Non-targeted liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS) is increasingly applied for the structure-resolved chemical analysis of dissolved organic matter (DOM). With new developments in MS instrumentation and analysis software, the approach has gained substantial momentum over the past decade. However, achieving high-quality analytical data that is reproducible and comparable across laboratories can be a bottleneck in non-targeted metabolomics and organic matter chemical analysis, especially for data reuse in repository-scale analyses. Understanding the capabilities as well as challenges of comparing LC-MS/MS data from different laboratories is necessary for inferring global trends from public data sets. To illuminate instrumentation factors that drive differences and variability, we used a standardized data analysis pipeline, including classical (CMN) and feature-based molecular networking (FBMN), to analyze data from a ring trial by 24 laboratories on identical sample sets of algal and DOM extracts that were mixed in predefined concentrations and spiked with standards. Our results showed that data sets from similar mass spectrometer types with unified instrument parameters were qualitatively comparable, resolving the same general trends and shared mass spectral features. Interlaboratory comparability was best for high-intensity features, while low-intensity features showed greater detection variability. Our analysis also highlights challenges when comparing data from instruments with different acquisition rates or operating with less standardized methods. Lastly, we provide recommendations for data integration, public data sharing, standardization, and best practices for standardized LC-MS/MS data acquisition, which will be critical for long-term time series and intercomparability of DOM chemical analyses.

Climatic changes in the physical environment modulate biogeochemical cycles, biodiversity, and trophic interactions in the Central Arctic Ocean (CAO). Physical processes and sea-ice conditions are highly seasonal in the CAO and dependent on interactions that occur throughout the evolution of the upper ocean–sea ice–lower atmosphere system. Understanding these seasonal interactions is critical to comprehending and predicting the long-term trends as the CAO moves towards ice-free summers and to informing future policy decisions at the core of ongoing discussions concerning the CAO Fisheries agreement, for example, at the Arctic Council and International Council for Exploration of the Sea working group on the CAO. Here, we review current knowledge of the physical environment, biogeochemical cycles, and biodiversity in the waters of the CAO, identify emerging research questions, and introduce the science plan for the first Tara Polaris drift onboard the Tara Polar Station to advance knowledge and address these questions. Despite increased observational programmes in the CAO over the past years, for example, the Nansen and Amundsen Basin Observational System (NABOS) and Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC), extensive knowledge gaps remain in relation to ocean stratification, sea ice and lightscape, nitrogen fixation and nutrient fluxes, carbon export and transfer, sympagic-pelagic coupling, aerosol production, contaminant transport and transformation, chronobiology, and fish distribution. Further knowledge on overall CAO biodiversity, ecosystem functionality and interannual variability is also critically needed. We describe a way forward to address these knowledge gaps using ice-tethered and profiling instruments coupled with multi-omics, culturing, and imagery approaches deployed from Tara Polar Station during the first of ten Tara Polaris drifts designed to facilitate detection of interannual variability and change over time.

Phytoplankton, the photosynthetic microalgae driving nearly half of Earth's primary production, are the foundation of marine food webs and central to climate regulation. Skeletonema marinoi is a globally distributed and often dominant diatom species in marine phytoplankton communities. It inhabits a dynamic and frequently hostile microbial environment in which interactions with bacteria can negatively affect its survival. Skeletonema marinoi is susceptible to the algicidal bacterium Kordia algicida that can lyse the algal cells and even terminate entire blooms. The extent to which resistance against such a lysis can evolve in diatoms exposed to biotic stress by algicidal bacteria remains unknown. Using adaptive laboratory evolution, we investigated how S. marinoi adapts to toxins produced by K. algicida. Skeletonema marinoi evolved resistance already after 11 growth cycles under sublethal exposure to algicides. This was accompanied by changes in DNA methylation. Untargeted comparative metabolomics of the original and the evolved population revealed the up-regulation of the oxylipins 5-hydroxyeicosapentaenoic acid, prostaglandin E2, and 17-hydroxydocosahexaenoic acid. These oxylipins significantly inhibited the growth of K. algicida, indicating their role in chemical defense. The metabolic plasticity of diatoms and the rapid evolution observed after exposure to bacteria open new perspectives on our understanding of diatom bloom dynamics in nature.

