MetaboLights

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mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/VN_250210_CI_Col_603_12C_5.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250210_CI_Col_1403_13C_2.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250210_CI_Col_603_12C_4.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250204_Col_Bn_lys_12C_1.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/VN_250207_CI_Bn_12C_1.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/VN_250207_CI_Col_Bn_13C_1.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/VN_250209_Col_603_12C_2.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250205_Col_Bn_lys_13C_1.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250209_Col_603_13C_2.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/VN_250205_Col_Bn_lys_13C_2.mzXMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592/FILES/DERIVED_FILES/VN_250204_Col_lys_12C_3.mzXMLprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12592GC-MS data processing and analysisRaw data obtained from GC-MS analysis with EI mode was converted into .mzXML format using the Proteowizard Suite (proteowizard.sourceforge.net) with vendor peak picking enabled. Data was processed with the R-based X13CMS package <a href='https://pubs.acs.org/doi/10.1021/ac403384n' rel='noopener noreferrer' target='_blank'>4</a> for isotopologues detection. Afterwards, significantly labeled isotopologue groups (p-value<0.05) were manually curated and only groups with at least three consecutive isotopologues were considered for further analysis. For compound identification, the same .mzXML files were processed with the previously established R-based pipeline <a href='https://www.mdpi.com/2218-1989/10/4/143' rel='noopener noreferrer' target='_blank'>5</a> with some modification (Appendix S2). The pipeline includes the XCMS package <a href='https://pubs.acs.org/doi/10.1021/ac051437y' rel='noopener noreferrer' target='_blank'>6</a>, CAMERA package <a href='https://pubs.acs.org/doi/10.1021/ac202450g' rel='noopener noreferrer' target='_blank'>7</a>, and MetaMS package <a href='https://www.sciencedirect.com/science/article/pii/S1570023214001548?via%3Dihub' rel='noopener noreferrer' target='_blank'>8</a>. The significantly labeled features from the X13CMS analysis were matched with the features from the analysis with the second pipeline based on the same retention time and m/z value. Identification of metabolites was conducted using the NIST 2011 Mass Spectral Library database (National Institute of Standards and Technology, USA). The final verification of compounds was done by comparison of mass spectra and retention index (accepted difference ≤ 3 units) against mass spectra and retention index of analytical standards deposited in an in-house library. LC-HR-MS data processing and analysisRaw data obtained from LC-HR-MS analysis was preprocessed using Compound Discoverer version 3.3 SP3 (Thermo Fisher Scientific, USA). The standard workflow (Stable Isotope Labeling w Metabolika Pathways and ID using Local Databases) with default settings was applied. The setting for Peak Rating Filter was changed to: Peak Rating Threshold – 5, number of files – 3. Within this workflow, a grouping of isotopes and adducts belonging to one molecule is performed. All incubation experiments were processed within one study. The compound identification was performed with an in-house library based on retention time and MS1/MS2 spectra (accepted difference ≤ 0.2 min, < 5 ppm).MetaboLightsPublicLiquid Chromatography MS - positive - hilicGas Chromatography MS - positiveGas Chromatography MS -GC-MS measurementsGC-HRMS measurements were performed on a Q-Exactive Orbitrap mass spectrometer connected to a Trace 1310 Gas Chromatograph with a TriPlus RSH Autosampler (Thermo Scientific, Bremen, Germany). The gas chromatograph was equipped with a Zebron ZB-SemiVolatiles column (30 m × 0.25 mm × 0.25 μm, Phenomenex, USA). Helium was used as a carrier gas with a flow rate of 1 mL min−1. Temperature of the injector was 250°C, of the transfer line 250°C, and of the auxiliary zone 280°C. The oven program was as followed: the initial temperature 80 °C was maintained for 2 min, rose to 120 °C at a rate of 20 °C/min, maintained for 1 min, rose to 250 °C at a rate of 5 °C/min, rose to 320 °C at a rate of 10 °C/min and maintained for 2 min. For the electron ionization (EI) mode the ion source temperature was 300°C and for the chemical ionization (CI) mode the ion source was set to 180°C. Methane was used as an ionization gas with a flow rate of 1.5 mL min−1. The injected volume of samples was 1 μL, for EI mode split ratio was 1 to 5, in CI mode samples were