Thermal stress restructures volatile gas emissions from the model cnidarian Aiptasia

Thermal stress restructures volatile gas emissions from the model cnidarian Aiptasia | 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 Research Article Thermal stress restructures volatile gas emissions from the model cnidarian Aiptasia Maggie Wuerz, Caitlin A. Lawson, Clinton A. Oakley, Malcolm Possell, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7991501/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Coral Reefs → Version 1 posted 11 You are reading this latest preprint version Abstract Coral bleaching events, in which symbionts are lost from host tissues, have become more frequent and severe because of climate change and specifically, elevated temperatures. How such events impact biogenic volatile organic compound (BVOC) emissions, compounds that can function as metabolic signalling elements, remains underexplored. Here we characterised the suite of BVOCs (collectively the “volatilome”) from the model sea anemone Exaiptasia diaphana (‘Aiptasia’) under three temperatures (control: 25°C; sub-bleaching: 30°C; and bleaching: 33.5°C), both without symbionts (aposymbiotic) and when populated by its native dinoflagellate symbiont, Breviolum minutum . The volatilome of symbiotic anemones during bleaching at an elevated temperature was distinct from that at lower temperatures, with high dimethyl sulphide (DMS), eucalyptol, and 1-iodododecane levels at the higher temperature. In comparison, the volatilome of aposymbiotic anemones was most distinct at the sub-bleaching temperature; the most abundant and significant BVOC differences included 2-phenyl-3-methyl-pyrrolo(2,3-b)pyrazine, acetone, and naphthalene. Symbiotic anemones had 12-fold more ‘core volatiles’ (BVOCs in all biological replicates across all temperature treatments) than aposymbiotic anemones (48 vs . 4 BVOCs); during thermal stress, the symbiotic anemone volatilomes retained their compound richness whereas the richness of aposymbiotic anemone volatilomes decreased. These observations suggest that symbiotic dinoflagellates enhance BVOC diversity and abundance and may confer a degree of metabolic stability to the intact symbiosis (i.e., ‘holobiont'). Such changes in metabolic outputs can inform our understanding of how coral holobionts respond to increasing seawater temperatures, enable targeted studies of BVOC function, and facilitate the development of biomarkers indicative of coral reef health. coral reefs coral bleaching volatilome BVOC Exaiptasia diaphana Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coral reefs are exceptionally diverse and ecologically and commercially valuable habitats that can harbour 25% of all marine species within a small oceanic footprint (~ 0.1% of total surface) (Hoegh-Guldberg et al., 2017 ). At the foundation of this diversity is the endosymbiosis between the cnidarian host and its dinoflagellate symbionts of the family Symbiodiniaceae (LaJeunesse et al., 2018 ). Efficient nutritional cycling between host and dinoflagellate symbionts allows for the growth and survival of reef-building corals in nutrient-poor waters (Muscatine and Porter, 1977 ; McIlroy et al., 2022 ), but this association is particularly vulnerable to thermal stress (Beyer et al., 2018 ; Cziesielski et al., 2022 ). When exposed to high temperature, the symbiosis becomes dysfunctional and the dinoflagellate symbionts are lost from the host tissue (Nielsen et al., 2018), resulting in a ‘bleached’ coral that dies if the thermal stress is prolonged (Oakley and Davy 2018 ). Coral bleaching is the result of perturbations to complex metabolic networks (Helgoe et al., 2024 ), with accumulation of reactive oxygen species (ROS) and nutritional imbalance considered the prevailing drivers of dysbiosis (Rädecker et al., 2023 ; Lesser, 2024 ). Synthesis of ROS is a natural by-product of photosynthetic and respiratory metabolisms (Foyer, 2018 ), although damage to the photosynthetic apparatus by high temperatures causes decreased photosynthetic efficiency and more excitation energy diverted to the generation of ROS. ROS are also generated by respiratory electron transport, with production again increasing when the organisms are heat stressed (Heise et al., 2003 ; Slimen et al., 2014 ), with surplus ROS potentially overwhelming repair mechanisms. Sustained oxidative stress leads to the loss of symbionts from the host tissues, potentially through mechanisms of apoptosis, autophagy, and exocytosis, although the relative importance of these different mechanisms remains unclear (Helgoe et al. 2024 ). Metabolic regulation and nutrient homeostasis are also crucial to the control of cnidarian-dinoflagellate symbiosis. The efficient cycling and use of nutrients is crucial in an oligotrophic environment; excess photosynthate by algae supports host metabolism, and in turn host respiration enhances CO 2 availability for algal photosynthesis. Nitrogen limitation restricts algal growth in a healthy symbiosis and ensures that excess carbon is available for translocation to the host. Failure to maintain a nitrogen-limited environment in hospite may therefore contribute to symbiosis breakdown under thermal stress (Rädecker et al., 2021 , 2023 ). In addition to traditional physiological and cell biological approaches (e.g., Gibbin & Davy, 2014 ; Matthews et al., 2023 ), various omics platforms are being applied to better understand the processes underpinning coral bleaching; notably, transcriptomics (Pinzón et al., 2015 ), proteomics (Ricaurte et al., 2016 ; Lust et al., 2025 ), lipidomics (Botana et al., 2024; Gamba et al., 2024), steady-state metabolomics (Hillyer et al., 2017a ; Lust et al., 2025 ), and volatilomics (Lawson et al., 2021 ). Volatilomics is the latest of the omics approaches applied to the study of reef ecosystems and involves identification of biogenic volatile organic compounds (BVOCs) that collectively comprise the “volatilome”. BVOCs are low molecular mass (< 200 Da) compounds with high vapour pressure (Mansurova et al., 2018 ) that are produced by all organisms on Earth (Laothawornkitkul et al., 2009 ; Tahir et al., 2017 ; Wuerz et al., 2022 ). BVOC functions are diverse: they can increase local cloud formation, thereby enhancing protection from UV radiation (Swan et al., 2016 ), as well as stabilise lipid membranes during thermal stress (Siwko et al., 2007 ) and elicit an immune response in neighbouring plants as a consequence of herbivory (Ton et al., 2007 ). Once thought to be simply by-products of primary metabolism, some BVOCs are now recognised as physiological agents that function across individuals (Wenig et al., 2019 ) or species (Minerdi et al., 2021 ), and which carry chemical messages. The nature and extent of BVOC emissions from an organism can be affected by environmental stressors, which is likely to be particularly important for understanding ecosystem function and regulation (Lawson et al., 2021 ; Okereke et al., 2022 , Lawson et al., 2022 , Dey et al., 2025 ). The symbiotic sea anemone Exaiptasia diaphana (hereafter, ‘Aiptasia’) is a model organism adopted for studying cnidarian-dinoflagellate symbiosis (Weis et al., 2008 ; Roberty et al., 2024 ). As with corals, Aiptasia forms a stable symbiosis with algae of the family Symbiodiniaceae (Nitschke et al., 2022 ) and similarly responds to environmental stressors that trigger the loss of algae from host tissues. Furthermore, Aiptasia can reproduce asexually, allowing the maintenance of genetically identical populations that can be used to examine the impact of environmental conditions on the animal’s physiological characteristics (Lehnert et al., 2012 ). While previous work has shown that thermal stress can alter volatile gas production in corals (Lawson et al., 2021 ) and that symbiotic state alone influences BVOC emissions (Wuerz et al., 2023 ), there is little known about how thermal stress alters BVOC emissions across symbiotic states and how these emissions interact and alter the responses of the host, dinoflagellate symbiont, and other organisms associated with the holobiont (e.g., the coral microbiome). Aiptasia provides an excellent model system with which to explore BVOC synthesis in a holobiont, aposymbiotic animals, and cultured endosymbiotic dinoflagellates, and how the quality and quantity of BVOCs are impacted by temperature, chemical treatment (Matthews et al., 2016 ), and symbiotic state (Matthews et al., 2017 ; Wuerz et al., 2022 & 2023 ). Coral reefs are major producers of BVOCs (Lawson et al., 2021 ), and characterization of BVOCs has shown promise in other ecosystems for providing a non-invasive snapshot of organism physiology (Manusurova et al., 2018). BVOC analysis therefore provides a promising avenue for understanding how coral reef metabolism is altered by climate change. Here we determine the impact of thermal stress on BVOC production by the Aiptasia holobiont. We specifically profiled BVOCs in two different trophic states (i.e., symbiotic versus aposymbiotic) at control, sub-bleaching, and bleaching temperatures. We hypothesised that thermal stress would shift the volatilome. This information will add to our understanding of physiological responses associated with the breakdown of symbiosis in this important model system, while also aiding in the identification potential of BVOC biomarkers that can be used to monitor the health of coral reefs. Materials and Methods Experimental organisms : A long-term (15 + years) clonal culture of Aiptasia (culture ID: NZ1) of unknown Pacific origin (Matthews et al., 2017 ) was maintained in the laboratory in 0.22-µm filtered seawater (FSW) at 25°C and 70 µmol photons m -2 s -1 on a 12:12 h light-dark cycle (GE Lighting T5 F54W/840). Clonal anemones (n = 100) were rendered aposymbiotic using menthol-induced bleaching, via exposure to menthol (20% w/v in ethanol; Sigma-Aldrich, Auckland, NZ) at a final concentration of 0.19 mmol L -1 in 0.22 µm FSW, as per Matthews et al. ( 2016 ). Anemones were incubated in menthol for 8 h during the 12-h light period, after which photosynthesis was inhibited by replacing menthol/FSW with FSW containing 5 µmol L -1 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 100 mmol L -1 dissolved in EtOH, Sigma-Aldrich) for 16 h to prevent repopulation by inhibiting photosynthesis of the remaining symbionts. After repeating this 24-h cycle for four consecutive days, anemones were maintained in 0.22 µm FSW without menthol or DCMU for an additional three days. Anemones were fed once weekly with Artemia sp. nauplii, with fresh FSW changes 8 h post-feeding: this culturing protocol was continued for six weeks. Aposymbiosis was confirmed using fluorescence microscopy (Olympus IX53 inverted microscope; 100× magnification). All aposymbiotic anemones were then maintained in 0.22 µm FSW in the dark at 25°C for 1.5 years prior to BVOC sampling, during which time they were fed with Artemia sp. nauplii and given fresh FSW twice weekly. A stock of symbiotic anemones maintained for > 1 year, initially generated via repopulation of aposymbiotic anemones with the homologous symbiont ( Breviolum minutum , ITS2 Type B1, culture ID ‘FLAp2’) as described in Wuerz et al. ( 2022 , 2023 ), was used for consistency. Symbiotic anemones were maintained at a constant temperature of 25°C and an irradiance of approximately 70 µmol photons m − 2 s − 1 on a 12:12 h light-dark cycle. This anemone stock culture was fed twice weekly, as described above. Thermal stress treatment : Prior to BVOC sampling, Aiptasia were kept in 250 mL glass jars each containing 20–30 anemones. The jars were maintained in temperature-controlled water baths as described in Wuerz et al. ( 2022 ). All anemones were fed three times weekly for the duration of the experiment with freshly-hatched Artemia sp. nauplii. Both aposymbiotic and symbiotic anemones were maintained at 130 µmol photons m − 2 s − 1 (light saturation for photosynthesis, Table S1 ) on a 12:12 h light:dark cycle during the thermal-ramping period. The control group was kept at 25°C throughout the experiment; the sub-bleaching group was subjected to a temperature increase of 1°C per day to a maximum temperature of 30°C, which was maintained for seven days prior to BVOC sampling. The bleaching group was subjected to a temperature increase of 1°C per day until 30°C, after which the temperature was increased by 0.5°C per day to a maximum temperature of 33.5°C, which was maintained for 24 h prior to BVOC sampling (Fig. 1 ). Photosystem II (PSII) maximum quantum yield ( F v /F m , dimensionless) was measured every day during thermal ramping and used as an indicator of stress associated with the symbiotic association (Levin et al., 2016 ). All anemones were dark-acclimated for 15 min before measuring various photosynthetic activities with an Imaging Pulse Amplitude Modulated Fluorometer (I-PAM, Walz, Effeltrich, Germany; settings: measuring light = 4, saturation intensity = 8, saturation width = 0.8 s, gain = 3 and damping = 3), as per Vergauwen et al. ( 2017 ). Symbiont cell density and protein determination Frozen anemones (n = 5, with 10–15 anemones per biological replicate) were thawed on ice and homogenised using a saw-tooth homogeniser in 500 µL milliQ water. Host and symbiont fractions were separated