Growing concerns regarding the spread of antibiotic resistance, together with increasing regulatory demands for advanced wastewater treatment, necessitate sensitive high-throughput analytical methods for monitoring antibiotic residues in the environment. In this study, an automated workflow for the determination of multi-class antibiotics in environmental waters was developed by combining a miniaturized 96-well solid-phase extraction (SPE) protocol with LC–HRMS detection. The SPE procedure enabled enrichment of 5 mL water samples to final extract volumes of 50 µL, corresponding to an enrichment factor of 100, while allowing direct injection of the SPE eluates into the LC–HRMS system without additional sample treatment. The automated workflow enabled parallel processing of up to 48 samples within 45 min while requiring less than 2 mL of organic solvent per sample. Comprehensive method validation demonstrated excellent analytical performance, including high linearity and method limits of quantification below 1 ng L-1 for all investigated antibiotics. Matrix effects, particularly in wastewater effluent, as well as variations in extraction recovery were effectively compensated using analyte-specific isotopically labeled internal standards. The applicability of the workflow for high-throughput environmental monitoring was demonstrated by analyzing wastewater effluent and river water samples, revealing the widespread occurrence of sulfamethoxazole, trimethoprim, ciprofloxacin, levofloxacin, azithromycin, clarithromycin, erythromycin, and roxithromycin, ranging from ultra-trace levels in river water to substantially elevated concentrations in wastewater effluent. Thus, the developed workflow represents a robust and sensitive high-throughput approach for the analysis of antibiotic residues in environmental waters.

IntroductionLC-MS system suitability test (SST) is crucial for reliable data acquisition especially in untargeted metabolomics.ObjectivesIdentification of best reference materials (RMs) to improve best quality assurance (QA) and quality control (QC) practices.MethodsInvestigations were performed using a C18 reversed-phase (RP) column LC-MS approach.ResultsTargeted cyanotoxin analysis revealed a performance loss of the used C18 RP column although the SST confirmed a fit for purpose instrument which prompted to test several additional RMs.ConclusionQA procedures for LC-MS can be improved by incorporating polar microcystins or arginine methyl ester as RMs for SST.

Abstract Collodaria (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 photosymbiosis are poorly understood. To uncover the metabolic dependencies within this photosymbiotic system, we develop a stable isotope labeling strategy that allows us to investigate the assimilation of inorganic and organic carbon in 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 observe the incorporation of inorganic carbon into glucose and fructose, followed by the enrichment of other metabolites including ribose. In contrast, osmotrophic uptake of dissolved organic compounds derived from labeled algal lysate does 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, are primarily acquired through osmotrophy implying a minor contribution of photosymbionts. Our findings thus reveal defined roles of phototrophic and osmotrophic metabolism in the Collodaria holobiont with consequences for our understanding of element fluxes in plankton.

Marine bacteria are integral components of planktonic communities, where they regulate algal growth, induce cell death, and contribute to bloom termination and species succession. They also play a key role in marine biogeochemical cycling by recycling algal-derived organic matter and releasing bioactive metabolites. Despite their ecological importance, bacterial-plankton interactions and their consequences for community structure and chemistry remain poorly understood. We investigated the impact of the algicidal marine bacterium Kordia algicida OT-1 on a natural plankton microbiome collected from a mesocosm experiment simulating present and future climate conditions. Plankton communities were exposed to ambient conditions or to a worst-case climate scenario, with a subset further subjected to a one-week heatwave. After 24 h of incubation, K. algicida significantly altered phytoplankton abundance and phylum-level community composition, independent of the applied abiotic conditions. Chemical changes induced by bacterial interactions were assessed by extracting filtrates from cocultures and analyzing them using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Four natural products, i.e., adenosylhomocysteine, two indole alkaloid derivatives, and 5-bromotryptophan, were identified among metabolites released in response to bacterial exposure. Overall, shifts in the planktonic chemical landscape were primarily driven by bacterial activity, rather than abiotic conditions.

