Thermal Stress During Embryogenesis Alters the Metabolome of Zebrafish Larvae

Thermal Stress During Embryogenesis Alters the Metabolome of Zebrafish Larvae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Thermal Stress During Embryogenesis Alters the Metabolome of Zebrafish Larvae Jeong Eun Kim, A hyun Park, Hyun-Hwan Jeong, Ki-Bae Hong, Jewon Jung, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7397518/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Global warming has led to continuous increases in marine surface temperatures, posing significant challenges to aquatic ecosystems and the aquaculture industry. Thermal stress during early development can profoundly alter physiological, morphological, and metabolic process in fish, with potential long-term consequences for growth, behavior, and survival. In this study, we investigated the effects of elevated temperature during zebrafish embryogenesis on developmental timing, morphological phenotypes, larval behavior, and metabolic regulation. Embryos were exposed to control and thermal stress conditions, and subsequent phenotypic and biochemical analyses were conducted from hatching through the larval stage. Thermal stress accelerated hatching to within 48 hpf, but this was accompanied by morphological abnormalities. At the late larval stage, larvae exposed to thermal stress exhibited significantly increased swimming velocity and distance with altered spatial occupancy patterns. Untargeted metabolomic profiling via UPLC-QTOF-MS elucidated alterations in purine metabolism, amino acid turnover, nucleotide metabolism, and phospholipid composition. Notably, phosphatidylethanolamines and phosphatidylserines were significantly depleted and phosphatidylinositols was elevated, implicating disruptions in pathways involved in membrane integrity, autophagy regulation, and ferroptosis. These findings demonstrate that thermal stress during embryogenesis elicits coordinated physiological, behavioral, and molecular responses in zebrafish, providing mechanistic insights into the vulnerability of aquatic organisms to climate change-driven temperature fluctuations. Biological sciences/Developmental biology Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Physiology Biological sciences/Zoology Zebrafish Thermal stress Metabolomics Lipidome Climate change Embryogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Global warming has driven a continuous rise in average sea surface temperatures with new record highs being reached almost every year 1 . Aquatic ecosystems are particularly sensitive to climate changes, which alters the distribution and abundance of marine organisms and affects fisheries and aquaculture production by modifying the physiological and phenotypic traits of cultured species 2 – 6 . Such changes pose a direct threat to the fisheries industry and global food security 2 – 4 . Elevated water temperatures can influence critical biological processes in aquatic animals, including reproduction, growth rate, and behavioral patterns, thereby challenging population sustainability 5 , 7 – 9 . To cope with temperature perturbations, aquatic animals adjust their physiological and behavioral responses to maintain homeostasis 10 , 11 . Zebrafish ( Danio rerio ) are on established vertebrate aquatic model for studying the effects of environmental stressors due to their genetic tractability, transparent embryos, and well-characterized developmental timeline 12 . Their sensitivity to thermal variation, coupled with conserved stress response pathways, makes them an ideal system for dissecting the multi-scale impacts of heat exposure. Previous studies in zebrafish and other fish have demonstrated that thermal stress during embryonic and larval stages can accelerate developmental timing 13 , 14 . These outcomes are often accompanied by metabolic reprogramming due to the alteration of microbiota 15 . However, the mechanistic links between early-life heat exposure, behavioral alterations, and underlying metabolic and lipidomic remodeling remain incompletely understood. Metabolites produced as intermediates or end products of metabolism serve as key indicators of the biochemical state of an organism. They provide a critical link among genotype, phenotype, and environmental conditions 16 , 17 . By profiling metabolite abundance and composition, it is possible to capture the physiological adjustments organism make in response to environmental stressors such as heat 18 . Thus, integrating metabolomics with phenotypic and behavioral assessments offers a powerful framework for elucidating how thermal stress impacts biological function from molecular pathways to whole-animal performance 16 , 18 . In this study, we investigated the effect of early-life thermal stress on zebrafish embryonic development, larval swimming behavior, and biochemical profiles using untargeted metabolomics. Consequently, this work will provide new insight into the coordinated physiological and biochemical mechanisms supporting thermal stress adaptation in aquatic animals. Results High temperature accelerates embryonic development and induces malformations. To investigate the physiological effects of thermal stress on fish, zebrafish embryos were exposed to either normal or high temperature conditions. Two temperature regimes were applied: 26°C as the control and 34°C as the thermal stress condition, based on previous findings from a fish treadmill assay 19 . The experimental design included multi-well plates for controlled temperature incubation and analysis at three developmental stages: embryo, early larva, and late larva (Figure 1a). Water parameters for the control group were maintained as 26.5 ± 0.4°C, with dissolved oxygen (DO) ranging from 3.6 to 6.3 mg/L and pH 7.6 ± 0.1. For the thermal stress group, conditions were maintained at 34.0 ± 0.3°C, DO 3.3 – 6.1 mg/L, and pH 7.6 ± 0.1 (Figure 1a). Exposed to 34°C led to accelerated development, resulting in premature hatching by 48 hours post-fertilization (hpf). Morphological assessments displayed pronounced developmental abnormalities in the high-temperature group, including pericardial edema (PE), spinal curvature (SC), yolk sac edema (YSE), and yolk-not depleted (YND), as indicated in representative images (Figure 1b, summarized in Table 1). In contrast, embryos incubated at 26°C developed normally with no malformations observed. Quantitatively, 15 out of 24 embryos in the thermal stress group hatched at 48 hpf, while two embryos degenerated during development (Table 1). All embryos in the control group hatched normally and exhibited typical morphology (Table 1). These findings confirm that elevated temperature accelerates the developmental timeline but impairs morphological integrity, suggesting disrupted developmental signaling or altered energy allocation under stress conditions. Table 1 . Morphological changes of zebrafish embryos altered by high-temperature stress (total 24 embryos per experimental group) High temperature enhances larval locomotor activity. To assess behavioral consequences of early-life thermal stress, swimming activity was analyzed in zebrafish larvae at 14 days post-fertilization (dpf) using an automated tracking system. The swimming patterns of individual larvae were quantified for total distance traveled and average velocity over a 5-min observation period. Larvae exposed to high temperature during development exhibited significantly increased locomotor activity compared to controls (Figure 2a, b). Specifically, both total distance and velocity were significantly elevated. Representative heat maps (top row of Figure 2c) showed intensified movement density, particularly in the central zone, while the track visualizations (bottom row) revealed increased frequency of circular and exploratory motion. This hyperactive phenotype is indicative of possible alterations in stress-related behavioral circuits caused by early developmental thermal stress. Metabolomic profiling reveals distinct thermal stress-associated signatures To elucidate the biochemical basis underlying the morphological and behavioral phenotypes induced by thermal stress, we conducted untargeted metabolomic profiling via UPLC-QTOF-MS on zebrafish larvae exposed to either 26°C (control) or 34°C (thermal stress) from the early larval stage (Figure 3a). A total 516 metabolites were significantly altered between the two groups. Principal component analysis (PCA) plot showed a distinct separation between the control and high-temperature groups with minimal intra-group variation (Figure 3b). This clear clustering indicates that thermal stress induced a consistent and substantial shift in the overall metabolomic profile. A volcano plot analysis identified numerous metabolites significantly up-regulated (red) or down-regulated (blue) in