by centrifugation at 400 x g for 2 min after which the symbiont fraction was washed of residual host material by one further resuspension and centrifugation with milliQ water. The supernatant fraction was analysed for protein content using the fluorometric Qubit Protein Assay Kit (Vergauwen et al 2017 ). Cell counts were performed using a haemocytometer (Improved Neubauer) with eight replicate counts per sample and normalised to protein content. Cell density (symbionts per mg host protein) was also calculated. Photosynthesis and respiration Holobiont physiology was assessed by measuring oxygen consumption in the dark by both aposymbiotic and symbiotic anemones, and photosynthetic oxygen evolution in the symbiotic anemones was evaluated by incubating anemones in a sealed Perspex chamber (13 mL) fitted with an internal stir bar and an oxygen optode (Fibox 4, PreSens Gmbh, Regensburg, Germany). The magnetic stir bar was situated underneath a perforated mesh floor to which the anemones were attached, and rotated at a speed at which the anemones did not appear visually stressed. The Perspex chamber vials were immersed in one of three temperature baths according to the treatment temperature (25°C, 30°C or 33.5°C), atop a magnetic stir table (Multistirrer Digital 6, VELP Scientifica Srl, Italy). The anemones were allowed to settle and attach to the bottom of the chambers for ~ 1 h prior to making measurements. To quantify respiratory rates, the lights were extinguished and oxygen consumption measured for 15 min. For symbiotic anemones, illumination was for 20 min at 130 µmol photons m –2 s –1 which, as previously mentioned, is sufficient to saturate holobiont photosynthesis. All measurements were taken during the photic period, with measurements of holobiont dark respiration calculated as moles O 2 consumed h − 1 . Gross photosynthesis values were calculated by adding dark respiration rates to net photosynthetic rates for symbiotic anemones (moles O 2 produced h − 1 during the photic period). Respiration and gross photosynthetic rates were normalised to soluble protein content of each anemone and per million symbiont cells, respectively. Immediately after sampling, the anemones were flash frozen at -80°C and stored for determination of algal cell density and protein concentration. BVOC sampling and volatilome characterisation : BVOC gases were collected and analysed from anemones in two different symbiotic states and three temperatures each: aposymbiotic anemones (n = 6 vials at 25°C and 33.5°C, n = 5 vials at 30°C; ~15 anemones per vial); symbiotic anemones (n = 6 vials at 25°C and 33.5°C, n = 5 vials at 30°C; ~10 anemones per vial). Different numbers of anemones were used for each sample to ensure a similar biomass among the groups; biological replicates varied among treatments due to technical instrument error, which resulted in fewer replicates in the sub-bleaching treatment. BVOC retrieval was performed using previously established methods (Ton et al., 2007 , Wuerz et al., 2022 , 2023 ). First, experimental organisms were transferred into sterile 150 mL, unsealed, crimp-cap serum glass vials (Wheaton, Millville, NJ, USA) containing 75 mL 0.22 µm FSW, where they were held overnight under conditions identical to those used to grow the cultures. This incubation allowed the organisms to settle, before the FSW medium was refreshed and the vials sealed using 20 mm PTFE/Si crimp caps (Agilent, USA). BVOCs were collected by passing instrument-grade air (100 mL min − 1 ; BOV Gases, Wellington, NZ) into gas-tight sampling vials for 20 min, with the outgoing air passed through open-ended thermal desorption tubes (TDTs; Markes International Ltd, Llantrisant, UK) containing the sorbent Tenax TA, onto which the BVOCs adhere (Ton et al., 2007 ; Guitton et al., 2017 ). After a 20 min sampling time, TDTs were immediately sealed with brass storage caps and stored at 4°C until processed. All TDTs were analysed within two weeks of sampling to minimise sample degradation, using gas chromatography coupled with single quadrupole mass spectrometry (GC×MS) as per Wenig & Odermatt ( 2010 ). TDT desorption was performed using a Marks Unity 2 Series Thermal Desorber and ULTRA multi-tube autosampler. Desorption occurred at 300°C for 6 min, after which BVOCs were concentrated in a cold trap at -30°C. The cold trap was subsequently flash-heated to 300°C, injecting the concentrated sample onto a 7890A GC (Agilent Technologies, Ltd., Melbourne) through a transfer line maintained at 150°C. The GC was equipped with a 30 m x 0.25 mm Rxi-624Sil MS column (Restek Corp.) with a film thickness of 1.40 µm. To encourage complete desorption of BVOCs from the column, the GC oven was heated at 35°C for 5 min and then increased to 240°C by increments of 5°C min − 1 ; once at 240°C, the temperature was maintained for 5 min. All samples were run in splitless mode at a flowrate of 1 mL min − 1 . The GC was attached to a Model 59745 mass-selective detector (Agilent Technologies, Ltd., Melbourne) with the scanning range set to 29—450 amu. Spectral data from the GC-MS were run through the open-source MS data processing program OpenChrom (Weing & Odermatt, 2010) to remove common contaminating ions (amu: 73, 84, 147, 149, 207, 221). Output files were imported to Galaxy (Guitton et al., 2017 ) and processed using the metaMS.runGC package (Galaxy version 2.1.1; (Wehrens et al., 2014 )) in Workflow4Metabolomics. Peaks were tentatively identified against a database in the National Institute of Standards and Technology (NIST) Mass Spectral library (NIST 14 library in NIST MS Search v2.2; NIST, Gaithersburg, MD). Blank seawater samples (n = 6 vials at 25°C and 33.5°C; n = 5 vials at 30°C) were run in conjunction with all analyses; average values of blanks were subtracted from each sample. Any compound that was not present in two or more biological replicates was considered to be contamination and removed. Unclassified compounds are denoted as UC; the number following ‘UC’ indicates the retention time of the compound. Prior to desorption, each TDT was injected with 0.2 µL of 150 ppm bromobenzene (GC grade, Sigma Aldrich, Castle Hill, NSW, Australia) in methanol (HPLC grade, Sigma Aldrich) which serves as an internal standard. Finally, peak abundances were normalised to the protein content of each replicate. Protein abundances were measured after the samples were lysed using an ultrasonication probe (VCX500; Sonics & Materials Inc., Newtown, CT, USA); the protein concentration in the lysate was measured using the fluorometric Qubit Protein Assay Kit (Vergauwen et al., 2017 ). Compounds identified as likely methodological artifacts, e.g. silicon-containing compounds, were also removed from the dataset, as they were suspected contaminants from the GC column dimethylpolysiloxane hydrolysis (Cella and Carpenter 1994 ). All BVOCs were grouped according to their chemical class. BVOC abundance data are dimensionless and were normalised to a chemical standard and total protein content, after BVOCs in blanks were subtracted from test TDTs. Data analysis Oxygen production/consumption, and cell density measurements were tested for normality and homoscedasticity and then tested with two-way ANOVA in R (version 4.0.3; Team RC 2021). PSII maximum quantum yield was analysed by linear regression using the stats package in R (version 4.4.1) and changes in this yield were considered as an indication of health of the organisms over the temperature treatment period. Differential abundance of BVOCs was estimated using the limma R package (Ritchie et al 2015 ); the voom function was used to convert counts to log 2 -counts- per -million and to assign weights to each observation based on the mean-variance trend. The counts assigned to each observation roughly correspond to the abundance of each BVOC, which can then be used to compare BVOC production between treatments. Functions lmFit, eBayes, and topTable were used to fit weighted linear regression models, calculate empirical Bayes moderated t-statistic, and calculate FDR-corrected p-values. Biological replicates, standardised using the decostand function in the vegan package (Oksanen et al 2020 ) in R (version 1.2.5033), were compared using the Bray-Curtis similarity measure, and this output was subjected to non-metric multidimensional scaling (NMDS) to visualise differences among groups based on BVOCs. To compare dispersion of biological replicates using a distance measure, PERMANOVA was performed to test if the distance between biological replicates was greater between treatments than within treatments. PERMANOVA was performed on NMDS scores using the adonis function, and post hoc tests were performed using the pairwise.adonis function in the vegan package in R (version 1.2.5033) to distinguish differences among treatments. All plots were created with ggplot2 (Wickham 2016 ) in RStudio. Results Photosystem II quantum yield, symbiont cell density, respiration, and oxygen production : PSII maximum quantum yield values for anemones in the sub-bleaching treatment increased over time but showed a sustained decrease in the bleaching thermal treatment (linear regression; Fig. 1 , sub-bleaching: F 1,37 : 7.971, p < 0.01; bleaching: F 1,37 , p < 0.001; fit models with confidence intervals shown in Figs. S1-S3 ), which suggests damage to PSII. By comparison, values in the control treatment remained constant. Symbiont density was also significantly impacted by elevated temperature (Fig. 2 A, one-way ANOVA, F 2,27 = 10.84, p < 0.01). In particular, algal cell density in the anemones at the putative bleaching temperature of 33.5°C (1.72 x 10 6 cells per mg host protein) was significantly lower than that in anemones at the control (25°C) and sub-bleaching (30°C) temperatures (2.29 and 2.63 x 10 6 cells per mg host protein, respectively; Tukey post hoc , p < 0.01 for both comparisons). Cell densities were not significantly different at 25°C vs. 30°C. Consistent with bleaching at the highest temperature was the sustained decrease in F v /F m at 33.5°C (Fig. 1 , Fig S3 ). In contrast, the symbiont cell-specific gross photosynthetic rate was not significantly impacted by thermal treatment, although there was a trend towards an elevated rate at 33.5°C (Fig. 2 B) that might (partially) compensate for the loss of symbionts at that temperature, resulting in little overall change in the rate of holobiont photosynthesis at the different temperatures (Fig. 2 C). The respiration rate of aposymbiotic anemones doubled at the bleaching temperature relative to the two lower temperatures (one-way ANOVA, F 2,11 = 6.94, p < 0.05; Tukey post hoc p < 0.05 for both comparisons; Fig. 2 D). Similarly, the respiration rate of symbiotic anemones increased by 50% at the bleaching temperature relative to the other two temperatures (Fig. 2 E) (one-way ANOVA, F 2,10 = 8.746, p < 0.05; Tukey post hoc p < 0.01 for both comparisons). BVOC emissions by aposymbiotic anemones are influenced by temperature : Non-metric multidimensional scaling demonstrated a shift in the volatilome of aposymbiotic Aiptasia across temperature treatments (Fig. 3 A). Specifically, the volatilome of anemones at the sub-bleaching temperature was different to that of anemones at the highest temperature (PERMANOVA: F 2,14 = 2.49, p < 0.005; Tukey post hoc : p < 0.005). In contrast, the volatilome of control aposymbiotic anemones was not significantly different from the volatilome of aposymbiotic anemones at either of the higher temperatures. A total of 148 BVOCs were detected in aposymbiotic anemones across the three temperatures (Fig. 3 B). Of these, 70 were detected solely at the control temperature, 15 at the sub-bleaching temperature, and 11 at the bleaching temperature. A total of 40 BVOCs were common to aposymbiotic anemones at the control and sub-bleaching temperatures, five were common to the sub-bleaching and bleaching temperatures, and three were common to the control and bleaching temperatures (Fig. 3 B). Only four BVOCs were present in aposymbiotic samples after all three temperature treatments; these are considered the ‘core aposymbiotic compounds’. Two of the four volatiles are aromatic compounds (styrene and (1-methylethyl)-benzene), one has diverse functional groups (3-ethenyl-2-ethoxypyrazine), and one is an unclassified compound (UC45.72). Exposure to elevated temperature decreased the number of BVOCs detected in the volatilome of aposymbiotic anemones. Aposymbiotic anemones produced 117 BVOCs at 25°C, the most common of which were aromatic compounds (29 BVOCs); 64 BVOCs were produced at 30°C, predominantly aromatic compounds; only 23 BVOCs were produced at 33.5°C, of which five were unclassified (Fig. 3 C). A total of 14 BVOCs were significantly differentially abundant in response to temperature in the volatilome of aposymbiotic anemones (Fig. 4 A; Table S2 ). Only one BVOC, 2,7,10-trimethyldodecane, was differentially abundant between control and heat-stressed anemones and specifically detected at higher abundance at 33.5°C than at either the control or sub-bleaching temperatures. The other 13 differentially abundant BVOCs were significantly different between 33.5°C and 30°C, with 12 detected at higher abundance in the sub-bleaching treatment, including dibromomethane, 1,2-dichloroethane, and 2-butanone. The one exception was hexadecane, which was detected in higher quantities at 33.5°C vs . 