measured in the splitless mode. LC-HR-MS measurementsSamples for LC-HR-MS analysis were measured with a Dionex UltiMate 3000 system equipped with UltiMate WPS-3000RS autosampler and coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). The column compartment temperature was set to 25°C. Injection volume of the samples was 1 µl. The auto-injector was not washed between samples, the carryover of the samples was controlled by solvent blank measurements.The chromatographic separation was performed on a SeQuant ZIC-HILIC column (5 μm, 200 Å, 150 × 2.1 mm, Merck, Germany), equipped with SeQuant ZIC-HILIC guard column (20 × 2.1 mm, Merck, Germany). The duration of the method was 14.5 min with an MS runtime from 0.5 min to 9 min. Eluent A consisted of water with 2% acetonitrile and 0.1% formic acid, eluent B of 90% acetonitrile with 10% aqueous ammonium acetate (1 mmol L−1). The flow rate was set to 0.6 mL min−1, the gradient started with 85% solvent B, which was held for 4.0 min, gradient to 0% of solvent B (4.0–5.0 min), hold time 0% B (5.0–9.0 min), gradient to 100% of B (9.0–10.0 min), hold time 100% B (10.0–12.0 min), gradient to 85% of B (12.0–12.5 min), hold time 85% B (12.5–14.5 min).Partitioning of Carbon Metabolism Between Photosynthesis and Osmotrophy in Collodaria Photosymbiotic Holobionts.Vera NikitashinaInstitute for Inorganic and Analytical Chemistry Bioorganic Analytics, Friedrich-Schiller-UniversitÃÂÃÂ¤t JenaLysateWhole Organismmass spectrometryBefore extraction, all samples were lyophilized. The extraction of the samples was performed according to a previously published method 47. The samples evaporated under vacuum were kept under argon and stored below –20°C.Dry samples were dissolved in 80 µL of methanol:acetonitrile:water (5:9:1, v:v:v). For GC-HRMS analysis, 25 µL of each sample were transferred into vials and 1 µL from each sample was combined to create the pooled quality-control (QC) sample . Samples and QC were subsequently dried under vacuum. Dried samples were dissolved in 35 µL and QC in 140 µL of pyridine (Chromasolv Plus, Sigma-Aldrich) containing 20 mg mL−1 methoxyamine monohydrochloride (Sigma-Aldrich). All samples were vortexed for 1 min, briefly spun down, sonicated in an ultrasonic cleaner for 10 min, vortexed for 1 min, briefly spun down again, heated at 60°C for 1 h, vortexed 1 min, briefly spun down, and incubated at room temperature overnight. The pooled QC was split into 4 replicates. To each sample 35 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (Macherey-Nagel) were added. To the QC samples, 1 μL of a C7–C40 alkane standard mix, 100 μg mL−1 in hexane (diluted 1:10 (v:v) from the 1 mg mL−1 standard from Sigma-Aldrich) was added. Samples were vortexed for 1 min, briefly spun down, ultrasonicated for 10 min, vortexed for 1 min, briefly spun down, heated at 60°C for 1 h, vortexed 1 min, briefly spun down, ultrasonicated for 10 min, transferred to 1.5 mL Eppendorf Safe-Lock tubes, and centrifuged at 16,000×g at 4°C for 15 min. Supernatants were transferred into glass vials with inserts and directly measured. For LC-HRMS analysis, 35 µL of each of sample dissolved in methanol:acetonitrile:water (5:9:1, v:v:v) were transferred into vials with inserts and 5.5 times diluted with the same solvent mixture to measure on a SeQuant ZIC-HILIC column. An extract of B. nutricula equivalent to 107 cells per sample was processed in the same way and used as QC sample.CollodariaBrandtodinium nutriculahttps://www.ebi.ac.uk/metabolights/MTBLS12592Vera Nikitashina. Friedrich Schiller University Jena Institute for Inorganic and Analytical Chemistry. Lessingstr. 8 07743 Jena. vera.nikitashina@uni-jena.de.Georg Pohnert. Friedrich Schiller University Jena Institute for Inorganic and Analytical Chemistry. Lessingstr. 8 07743 Jena Germany. georg.pohnert@uni-jena.de.GC-MS data processing Raw data obtained from GC-MS analysis with EI mode was converted into .mzXML format using the Proteowizard Suite (proteowizard.sourceforge.net) with vendor peak picking enabled. LabelingTimepointveranikitashina@gmail.comStrains and culture conditions of the microalgaeA strain of Brandtodinium nutricula RCC 3387 was obtained from the Roscoff Culture Collection (Roscoff, France). The strain was grown under sterile conditions, the associated microbiota were not removed. The culture medium was artificial seawater (ASW) <a href='https://onlinelibrary.wiley.com/doi/10.1111/j.1438-8677.1994.tb00820.x' rel='noopener noreferrer' target='_blank'>1</a> supplemented with an L1 culture medium kit from the Bigelow National Center for Marine Algae and Microbiota (Maine, USA) and 3 g L−1 sea salts (Sigma-Aldrich, USA). To obtain the algae with 