the high-temperature group compared to controls (Figure 3c). These differences highlight specific biochemical features that are responsive to thermal stress. Hierarchical clustering heatmap analysis demonstrated distinct metabolite expression patterns between the two groups (Figure 3d). The high-temperature group exhibited a characteristic profile with coordinated changes across multiple metabolite clusters, supporting the robustness of the group separation observed in Figure 3b. Pathway enrichment analysis indicated that the most significantly affected pathways including metabolism of energy carrying molecules and cell-cell interaction-related molecules (purine metabolism; amino sugar and nucleotide sugar metabolism), amino acid metabolism (β-alanine metabolism; histidine metabolism; lysine degradation), and lipid metabolism (glycerophospholipid metabolism, ether lipid metabolism) (Figure 3e). Bubble plot visualization of pathway analysis further confirmed that these pathways as the most significantly affected, with high enrichment ratios and statistical significance. Larger bubble sizes indicate pathways with greater numbers of altered metabolites, emphasizing their central role in the thermal stress response (Figure 3f). These results suggest that thermal stress disrupts both nucleic acid production and amino acid processing. Taken together, thermal stress during early zebrafish development results in a metabolic shift, characterized by altered purine metabolism, amino acid processing, and lipid biosynthesis. These biochemical changes are likely contributors to the observed morphological abnormalities and hyperactive swimming behavior in thermally stressed larvae. Thermal stress modulates key metabolic pathways Pathway mapping of significantly altered metabolites revealed that thermal stress strongly affected folate biosynthesis, purine degradation, and amino sugar and nucleotide sugar metabolism. In the folate biosynthesis pathways, levels of lactoyltetrahydropterin were elevated in the thermal stress group, suggesting altered cofactor availability for enzymatic reactions linked to nucleotide and amino acid metabolism (Figure 4a, c). Within the purine degradation pathway, significant increases were detected in guanosine, guanine, and hypoxanthine. These metabolites are intermediates in purine catabolism, and their accumulation suggests enhanced nucleotide turnover or altered nucleic acid metabolism under elevated temperature conditions (Figure 4b, c). Such changes may reflect increased energy demand and accelerated cellular processes, constituent with the observed early hatching and hyperactivity in thermally stressed larvae. In amino sugar and nucleotide sugar metabolism, the level of galactose-1-phosphate (Gal-1-P) was reduced in the thermal stress group. This decrease could impair glycosylation processes and structural carbohydrate synthesis, potentially contributing to the morphological abnormalities observed during development (Figure 4d, e). Collectively, the targeted pathway analysis supports the global metabolomic findings, indicating that thermal stress induces a metabolic state characterized by elevated nucleotide turnover, disrupted carbohydrate metabolism, and altered cofactor biosynthesis. These molecular alterations align with the phenotypic outcomes, such as accelerated development, morphological malformations, and heightened locomotor activity, suggesting that the metabolic shifts may underlie the physiological behavioral consequences of early-life heat exposure. Thermal stress induces profound alterations in the lipidome of zebrafish larvae Lipidomic profiling revealed that early-life exposure to elevated temperature caused marked remodeling of the zebrafish larval lipidome compared to the control condition. Differential lipid profiles were visualized by heatmap analysis, which demonstrated clear separation between the control and thermal stress groups, with widespread reductions in multiple lipid species under high temperature (Figure 5a). Notably, lipid classes most affected included glycerophosphoethanolamines (PEs), phosphatidylinositols (PIs), and phosphatidylserines (PSs) (Figure 5a). Classification of altered lipids indicated that the majority of significantly changed metabolites belonged to PE and glycerophosphoinositols, followed by amino acids, peptides, and analogues; cholestane steroids; and fatty acids and conjugates (Figure 5b). This distribution highlights glycerophospholipid metabolism as a major target of thermal stress-induced lipid alterations. Pathway enrichment analysis revealed that these lipid alterations were significantly associated with glycerophosphatidylinositol (GPI)-anchor biosynthesis, autophagy pathways, glycerophospholipid metabolism, ferroptosis, and glycine, serine, and threonine metabolism (Figure 5c). The enrichment of ferroptosis-related pathways suggests that oxidative lipid damage may contribute to the cellular stress response induced by elevated temperature. Quantitative analysis of PEs showed significant increases in PE(15:0/24:1) and PE(17:2/20:0), decreases in PE(17:1/0:0), PE(17:2/22:6), and PE(22:4/20:2) in the thermal stress group, with some species nearly undetectable under high temperature (Figure 5d). Similarly, PI levels for PI(16:0/20:5) and PI(20:3/18:2) were drastically altered in the high-temperature group, suggesting a potential impairment in phosphoinositide-mediated signaling and membrane dynamics (Figure 5e). PS species including PS(20:5/22:6) and PS(22:2/22:1) were also significantly elevated under thermal stress, further indicating disruption in membrane phospholipid homeostasis (Figure 5f). Collectively, these data demonstrate that early-life thermal stress leads to extensive depletion of key membrane phospholipids, particularly PEs and PIs, and perturbs multiple lipid-associated pathways critical for membrane integrity, signaling, and stress adaptation. Such lipidomic shifts are likely to account for or exacerbate the morphological abnormalities, altered swimming behavior, and metabolic reprogramming observed in thermally stressed zebrafish larvae. Discussion This study provides compelling evidence that thermal stress during embryogenesis profoundly disrupts zebrafish development, behavior, and metabolism, culminating in large-scale lipidome remodeling. By integrating morphological analysis, behavioral assays, untargeted metabolomics, and targeted pathway mapping, we reveal a coherent physiological and biochemical responses to elevated temperature that spans from phenotypes to molecular pathways. Exposure to high temperature during embryogenesis significantly accelerated developmental timing, leading to premature hatching within 48 hpf. While rapid development can be an adaptive response to environmental stress, it occurred at the cost of normal morphology: morphological abnormalities such as PE, SC, YSE, and YND were frequent in the thermal stress group. These phenotypes suggest disruptions in cellular differentiation and tissue morphogenesis, potentially mediated by altered metabolic allocation during embryogenesis. By 14 dpf, larvae previously exposed to early-life heat stress exhibited hyperactivity, with significantly increased swimming velocity and total distance traveled. Heatmap and trajectory plots showed altered spatial occupancy patterns, including greater central zone usage and more erratic movement. These behavioral changes may reflect heightened metabolic rate, neuromuscular alterations, or shifts in anxiety-related behavior, all of which could be linked to early metabolic reprogramming 23 , 24 . Our findings are consistent with prior research across diverse fish species, which collectively demonstrate that thermal stress induces oxidative damage, structural tissue impairment, and systemic metabolic reprogramming 15 , 25 – 27 . Similar to our zebrafish results, catfish and seabass studies reported elevated oxidative stress and activation of heat shock responses, while Antarctic species exhibited functional tissue damage in critical organs such as gills 25 – 27 . Moreover, the rainbow trout study highlights that thermal stress-induced shifts in metabolism are often linked to microbial dysbiosis, supporting the idea that our observed metabolomic and lipidomic alterations may be part of a conserved multi-organ stress response 15 . Untargeted UPLC-QTOF-MS analysis showed clear separation between control and thermal stress groups, indicating robust metabolic divergence. Volcano plot and heatmap analyses identified broad changes in metabolite abundance, with pathway enrichment highlighting perturbations in purine metabolism, amino acid turnover, and lipid metabolism. The consistent clustering patterns suggest that thermal stress drives a coordinated biochemical adaptation, rather than random