30°C. All other differentially abundant BVOCs are listed in Supplementary Table S2 . BVOC emissions by symbiotic anemones are also influenced by temperature : Among the symbiotic anemones exposed to different temperatures, only seven BVOCs were significantly differentially abundant (Fig. 4 B; Table S3 ). The only BVOC more abundant at the sub-bleaching temperature relative to the bleaching temperature was di-tert-butyl peroxide. The other six BVOCs were more abundant in anemones exposed to the bleaching than the sub-bleaching temperature, and included dimethyl sulphide, eucalyptol, methyl N-hydroxybenzenecarboximidoate, 1-(2-pyridyl)piperazine, 1-iodododecane, and 2,7,10-trimethyldodecane. DMS was also more abundant in the volatilome of symbiotic anemones at the bleaching than at the control temperature. Non-metric multidimensional scaling also demonstrated changes in the volatilomes produced by symbiotic anemones across temperature treatments (Fig. 5 A). Symbiotic anemones exposed to the highest temperature (33.5°C) produced a volatilome that differed from those of anemones exposed to 25°C or 30°C (PERMANOVA F 2,14 = 3.25, p < 0.005; Tukey post hoc , p < 0.05 for both comparisons), with the 25°C and 30°C volatilomes being statistically similar. A total of 170 BVOCs were detected in the volatilomes of symbiotic anemones across the three temperature treatments (Fig. 5 B). Of these, 21 were produced solely at the control temperature, eight at the sub-bleaching temperature, and 39 at the bleaching temperature. A total of 17 BVOCs were common between symbiotic anemones at control and sub-bleaching temperatures, 16 were common between sub-bleaching and bleaching temperatures, and 21 were common between control and bleaching temperatures. A substantial number of BVOCs (48) were present in the volatilome of all symbiotic anemones – irrespective of temperature treatment – and are therefore designated as ‘core symbiotic compounds’. Of these 48 core BVOCs, the most abundant chemical group were aromatic compounds (14), followed by halogenated hydrocarbons (8), including dibromomethane, bromodichloromethane, dibromochloromethane and bromochlorodifluoromethane. As observed for aposymbiotic anemones, temperature influenced the number of BVOCs detected in symbiotic anemones. However, intriguingly – and in contrast to aposymbiotic anemones – the richest volatilome (124) was detected at the highest temperature (33.5°C), with aromatic compounds (29) being the most abundant chemical class. Symbiotic anemones produced the fewest BVOCs (89) at 30°C, with aromatic compounds again being the most abundant (21), whereas symbiotic anemones at 25°C produced 107 BVOCs, comprised primarily of volatiles with diverse functional groups (Fig. 5 C). Discussion Core BVOCs : Between symbiotic states and across the three temperature treatments, three BVOCs were common to all Aiptasia samples: (1-methylethyl)-benzene, 3-ethenyl-2-ethoxypyrazine, and styrene. None of these core compounds were detected at differentially abundant concentrations between temperature treatments, suggesting that they play an important role in central metabolism across temperatures. One unclassified BVOC, UC45.72, was identified as a core BVOC in aposymbiotic anemones and only present in symbiotic anemones when undergoing bleaching. Such a response would suggest that UC45.72 is produced in response to stress, or suppressed by the presence of symbionts. Indeed, upregulation of pathways for mediating oxidative stress and apoptosis in aposymbiotic Aiptasia has been reported (Rodriguez-Lanetty et al., 2006 ; Oakley et al., 2016 ; Gorman et al., 2022 ), suggesting that symbiosis protects the host from oxidative stress and cellular damage (Oakley et al., 2016 ). Oxidative stress is a well-known feature of coral bleaching (reviewed by Sun et al., 2022 ), so the production of UC45.72 in the volatilome of thermally-stressed symbiotic anemones could reflect a direct impact of high temperature and/or a marked loss of symbionts and hence a shift towards a state closer to that of aposymbiotitc anemones. Either way, this unclassified BVOC is likely an important candidate for future studies. Interestingly, only four BVOCs were detected consistently in the volatilome of aposymbiotic anemones irrespective of thermal treatment, while for symbiotic anemones, 48 BVOCs met this criterion. As such, physiological stability may be greater for symbiotic than for aposymbiotic anemones, once again suggesting that symbiosis may confer a degree of protection to the host and holobiont. In particular, given that aposymbiotic anemones already appear to be experiencing some degree of stress (Oakley et al., 2016 ), additional stressful environmental conditions may trigger a more rapid shutdown of central metabolism, resulting in the release of fewer BVOCs as by-products. By contrast, despite previously observed transcriptomic, proteomic and metabolomic impacts of thermal stress on symbiotic cnidarians (Desalvo et al., 2010 ; Oakley et al., 2017 ; Hillyer et al., 2017b ; Cziesielski et al., 2018 ), the relative consistency of the volatilome at high temperature observed here suggests that central metabolism is altered less dramatically than in the aposymbiotic state. Elucidation of the underlying cellular processes that control BVOC synthesis, and how the different members of the holobiont (including other members of the microbiome such as bacteria) interact to facilitate metabolic homeostasis, are key questions for future research. Temperature affects the Aiptasia volatilome Aposymbiotic anemones : In the aposymbiotic state, progressively higher temperatures generated increasingly diminished volatilome richness, with a consistent core volatilome of just four BVOCs. As observed in other organisms (e.g., Taris et al., 2010 ), the respiration rate in aposymbiotic anemones increased at the highest temperature (Fig. 2 D). As respiration and metabolism are intrinsically linked processes (Weis, 2014 ), increased metabolism, and therefore BVOC output, may be expected. However, our results demonstrated the contrary: the decline in BVOC richness at 33.5°C may be related to the onset of metabolic dysfunction, which is typical for symbiotic cnidarians at such a high temperature (Roach et al., 2021 ; Sun et al., 2022 ;). It is, however, important to exercise caution when drawing such conclusions, as little is known about the physiology of the associated microbiome. Previous studies have shown temperature-induced shifts towards pathogenic microbiota in corals (Bourne et al., 2009 ). Additionally, Aiptasia-associated microbiota have been observed to change in response to different symbiotic states (Curtis et al., 2023 ; Wuerz et al., 2023 ) and thermal stress (Ahmed et al., 2019 , Sydnor et al., 2023 ); thus, it is plausible that a thermally-altered microbiome may have contributed to the observed differences between the volatilomes in our current study. Fourteen BVOCs were differentially expressed in the volatilome of aposymbiotic anemones in response to thermal change. Interestingly, 12 of these were detected at higher abundance at the sub-bleaching relative to the bleaching temperature. The most abundant BVOCs in the volatilome of aposymbiotic anemones at the elevated, sub-bleaching temperature are known to have diverse functions in other systems and include naphthalene and dibromomethane. Naphthalene is produced by termites (Chen et al., 1998 ; Wilcke et al., 2000 ), inhibiting the growth of pathogenic fungi and forming part of the termite’s defence system (Wright et al., 2022 ). It has also been shown to act as an apoptosis inhibitor in the model nematode Caenorhabditis elegans , suggesting that it may promote survival and proliferation of tumour cells (Kokel et al., 2006 ). Dibromomethane induces metamorphosis in the mollusc Crepidula fornicata (Taris et al., 2010 ). These functions suggest that BVOCs have the potential to play roles in developmental processes and cellular signalling in Aiptasia and other symbiotic cnidarians, although this requires detailed confirmation. Just two BVOCs – hexadecane and 2,7,10-trimethyldodecane – were produced at higher abundances by anemones at the bleaching temperature relative to the sub-bleaching temperature. Given the physiological stress that occurs in Aiptasia at such a high temperature, as noted both in our current study and previous work (Oakley et al., 2017 ; Hawkins et al., 2016 ; Cleves et al., 2020 ), it is possible that these BVOCs are downstream products of stress pathways. Moreover, hexadecane has been previously shown to induce cell death in both rat and human cell cultures (Herman et al., 2012 ). Hexadecane may be an element of the bleaching process, as apoptosis is thought to be an important cellular end-point of the cnidarian bleaching cascade (Helgoe et al., 2024 ). Symbiotic anemones In contrast to aposymbiotic anemones, the volatilome of symbiotic anemones was more stable in response to thermal change, with the core volatilome consisting of 48 BVOCs, with only seven that were differentially abundant at the different temperatures. There was also a trend of increasing BVOC diversity at increasing temperature. This is in contrast to the findings of Lawson et al. ( 2021 ), who showed a decrease in BVOC richness and abundance at higher temperatures in corals. These differences perhaps indicate species-specific differences in BVOC synthesis among cnidarians in response to elevated temperature, though they could also reflect the different maximum temperatures used in the two studies (32°C vs . 33.5°C). The increased volatilome diversity at the highest temperature seen in our study suggests a proliferation of metabolic downstream products associated with cellular dysfunction in the host or symbionts, and/or altered microbiome metabolism during the bleaching process. It is not possible from this approach to determine the member(s) of the holobiont responsible for the production of each BVOC, or how the metabolism of various partners interacts under stress, but future work with isolated partners (e.g., axenic Symbiodiniaceae, or microbes associated with the cnidarian surface) would help shed light on this matter. In contrast, the volatilome was least diverse at the intermediate, sub-bleaching temperature. One interpretation of this is that metabolic integration of the holobiont was greatest at this temperature, resulting in the release of fewer BVOCs to the environment. It is somewhat surprising that this would be the case at the intermediate rather than at the control temperature, but we note that there were no obvious signs of physiological stress at the intermediate temperature used in this study. Of the seven differentially abundant BVOCs in symbiotic anemones, most were detected at higher abundance at the bleaching relative to the sub-bleaching (five BVOC) or control temperatures (one BVOC). Of these BVOCs, dimethyl sulphide (DMS) and eucalyptol have roles as antioxidants in other systems (Guan et al., 2017 ; Xu et al., 2021 ), suggesting that their production in the cnidarian-dinoflagellate symbiosis may involve the amelioration of free radicals or toxic metabolic by-products. The sources of these compounds (i.e., host, symbiont or microbiome) are difficult to determine from our current approach, but it is notable that 2,7,10-trimethyldodecane was also conspicuous in the volatilome of heat-stressed aposymbiotic anemones, suggesting that it may originate from the host or members of the microbiome that are common to symbiotic and aposymbiotic anemones. Only one BVOC was more abundant at the sub-bleaching vs bleaching temperature - di-tert-butyl peroxide - suggesting that this compound is involved in or is a by-product of a fully functional symbiosis. However, it is not clear why di-tert-butyl peroxide was more abundant at the intermediate than at the control temperature. Notably, DMS was the BVOC produced in highest abundance by symbiotic anemones at the bleaching temperature. DMS is a widely studied, multi-functional BVOC with established roles in the global sulphur cycle (Brimblecombe, 2013 ) and stress physiology (Sunda et al., 2002 ). At the cellular level, dimethylsulphoniopropionate (DMSP) and its breakdown products (DMS, acrylate, dimethyl sulphoxide and methane sulphinic acid) represent a powerful antioxidant system (Sunda et al., 2002 ; Hopkins et al., 2016 ) that increases in both corals (Hopkins et al., 2016 ) and Symbiodiniaceae (Deschaseaux et al., 2014 ) in response to thermal stress. This pattern of DMS production can be considered in the context of physiological responses associated with elevated temperature, including an increase in the holobiont respiratory rate and a decrease in symbiont density in the host. Such gross physiological changes typically lead to metabolic shifts in both the host and symbiont that might, for example, compensate for nutritional deficits by mobilising carbohydrate and lipid stores (Hillyer et al., 2017a , 2017b ). Furthermore, high temperature can induce oxidative stress and potentially elicit activation of immunity and inter-partner signalling pathways involved in symbiosis dysfunction (Dunn et al., 2007 ; Krueger et al., 