ABSTRACT Microalgal fitness in nature is shaped by interactions within a diverse microbial community, yet most experimental studies have examined algal-bacterial interactions in pairwise systems. It is well established that bacteria can exhibit growth promoting or inhibiting effects on co-existing algae. Comparatively little information is available about how additional partners can alter the outcome of diatom-bacteria interactions. In the present study, we screened the pairwise interaction of the marine diatom Skeletonema marinoi with ten different bacteria. This screening identified Marinobacter adhaerens as a growth promoting and Vibrio cyclitrophicus HSW24 as growth inhibiting partner. Growth inhibition of V. cyclitrophicus was associated with cell lysis, chain fragmentation and altered pigmentation whereas M. adhaerens supported increased chlorophyll a fluorescence, uniform pigmentation, intact chains and healthy cell morphology. In a tripartite community containing both bacteria and the alga, M. adhaerens protected S. marinoi from the inhibitory effect of V. cyclitrophicus in a density dependent manner. Comparative metabolomics revealed distinct metabolic profiles between the pairwise and tripartite interactions. This allowed to identify metabolites that were up-regulated in the tripartite community and therefore candidates for the observed protection. Among these, kynurenic acid and N -acetyltyramine were identified in bioassays as protective molecules, thus clearly highlighting the importance of secondary metabolites in this interaction. The present findings demonstrate that a third bacterial partner can alter the outcome of an antagonistic algal-bacterial interaction by means of chemical mediators. This work has implications for our understanding of microbial community functioning that cannot only be derived from the investigation of pairwise interactions.

DNA (deoxyribonucleic acid) methylation is a key epigenetic mechanism that regulates gene expression and developmental transitions in eukaryotes. Interestingly, algal growth- and morphogenesis-promoting bacteria can reduce global DNA methylation levels in the green seaweed Ulva (Chlorophyta), highlighting DNA methylation as a dynamic and environmentally responsive epigenetic mechanism in marine macroalgae. We hypothesized that DNA methylation serves as a rapid and essential epigenetic regulatory mechanism during gametogenesis in the model organism Ulva compressa (cultivar U. mutabilis slender). We further propose that this process is controlled by sporulation inhibitors - extracellular compounds long known to regulate reproduction in Ulva, yet whose molecular mode of action has remained elusive. Using ultra-high-performance liquid chromatography-mass spectrometry (HPLC–HRMS), we quantified global levels of 5-methylcytosine (5mC) and N6-methyladenine (6mA) across defined life-cycle stages of Ulva, including gametogenesis induction and gamete release. We observed a rapid and pronounced increase in 5mC immediately following removal of sporulation inhibitors, consistent with active epigenetic reprogramming. In contrast, 6mA was detected only in mature thallus (vegetative tissue of macroalgae) and gametes, and not throughout gametogenesis, indicating a distinct yet minor role for this DNA modification in Ulva development. Re-addition of sporulation inhibitors prevented both gametogenesis and the characteristic methylation dynamics, indicating that these not-yet-characterized inhibitors act as epigenetic modulators. Together, our findings identify DNA methylation as a key regulatory layer linking bacterial cues, sporulation control, and reproductive development in Ulva, with direct implications for the epigenetic management of growth and reproduction in marine algal biotechnology.