metabolic noise. Mapping of significantly altered metabolites revealed up-regulation of hypoxanthine, guanine, and guanosine, pointing to accelerated purine degradation. Additionally, elevated 6-lactoyltetrahydropterin implicates folate biosynthesis in the thermal stress response, potentially reflecting increased demand for nucleotide synthesis and methylation reactions during rapid development 28 , 29 . Conversely, depletion of galactose-1-phosphate suggests impaired amino sugar and nucleotide sugar metabolism, which could compromise glycoprotein and glycolipid synthesis, thereby contributing to developmental malformations 30 , 31 . Our metabolomic findings are in line with broader evidence showing that thermal stress triggers coordinated metabolic reprogramming to maintain energy balance, redox homeostasis, and biosynthetic capacity. Alterations in folate-related metabolites are consistent with multi-omics evidence that stress states, including heat stress, modulate the one-carbon folate cycle to support nucleotide biosynthesis and redox control, while influencing longevity-linked pathways through its downregulation 32 , 33 . Moreover, systemic metabolomic changes in amino acid, lipid, and microbial metabolism reported in livestock under diurnal heat stress closely mirror the patterns identified here, suggesting conserved mechanisms of thermal adaptation across vertebrates 34 . These patterns are further supported by studies in zebrafish showing that acute environmental temperature changes rapidly alter whole-organism metabolism and gene expression, particularly in pathways linked to energy turnover, redox regulation, and membrane remodeling 35 . In addition, thermal stress in zebrafish embryos has been shown to induce a positive phenotypic and molecular feedback loop that amplifies stress responses over developmental time, with long-term consequences for growth and behavior 14 . Together with previous findings across fish and other vertebrates, these results reinforce the view that thermal stress elicits a tightly coordinated, multi-level adaptive response spanning from molecular pathways to physiological and behavioral outcomes. Lipid profiles demonstrated significant changes of PEs, PIs, and PSs under thermal stress, lipid classes that are critical for membrane fluidity, signaling, and protein anchoring 36 . Pathway enrichment linked these changes to GPI-anchor biosynthesis, autophagy regulation, and ferroptosis. Reduced availability of structural phospholipids could destabilize cellular membranes, while ferroptosis-related signatures suggest susceptibility to oxidative lipid damage 37 – 39 . These alterations are consistent with the metabolic data, which indicated disrupted lipid metabolism and increased oxidative stress potential. These findings align with previous studies showing that thermal stress disrupts structural and signaling lipid homeostasis across diverse aquatic species. In black rockfish, acute heat exposure rapidly reprogrammed amino acid and lipid metabolism, paralleling the widespread phospholipid remodeling we observed 40 . In juvenile turbot, transcriptome-lipidome integration clarified suppression of lipid biosynthesis genes and activation of degradation pathways, matching our depletion of PE, PI, and PS 41 . Zebrafish embryo multi-stressor lipidomics demonstrated that heat stress specifically reduces structural phospholipids while enriching oxidized lipids, supporting the link between our observed phospholipid loss and oxidative susceptibility 42 . Multi-omics work in zebrafish embryos further showed that heat-induced metabolic shifts propagate to later life stages, consistent with our behavioral findings at 14 dpf 13 . Mechanistically, the connection between phospholipid depletion and ferroptosis, amplified by autophagy, offers a plausible pathway for heat-induced membrane destabilization 43 . Additionally, temperature-driven changes in neural lipid composition, as seen in adult zebrafish brains, implicates a role for lipid remodeling in behavioral alterations 44 . Finally, the dynamic lipidome reorganization observed under thermal stress in mammalian systems emphasizes the conserved nature of glycerophospholipid and sphingolipid remodeling, pointing out their central role in thermal adaptation 45 . Conclusion These findings support a model in which thermal stress during early development of zebrafish accelerates growth but imposes substantial energetic and biosynthetic demands. This reallocation of metabolic resources disrupts nucleotide and sugar metabolism, undermines structural glycosylation, and depletes essential membrane lipids. The resulting cellular instability manifests as morphological abnormalities, altered swimming behavior, and a systemic shift in biochemical pathways. Such changes may have ecological consequences, reducing survival and fitness under natural warming scenarios. Given the sensitivity of early developmental stages, the observed metabolic and lipidomic disruptions could serve as biomarkers for thermal stress in aquatic species. Furthermore, the identified link between elevated temperature, nucleotide metabolism, and lipid depletion highlights potential targets for intervention in aquaculture, where temperature fluctuations are increasingly common. Collectively, these findings illustrate that early metabolic and lipidomic alterations under thermal stress may provide critical indicators for assessing resilience and optimizing management strategies in aquaculture. Methods Animal Maintenance All animal experiments were conducted in accordance with the guidelines approved by Animal Care and Use Committee at SouthEast Medical Medi-chem Institute (SEMI-24-010). Zebrafish ( Danio rerio ) distributed from Zebrafish Center for Disease Modeling (ZCDM) were reared and acclimated at 26˚C ± 2 under a 14 h light and 10 h dark cycle in UV-sterilized culture water with pH 7.1–7.3 and 600–650 µS/cm of electrical conductivity. They were feed with Artermia (INVE SEP-ART, Australia) and Gemma Micro ZF 300 (Skeretting, USA) once a day. All animals were maintained at/under the automatic flow-through culture system (21C HighTech, Korea). For fertilization, male and female zebrafish in a 2:1 ratio were transferring to a new water tank installed with spawning tray, and they were kept in the dark. Embryos were produced within 30 min after light cycle beginning on the morning of the test day. To avoid genetic bias, the fertilized eggs from at least five different spawning tanks were mixed together and then randomly selected. The embryos were washed with culture water twice and E2 medium containing 7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO 4 , 75 µM KH 2 PO 4 , 25 µM Na 2 HPO 4 , 0.5 mM CaCl 2 , 0.35 mM NaHCO 3 . They were cultured at 26°C incubator (Daeyang, Korea) maximally for 2 h post-fertilization before test. Embryo Exposure Zebrafish embryos were collected at 2 hpf and randomly distributed into 6-well plates with 25 embryos per well in 10 ml of E2 medium. Embryos were incubated under two temperature conditions: 26˚C (control) and 34˚C (thermal stress). At 24 hpf, embryos were transferred to 24-well plates with one embryo per well in 2 ml of E2 medium, and maintained under the same temperature conditions until 72 hpf. The E2 medium was replaced with breeding water without additional supplementation. To minimize nutritional effects on early development, feeding was withheld until 6 hpf. At this stage, larvae were transferred to 1 L tanks (50 larvae per tank), acclimated for 8 h, and then fed twice daily with standard diet the end of the experiment. From 7 dpf onward, breeding water was partially replaced daily (20–30% volume change) to maintain water quality. Uneaten food and waste were removed daily. All experimental solutions were monitored for temperature, D.O. and pH twice daily to ensure stable conditions. Developmental and Morphological Assessments Embryonic development was monitored up to 72 hpf. For each experimental groups, 24 embryos were randomly selected and examined under on optical stereomicroscope (Nikon SMZ1500, Japan) at 24 h intervals to evaluate morphological progression. At 24 hpf, spontaneous movement was quantified by counting tail flicks during a 20 s interval. The hatching rate was determined at 72 hpf. Larval Swimming Behavior Analysis Developmental and behavioral assessments were conducted between 7 and 14 dpf. At 7 dpf, larvae were transferred individually into 14-well plates, and their locomotor activity was recorded for 5 min to characterize behavioral patterns. At 14 dpf, body weight and length were measured following anesthesia with tricaine methanesulfonate (MS-222, 200 mg/L) in accordance with ARRIVE guidelines, and these individuals were subsequently excluded from molecular analyses to prevent bias introduced