2015 ; Hillyer et al., 2017b ). In symbiotic Aiptasia, it is reasonable to suggest that the observed increase in DMS at the bleaching temperature acts to ameliorate ROS produced in response to thermal stress (Perez and Weis, 2006 ). Although the total number of symbionts in the holobiont decreased during bleaching, DMS production was higher such that the remaining symbionts produced much more DMS per cell than at the lower temperatures. The ability to produce copious amounts of DMS may be particularly advantageous during periods of thermal stress; it may contribute to sustaining the symbiosis as a consequence of its antioxidant activity. Eucalyptol, which was also most abundant in the holobiont at the highest temperature, may have a similar protective function. Like DMS, this volatile has known antioxidant activity (Kennedy-Feitosa et al., 2016 ), but also has anti-inflammatory (Kim et al., 2015 ) and anti-bacterial (Li et al., 2016 ) properties in mammalian models, although it can also induce DNA damage in mammals (Dörsam et al., 2015 ). While identification of BVOCs in other systems provides a framework for exploring their role in the cnidarian-dinoflagellate symbiosis, much more work is needed to elucidate their impact on the establishment, maintenance, and dysfunction of this ecologically important symbiosis. Conclusion Elevated temperature impacts the volatilome of both aposymbiotic and symbiotic Aiptasia. The largest temperature-induced alterations in the volatilome were observed in aposymbiotic anemones, suggesting that the presence of Symbiodiniaceae provides a stabilising effect on holobiont function, at least until the upper thermal threshold is reached. More work is now needed to identify all BVOCs, and the sources and functions of BVOCs associated with the cnidarian-dinoflagellate symbiosis. Focused future efforts are required to characterise the volatilome of individual components of the holobiont (cnidarian host, dinoflagellate symbionts, associated microbes) and to elucidate BVOC biosynthetic pathways and their control. Nevertheless, our observations in this study on the model organism Aiptasia provide a first step towards understanding how heat stress can influence BVOC emissions across symbiotic states and at different temperatures. Additionally, identifying various BVOCs (e.g., DMS, eucalyptol) and their abundances in symbioses may facilitate their use as non-invasive biomarkers for thermal and cellular stress and for evaluating the overall health of coral reef ecosystems. Declarations Funding: This work was funded by a Victoria University doctoral scholarship awarded to M.W. and the Royal Society of New Zealand Te Aparangi Marsden Fund (19-VUW-086, awarded to S.K.D., A.R.G., C.A.O., D.J.S., and V.M.W.). The contributions of D.J.S and C.A.L. were furthermore supported by an Australian Research Council discovery project (DP200100091, awarded to D.J.S.). Author Contribution Statement: MW and SKD conceived the study. MW, CAL, CAO, ARG, VMW, DJS, and SKD conceptualized the study. MW, CAL, CAO, DJS, and SKD conceptualized the methods. MW, CAO, ARG, VMW, DJS, and SKD provided funding. MW administered the project and performed laboratory work. MW and MP conducted data analysis. MW, CAL, CAO, and MP conducted data analysis, interpretation, and visualization. MW wrote the original draft of the manuscript, with input from all authors. All authors approved the final manuscript. Competing interests : The authors have no conflicts of interest to declare. 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Supplementary Files Thermalstresssupplementarymaterialfinal.docx Cite Share Download PDF Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Coral Reefs → Version 1 posted Editorial decision: Revision requested 26 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviews received at journal 02 Dec, 2025 Reviews received at journal 26 Nov, 2025 Reviewers agreed at journal 15 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers invited by journal 12 Nov, 2025 Editor assigned by journal 04 Nov, 2025 Submission checks completed at journal 01 Nov, 2025 First submitted to journal 30 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":77416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotosystem II quantum yield (F\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ev\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e/F\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, dimensionless, ± standard error) measurements for Aiptasia symbiotic with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBreviolum minutum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e at three temperature treatments (control: 25 °C; sub-bleaching: 30 °C; bleaching: 33.5 °C), prior to BVOC sampling.\u003c/strong\u003e Anemones (n = 3 for each temperature treatment \u003cem\u003eper\u003c/em\u003e day) were dark acclimated for 15 min before measurement. Data points are offset for clarity.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/08e821a9af0cdcdcb3193639.png"},{"id":96640510,"identity":"fb9cf39e-4cf6-4d82-a02d-d6a301723a8b","added_by":"auto","created_at":"2025-11-24 14:25:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of temperature on symbiosis stability and physiology.\u003c/strong\u003e Anemones were acclimated to each temperature treatment (control (25 °C), sub-bleaching (30 °C) and bleaching (33.5 °C)) over 14 days, with oxygen measurements taken on Day 14. \u003cstrong\u003eA\u003c/strong\u003e) Symbiont cell densities in Aiptasia (n = 10); \u003cstrong\u003eB\u003c/strong\u003e) Symbiont cell-specific rate of gross photosynthesis (n = 5); \u003cstrong\u003eC\u003c/strong\u003e) Overall photosynthetic rate of symbiotic anemones (n = 5); \u003cstrong\u003eD\u003c/strong\u003e) Respiration rate of aposymbiotic anemones (n = 5); \u003cstrong\u003eE\u003c/strong\u003e) Respiration rate of symbiotic anemones (n = 5). Lines around box plot indicate interquartile ranges from 25-75%; statistically significant groupings among temperature treatments are indicated by letters above error bars.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/ba04b436ecaa90bb602f8243.png"},{"id":96640512,"identity":"28a56e82-49ce-439b-ab1e-f6c6cd6f3f1b","added_by":"auto","created_at":"2025-11-24 14:25:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155709,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of BVOCs among the volatilomes of aposymbiotic anemones maintained at three different temperatures (25 °C, 30 °C, 33.5 °C). BVOCs had to be present in at least three biological replicates in at least one thermal treatment to be included. (A) Non-metric multidimensional scaling (NMDS; stress = 0.187) plot of BVOCs produced by aposymbiotic anemones. Displayed BVOCs were chosen based on the top five loading scores for each NMDS dimension. Volatilomes for anemones at 30 °C and 33.5 °C were significantly different from one another (PERMANOVA, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01); volatilomes for anemones at 25 °C were not significantly different from of the higher temperature treatments (PERMANOVA, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05 for both temperatures). (B) Venn diagram showing presence of 148 BVOCs detected across the aposymbiotic anemone dataset. (C) Abundance of BVOCs across differing temperature treatments grouped by chemical class. CPDH = Cyclopropa[3,4]pentaleno[1,2-d][1,3]dioxole, 2a,2b,2c,5a,5b,5c-hexahydro-, (2aa,2ba,2ca,5aa,5ba,5ca)-; DFG = diverse functional group; HC = hydrocarbon; N = nitrogen; S = sulphur.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/008f33b940e8d03576f54bb6.png"},{"id":96709428,"identity":"460cdbad-4907-48c3-9dfa-eb84761b58d1","added_by":"auto","created_at":"2025-11-25 10:09:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially abundant BVOCs (+ standard error) in aposymbiotic and symbiotic anemones in three thermal treatments (control: 25 °C; sub-bleaching: 30 °C; bleaching: 33.5 °C).\u003c/strong\u003e BVOCs had to be present in at least three biological replicates in at least one thermal treatment to be included in the volatilome of: A) aposymbiotic Aiptasia; and B) symbiotic Aiptasia. Letters beside error bars indicate statistical groupings among temperature treatments (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). Abundance data are dimensionless and were normalised to a chemical standard, seawater blanks and holobiont protein content.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/a506834560d6151403383baf.png"},{"id":96708987,"identity":"80f49763-b4ba-461b-b85c-d058e2309a6a","added_by":"auto","created_at":"2025-11-25 10:06:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":185063,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of BVOCs in the volatilomes of symbiotic anemones in response to thermal treatment (25 °C, 30 °C, 33.5 °C). BVOCs had to be present in at least three biological replicates in at least one temperature treatment to be included. (A) Non-metric multidimensional scaling (NMDS; stress = 0.152) plot of BVOCs produced by symbiotic anemones. Displayed BVOCs were chosen based on the top five loading scores for each NMDS dimension. Volatilomes for anemones at 33.5 °C were distinct from those at 25 and 30 °C (PERMANOVA, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 for both temperatures). Volatilomes for anemones at 25 °C were not distinct from those at 30 °C (PERMAOVA, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). (B) Venn diagram showing overlap of 170 BVOCs detected across the symbiotic anemone datasets. (C) Abundance of BVOCs at each of the three temperature grouped according to chemical class. DFG = diverse functional group; HC = hydrocarbon; N = nitrogen; S = sulphur.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/69f1969c283db0dc14b3d63a.png"},{"id":106809089,"identity":"3ae378df-a2bf-4b80-acc7-07ec19271f1c","added_by":"auto","created_at":"2026-04-13 16:06:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1592622,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/289877d9-fb82-4e93-aeb3-4cdd035b589f.pdf"},{"id":96640513,"identity":"4a5f0890-fe56-485a-8059-8305be49dd35","added_by":"auto","created_at":"2025-11-24 14:25:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":459990,"visible":true,"origin":"","legend":"","description":"","filename":"Thermalstresssupplementarymaterialfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7991501/v1/4d1d6e109406558470fadc2d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thermal stress restructures volatile gas emissions from the model cnidarian Aiptasia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoral reefs are exceptionally diverse and ecologically and commercially valuable habitats that can harbour 25% of all marine species within a small oceanic footprint (~\u0026thinsp;0.1% of total surface) (Hoegh-Guldberg et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At the foundation of this diversity is the endosymbiosis between the cnidarian host and its dinoflagellate symbionts of the family Symbiodiniaceae (LaJeunesse et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Efficient nutritional cycling between host and dinoflagellate symbionts allows for the growth and survival of reef-building corals in nutrient-poor waters (Muscatine and Porter, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; McIlroy et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but this association is particularly vulnerable to thermal stress (Beyer et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cziesielski et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When exposed to high temperature, the symbiosis becomes dysfunctional and the dinoflagellate symbionts are lost from the host tissue (Nielsen et al., 2018), resulting in a \u0026lsquo;bleached\u0026rsquo; coral that dies if the thermal stress is prolonged (Oakley and Davy \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Coral bleaching is the result of perturbations to complex metabolic networks (Helgoe et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with accumulation of reactive oxygen species (ROS) and nutritional imbalance considered the prevailing drivers of dysbiosis (R\u0026auml;decker et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lesser, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Synthesis of ROS is a natural by-product of photosynthetic and respiratory metabolisms (Foyer, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), although damage to the photosynthetic apparatus by high temperatures causes decreased photosynthetic efficiency and more excitation energy diverted to the generation of ROS. ROS are also generated by respiratory electron transport, with production again increasing when the organisms are heat stressed (Heise et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Slimen et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with surplus ROS potentially overwhelming repair mechanisms. Sustained oxidative stress leads to the loss of symbionts from the host tissues, potentially through mechanisms of apoptosis, autophagy, and exocytosis, although the relative importance of these different mechanisms remains unclear (Helgoe et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Metabolic regulation