13C-enriched metabolome, the NaH12CO3 in the medium was replaced with NaH13CO3 (SIGMA-ALDRICH, USA). To maximize 13C label incorporation in the algal metabolome, at least three passages of cultivation were performed in NaH13CO3-containing medium <a href='https://www.frontiersin.org/journals/marine-science/articles/10.3389/fmars.2022.821167/full' rel='noopener noreferrer' target='_blank'>2</a>. The inoculation of the new cultures was performed from the stationary phase cultures grown in NaH13CO3-containing media. Starting cell density was 2.3×104 cells mL−1. Control cultures were grown in normal medium. The culturing temperature was 18°C and the light was adjusted to 90–110 μmol photons m−2 s−1 with a 14/10 light/dark cycle.Preparation of algal lysateFor the preparation of algal lysate, B. nutricula was grown in 250 mL culture flasks with 265 mL of the 13C-enriched culture medium (filled to the top of the rim), to minimize the surface area where air exchange occurs. 12C control cultures were grown in 250 mL culture flasks containing 180 mL of culture medium. When the cultures reached the stationary phase, cells were harvested by centrifugation at 3,000×g at 18°C for 5 min, washed and re-centrifuged three times with non-labeled medium, and frozen at –80°C. To prepare the algal lysate, frozen samples were lyophilized, resuspended in the non-labeled medium, and sonicated for 10 min in an ultrasonic cleaner USC1200TH (VWR, Malaysia). The samples were then vortexed for 30 s, frozen at –80°C, thawed, frozen again and subsequently lyophilized and stored below –20°C. Collection of CollodariaCollodaria were collected in the bay of Villefranche-sur-Mer (France, 43°41´10´´N, 7°18´50´´E) in March 2024 using a plankton net with a 200 µm mesh size. Samples were obtained by slowly towing the net just below the water subsurface.The samples were diluted in buckets with freshly collected seawater from the same sampling site. Individual morphologically similar Collodaria colonies containing symbiotic algae were manually selected under a microscope and transferred into filtered seawater (FSW, 0.2 µm pore size) from the same location. After at least 1 hour of incubation, Collodaria colonies were transferred to fresh FSW, this transfer protocol was repeated at least three times to remove attached particles. This and all subsequent incubations were performed at the same temperature as the ambient seawater, with illumination set to 85–95 μmol photons m−2 s−1 with various light regimes (see below).Incubation of CollodariaCleaned colonies were incubated either with NaH13CO3-enriched medium or 13C-enriched B. nutricula lysate. Corresponding non-labeled (12C) controls were prepared and processed for each experiment. A minimum of three replicates were processed per treatment. For incubation with 13C-bicarbonate, NaH13CO3 (SIGMA-ALDRICH, USA) was dissolved in FSW to the final concentration of 0.2 g L−1. For controls, solution of NaH12CO3 (Merck KGaA, Germany) in FSW with the same final concentration was prepared. The incubation of Collodaria colonies was performed in either 6-well plates with 10 mL of FSW supplemented with NaH12/13CO3 per well (12h incubation), or in 50 mL cell culture flasks containing 55 mL of FSW supplemented with NaH12/13CO3 per flask (24h incubation). Cleaned colonies were randomly distributed among the wells when incubated in the well plates (two colonies per well), or among the flasks when incubated in the flasks (nine colonies per flask). Three setups with different time regimes were performed: i) incubation for 12h in the light, ii) incubation for 12h in the light followed by 12h in the darkness,iii) incubation for 24h in the light. The incubations were terminated by transfer of all colonies with a pipet from the treatment into fresh FSW. For washing off residual labeled medium, these colonies were transferred two more times to fresh FSW. The colonies were checked for their integrity under the microscope, transferred into 1.5 mL Micro tubes (SARSTEDT AG &Co. KG, Germany) with a minimal amount of FSW, immediately frozen in liquid nitrogen, and stored below –70°C until further processing. Each replicate contained seven to nine colonies, each replicate had the same number of colonies across one setup.iv) incubation with algal lysateFor incubation with the algal lysate, lyophilized lysates of B. nutricula grown in either normal or 13C-labeled media as described above, were used. The lyophilized lysates from two 250 mL cultures for each treatment were resuspended in 2.5 mL of double-distilled, sterile-filtered H2O and pooled together. One mL samples corresponding to approximately 3×106 B. nutricula cells mL−1 were aliquoted for LC-/GC-HRMS analysis