by anesthetic exposure. Following measurement, the anesthetized animals were allowed to recover and were then reared to adulthood under standard conditions for subsequent studies. Locomotor activity was analyzed using the EthoVision-XT tracking system (Noldus Information Technology, Netherlands). Four behavioral parameters were evaluated: (i) total distance moved, defined as the cumulative distance traveled by each larva during the 5 min recording; (ii) mean velocity, calculated as the average swimming speed across the recording period; (iii) cumulative duration of movement, representing the total time the larva was actively swimming; and (iv) cumulative duration of inactivity, defined as the total time spent immobile. These parameters collectively provided a quantitative assessment of larval swimming performance under different thermal conditions. Sample Preparation for Untargeted Metabolomics For metabolomic analysis, 3 dpf zebrafish larvae were euthanized by rapid hypothermic shock through immersion in liquid nitrogen, and death was confirmed by the absence of heartbeat and movement, in accordance with ARRIVE guidelines. Metabolites were extracted from male zebrafish larvae using ice-cold extraction solvent mixture consisting of 40% acetonitrile, 40% methanol, and 20% distilled water (v/v/v). Samples were snap-frozen in liquid nitrogen, and subsequently homogenized on ice using a motorized homogenizer with a sterilized pestle in extraction solvent. Homogenates were further disrupted in an ultrasonic water bath with three cycles of 30 s sonication followed by 30 s rest on ice (total 3 min). The lysates were then incubated on ice for 10 min and centrifuged at 16,000 x g for 15 min at 4°C. The resulting supernatants were filtered through 0.2 µm PVDF membranes (Whatman, Cytiva, UK), and the filtrates were transferred into screw-cap glass vials with inserts (Agilent Technologies, Santa Clara, CA, USA) for subsequent UPLC-QTOF-MS analysis. Ultrahigh Performance Liquid Chromatography and Mass Spectrometry (UPLC-QTOF MS) Untargeted metabolomic profiling was performed using a quadrupole time-of-flight mass spectrometer coupled to an ultrahigh-performance liquid chromatography system (UPLC-QTOF-MS; Agilent Technologies, USA). Samples were analyzed on an Agilent 1290 Infinity LC system equipped with an InfinityLab Poroshell 120 HILIC-Z column (2.1 x 100 mm, 2.7 µm; Agilent Technologies) maintained at 25°C. A 3 µL aliquot of each sample was injected for analysis. The mobile phases consisted of (A) 10 mM ammonium formate in water with 0.1% formic acid and (B) 10 mM ammonium formate in 90% acetonitrile and 10% water with 0.1% formic acid. Chromatographic separation was achieved with a gradient elution at a flow rate of 0.25 mL/min as follows: 0–3 min, 2% A; 3–11 min, linear increase to 30% A; 11–12 min, 40% A; 12–16 min, 95% A; 16–18 min, held at 95% A; 18–19 min, returned to 2% A; and 19–20 min, re-equilibration at 2% A. A port-run time of 4 min was applied for column re-equilibration. The QTOF-MS was operated in positive ionization mode using a Dual AJS ESI source. The capillary voltage was set at 3,000 V with a fragmentor voltage of 125 V and skimmer voltage of 65 V. The drying gas maintained at 225°C with a flow of 6 L/min, while the nebulizer was set at 40 psi. The sheath gas was supplied at 10 L/min at 225°C. The RF voltage was 450 V. Full-scan mass spectra were acquired over the m/z 50–1,000 range. Data Processing and Analysis Raw data files (*.d) were first converted to *.cef format using Agilent Profinder 10.0. Feature extraction, peak alignment, and metabolite annotation were subsequently carried out wianth Agilent MassHunter Mass Profiler Profession 15.0. The resulting data matrix of identified metabolites was exported for downstream analysis. Metabolite enrichment and pathway analyses were performed using MetaboAnalyst 6.0 ( http://www.metaboanalyst.ca ; accessed January 27, 2025). Multivariate statistical methods, including PCA for unsupervised clustering, were applied to assess global metabolomic differences between groups. Pathway enrichment and topology analyses were conducted within MetaboAnalyst based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify significantly perturbed metabolic pathways Statistical Analysis Data analyses were performed in GraphPad Prism 10 (GraphPad Software, USA) and MetaboAnalyst 6.0 ( http://www.metaboanalyst.ca ). Data are expressed as mean ± standard error of the mean (SEM) for continuous variables or proportions (%) with 95% confidence intervals (Cis) for categorical outcome such as hatching and malformation rates. Normality of data distribution was assessed using the Shapiro-Wilk test. Differences between control and treatment groups were evaluated using unpaired two-tailed Student’s t -tests for continuous variable and Fisher’s exact test for categorical variables. A p -value < 0.05 was considered statistically significant. Declarations Competing interests The authors declare no competing interests. Fund Declaration This work was supported by the Pukyong National University Industry-university Cooperation Foundation’s 2024 (202418860001), the Global Joint Research Program funded by the Pukyong National University (202506320001), and a grant from the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (Project No.: RS-2023-00232749). Author Contribution Conceptualization: JJ, BK; Methodology: JEK, AHP, JJ, BK; Investigation (Animal maintenance & Experiments): JEK, AHP, BK; Formal Analysis (Metabolomics): HHJ, JJ, BK; Formal Analysis (Behavioral/Locomotor Activity): JEK, AHP, KBH; Writing – Original Draft: JJ, BK; Writing – Review & Editing: JJ, BK, HHJ; Funding Acquisition: JJ, BK. All authors read and approved the final manuscript. 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J. Lipidomics reveals multiple stressor effects (temperature x mitochondrial toxicant) in the zebrafish embryo toxicity test. Chemosphere 264 , 128472. 10.1016/j.chemosphere.2020.128472 (2021). Lee, S. et al. Autophagy mediates an amplification loop during ferroptosis. Cell. Death Dis. 14 10.1038/s41419-023-05978-8 (2023). Maffioli, E. et al. Environmental Temperature Variation Affects Brain Lipid Composition in Adult Zebrafish (Danio rerio). Int. J. Mol. Sci. 25 10.3390/ijms25179629 (2024). Solano, L. E. et al. Dynamic Lipidome Reorganization in Response to Heat Shock Stress. Int. J. Mol. Sci. 26 10.3390/ijms26072843 (2025). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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":1066785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of elevated temperature on zebrafish development and morphology.\u003c/strong\u003e (a) Experimental design schematic. Zebrafish embryos were exposed to control (26°C) or high-temperature (34°C) conditions from fertilized through early larval stages, followed by developmental and behavioral assessments. (b) Representative images illustrating normal development under control conditions and temperature-induced abnormalities at elevated temperature. Observed phenotypes include early coloration, premature hatching, and morphological deformities such as PE, YSE, YND, and SC. Scale bars: 200 μm (embryo images) and 500 μm (larva images)\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/8c2b5eccf42c0452e6d24e94.png"},{"id":92509421,"identity":"80b2d000-f28a-4f86-8c44-68842fd8cf5f","added_by":"auto","created_at":"2025-09-30 13:09:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1002592,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral consequences of thermal stress in zebrafish larvae. (a, b) Quantitative analyses of locomotor behavior, including total distance traveled (a) and mean swimming velocity (b). (c) Representative locomotor heatmaps and corresponding swim trajectories of zebrafish larvae reared at 26°C (control) and 34°C (thermal stress). Data are presented as mean ± SEM. Statistical significance was assessed using unpaired two-tailed Student t-tests; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/50e8bf3b0326aef690f1da35.png"},{"id":92509442,"identity":"70a2ee86-3323-465c-83c8-74cbf912a58d","added_by":"auto","created_at":"2025-09-30 13:09:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":427338,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolomic profiling of zebrafish larvae under thermal stress.\u003c/strong\u003e (a) Schematic overview of the experimental design showing zebrafish embryos exposed to control and high temperature from fertilization through early larval stages, followed by phenotypic assessment and metabolomic profiling using UPLC-QTOF-MS. (b) PCA score plots displaying clear separation between control and thermal stress groups, indicating distinct metabolomic signatures. (c) Volcano plot highlighting significantly upregulated (red) and downregulated (blue) metabolites between groups (d) Hierarchical clustering heatmap of differentially abundant metabolites, demonstrating distinct temperature-dependent metabolomic profiles. (e) Metabolite set enrichment analysis identifying pathways significantly enriched in the high-temperature groups. (e) Pathway impact analysis based on KEGG topology, emphasizing key metabolic pathways most perturbed by thermal stress.