The algal holobiont, a symbiotic association of algae and bacteria, provides strategies to cope with environmental stresses. Bacteria associated with the green alga Ulva (Chlorophyta) are known to produce essential bioactive compounds, such as thallusin and ectoine, that promote algal growth; however, the metabolic mechanisms underlying stress adaptation in these mutualistic interactions remain poorly understood. Specifically, research is still emerging on the metabolome of cold-adapted Ulva holobionts from remote regions, such as Antarctica, and on how the metabolic profiles respond to environmental changes. This study thus examined heat-induced changes in osmolyte and zwitterion molecules bearing balanced positive and negative charges in Ulva sp. (strain FSU-UPC-109) collected from the Antarctic ecosystem of Potter Cove, South Shetland Islands, and maintained under laboratory conditions at 2°C. Using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC–HRMS), we identified highly polar low molecular weight compounds, including cysteinolic acid, ectoine, glutamine, glycerol, and proline. Their spatial distribution on the thallus and rhizoids was determined with atmospheric-pressure matrix-assisted laser desorption/ionization imaging (AP–SMALDI). We demonstrated that the metabolite levels shifted significantly under heat stress, indicating that environmental temperature changes strongly influence the metabolome of Antarctic Ulva seaweed and may support their capacity for thermal acclimation. Using high-resolution (imaging) mass spectrometry, we studied the heat-induced changes in abundance of the osmolytes and zwitterions, molecules bearing balanced positive and negative charges, in Ulva collected from the Antarctic ecosystem of Potter Cove, South Shetland Islands.

Harmful algal blooms (HABs) can be caused by dinoflagellates like Alexandrium minutum, which produces Paralytic Shellfish Poison toxins derived from saxitoxins. The abundance of these toxic algae is shaped not only by abiotic factors, but also by microbial interactions such as parasitism by the alveolate Parvilucifera infectans. These parasites can infect algal cells, inhibit growth, and induce death, thereby contributing to the termination of HABs. Salinity can affect algal physiology and may modulate host-parasite dynamics by altering the parasite's ability to infect and kill HAB-forming algae. Despite their ecological relevance, the interactions between algae and parasites, and the modulation of algal toxins during cell infection at varying salinity, remain poorly understood. Here, we investigated the impact of the parasite P. infectans, a well-recognized model organism that preys on A. minutum under different salinity conditions. We monitored infection success in cultures and recorded metabolome changes during parasite cell invasion using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLCHRMS). P. infectans tolerated salinity from 20 PSU to 32 PSU and induced the death of whole algal cultures within 3 weeks in laboratory-controlled experiments. Comparative metabolomics revealed that parasite infection decreased the abundance of algal toxins in A. minutum. The parasite infection, rather than salinity, induced significant shifts in the host cell metabolome. Several polar metabolites and osmolytes, such as ectoine, dimethylsulfoniopropionate, glycine betaine, choline, and carnitine, were identified at elevated levels in parasite-infected cells, suggesting potential roles in parasite development and reproduction.

Marine alveolates (MALVs) are diverse, primarily parasitic micro-eukaryotes that significantly impact marine ecosystems. The life cycles of most MALVs remain elusive and the role of sexual reproduction in these organisms is a key question that may determine their ecological success. In this study we focus on a widespread dinoflagellate parasite of bloom-forming dinoflagellates, Amoebophrya . After infection, we identified two distinct spores, differing in size, ultrastructure, swimming behavior, lifespan, gene expression, and metabolite composition. The smaller spores serve as infectious propagules, equipped with an apical complex for host invasion. They exhibit a distinct, shorter, and straighter swimming pattern, likely optimized for an extended lifespan while enhancing dispersion and chance for host encounters. Transcriptomic analysis reveals that these smaller spores are primed for efficient protein synthesis upon initiating a new infection. Conversely, the larger spores cannot infect new hosts and are characterized by the expression of meiotic genes, underscoring their sexual nature. They have a shorter lifespan, exhibit more tortuous movement, along display condensed chromosomes, signaling readiness for mating. Interestingly, infected hosts already express meiotic genes, and a single infected host only produces progeny of the same spore type, suggesting that cell fate is determined prior to spore release. Our study provides one of the first formal demonstrations of a sexually specialized cell in MALVs. Isolating compatible strains for cross-breeding and understanding how environmental conditions favor each reproductive route are the next key questions for elucidating the ecological success of MALVs in marine waters. Significance Statement Marine alveolates (MALVs) are ecologically significant parasites that impact carbon cycling, causing major disease outbreaks affecting fisheries and aquaculture, and influencing the dynamics of harmful algal blooms. Despite their diversity and wide host range, much of our knowledge comes from environmental DNA, leaving important aspects of their biology, such as their life cycles, largely unknown. This study provides the first evidence of sexual reproduction in MALVs, linking spore polymorphism to infective or sexual routes. This discovery is crucial as sexual reproduction increases genetic diversity and adaptability, aiding MALVs’ resilience in changing environments. Understanding MALVs’ reproductive strategies deepens our insight into their ecological roles and their broader impact on marine ecosystems. ### Competing Interest Statement The authors have declared no competing interest.