and nutrient homeostasis are also crucial to the control of cnidarian-dinoflagellate symbiosis. The efficient cycling and use of nutrients is crucial in an oligotrophic environment; excess photosynthate by algae supports host metabolism, and in turn host respiration enhances CO\u003csub\u003e2\u003c/sub\u003e availability for algal photosynthesis. Nitrogen limitation restricts algal growth in a healthy symbiosis and ensures that excess carbon is available for translocation to the host. Failure to maintain a nitrogen-limited environment \u003cem\u003ein hospite\u003c/em\u003e may therefore contribute to symbiosis breakdown under thermal stress (R\u0026auml;decker et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to traditional physiological and cell biological approaches (e.g., Gibbin \u0026amp; Davy, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Matthews et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), various omics platforms are being applied to better understand the processes underpinning coral bleaching; notably, transcriptomics (Pinz\u0026oacute;n et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), proteomics (Ricaurte et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lust et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), lipidomics (Botana et al., 2024; Gamba et al., 2024), steady-state metabolomics (Hillyer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Lust et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and volatilomics (Lawson et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Volatilomics is the latest of the omics approaches applied to the study of reef ecosystems and involves identification of biogenic volatile organic compounds (BVOCs) that collectively comprise the \u0026ldquo;volatilome\u0026rdquo;. BVOCs are low molecular mass (\u0026lt;\u0026thinsp;200 Da) compounds with high vapour pressure (Mansurova et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) that are produced by all organisms on Earth (Laothawornkitkul et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tahir et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wuerz et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). BVOC functions are diverse: they can increase local cloud formation, thereby enhancing protection from UV radiation (Swan et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), as well as stabilise lipid membranes during thermal stress (Siwko et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and elicit an immune response in neighbouring plants as a consequence of herbivory (Ton et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Once thought to be simply by-products of primary metabolism, some BVOCs are now recognised as physiological agents that function across individuals (Wenig et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or species (Minerdi et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and which carry chemical messages. The nature and extent of BVOC emissions from an organism can be affected by environmental stressors, which is likely to be particularly important for understanding ecosystem function and regulation (Lawson et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Okereke et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Lawson et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Dey et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe symbiotic sea anemone \u003cem\u003eExaiptasia diaphana\u003c/em\u003e (hereafter, \u0026lsquo;Aiptasia\u0026rsquo;) is a model organism adopted for studying cnidarian-dinoflagellate symbiosis (Weis et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Roberty et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As with corals, Aiptasia forms a stable symbiosis with algae of the family Symbiodiniaceae (Nitschke et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and similarly responds to environmental stressors that trigger the loss of algae from host tissues. Furthermore, Aiptasia can reproduce asexually, allowing the maintenance of genetically identical populations that can be used to examine the impact of environmental conditions on the animal\u0026rsquo;s physiological characteristics (Lehnert et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). While previous work has shown that thermal stress can alter volatile gas production in corals (Lawson et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and that symbiotic state alone influences BVOC emissions (Wuerz et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), there is little known about how thermal stress alters BVOC emissions across symbiotic states and how these emissions interact and alter the responses of the host, dinoflagellate symbiont, and other organisms associated with the holobiont (e.g., the coral microbiome). Aiptasia provides an excellent model system with which to explore BVOC synthesis in a holobiont, aposymbiotic animals, and cultured endosymbiotic dinoflagellates, and how the quality and quantity of BVOCs are impacted by temperature, chemical treatment (Matthews et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and symbiotic state (Matthews et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wuerz et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e \u0026amp; \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCoral reefs are major producers of BVOCs (Lawson et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and characterization of BVOCs has shown promise in other ecosystems for providing a non-invasive snapshot of organism physiology (Manusurova et al., 2018). BVOC analysis therefore provides a promising avenue for understanding how coral reef metabolism is altered by climate change. Here we determine the impact of thermal stress on BVOC production by the Aiptasia holobiont. We specifically profiled BVOCs in two different trophic states (i.e., symbiotic \u003cem\u003eversus\u003c/em\u003e aposymbiotic) at control, sub-bleaching, and bleaching temperatures. We hypothesised that thermal stress would shift the volatilome. This information will add to our understanding of physiological responses associated with the breakdown of symbiosis in this important model system, while also aiding in the identification potential of BVOC biomarkers that can be used to monitor the health of coral reefs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eExperimental organisms\u003c/b\u003e: A long-term (15\u0026thinsp;+\u0026thinsp;years) clonal culture of Aiptasia (culture ID: NZ1) of unknown Pacific origin (Matthews et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) was maintained in the laboratory in 0.22-\u0026micro;m filtered seawater (FSW) at 25\u0026deg;C and 70 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e on a 12:12 h light-dark cycle (GE Lighting T5 F54W/840). Clonal anemones (n\u0026thinsp;=\u0026thinsp;100) were rendered aposymbiotic using menthol-induced bleaching, \u003cem\u003evia\u003c/em\u003e exposure to menthol (20% w/v in ethanol; Sigma-Aldrich, Auckland, NZ) at a final concentration of 0.19 mmol L\u003csup\u003e-1\u003c/sup\u003e in 0.22 \u0026micro;m FSW, as \u003cem\u003eper\u003c/em\u003e Matthews et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Anemones were incubated in menthol for 8 h during the 12-h light period, after which photosynthesis was inhibited by replacing menthol/FSW with FSW containing 5 \u0026micro;mol L\u003csup\u003e-1\u003c/sup\u003e 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 100 mmol L\u003csup\u003e-1\u003c/sup\u003e dissolved in EtOH, Sigma-Aldrich) for 16 h to prevent repopulation by inhibiting photosynthesis of the remaining symbionts. After repeating this 24-h cycle for four consecutive days, anemones were maintained in 0.22 \u0026micro;m FSW without menthol or DCMU for an additional three days. Anemones were fed once weekly with \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii, with fresh FSW changes 8 h post-feeding: this culturing protocol was continued for six weeks. Aposymbiosis was confirmed using fluorescence microscopy (Olympus IX53 inverted microscope; 100\u0026times; magnification). All aposymbiotic anemones were then maintained in 0.22 \u0026micro;m FSW in the dark at 25\u0026deg;C for 1.5 years prior to BVOC sampling, during which time they were fed with \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii and given fresh FSW twice weekly.\u003c/p\u003e\u003cp\u003eA stock of symbiotic anemones maintained for \u0026gt;\u0026thinsp;1 year, initially generated \u003cem\u003evia\u003c/em\u003e repopulation of aposymbiotic anemones with the homologous symbiont (\u003cem\u003eBreviolum minutum\u003c/em\u003e, ITS2 Type B1, culture ID \u0026lsquo;FLAp2\u0026rsquo;) as described in Wuerz et al. (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), was used for consistency. Symbiotic anemones were maintained at a constant temperature of 25\u0026deg;C and an irradiance of approximately 70 \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. This anemone stock culture was fed twice weekly, as described above.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermal stress treatment\u003c/b\u003e: Prior to BVOC sampling, Aiptasia were kept in 250 mL glass jars each containing 20\u0026ndash;30 anemones. The jars were maintained in temperature-controlled water baths as described in Wuerz et al. (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All anemones were fed three times weekly for the duration of the experiment with freshly-hatched \u003cem\u003eArtemia\u003c/em\u003e sp. nauplii. Both aposymbiotic and symbiotic anemones were maintained at 130 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (light saturation for photosynthesis, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) on a 12:12 h light:dark cycle during the thermal-ramping period. The control group was kept at 25\u0026deg;C throughout the experiment; the sub-bleaching group was subjected to a temperature increase of 1\u0026deg;C \u003cem\u003eper\u003c/em\u003e day to a maximum temperature of 30\u0026deg;C, which was maintained for seven days prior to BVOC sampling. The bleaching group was subjected to a temperature increase of 1\u0026deg;C \u003cem\u003eper\u003c/em\u003e day until 30\u0026deg;C, after which the temperature was increased by 0.5\u0026deg;C \u003cem\u003eper\u003c/em\u003e day to a maximum temperature of 33.5\u0026deg;C, which was maintained for 24 h prior to BVOC sampling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhotosystem II (PSII) maximum quantum yield (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003ev\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e/F\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e, dimensionless) was measured every day during thermal ramping and used as an indicator of stress associated with the symbiotic association (Levin et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All anemones were dark-acclimated for 15 min before measuring various photosynthetic activities with an Imaging Pulse Amplitude Modulated Fluorometer (I-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), as \u003cem\u003eper\u003c/em\u003e Vergauwen et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSymbiont cell density and protein determination\u003c/strong\u003e\u003cp\u003eFrozen anemones (n\u0026thinsp;=\u0026thinsp;5, with 10\u0026ndash;15 anemones \u003cem\u003eper\u003c/em\u003e biological replicate) were thawed on ice and homogenised using a saw-tooth homogeniser in 500 \u0026micro;L milliQ water. Host and symbiont fractions were separated by centrifugation at 400 x g for 2 min after which the symbiont fraction was washed of residual host material by one further resuspension and centrifugation with milliQ water. The supernatant fraction was analysed for protein content using the fluorometric Qubit Protein Assay Kit (Vergauwen et al \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cell counts were performed using a haemocytometer (Improved Neubauer) with eight replicate counts \u003cem\u003eper\u003c/em\u003e sample and normalised to protein content. Cell density (symbionts \u003cem\u003eper\u003c/em\u003e mg host protein) was also calculated.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePhotosynthesis and respiration\u003c/strong\u003e\u003cp\u003eHolobiont physiology was assessed by measuring oxygen consumption in the dark by both aposymbiotic and symbiotic anemones, and photosynthetic oxygen evolution in the symbiotic anemones was evaluated by incubating anemones in a sealed Perspex chamber (13 mL) fitted with an internal stir bar and an oxygen optode (Fibox 4, PreSens Gmbh, Regensburg, Germany). The magnetic stir bar was situated underneath a perforated mesh floor to which the anemones were attached, and rotated at a speed at which the anemones did not appear visually stressed. The Perspex chamber vials were immersed in one of three temperature baths according to the treatment temperature (25\u0026deg;C, 30\u0026deg;C or 33.5\u0026deg;C), atop a magnetic stir table (Multistirrer Digital 6, VELP Scientifica Srl, Italy). The anemones were allowed to settle and attach to the bottom of the chambers for ~\u0026thinsp;1 h prior to making measurements. To quantify respiratory rates, the lights were extinguished and oxygen consumption measured for 15 min.