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/f7e60b0a57773774e3482e8e.png"},{"id":92509438,"identity":"4c489e13-3ffb-4141-a170-4d85d670992a","added_by":"auto","created_at":"2025-09-30 13:09:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":282376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered metabolic pathways in zebrafish larvae exposed to elevated temperature.\u003c/strong\u003e (a, b, d) Significantly altered metabolites, including 6-lactoyltetrahydroptein (a) and hypoxanthine, guanine, and guanosine (b), and galactose 1-phosphate (d). (c, e) Pathway analyses revealed significant perturbations in folate biosynthesis, purine degradation, and nucleotide sugar biosynthesis. Metabolites shown in red indicating upregulation, and blue denoting downregulation under thermal stress. Statistical analysis was performed using an unpaired two-tailed Student’s t-test with significance indicated as ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001. Pathway images in this figure are adapted from KEGG: Kyoto Encyclopedia of Genes and Genomes (\u003ca href=\"http://www.kegg.jp\"\u003ewww.kegg.jp\u003c/a\u003e), Kanehisa Laboratories, and are reproduced with permission \u003csup\u003e20-22\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/657efa025cc0f317ae3fef57.png"},{"id":92509887,"identity":"be37def8-c768-4474-84c3-2c7c598e85e7","added_by":"auto","created_at":"2025-09-30 13:17:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":272327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipid metabolism alterations in zebrafish larvae exposed to thermal stress.\u003c/strong\u003e (a) Heatmap illustrating the relative abundance of lipid metabolites between the control and high-temperature groups. (b) Pie chart showing the distribution of lipid classes detected in the metabolomic dataset. (c) Pathway enrichment analysis indicating significant perturbations in lipid-related pathways. (d – f) Quantitative comparisons of selected lipid species, showing significant differences in abundance between control and thermal stress groups. Statistical analysis was performed using an unpaired two-tailed Student’s t-test with significance indicated as **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/67558dfd0b45d126dc06ba73.png"},{"id":107483656,"identity":"51382aed-1326-46dc-ac4b-50069053d03a","added_by":"auto","created_at":"2026-04-22 02:28:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3522616,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7397518/v1/58d53088-1d9a-418c-a74b-923f66e1c082.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thermal Stress During Embryogenesis Alters the Metabolome of Zebrafish Larvae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobal warming has driven a continuous rise in average sea surface temperatures with new record highs being reached almost every year\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Aquatic ecosystems are particularly sensitive to climate changes, which alters the distribution and abundance of marine organisms and affects fisheries and aquaculture production by modifying the physiological and phenotypic traits of cultured species\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e–\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Such changes pose a direct threat to the fisheries industry and global food security\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e–\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Elevated water temperatures can influence critical biological processes in aquatic animals, including reproduction, growth rate, and behavioral patterns, thereby challenging population sustainability\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e–\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To cope with temperature perturbations, aquatic animals adjust their physiological and behavioral responses to maintain homeostasis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) are on established vertebrate aquatic model for studying the effects of environmental stressors due to their genetic tractability, transparent embryos, and well-characterized developmental timeline\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Their sensitivity to thermal variation, coupled with conserved stress response pathways, makes them an ideal system for dissecting the multi-scale impacts of heat exposure. Previous studies in zebrafish and other fish have demonstrated that thermal stress during embryonic and larval stages can accelerate developmental timing\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These outcomes are often accompanied by metabolic reprogramming due to the alteration of microbiota\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the mechanistic links between early-life heat exposure, behavioral alterations, and underlying metabolic and lipidomic remodeling remain incompletely understood.\u003c/p\u003e\u003cp\u003eMetabolites produced as intermediates or end products of metabolism serve as key indicators of the biochemical state of an organism. They provide a critical link among genotype, phenotype, and environmental conditions\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. By profiling metabolite abundance and composition, it is possible to capture the physiological adjustments organism make in response to environmental stressors such as heat\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Thus, integrating metabolomics with phenotypic and behavioral assessments offers a powerful framework for elucidating how thermal stress impacts biological function from molecular pathways to whole-animal performance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this study, we investigated the effect of early-life thermal stress on zebrafish embryonic development, larval swimming behavior, and biochemical profiles using untargeted metabolomics. Consequently, this work will provide new insight into the coordinated physiological and biochemical mechanisms supporting thermal stress adaptation in aquatic animals.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh temperature accelerates embryonic development and induces malformations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To investigate the physiological effects of thermal stress on fish, zebrafish embryos were exposed to either normal or high temperature conditions. Two temperature regimes were applied: 26\u0026deg;C as the control and 34\u0026deg;C as the thermal stress condition, based on previous findings from a fish treadmill assay\u003csup\u003e19\u003c/sup\u003e. The experimental design included multi-well plates for controlled temperature incubation and analysis at three developmental stages: embryo, early larva, and late larva (Figure 1a). Water parameters for the control group were maintained as 26.5 \u0026plusmn; 0.4\u0026deg;C, with dissolved oxygen (DO) ranging from 3.6 to 6.3 mg/L and pH 7.6 \u0026plusmn; 0.1. For the thermal stress group, conditions were maintained at 34.0 \u0026plusmn; 0.3\u0026deg;C, DO 3.3 \u0026ndash; 6.1 mg/L, and pH 7.6 \u0026plusmn; 0.1 (Figure 1a). Exposed to 34\u0026deg;C led to accelerated development, resulting in premature hatching by 48 hours post-fertilization (hpf). Morphological assessments displayed pronounced developmental abnormalities in the high-temperature group, including pericardial edema (PE), spinal curvature (SC), yolk sac edema (YSE), and yolk-not depleted (YND), as indicated in representative images (Figure 1b, summarized in Table 1). In contrast, embryos incubated at 26\u0026deg;C developed normally with no malformations observed. Quantitatively, 15 out of 24 embryos in the thermal stress group hatched at 48 hpf, while two embryos degenerated during development (Table 1). All embryos in the control group hatched normally and exhibited typical morphology (Table 1). These findings confirm that elevated temperature accelerates the developmental timeline but impairs morphological integrity, suggesting disrupted developmental signaling or altered energy allocation under stress conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e. Morphological changes of zebrafish embryos altered by high-temperature stress\u003c/strong\u003e (total 24 embryos per experimental group)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1759237617.png\" alt=\"image\"\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh temperature enhances larval locomotor activity. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To assess behavioral consequences of early-life thermal stress, swimming activity was analyzed in zebrafish larvae at 14 days post-fertilization (dpf) using an automated tracking system. The swimming patterns of individual larvae were quantified for total distance traveled and average velocity over a 5-min observation period. Larvae exposed to high temperature during development exhibited significantly increased locomotor activity compared to controls (Figure 2a, b). Specifically, both total distance and velocity were significantly elevated. Representative heat maps (top row of Figure 2c) showed intensified movement density, particularly in the central zone, while the track visualizations (bottom row) revealed increased frequency of circular and exploratory motion. This hyperactive phenotype is indicative of possible alterations in stress-related behavioral circuits caused by early developmental thermal stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomic profiling reveals distinct thermal stress-associated signatures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To elucidate the biochemical basis underlying the morphological and behavioral phenotypes induced by thermal stress, we conducted untargeted metabolomic profiling via UPLC-QTOF-MS on zebrafish larvae exposed to either 26\u0026deg;C (control) or 34\u0026deg;C (thermal stress) from the early larval stage (Figure 3a). A total 516 metabolites were significantly altered between the two groups. Principal component analysis (PCA) plot showed a distinct separation between the control and high-temperature groups with minimal intra-group variation (Figure 3b). This clear clustering indicates that thermal stress induced a consistent and substantial shift in the overall metabolomic profile. A volcano plot analysis identified numerous metabolites significantly up-regulated (red) or down-regulated (blue) in the high-temperature group compared to controls (Figure 3c). These differences highlight specific biochemical features that are responsive to thermal stress. Hierarchical clustering heatmap analysis demonstrated distinct metabolite expression patterns between the two groups (Figure 3d). The high-temperature group exhibited a characteristic profile with coordinated changes across multiple metabolite clusters, supporting the robustness of the group separation observed in Figure 3b. Pathway enrichment analysis indicated that the most significantly affected pathways including metabolism of energy carrying molecules and cell-cell interaction-related molecules (purine metabolism; amino sugar and nucleotide sugar metabolism), amino acid metabolism (\u0026beta;-alanine metabolism; histidine metabolism; lysine degradation), and lipid metabolism (glycerophospholipid metabolism, ether lipid metabolism) (Figure 3e). Bubble plot visualization of pathway analysis further confirmed that these pathways as the most significantly affected, with high enrichment ratios and statistical significance. Larger bubble sizes indicate pathways with greater numbers of altered metabolites, emphasizing their central role in the thermal stress response (Figure 3f). These results suggest that thermal stress disrupts both nucleic acid production and amino acid processing. Taken together, thermal stress during early zebrafish development results in a metabolic shift, characterized by altered purine metabolism, amino acid processing, and lipid biosynthesis. These biochemical changes are likely contributors to the observed morphological abnormalities and hyperactive swimming behavior in thermally stressed larvae. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermal stress modulates key metabolic pathways\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Pathway mapping of significantly altered metabolites revealed that thermal stress strongly affected folate biosynthesis, purine degradation, and amino sugar and nucleotide sugar metabolism. In the folate biosynthesis pathways, levels of lactoyltetrahydropterin were elevated in the thermal stress group, suggesting altered cofactor availability for enzymatic reactions linked to nucleotide and amino acid metabolism (Figure 4a, c). Within the purine degradation pathway, significant increases were detected in guanosine, guanine, and hypoxanthine. These metabolites are intermediates in purine catabolism, and their accumulation suggests enhanced nucleotide turnover or altered nucleic acid metabolism under elevated temperature conditions (Figure 4b, c). Such changes may reflect increased energy demand and accelerated cellular processes, constituent with the observed early hatching and hyperactivity in thermally stressed larvae. In amino sugar and nucleotide sugar metabolism, the level of galactose-1-phosphate (Gal-1-P) was reduced in the thermal stress group. This decrease could impair glycosylation processes and structural carbohydrate synthesis, potentially contributing to the morphological abnormalities observed during development (Figure 4d, e). Collectively, the targeted pathway analysis supports the global metabolomic findings, indicating that thermal stress induces a metabolic state characterized by elevated nucleotide turnover, disrupted carbohydrate metabolism, and altered cofactor biosynthesis. These molecular alterations align with the phenotypic outcomes, such as accelerated development, morphological malformations, and heightened locomotor activity, suggesting that the metabolic shifts may underlie the physiological behavioral consequences of early-life heat exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermal stress induces profound alterations in the lipidome of zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Lipidomic profiling revealed that early-life exposure to elevated temperature caused marked remodeling of the zebrafish larval lipidome compared to the control condition. Differential lipid profiles were visualized by heatmap analysis, which demonstrated clear separation between the control and thermal stress groups, with widespread reductions in multiple lipid species under high temperature (Figure 5a). Notably, lipid classes most affected included glycerophosphoethanolamines (PEs), phosphatidylinositols (PIs), and phosphatidylserines (PSs) (Figure 5a). Classification of altered lipids indicated that the majority of significantly changed metabolites belonged to PE and glycerophosphoinositols, followed by amino acids, peptides, and analogues; cholestane steroids; and fatty acids and conjugates (Figure 5b). This distribution highlights glycerophospholipid metabolism as a major target of thermal stress-induced lipid alterations. Pathway enrichment analysis revealed that these lipid alterations were significantly associated with glycerophosphatidylinositol (GPI)-anchor biosynthesis, autophagy pathways, glycerophospholipid metabolism, ferroptosis, and glycine, serine, and threonine metabolism (Figure 5c). The enrichment of ferroptosis-related pathways suggests that oxidative lipid damage may contribute to the cellular stress response induced by elevated temperature. Quantitative analysis of PEs showed significant increases in PE(15:0/24:1) and PE(17:2/20:0), decreases in PE(17:1/0:0), PE(17:2/22:6), and PE(22:4/20:2) in the thermal stress group, with some species nearly undetectable under high temperature (Figure 5d). Similarly, PI levels for PI(16:0/20:5) and PI(20:3/18:2) were drastically altered in the high-temperature group, suggesting a potential impairment in phosphoinositide-mediated signaling and membrane dynamics (Figure 5e). PS species including PS(20:5/22:6) and PS(22:2/22:1) were also significantly elevated under thermal stress, further indicating disruption in membrane phospholipid homeostasis (Figure 5f). Collectively, these data demonstrate that early-life thermal stress leads to extensive depletion of key membrane phospholipids, particularly PEs and PIs, and perturbs multiple lipid-associated pathways critical for membrane integrity, signaling, and stress adaptation. Such lipidomic shifts are likely to account for or exacerbate the morphological abnormalities, altered swimming behavior, and metabolic reprogramming observed in thermally stressed zebrafish larvae.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides compelling evidence that thermal stress during embryogenesis profoundly disrupts zebrafish development, behavior, and metabolism, culminating in large-scale lipidome remodeling. By integrating morphological analysis, behavioral assays, untargeted metabolomics, and targeted pathway mapping, we reveal a coherent physiological and biochemical responses to elevated temperature that spans from phenotypes to molecular pathways.