Diatoms are prolific unicellular microalgae, contributing ≈20% of global photosynthetic CO2 fixation. These algae form the base of the marine food web. They are also widely distributed in freshwater and utilized in aquaculture. Despite their ecological and economic significance, many fundamental aspects of diatom biology, including their sexual reproduction, remain poorly understood. This review highlights recent advances in unraveling the chemical signaling essential for diatom sexual reproduction and introduces how natural products chemistry and marine microbiology synergize to unravel novel chemical communication strategies. Diatoms communicate through a sophisticated pheromone-based "language" that synchronizes cell cycles, regulates the physiology, and guides cells to their mating partners. The structural elucidation of diatom pheromones becomes possible with emerging model organisms and the development of analytical approaches to detect these compounds at extremely low active concentrations. Breakthroughs in comparative metabolomics, combined with reliable bioassays, have enabled the identification of the first pheromones. However, only the adaptation of novel labeling techniques and sensitive NMR experiments allows the determination of the pheromone structures. With these structural insights and the availability of genetic resources, a new research field is emerging-spanning evolutionary studies to the potential manipulation of natural diatom populations and applications in aquaculture.

A key aspect of algal biology is the production of chemicals used for communication and regulation of interactions. Allelopathic chemicals can facilitate competition between algae, influence community composition and drive succession in the phytoplankton. Much is still unknown about the identities of these allelochemicals, and even the ecological relevance of laboratory derived results is often unclear. We sought to evaluate the variability of allelopathy employing a systematic approach. We used Prymnesium parvum as a model to assess species-specificity of allelopathic interactions in laboratory co-cultivations. We assessed the influence of media, ratio of cell densities of the interaction partners and evaluation methods (monitoring of chlorophyll a versus cell density) on the outcome of co-cultivation experiments. Five tested ecologically relevant phytoplankton species, Dunaliella tertiolecta, Emiliania (presently Gephyrocapsa) huxleyi, Florenciella parvula, Ochromonas triangulata, and Pavlova gyrans, exhibited and experienced varying positive and negative allelopathic effects in interactions with P. parvum. Further, we document pronounced effects of variations of laboratory parameters. Among the ten possible bidirectional allelopathic relationships, half of the allelopathic effects were changed by one or more of the variable parameters tested. Allelopathy is thus not only a species-specific trait but additionally depends on the external factors present during the interactions. We support our findings through the identification of interaction patterns from mining the Tara Oceans database for reoccurring trends in natural and laboratory communities. This study strongly supports the notion that allelopathic activity of an alga is not only a species-specific property but also depends on many external parameters that have to be considered and thoroughly documented in future studies of allelopathy.

In marine ecosystems, microbial communities often interact using specialised metabolites, which play a central role in shaping the dynamics of the ecological networks and maintaining the balance of the ecosystem. With metabolomics and transcriptomics analyses, this study explores the interactions between two marine microalgae, Skeletonema marinoi and Prymnesium parvum, grown in mono-cultures and non-contact co-cultures. As a growth indicator, the photosynthetic potential, measured via fluorescence, suggested chemical interaction between S. marinoi and P. parvum. Using Liquid Chromatography-Mass Spectrometry (LC-MS) data, we identified 346 and 521 differentially produced features in the endo- and exometabolome of S. marinoi and P. parvum, respectively. Despite limited tandem mass spectrometry data (MS2) for these features, we structurally annotated 14 compounds, most of which were previously under-studied specialised metabolites. Differential gene expression analysis was then performed on the transcriptomes of the microalgae, which uncovered differentially expressed genes involved in energy metabolism and cellular repair for both species. These metabolic changes depict the adaptation of both species in the co-culture. However, further data acquisition and investigation will be necessary to confirm the type of interaction and the underlying mechanisms.

