eS\u003c/em\u003e)-LOX candidates was constructed from MUSCLE multiple sequence alignments, together with the other LOX references\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. The resulting distance matrix provided the information to generate a phylogenetic tree using the neighbor-joining clustering method, with branch lengths optimized \u003cem\u003evia\u003c/em\u003e a phylogenetic maximum likelihood criterion\u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e, with Le and Gascuel model parameters\u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. The fitted tree was then bootstrapped 100 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eBefore evaluating the alterations caused by symbiotic state in both the host and symbiont, oxylipin profiles of both aposymbiotic anemones and cultured dinoflagellates were characterized, and symbiont species-specific compounds and the potential origins of octadecanoid enantiomer biosynthesis patterns (\u003cem\u003ei.e., R\u003c/em\u003e and/or \u003cem\u003eS\u003c/em\u003e) were assessed by \u003cb\u003eee\u003c/b\u003e (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Principal component analysis (PCA) was used to identify the main differences in the octadecanoid profiles associated with symbiotic state and symbiont identity, in the host and symbiont separately. A two-way crossed ANOVA using symbiotic state (Symb; two levels in the host: aposymbiotic and symbiotic; two levels in the symbiont: cultured and symbiotic) orthogonal to the dinoflagellate species (Symb; two levels: \u003cem\u003eB. minutum\u003c/em\u003e and \u003cem\u003eD. trenchii\u003c/em\u003e) was further applied for univariate validation of octadecanoids highlighted in the PCA. Additionally, pairwise comparisons were analyzed with Tukey's HSD test, and the significance threshold set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with false discovery rate corrections. A volcano plot analysis that considered pairwise fold-change differences of greater than two was also applied to the whole octadecanoid matrix, to investigate whether oxidation pathways were altered due to the symbiotic state in the host and symbiont. MetaboAnalyst version 6.0\u003csup\u003e99\u003c/sup\u003e was used for all statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Miguel Mies (University of Sao Paulo, Brazil), Dr. Matthias Kellerman (Carl-von-Ossietzky University, Germany), Dr. Matthew Nitschke, Dr. Gerhard Hagn and Prof. Robert Keyzers for reviewing the manuscript. We also thank Prof. Blair Paul (Marine Biological Laboratory, Woods Hole, USA) for the assistance provided with the bioinformatics pipeline and Dr. Ellen Hornung (Georg-August-University, Germany) for the assistance with the LOX analysis. This manuscript is in memory of Professor Raymond Valentine (University of California, Davis, USA), may his incessant curiosity in understanding life always remains with us.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Marsden Fund of the Royal Society Te Apārangi, grant number 19VUW086, awarded to S.K.D., C.A.O., A.R.G., D.J.S. and V.M.W., including a postgraduate scholarship awarded to M.T.B. I.F. acknowledges funding from the German Research Foundation (DFG) grant numbers GRK 1422 and GRK 2172. C.E.W. acknowledges support from the Swedish Research Council (2022-00796) and the Cayman Biochemical Research Institute (CABRI).\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eM.T.B. and S.K.D. conceptualization; M.T.B. experimental design and sample processing, data analysis and manuscript writing; R.E.L. bioinformatics and data analysis; A.Q. and O.S. analytical sample runs; J.R.C. standards synthesis, M.H. standards synthesis and pathway analysis supervision; I.F. LOX analysis and pathway analysis supervision; I.F., C.A.O., A.R.G., D.J.S., V.M.W. manuscript editing; C.E.W. and S.K.D. funding, supervision and manuscript editing.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCraig Wheelock: [email protected]\u003c/p\u003e\n\u003cp\u003eSimon Davy: [email protected]\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaker, A. C. Flexibility and Specificity in Coral-Algal Symbiosis: Diversity, Ecology, and Biogeography of \u003cem\u003eSymbiodinium\u003c/em\u003e. \u003cem\u003eAnnu. Rev. Ecol. Evol. 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A Polyp-on-Chip for Coral Long-Term Culture. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e (1), 6964. https://doi.org/10.1038/s41598-020-63829-4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cell signaling, coral reef, Aiptasia, Exaiptasia diaphana, Symbiodiniaceae, zooxanthellae, lipoxygenase, octadecanoid, mass spectrometry, supercritical fluid chromatography","lastPublishedDoi":"10.21203/rs.3.rs-5690019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5690019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxylipin signaling has been suggested as a potential mechanism for the inter-partner recognition and homeostasis regulation of cnidarian-dinoflagellate symbiosis, which maintains the ecological viability of coral reefs. Here we assessed the effects of symbiosis and symbiont identity on a model cnidarian, the sea anemone \u003cem\u003eExaiptasia diaphana\u003c/em\u003e, using mass spectrometry to quantify octadecanoid oxylipins (\u003cem\u003ei.e.\u003c/em\u003e, 18-carbon-derived oxygenated fatty acids). A total of 84 octadecanoids were reported, and distinct stereospecificity was observed for the synthesis of \u003cem\u003eR-\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e-enantiomers for symbiont-free anemones and free-living cultured dinoflagellate symbionts, respectively. Symbiont-derived 13(\u003cem\u003eS\u003c/em\u003e)-hydroxy-octadecatetraenoic acid (13(\u003cem\u003eS\u003c/em\u003e)-HOTE) linked to a 13(\u003cem\u003eS\u003c/em\u003e)-lipoxygenase was translocated to the host anemone with a 32-fold increase, suggesting it as a biomarker of symbiosis and as a potential agonist of host receptors that regulate inflammatory transcription. Only symbiosis with the native symbiont \u003cem\u003eBreviolum minutum\u003c/em\u003e decreased the abundance of pro-inflammatory 9(\u003cem\u003eR\u003c/em\u003e)-hydroxy-octadecadienoic acid (9(\u003cem\u003eR\u003c/em\u003e)-HODE) in the host. In contrast, symbiosis with the non-native symbiont \u003cem\u003eDurusdinium trenchii\u003c/em\u003e was marked by higher abundance of autoxidation-derived octadecanoids, corroborating previous evidence for cellular stress in this association. The putative octadecanoid signaling pathways reported here suggest foundational knowledge gaps that can support the bioengineering and selective breeding of more optimal host-symbiont pairings to enhance resilience and survival of coral reefs.\u003c/p\u003e","manuscriptTitle":"Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 11:56:35","doi":"10.21203/rs.3.rs-5690019/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bbb972df-fdf2-48bc-9401-677d4d59d2ac","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T08:09:05+00:00","versionOfRecord":{"articleIdentity":"rs-5690019","link":"https://doi.org/10.1038/s42003-025-09104-6","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-11-04 05:00:00","publishedOnDateReadable":"November 4th, 2025"},"versionCreatedAt":"2025-01-13 11:56:35","video":"","vorDoi":"10.1038/s42003-025-09104-6","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09104-6","workflowStages":[]},"version":"v1","identity":"rs-5690019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5690019","identity":"rs-5690019","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}