\u003c/p\u003e\u003cp\u003eExposure to high temperature during embryogenesis significantly accelerated developmental timing, leading to premature hatching within 48 hpf. While rapid development can be an adaptive response to environmental stress, it occurred at the cost of normal morphology: morphological abnormalities such as PE, SC, YSE, and YND were frequent in the thermal stress group. These phenotypes suggest disruptions in cellular differentiation and tissue morphogenesis, potentially mediated by altered metabolic allocation during embryogenesis. By 14 dpf, larvae previously exposed to early-life heat stress exhibited hyperactivity, with significantly increased swimming velocity and total distance traveled. Heatmap and trajectory plots showed altered spatial occupancy patterns, including greater central zone usage and more erratic movement. These behavioral changes may reflect heightened metabolic rate, neuromuscular alterations, or shifts in anxiety-related behavior, all of which could be linked to early metabolic reprogramming\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Our findings are consistent with prior research across diverse fish species, which collectively demonstrate that thermal stress induces oxidative damage, structural tissue impairment, and systemic metabolic reprogramming\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Similar to our zebrafish results, catfish and seabass studies reported elevated oxidative stress and activation of heat shock responses, while Antarctic species exhibited functional tissue damage in critical organs such as gills\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Moreover, the rainbow trout study highlights that thermal stress-induced shifts in metabolism are often linked to microbial dysbiosis, supporting the idea that our observed metabolomic and lipidomic alterations may be part of a conserved multi-organ stress response\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUntargeted UPLC-QTOF-MS analysis showed clear separation between control and thermal stress groups, indicating robust metabolic divergence. Volcano plot and heatmap analyses identified broad changes in metabolite abundance, with pathway enrichment highlighting perturbations in purine metabolism, amino acid turnover, and lipid metabolism. The consistent clustering patterns suggest that thermal stress drives a coordinated biochemical adaptation, rather than random metabolic noise. Mapping of significantly altered metabolites revealed up-regulation of hypoxanthine, guanine, and guanosine, pointing to accelerated purine degradation. Additionally, elevated 6-lactoyltetrahydropterin implicates folate biosynthesis in the thermal stress response, potentially reflecting increased demand for nucleotide synthesis and methylation reactions during rapid development\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Conversely, depletion of galactose-1-phosphate suggests impaired amino sugar and nucleotide sugar metabolism, which could compromise glycoprotein and glycolipid synthesis, thereby contributing to developmental malformations\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our metabolomic findings are in line with broader evidence showing that thermal stress triggers coordinated metabolic reprogramming to maintain energy balance, redox homeostasis, and biosynthetic capacity. Alterations in folate-related metabolites are consistent with multi-omics evidence that stress states, including heat stress, modulate the one-carbon folate cycle to support nucleotide biosynthesis and redox control, while influencing longevity-linked pathways through its downregulation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, systemic metabolomic changes in amino acid, lipid, and microbial metabolism reported in livestock under diurnal heat stress closely mirror the patterns identified here, suggesting conserved mechanisms of thermal adaptation across vertebrates\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These patterns are further supported by studies in zebrafish showing that acute environmental temperature changes rapidly alter whole-organism metabolism and gene expression, particularly in pathways linked to energy turnover, redox regulation, and membrane remodeling\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In addition, thermal stress in zebrafish embryos has been shown to induce a positive phenotypic and molecular feedback loop that amplifies stress responses over developmental time, with long-term consequences for growth and behavior\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Together with previous findings across fish and other vertebrates, these results reinforce the view that thermal stress elicits a tightly coordinated, multi-level adaptive response spanning from molecular pathways to physiological and behavioral outcomes.\u003c/p\u003e\u003cp\u003eLipid profiles demonstrated significant changes of PEs, PIs, and PSs under thermal stress, lipid classes that are critical for membrane fluidity, signaling, and protein anchoring\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Pathway enrichment linked these changes to GPI-anchor biosynthesis, autophagy regulation, and ferroptosis. Reduced availability of structural phospholipids could destabilize cellular membranes, while ferroptosis-related signatures suggest susceptibility to oxidative lipid damage\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These alterations are consistent with the metabolic data, which indicated disrupted lipid metabolism and increased oxidative stress potential. These findings align with previous studies showing that thermal stress disrupts structural and signaling lipid homeostasis across diverse aquatic species. In black rockfish, acute heat exposure rapidly reprogrammed amino acid and lipid metabolism, paralleling the widespread phospholipid remodeling we observed\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In juvenile turbot, transcriptome-lipidome integration clarified suppression of lipid biosynthesis genes and activation of degradation pathways, matching our depletion of PE, PI, and PS\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Zebrafish embryo multi-stressor lipidomics demonstrated that heat stress specifically reduces structural phospholipids while enriching oxidized lipids, supporting the link between our observed phospholipid loss and oxidative susceptibility\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Multi-omics work in zebrafish embryos further showed that heat-induced metabolic shifts propagate to later life stages, consistent with our behavioral findings at 14 dpf\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Mechanistically, the connection between phospholipid depletion and ferroptosis, amplified by autophagy, offers a plausible pathway for heat-induced membrane destabilization\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Additionally, temperature-driven changes in neural lipid composition, as seen in adult zebrafish brains, implicates a role for lipid remodeling in behavioral alterations\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Finally, the dynamic lipidome reorganization observed under thermal stress in mammalian systems emphasizes the conserved nature of glycerophospholipid and sphingolipid remodeling, pointing out their central role in thermal adaptation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThese findings support a model in which thermal stress during early development of zebrafish accelerates growth but imposes substantial energetic and biosynthetic demands. This reallocation of metabolic resources disrupts nucleotide and sugar metabolism, undermines structural glycosylation, and depletes essential membrane lipids. The resulting cellular instability manifests as morphological abnormalities, altered swimming behavior, and a systemic shift in biochemical pathways. Such changes may have ecological consequences, reducing survival and fitness under natural warming scenarios. Given the sensitivity of early developmental stages, the observed metabolic and lipidomic disruptions could serve as biomarkers for thermal stress in aquatic species. Furthermore, the identified link between elevated temperature, nucleotide metabolism, and lipid depletion highlights potential targets for intervention in aquaculture, where temperature fluctuations are increasingly common. Collectively, these findings illustrate that early metabolic and lipidomic alterations under thermal stress may provide critical indicators for assessing resilience and optimizing management strategies in aquaculture.