Groundwater health is increasingly threatened by climate change, which alters precipitation patterns, leading to groundwater recharge shifts. These shifts impact subsurface microbial communities, crucial for maintaining ecosystem functions. In this decade-long study of carbonate aquifers, we analyzed 815 bacterial 16S rRNA gene datasets, 226 dissolved organic matter (DOM) profiles, 387 metabolomic datasets, and 174 seepage microbiome sequences. Our findings reveal distinct short- and long-term temporal patterns of groundwater microbiomes driven by environmental fluctuations. Microbiomes of hydrologically connected aquifers exhibit lower temporal stability due to stochastic processes and greater susceptibility to surface disturbances, yet they demonstrate remarkable resilience. Conversely, isolated aquifer microbiomes show resistance to short-term changes, governed by deterministic processes, but exhibit reduced stability under prolonged stress. Variability in seepage-associated microorganisms, DOM, and metabolic diversity further drive microbiome dynamics. These findings highlight the dual vulnerability of groundwater systems to acute and chronic pressures, emphasizing the critical need for sustainable management strategies to mitigate the impacts of hydroclimatic extremes. ### Competing Interest Statement The authors have declared no competing interest.

Fucoxanthin, a dietary component recognized for its benefits in weight loss and liver fat reduction, can be efficiently synthesized and accumulated in the marine diatom Phaeodactylum tricornutum. This study demonstrates that the combination of arginine and urea as a mixed nitrogen source exhibits a synergistic effect, increasing fucoxanthin productivity by over 141 % in mixotrophic P. tricornutum compared to arginine as single nitrogen source. Multi-omics approaches were employed to analyze the metabolic regulatory mechanisms underlying fucoxanthin synthesis in response to this synergistic effect. The intracellular amino acid analysis revealed a marked increase in arginine consumption, primarily accelerating the urea cycle and the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway. During the early stages of cultivation, cell respiration and photosynthesis were significantly enhanced, providing ample precursor substances for active carbon and nitrogen transfer and energy exchange, which supported rapid cell proliferation and division. In the later stages, the pathways of gluconeogenesis and fucoxanthin biosynthesis remained highly active, demonstrating a significant upregulation of intermediate metabolites in both pathways. Collectively, this achieved the synchronous accumulation of biomass and fucoxanthin in mixotrophic cells under the synergistic effect. These findings underscore critical pathways for enhanced utilization of glycerol, arginine, and urea, thereby promoting fucoxanthin production in a photo-fermentation system.

Marine planktonic Radiolaria harboring symbiotic microalgae are ubiquitous in the oceans and abundant in oligotrophic areas. In these low-nutrient environments they are among the most important primary producers. Systematic studies of radiolarian biology are limited because Radiolaria are non-culturable and prone to damage during sampling. To obtain insight into the mechanistic basis of radiolarian photosymbiosis we address here the metabolic contributions of the partners to the performance of the holobiont. Therefore, we describe the metabolic inventory of two highly abundant photosymbiotic Radiolaria - colony-forming Collodaria and single-celled Acantharia and compare their metabolomes to metabolomes of respective free-living algae. Most of the metabolites detected in the symbiosis are not present in the free-living algae, suggesting a significant transformation of symbionts' metabolites by the host. The metabolites identified in both the holobiont and the free-living algae encompass molecules of primary metabolism and a number of osmolytes, including dimethylsulfoniopropionate. Mass spectrometry imaging revealed the presence of dimethylsulfoniopropionate in both the symbionts and host cells, indicating that the algae provide osmolytic protection to the host. Furthermore, our findings suggest a possible dependence of Collodaria on symbiotic vitamin B3. Distinctive differences in phospholipid composition between free-living and symbiotic stages indicate that the algal cell membrane may undergo rearrangement in the symbiosis. Our results demonstrate a strong interdependence and rewiring of the algal metabolism underlying Radiolaria-microalgae photosymbioses. ### Competing Interest Statement The authors have declared no competing interest.