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eFor symbiotic anemones, illumination was for 20 min at 130 \u0026micro;mol photons m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e which, as previously mentioned, is sufficient to saturate holobiont photosynthesis. All measurements were taken during the photic period, with measurements of holobiont dark respiration calculated as moles O\u003csub\u003e2\u003c/sub\u003e consumed h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Gross photosynthesis values were calculated by adding dark respiration rates to net photosynthetic rates for symbiotic anemones (moles O\u003csub\u003e2\u003c/sub\u003e produced h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during the photic period). Respiration and gross photosynthetic rates were normalised to soluble protein content of each anemone and \u003cem\u003eper\u003c/em\u003e million symbiont cells, respectively. Immediately after sampling, the anemones were flash frozen at -80\u0026deg;C and stored for determination of algal cell density and protein concentration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBVOC sampling and volatilome characterisation\u003c/b\u003e: BVOC gases were collected and analysed from anemones in two different symbiotic states and three temperatures each: aposymbiotic anemones (n\u0026thinsp;=\u0026thinsp;6 vials at 25\u0026deg;C and 33.5\u0026deg;C, n\u0026thinsp;=\u0026thinsp;5 vials at 30\u0026deg;C; ~15 anemones \u003cem\u003eper\u003c/em\u003e vial); symbiotic anemones (n\u0026thinsp;=\u0026thinsp;6 vials at 25\u0026deg;C and 33.5\u0026deg;C, n\u0026thinsp;=\u0026thinsp;5 vials at 30\u0026deg;C; ~10 anemones \u003cem\u003eper\u003c/em\u003e vial). Different numbers of anemones were used for each sample to ensure a similar biomass among the groups; biological replicates varied among treatments due to technical instrument error, which resulted in fewer replicates in the sub-bleaching treatment. BVOC retrieval was performed using previously established methods (Ton et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Wuerz et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). First, experimental organisms were transferred into sterile 150 mL, unsealed, crimp-cap serum glass vials (Wheaton, Millville, NJ, USA) containing 75 mL 0.22 \u0026micro;m FSW, where they were held overnight under conditions identical to those used to grow the cultures. This incubation allowed the organisms to settle, before the FSW medium was refreshed and the vials sealed using 20 mm PTFE/Si crimp caps (Agilent, USA). BVOCs were collected by passing instrument-grade air (100 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; BOV Gases, Wellington, NZ) into gas-tight sampling vials for 20 min, with the outgoing air passed through open-ended thermal desorption tubes (TDTs; Markes International Ltd, Llantrisant, UK) containing the sorbent Tenax TA, onto which the BVOCs adhere (Ton et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guitton et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). After a 20 min sampling time, TDTs were immediately sealed with brass storage caps and stored at 4\u0026deg;C until processed. All TDTs were analysed within two weeks of sampling to minimise sample degradation, using gas chromatography coupled with single quadrupole mass spectrometry (GC\u0026times;MS) as \u003cem\u003eper\u003c/em\u003e Wenig \u0026amp; Odermatt (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). TDT desorption was performed using a Marks Unity 2 Series Thermal Desorber and ULTRA multi-tube autosampler. Desorption occurred at 300\u0026deg;C for 6 min, after which BVOCs were concentrated in a cold trap at -30\u0026deg;C. The cold trap was subsequently flash-heated to 300\u0026deg;C, injecting the concentrated sample onto a 7890A GC (Agilent Technologies, Ltd., Melbourne) through a transfer line maintained at 150\u0026deg;C. The GC was equipped with a 30 m x 0.25 mm Rxi-624Sil MS column (Restek Corp.) with a film thickness of 1.40 \u0026micro;m. To encourage complete desorption of BVOCs from the column, the GC oven was heated at 35\u0026deg;C for 5 min and then increased to 240\u0026deg;C by increments of 5\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; once at 240\u0026deg;C, the temperature was maintained for 5 min. All samples were run in splitless mode at a flowrate of 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The GC was attached to a Model 59745 mass-selective detector (Agilent Technologies, Ltd., Melbourne) with the scanning range set to 29\u0026mdash;450 amu.\u003c/p\u003e\u003cp\u003eSpectral data from the GC-MS were run through the open-source MS data processing program OpenChrom (Weing \u0026amp; Odermatt, 2010) to remove common contaminating ions (amu: 73, 84, 147, 149, 207, 221). Output files were imported to Galaxy (Guitton et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and processed using the metaMS.runGC package (Galaxy version 2.1.1; (Wehrens et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)) in Workflow4Metabolomics. Peaks were tentatively identified against a database in the National Institute of Standards and Technology (NIST) Mass Spectral library (NIST 14 library in NIST MS Search v2.2; NIST, Gaithersburg, MD). Blank seawater samples (n\u0026thinsp;=\u0026thinsp;6 vials at 25\u0026deg;C and 33.5\u0026deg;C; n\u0026thinsp;=\u0026thinsp;5 vials at 30\u0026deg;C) were run in conjunction with all analyses; average values of blanks were subtracted from each sample. Any compound that was not present in two or more biological replicates was considered to be contamination and removed. Unclassified compounds are denoted as UC; the number following \u0026lsquo;UC\u0026rsquo; indicates the retention time of the compound.\u003c/p\u003e\u003cp\u003ePrior to desorption, each TDT was injected with 0.2 \u0026micro;L of 150 ppm bromobenzene (GC grade, Sigma Aldrich, Castle Hill, NSW, Australia) in methanol (HPLC grade, Sigma Aldrich) which serves as an internal standard. Finally, peak abundances were normalised to the protein content of each replicate. Protein abundances were measured after the samples were lysed using an ultrasonication probe (VCX500; Sonics \u0026amp; Materials Inc., Newtown, CT, USA); the protein concentration in the lysate was measured using the fluorometric Qubit Protein Assay Kit (Vergauwen et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Compounds identified as likely methodological artifacts, e.g. silicon-containing compounds, were also removed from the dataset, as they were suspected contaminants from the GC column dimethylpolysiloxane hydrolysis (Cella and Carpenter \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). All BVOCs were grouped according to their chemical class. BVOC abundance data are dimensionless and were normalised to a chemical standard and total protein content, after BVOCs in blanks were subtracted from test TDTs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003cp\u003eOxygen production/consumption, and cell density measurements were tested for normality and homoscedasticity and then tested with two-way ANOVA in R (version 4.0.3; Team RC 2021). PSII maximum quantum yield was analysed by linear regression using the stats package in R (version 4.4.1) and changes in this yield were considered as an indication of health of the organisms over the temperature treatment period. Differential abundance of BVOCs was estimated using the limma R package (Ritchie et al \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); the voom function was used to convert counts to log\u003csub\u003e2\u003c/sub\u003e-counts-\u003cem\u003eper\u003c/em\u003e-million and to assign weights to each observation based on the mean-variance trend. The counts assigned to each observation roughly correspond to the abundance of each BVOC, which can then be used to compare BVOC production between treatments. Functions lmFit, eBayes, and topTable were used to fit weighted linear regression models, calculate empirical Bayes moderated t-statistic, and calculate FDR-corrected p-values. Biological replicates, standardised using the decostand function in the vegan package (Oksanen et al \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in R (version 1.2.5033), were compared using the Bray-Curtis similarity measure, and this output was subjected to non-metric multidimensional scaling (NMDS) to visualise differences among groups based on BVOCs. To compare dispersion of biological replicates using a distance measure, PERMANOVA was performed to test if the distance between biological replicates was greater between treatments than within treatments. PERMANOVA was performed on NMDS scores using the adonis function, and \u003cem\u003epost hoc\u003c/em\u003e tests were performed using the pairwise.adonis function in the vegan package in R (version 1.2.5033) to distinguish differences among treatments. All plots were created with ggplot2 (Wickham \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in RStudio.\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePhotosystem II quantum yield, symbiont cell density, respiration, and oxygen production\u003c/b\u003e: PSII maximum quantum yield values for anemones in the sub-bleaching treatment increased over time but showed a sustained decrease in the bleaching thermal treatment (linear regression; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, sub-bleaching: F\u003csub\u003e1,37\u003c/sub\u003e: 7.971, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; bleaching: F\u003csub\u003e1,37\u003c/sub\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; fit models with confidence intervals shown in \u003cb\u003eFigs. S1-S3\u003c/b\u003e), which suggests damage to PSII. By comparison, values in the control treatment remained constant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSymbiont density was also significantly impacted by elevated temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, one-way ANOVA, F\u003csub\u003e2,27\u003c/sub\u003e = 10.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In particular, algal cell density in the anemones at the putative bleaching temperature of 33.5\u0026deg;C (1.72 x 10\u003csup\u003e6\u003c/sup\u003e cells \u003cem\u003eper\u003c/em\u003e mg host protein) was significantly lower than that in anemones at the control (25\u0026deg;C) and sub-bleaching (30\u0026deg;C) temperatures (2.29 and 2.63 x 10\u003csup\u003e6\u003c/sup\u003e cells \u003cem\u003eper\u003c/em\u003e mg host protein, respectively; Tukey \u003cem\u003epost hoc\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for both comparisons). Cell densities were not significantly different at 25\u0026deg;C \u003cem\u003evs.\u003c/em\u003e 30\u0026deg;C. Consistent with bleaching at the highest temperature was the sustained decrease in F\u003csub\u003ev\u003c/sub\u003e/F\u003csub\u003em\u003c/sub\u003e at 33.5\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eFig S3\u003c/b\u003e). In contrast, the symbiont cell-specific gross photosynthetic rate was not significantly impacted by thermal treatment, although there was a trend towards an elevated rate at 33.5\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) that might (partially) compensate for the loss of symbionts at that temperature, resulting in little overall change in the rate of holobiont photosynthesis at the different temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThe respiration rate of aposymbiotic anemones doubled at the bleaching temperature relative to the two lower temperatures (one-way ANOVA, F\u003csub\u003e2,11\u003c/sub\u003e = 6.94, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Tukey \u003cem\u003epost hoc\u003c/em\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both comparisons; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similarly, the respiration rate of symbiotic anemones increased by 50% at the bleaching temperature relative to the other two temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) (one-way ANOVA, F\u003csub\u003e2,10\u003c/sub\u003e = 8.746, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Tukey \u003cem\u003epost hoc\u003c/em\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for both comparisons).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBVOC emissions by aposymbiotic anemones are influenced by temperature\u003c/b\u003e: Non-metric multidimensional scaling demonstrated a shift in the volatilome of aposymbiotic Aiptasia across temperature treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Specifically, the volatilome of anemones at the sub-bleaching temperature was different to that of anemones at the highest temperature (PERMANOVA: F\u003csub\u003e2,14\u003c/sub\u003e = 2.49, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; Tukey \u003cem\u003epost hoc\u003c/em\u003e: p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). In contrast, the volatilome of control aposymbiotic anemones was not significantly different from the volatilome of aposymbiotic anemones at either of the higher temperatures.