and transferred into 1.5 mL Eppendorf Safe-Lock tubes (Eppendorf AG, Germany), frozen in liquid nitrogen, and stored below –70°C until further processing. Aliquots corresponding to the final concentration of 3.5×104 cells mL−1 were added to FSW. This concentration is approximately ten times lower than both, the symbiont density within a colony 15,46 and the density of an exponentially growing algal culture,  and was chosen to avoid any potentially harmful effects of the lysate on the colony. Incubation of Collodaria colonies was performed in 50 mL cell culture flasks with 50 mL of algal lysate solution in the light. After 22h, Collodaria colonies were transferred into fresh FSW without lysate. To ensure residual lysate was removed, the isolation and transfer was repeated three times. After a total time of 24h, Collodaria colonies were checked for their integrity under microscopy, transferred into 5 mL Eppendorf tubes (Eppendorf AG, Germany) with a minimal amount of FSW, immediately frozen in liquid nitrogen, and stored below –70°C until further processing. Each replicate contained eleven colonies. MetabolomicsosmolytesCarbohydratesradiolariaphotosymbiosisCollodariauntargeted metabolitesmetabolic fluxesosmolytesCarbohydratesradiolariaphotosymbiosisCollodariauntargeted metabolitesmetabolic fluxesGC-MS measurementsGC-MS measurements were performed on a Q-Exactive Orbitrap mass spectrometer connected to a Trace 1310 Gas Chromatograph with a TriPlus RSH Autosampler (Thermo Scientific, Bremen, Germany). Data were recorded in full scan profile mode across a mass range from 50 to 600 m/z in EI mode, and mass range from 80 to 1000 m/z in CI mode, the ionization energy was 70 eV. LC-HR-MS measurementsSamples for LC-HR-MS analysis were measured with a Dionex UltiMate 3000 system equipped with UltiMate WPS-3000RS autosampler and coupled to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). The MS measurements were performed in positive mode, with a resolution set to 280,000 within a scan range from 80 to 1200 m/z, heated electrospray ionization was used to generate molecular ions. TrigonellineAdenosineL-phenylalanineglycine betaineAdeninetaurineβ-alanine betaineCreatineN(omega),N(omega)-dimethyl-L-arginineL-arginineGuanineL-proline betaineAlanine betaineL-prolineL-valineS,S-dimethyl-beta-propiothetinL-tyrosine(16:0)1-palmitoyl-sn-glycero-3-phosphocholineectoineHomarinefalseSymbiont photosynthesis supplies carbohydrates whereas osmotrophy provides osmolytes in Collodaria photosymbiotic associationCollodaria (Radiolaria) are widespread marine planktonic protists that host photosynthetic microalgal symbionts. Within this photosymbiosis, they can contribute substantially to primary production, particularly in oligotrophic ocean regions. Despite their ecological importance, the mechanisms underlying the formation and maintenance of the photosymbiosis are poorly understood. To uncover the metabolic dependencies within this photosymbiotic system, we developed a stable isotope labeling strategy that allows us to investigate the assimilation and exchange of inorganic and organic carbon within the Collodaria holobiont. By integrating metabolomic profiling with isotopic labeling analyses, we demonstrate that algal photosynthesis makes a substantial contribution to the holobiont carbohydrate pool. In particular, we observed the incorporation of inorganic carbon into glucose and fructose, followed by the enrichment of other metabolites including ribose. In contrast, osmotrophic uptake of labeled algal lysate did not lead to the incorporation of any of these carbohydrates, suggesting no significant contribution of osmotrophy to the holobiont's carbohydrate pool. In contrast, specialized metabolites, such as dimethylsulfoniopropionate (DMSP) and betaines, were primarily acquired through osmotrophy implying a minor contribution of photosymbionts. Our findings thus reveal defined roles of phototrophic and osmotrophic metabolism for the formation of distinct metabolites in the Collodaria holobiont with consequences for our understanding of element fluxes in the plankton. 2026-05-262025-06-13MTBLS12592MTBLC17234MTBLC10642MTBLC28757MTBLC69187MTBLC33942MTBLC17268MTBLC27371MTBLC26984MTBLC72998MTBLC16708MTBLC16335MTBLC28825MTBLC16467MTBLC17750MTBLC16919MTBLC27592MTBLC16235MTBLC69061MTBLC17929MTBLC17295MTBLC17203MTBLC35280MTBLC15891MTBLC18123MTBLC17895MTBLC16414MTBLC16457CHEBI:17234CHEBI:10642CHEBI:28757CHEBI:69187CHEBI:33942CHEBI:17268CHEBI:27371CHEBI:26984CHEBI:72998CHEBI:16708CHEBI:16335CHEBI:28825CHEBI:16467CHEBI:17750CHEBI:16919CHEBI:27592CHEBI:16235CHEBI:69061CHEBI:17929CHEBI:17295CHEBI:17203CHEBI:35280CHEBI:15891CHEBI:18123CHEBI:17895CHEBI:16414CHEBI:16457