\u003c/p\u003e\u003cp\u003eA total of 148 BVOCs were detected in aposymbiotic anemones across the three temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Of these, 70 were detected solely at the control temperature, 15 at the sub-bleaching temperature, and 11 at the bleaching temperature. A total of 40 BVOCs were common to aposymbiotic anemones at the control and sub-bleaching temperatures, five were common to the sub-bleaching and bleaching temperatures, and three were common to the control and bleaching temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Only four BVOCs were present in aposymbiotic samples after all three temperature treatments; these are considered the \u0026lsquo;core aposymbiotic compounds\u0026rsquo;. Two of the four volatiles are aromatic compounds (styrene and (1-methylethyl)-benzene), one has diverse functional groups (3-ethenyl-2-ethoxypyrazine), and one is an unclassified compound (UC45.72).\u003c/p\u003e\u003cp\u003eExposure to elevated temperature decreased the number of BVOCs detected in the volatilome of aposymbiotic anemones. Aposymbiotic anemones produced 117 BVOCs at 25\u0026deg;C, the most common of which were aromatic compounds (29 BVOCs); 64 BVOCs were produced at 30\u0026deg;C, predominantly aromatic compounds; only 23 BVOCs were produced at 33.5\u0026deg;C, of which five were unclassified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA total of 14 BVOCs were significantly differentially abundant in response to temperature in the volatilome of aposymbiotic anemones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; \u003cb\u003eTable S2\u003c/b\u003e). Only one BVOC, 2,7,10-trimethyldodecane, was differentially abundant between control and heat-stressed anemones and specifically detected at higher abundance at 33.5\u0026deg;C than at either the control or sub-bleaching temperatures. The other 13 differentially abundant BVOCs were significantly different between 33.5\u0026deg;C and 30\u0026deg;C, with 12 detected at higher abundance in the sub-bleaching treatment, including dibromomethane, 1,2-dichloroethane, and 2-butanone. The one exception was hexadecane, which was detected in higher quantities at 33.5\u0026deg;C \u003cem\u003evs\u003c/em\u003e. 30\u0026deg;C. All other differentially abundant BVOCs are listed in \u003cb\u003eSupplementary Table S2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBVOC emissions by symbiotic anemones are also influenced by temperature\u003c/b\u003e: Among the symbiotic anemones exposed to different temperatures, only seven BVOCs were significantly differentially abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; \u003cb\u003eTable S3\u003c/b\u003e). The only BVOC more abundant at the sub-bleaching temperature relative to the bleaching temperature was di-tert-butyl peroxide. The other six BVOCs were more abundant in anemones exposed to the bleaching than the sub-bleaching temperature, and included dimethyl sulphide, eucalyptol, methyl N-hydroxybenzenecarboximidoate, 1-(2-pyridyl)piperazine, 1-iodododecane, and 2,7,10-trimethyldodecane. DMS was also more abundant in the volatilome of symbiotic anemones at the bleaching than at the control temperature.\u003c/p\u003e\u003cp\u003eNon-metric multidimensional scaling also demonstrated changes in the volatilomes produced by symbiotic anemones across temperature treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Symbiotic anemones exposed to the highest temperature (33.5\u0026deg;C) produced a volatilome that differed from those of anemones exposed to 25\u0026deg;C or 30\u0026deg;C (PERMANOVA F\u003csub\u003e2,14\u003c/sub\u003e = 3.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; Tukey \u003cem\u003epost hoc\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both comparisons), with the 25\u0026deg;C and 30\u0026deg;C volatilomes being statistically similar.\u003c/p\u003e\u003cp\u003eA total of 170 BVOCs were detected in the volatilomes of symbiotic anemones across the three temperature treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Of these, 21 were produced solely at the control temperature, eight at the sub-bleaching temperature, and 39 at the bleaching temperature. A total of 17 BVOCs were common between symbiotic anemones at control and sub-bleaching temperatures, 16 were common between sub-bleaching and bleaching temperatures, and 21 were common between control and bleaching temperatures. A substantial number of BVOCs (48) were present in the volatilome of all symbiotic anemones \u0026ndash; irrespective of temperature treatment \u0026ndash; and are therefore designated as \u0026lsquo;core symbiotic compounds\u0026rsquo;. Of these 48 core BVOCs, the most abundant chemical group were aromatic compounds (14), followed by halogenated hydrocarbons (8), including dibromomethane, bromodichloromethane, dibromochloromethane and bromochlorodifluoromethane. As observed for aposymbiotic anemones, temperature influenced the number of BVOCs detected in symbiotic anemones. However, intriguingly \u0026ndash; and in contrast to aposymbiotic anemones \u0026ndash; the richest volatilome (124) was detected at the highest temperature (33.5\u0026deg;C), with aromatic compounds (29) being the most abundant chemical class. Symbiotic anemones produced the fewest BVOCs (89) at 30\u0026deg;C, with aromatic compounds again being the most abundant (21), whereas symbiotic anemones at 25\u0026deg;C produced 107 BVOCs, comprised primarily of volatiles with diverse functional groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eCore BVOCs\u003c/strong\u003e: Between symbiotic states and across the three temperature treatments, three BVOCs were common to all Aiptasia samples: (1-methylethyl)-benzene, 3-ethenyl-2-ethoxypyrazine, and styrene. None of these core compounds were detected at differentially abundant concentrations between temperature treatments, suggesting that they play an important role in central metabolism across temperatures. One unclassified BVOC, UC45.72, was identified as a core BVOC in aposymbiotic anemones and only present in symbiotic anemones when undergoing bleaching. Such a response would suggest that UC45.72 is produced in response to stress, or suppressed by the presence of symbionts. Indeed, upregulation of pathways for mediating oxidative stress and apoptosis in aposymbiotic Aiptasia has been reported (Rodriguez-Lanetty et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Oakley et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gorman et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that symbiosis protects the host from oxidative stress and cellular damage (Oakley et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Oxidative stress is a well-known feature of coral bleaching (reviewed by Sun et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e), so the production of UC45.72 in the volatilome of thermally-stressed symbiotic anemones could reflect a direct impact of high temperature and/or a marked loss of symbionts and hence a shift towards a state closer to that of aposymbiotitc anemones. Either way, this unclassified BVOC is likely an important candidate for future studies.\u003c/p\u003e\n\u003cp\u003eInterestingly, only four BVOCs were detected consistently in the volatilome of aposymbiotic anemones irrespective of thermal treatment, while for symbiotic anemones, 48 BVOCs met this criterion. As such, physiological stability may be greater for symbiotic than for aposymbiotic anemones, once again suggesting that symbiosis may confer a degree of protection to the host and holobiont. In particular, given that aposymbiotic anemones already appear to be experiencing some degree of stress (Oakley et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e), additional stressful environmental conditions may trigger a more rapid shutdown of central metabolism, resulting in the release of fewer BVOCs as by-products. By contrast, despite previously observed transcriptomic, proteomic and metabolomic impacts of thermal stress on symbiotic cnidarians (Desalvo et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Oakley et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hillyer et al., \u003cspan class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Cziesielski et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), the relative consistency of the volatilome at high temperature observed here suggests that central metabolism is altered less dramatically than in the aposymbiotic state. Elucidation of the underlying cellular processes that control BVOC synthesis, and how the different members of the holobiont (including other members of the microbiome such as bacteria) interact to facilitate metabolic homeostasis, are key questions for future research.\u003c/p\u003e\n\u003ch3\u003eTemperature affects the Aiptasia volatilome\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eAposymbiotic anemones\u003c/strong\u003e: In the aposymbiotic state, progressively higher temperatures generated increasingly diminished volatilome richness, with a consistent core volatilome of just four BVOCs. As observed in other organisms (e.g., Taris et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), the respiration rate in aposymbiotic anemones increased at the highest temperature (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). As respiration and metabolism are intrinsically linked processes (Weis, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), increased metabolism, and therefore BVOC output, may be expected. However, our results demonstrated the contrary: the decline in BVOC richness at 33.5\u0026deg;C may be related to the onset of metabolic dysfunction, which is typical for symbiotic cnidarians at such a high temperature (Roach et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sun et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e;). It is, however, important to exercise caution when drawing such conclusions, as little is known about the physiology of the associated microbiome. Previous studies have shown temperature-induced shifts towards pathogenic microbiota in corals (Bourne et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, Aiptasia-associated microbiota have been observed to change in response to different symbiotic states (Curtis et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wuerz et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) and thermal stress (Ahmed et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, Sydnor et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e); thus, it is plausible that a thermally-altered microbiome may have contributed to the observed differences between the volatilomes in our current study.\u003c/p\u003e\n\u003cp\u003eFourteen BVOCs were differentially expressed in the volatilome of aposymbiotic anemones in response to thermal change. Interestingly, 12 of these were detected at higher abundance at the sub-bleaching relative to the bleaching temperature. The most abundant BVOCs in the volatilome of aposymbiotic anemones at the elevated, sub-bleaching temperature are known to have diverse functions in other systems and include naphthalene and dibromomethane. Naphthalene is produced by termites (Chen et al., \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Wilcke et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e), inhibiting the growth of pathogenic fungi and forming part of the termite\u0026rsquo;s defence system (Wright et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has also been shown to act as an apoptosis inhibitor in the model nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, suggesting that it may promote survival and proliferation of tumour cells (Kokel et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Dibromomethane induces metamorphosis in the mollusc \u003cem\u003eCrepidula fornicata\u003c/em\u003e (Taris et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). These functions suggest that BVOCs have the potential to play roles in developmental processes and cellular signalling in Aiptasia and other symbiotic cnidarians, although this requires detailed confirmation.\u003c/p\u003e\n\u003cp\u003eJust two BVOCs \u0026ndash; hexadecane and 2,7,10-trimethyldodecane \u0026ndash; were produced at higher abundances by anemones at the bleaching temperature relative to the sub-bleaching temperature. Given the physiological stress that occurs in Aiptasia at such a high temperature, as noted both in our current study and previous work (Oakley et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hawkins et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cleves et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), it is possible that these BVOCs are downstream products of stress pathways. Moreover, hexadecane has been previously shown to induce cell death in both rat and human cell cultures (Herman et al., \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). Hexadecane may be an element of the bleaching process, as apoptosis is thought to be an important cellular end-point of the cnidarian bleaching cascade (Helgoe et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSymbiotic anemones\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn contrast to aposymbiotic anemones, the volatilome of symbiotic anemones was more stable in response to thermal change, with the core volatilome consisting of 48 BVOCs, with only seven that were differentially abundant at the different temperatures. There was also a trend of increasing BVOC diversity at increasing temperature. This is in contrast to the findings of Lawson et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), who showed a decrease in BVOC richness and abundance at higher temperatures in corals. These differences perhaps indicate species-specific differences in BVOC synthesis among cnidarians in response to elevated temperature, though they could also reflect the different maximum temperatures used in the two studies (32\u0026deg;C \u003cem\u003evs\u003c/em\u003e. 33.5\u0026deg;C). The increased volatilome diversity at the highest temperature seen in our study suggests a proliferation of metabolic downstream products associated with cellular dysfunction in the host or symbionts, and/or altered microbiome metabolism during the bleaching process. It is not possible from this approach to determine the member(s) of the holobiont responsible for the production of each BVOC, or how the metabolism of various partners interacts under stress, but future work with isolated partners (e.g., axenic Symbiodiniaceae, or microbes associated with the cnidarian surface) would help shed light on this matter. In contrast, the volatilome was least diverse at the intermediate, sub-bleaching temperature. One interpretation of this is that metabolic integration of the holobiont was greatest at this temperature, resulting in the release of fewer BVOCs to the environment. It is somewhat surprising that this would be the case at the intermediate rather than at the control temperature, but we note that there were no obvious signs of physiological stress at the intermediate temperature used in this study.\u003c/p\u003e\n\u003cp\u003eOf the seven differentially abundant BVOCs in symbiotic anemones, most were detected at higher abundance at the bleaching relative to the sub-bleaching (five BVOC) or control temperatures (one BVOC). Of these BVOCs, dimethyl sulphide (DMS) and eucalyptol have roles as antioxidants in other systems (Guan et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xu et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggesting that their production in the cnidarian-dinoflagellate symbiosis may involve the amelioration of free radicals or toxic metabolic by-products. The sources of these compounds (i.e., host, symbiont or microbiome) are difficult to determine from our current approach, but it is notable that 2,7,10-trimethyldodecane was also conspicuous in the volatilome of heat-stressed aposymbiotic anemones, suggesting that it may originate from the host or members of the microbiome that are common to symbiotic and aposymbiotic anemones. Only one BVOC was more abundant at the sub-bleaching \u003cem\u003evs\u003c/em\u003e bleaching temperature - di-tert-butyl peroxide - suggesting that this compound is involved in or is a by-product of a fully functional symbiosis. However, it is not clear why di-tert-butyl peroxide was more abundant at the intermediate than at the control temperature.\u003c/p\u003e\n\u003cp\u003eNotably, DMS was the BVOC produced in highest abundance by symbiotic anemones at the bleaching temperature. DMS is a widely studied, multi-functional BVOC with established roles in the global sulphur cycle (Brimblecombe, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) and stress physiology (Sunda et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e). At the cellular level, dimethylsulphoniopropionate (DMSP) and its breakdown products (DMS, acrylate, dimethyl sulphoxide and methane sulphinic acid) represent a powerful antioxidant system (Sunda et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hopkins et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) that increases in both corals (Hopkins et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Symbiodiniaceae (Deschaseaux et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) in response to thermal stress. This pattern of DMS production can be considered in the context of physiological responses associated with elevated temperature, including an increase in the holobiont respiratory rate and a decrease in symbiont density in the host. Such gross physiological changes typically lead to metabolic shifts in both the host and symbiont that might, for example, compensate for nutritional deficits by mobilising carbohydrate and lipid stores (Hillyer et al., \u003cspan class=\"CitationRef\"\u003e2017a\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2017b\u003c/span\u003e). Furthermore, high temperature can induce oxidative stress and potentially elicit activation of immunity and inter-partner signalling pathways involved in symbiosis dysfunction (Dunn et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Krueger et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hillyer et al., \u003cspan class=\"CitationRef\"\u003e2017b\u003c/span\u003e). In symbiotic Aiptasia, it is reasonable to suggest that the observed increase in DMS at the bleaching temperature acts to ameliorate ROS produced in response to thermal stress (Perez and Weis, \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Although the total number of symbionts in the holobiont decreased during bleaching, DMS production was higher such that the remaining symbionts produced much more DMS \u003cem\u003eper\u003c/em\u003e cell than at the lower temperatures. The ability to produce copious amounts of DMS may be particularly advantageous during periods of thermal stress; it may contribute to sustaining the symbiosis as a consequence of its antioxidant activity. Eucalyptol, which was also most abundant in the holobiont at the highest temperature, may have a similar protective function. Like DMS, this volatile has known antioxidant activity (Kennedy-Feitosa et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e), but also has anti-inflammatory (Kim et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) and anti-bacterial (Li et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) properties in mammalian models, although it can also induce DNA damage in mammals (D\u0026ouml;rsam et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). While identification of BVOCs in other systems provides a framework for exploring their role in the cnidarian-dinoflagellate symbiosis, much more work is needed to elucidate their impact on the establishment, maintenance, and dysfunction of this ecologically important symbiosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eElevated temperature impacts the volatilome of both aposymbiotic and symbiotic Aiptasia. The largest temperature-induced alterations in the volatilome were observed in aposymbiotic anemones, suggesting that the presence of Symbiodiniaceae provides a stabilising effect on holobiont function, at least until the upper thermal threshold is reached. More work is now needed to identify all BVOCs, and the sources and functions of BVOCs associated with the cnidarian-dinoflagellate symbiosis. Focused future efforts are required to characterise the volatilome of individual components of the holobiont (cnidarian host, dinoflagellate symbionts, associated microbes) and to elucidate BVOC biosynthetic pathways and their control. Nevertheless, our observations in this study on the model organism Aiptasia provide a first step towards understanding how heat stress can influence BVOC emissions across symbiotic states and at different temperatures. Additionally, identifying various BVOCs (e.g., DMS, eucalyptol) and their abundances in symbioses may facilitate their use as non-invasive biomarkers for thermal and cellular stress and for evaluating the overall health of coral reef ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was funded by a Victoria University doctoral scholarship awarded to M.W. and the Royal Society of New Zealand Te Aparangi Marsden Fund (19-VUW-086, awarded to S.K.D., A.R.G., C.A.O., D.J.S., and V.M.W.). The contributions of D.J.S and C.A.L. were furthermore supported by an Australian Research Council discovery project (DP200100091, awarded to D.J.S.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement:\u0026nbsp;\u003c/strong\u003eMW and SKD conceived the study. MW, CAL, CAO, ARG, VMW, DJS, and SKD conceptualized the study. MW, CAL, CAO, DJS, and SKD conceptualized the methods. MW, CAO, ARG, VMW, DJS, and SKD provided funding. MW administered the project and performed laboratory work. MW and MP conducted data analysis. MW, CAL, CAO, and MP conducted data analysis, interpretation, and visualization. MW wrote the original draft of the manuscript, with input from all authors. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eVolatilome dataset is available at: https://github.com/maggiewuerz/thermal_stress_volatilome.git\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed HI, Herrera M, Liew YJ, Aranda M (2019). Long-term temperature stress in the coral model Aiptasia supports the \u0026ldquo;Anna Karenina principle\u0026rdquo; for bacterial microbiomes. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/13880209.2021.1876101\u003c/span\u003e\u003cspan address=\"10.1080/13880209.2021.1876101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"coral reefs, coral bleaching, volatilome, BVOC, Exaiptasia diaphana","lastPublishedDoi":"10.21203/rs.3.rs-7991501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7991501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoral bleaching events, in which symbionts are lost from host tissues, have become more frequent and severe because of climate change and specifically, elevated temperatures. How such events impact biogenic volatile organic compound (BVOC) emissions, compounds that can function as metabolic signalling elements, remains underexplored. Here we characterised the suite of BVOCs (collectively the \u0026ldquo;volatilome\u0026rdquo;) from the model sea anemone \u003cem\u003eExaiptasia diaphana\u003c/em\u003e (\u0026lsquo;Aiptasia\u0026rsquo;) under three temperatures (control: 25\u0026deg;C; sub-bleaching: 30\u0026deg;C; and bleaching: 33.5\u0026deg;C), both without symbionts (aposymbiotic) and when populated by its native dinoflagellate symbiont, \u003cem\u003eBreviolum minutum\u003c/em\u003e. The volatilome of symbiotic anemones during bleaching at an elevated temperature was distinct from that at lower temperatures, with high dimethyl sulphide (DMS), eucalyptol, and 1-iodododecane levels at the higher temperature. In comparison, the volatilome of aposymbiotic anemones was most distinct at the sub-bleaching temperature; the most abundant and significant BVOC differences included 2-phenyl-3-methyl-pyrrolo(2,3-b)pyrazine, acetone, and naphthalene. Symbiotic anemones had 12-fold more \u0026lsquo;core volatiles\u0026rsquo; (BVOCs in all biological replicates across all temperature treatments) than aposymbiotic anemones (48 \u003cem\u003evs\u003c/em\u003e. 4 BVOCs); during thermal stress, the symbiotic anemone volatilomes retained their compound richness whereas the richness of aposymbiotic anemone volatilomes decreased. These observations suggest that symbiotic dinoflagellates enhance BVOC diversity and abundance and may confer a degree of metabolic stability to the intact symbiosis (i.e., \u0026lsquo;holobiont'). Such changes in metabolic outputs can inform our understanding of how coral holobionts respond to increasing seawater temperatures, enable targeted studies of BVOC function, and facilitate the development of biomarkers indicative of coral reef health.\u003c/p\u003e","manuscriptTitle":"Thermal stress restructures volatile gas emissions from the model cnidarian Aiptasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 14:25:41","doi":"10.21203/rs.3.rs-7991501/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-26T23:04:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T22:54:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T12:29:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T05:37:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120035743941715341488399152812219712933","date":"2025-11-15T17:49:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180610732651387975547450715996364620226","date":"2025-11-13T12:48:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243988037610211355555842462879860923580","date":"2025-11-13T02:06:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T19:10:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-04T06:43:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-01T07:05:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Coral Reefs","date":"2025-10-30T16:44:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7344a66a-2322-4d71-9017-fbdfff2af0d0","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:02:15+00:00","versionOfRecord":{"articleIdentity":"rs-7991501","link":"https://doi.org/10.1007/s00338-026-02866-3","journal":{"identity":"coral-reefs","isVorOnly":false,"title":"Coral Reefs"},"publishedOn":"2026-04-08 15:59:02","publishedOnDateReadable":"April 8th, 2026"},"versionCreatedAt":"2025-11-24 14:25:41","video":"","vorDoi":"10.1007/s00338-026-02866-3","vorDoiUrl":"https://doi.org/10.1007/s00338-026-02866-3","workflowStages":[]},"version":"v1","identity":"rs-7991501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7991501","identity":"rs-7991501","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}