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAnimal Maintenance\u003c/h2\u003e\u003cp\u003e All animal experiments were conducted in accordance with the guidelines approved by Animal Care and Use Committee at SouthEast Medical Medi-chem Institute (SEMI-24-010). Zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) distributed from Zebrafish Center for Disease Modeling (ZCDM) were reared and acclimated at 26˚C ± 2 under a 14 h light and 10 h dark cycle in UV-sterilized culture water with pH 7.1–7.3 and 600–650 µS/cm of electrical conductivity. They were feed with Artermia (INVE SEP-ART, Australia) and Gemma Micro ZF 300 (Skeretting, USA) once a day. All animals were maintained at/under the automatic flow-through culture system (21C HighTech, Korea). For fertilization, male and female zebrafish in a 2:1 ratio were transferring to a new water tank installed with spawning tray, and they were kept in the dark. Embryos were produced within 30 min after light cycle beginning on the morning of the test day. To avoid genetic bias, the fertilized eggs from at least five different spawning tanks were mixed together and then randomly selected. The embryos were washed with culture water twice and E2 medium containing 7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 75 µM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 µM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.35 mM NaHCO\u003csub\u003e3\u003c/sub\u003e. They were cultured at 26°C incubator (Daeyang, Korea) maximally for 2 h post-fertilization before test.\u003c/p\u003e\u003ch3\u003eEmbryo Exposure\u003c/h3\u003e\u003cp\u003eZebrafish embryos were collected at 2 hpf and randomly distributed into 6-well plates with 25 embryos per well in 10 ml of E2 medium. Embryos were incubated under two temperature conditions: 26˚C (control) and 34˚C (thermal stress). At 24 hpf, embryos were transferred to 24-well plates with one embryo per well in 2 ml of E2 medium, and maintained under the same temperature conditions until 72 hpf. The E2 medium was replaced with breeding water without additional supplementation. To minimize nutritional effects on early development, feeding was withheld until 6 hpf. At this stage, larvae were transferred to 1 L tanks (50 larvae per tank), acclimated for 8 h, and then fed twice daily with standard diet the end of the experiment. From 7 dpf onward, breeding water was partially replaced daily (20–30% volume change) to maintain water quality. Uneaten food and waste were removed daily. All experimental solutions were monitored for temperature, D.O. and pH twice daily to ensure stable conditions.\u003c/p\u003e\u003ch2\u003eDevelopmental and Morphological Assessments\u003c/h2\u003e\u003cp\u003eEmbryonic development was monitored up to 72 hpf. For each experimental groups, 24 embryos were randomly selected and examined under on optical stereomicroscope (Nikon SMZ1500, Japan) at 24 h intervals to evaluate morphological progression. At 24 hpf, spontaneous movement was quantified by counting tail flicks during a 20 s interval. The hatching rate was determined at 72 hpf.\u003c/p\u003e\u003ch2\u003eLarval Swimming Behavior Analysis\u003c/h2\u003e\u003cp\u003eDevelopmental and behavioral assessments were conducted between 7 and 14 dpf. At 7 dpf, larvae were transferred individually into 14-well plates, and their locomotor activity was recorded for 5 min to characterize behavioral patterns. At 14 dpf, body weight and length were measured following anesthesia with tricaine methanesulfonate (MS-222, 200 mg/L) in accordance with ARRIVE guidelines, and these individuals were subsequently excluded from molecular analyses to prevent bias introduced by anesthetic exposure. Following measurement, the anesthetized animals were allowed to recover and were then reared to adulthood under standard conditions for subsequent studies. Locomotor activity was analyzed using the EthoVision-XT tracking system (Noldus Information Technology, Netherlands). Four behavioral parameters were evaluated: (i) total distance moved, defined as the cumulative distance traveled by each larva during the 5 min recording; (ii) mean velocity, calculated as the average swimming speed across the recording period; (iii) cumulative duration of movement, representing the total time the larva was actively swimming; and (iv) cumulative duration of inactivity, defined as the total time spent immobile. These parameters collectively provided a quantitative assessment of larval swimming performance under different thermal conditions.\u003c/p\u003e\u003ch2\u003eSample Preparation for Untargeted Metabolomics\u003c/h2\u003e\u003cp\u003e For metabolomic analysis, 3 dpf zebrafish larvae were euthanized by rapid hypothermic shock through immersion in liquid nitrogen, and death was confirmed by the absence of heartbeat and movement, in accordance with ARRIVE guidelines. Metabolites were extracted from male zebrafish larvae using ice-cold extraction solvent mixture consisting of 40% acetonitrile, 40% methanol, and 20% distilled water (v/v/v). Samples were snap-frozen in liquid nitrogen, and subsequently homogenized on ice using a motorized homogenizer with a sterilized pestle in extraction solvent. Homogenates were further disrupted in an ultrasonic water bath with three cycles of 30 s sonication followed by 30 s rest on ice (total 3 min). The lysates were then incubated on ice for 10 min and centrifuged at 16,000 x g for 15 min at 4°C. The resulting supernatants were filtered through 0.2 µm PVDF membranes (Whatman, Cytiva, UK), and the filtrates were transferred into screw-cap glass vials with inserts (Agilent Technologies, Santa Clara, CA, USA) for subsequent UPLC-QTOF-MS analysis.\u003c/p\u003e\u003ch2\u003eUltrahigh Performance Liquid Chromatography and Mass Spectrometry (UPLC-QTOF MS)\u003c/h2\u003e\u003cp\u003eUntargeted metabolomic profiling was performed using a quadrupole time-of-flight mass spectrometer coupled to an ultrahigh-performance liquid chromatography system (UPLC-QTOF-MS; Agilent Technologies, USA). Samples were analyzed on an Agilent 1290 Infinity LC system equipped with an InfinityLab Poroshell 120 HILIC-Z column (2.1 x 100 mm, 2.7 µm; Agilent Technologies) maintained at 25°C. A 3 µL aliquot of each sample was injected for analysis. The mobile phases consisted of (A) 10 mM ammonium formate in water with 0.1% formic acid and (B) 10 mM ammonium formate in 90% acetonitrile and 10% water with 0.1% formic acid. Chromatographic separation was achieved with a gradient elution at a flow rate of 0.25 mL/min as follows: 0–3 min, 2% A; 3–11 min, linear increase to 30% A; 11–12 min, 40% A; 12–16 min, 95% A; 16–18 min, held at 95% A; 18–19 min, returned to 2% A; and 19–20 min, re-equilibration at 2% A. A port-run time of 4 min was applied for column re-equilibration. The QTOF-MS was operated in positive ionization mode using a Dual AJS ESI source. The capillary voltage was set at 3,000 V with a fragmentor voltage of 125 V and skimmer voltage of 65 V. The drying gas maintained at 225°C with a flow of 6 L/min, while the nebulizer was set at 40 psi. The sheath gas was supplied at 10 L/min at 225°C. The RF voltage was 450 V. Full-scan mass spectra were acquired over the m/z 50–1,000 range.\u003c/p\u003e\u003ch2\u003eData Processing and Analysis\u003c/h2\u003e\u003cp\u003eRaw data files (*.d) were first converted to *.cef format using Agilent Profinder 10.0. Feature extraction, peak alignment, and metabolite annotation were subsequently carried out wianth Agilent MassHunter Mass Profiler Profession 15.0. The resulting data matrix of identified metabolites was exported for downstream analysis. Metabolite enrichment and pathway analyses were performed using MetaboAnalyst 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed January 27, 2025). Multivariate statistical methods, including PCA for unsupervised clustering, were applied to assess global metabolomic differences between groups. Pathway enrichment and topology analyses were conducted within MetaboAnalyst based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify significantly perturbed metabolic pathways\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData analyses were performed in GraphPad Prism 10 (GraphPad Software, USA) and MetaboAnalyst 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Data are expressed as mean ± standard error of the mean (SEM) for continuous variables or proportions (%) with 95% confidence intervals (Cis) for categorical outcome such as hatching and malformation rates. Normality of data distribution was assessed using the Shapiro-Wilk test. Differences between control and treatment groups were evaluated using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests for continuous variable and Fisher’s exact test for categorical variables. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFund Declaration\u003c/h2\u003e\u003cp\u003eThis work was supported by the Pukyong National University Industry-university Cooperation Foundation\u0026rsquo;s 2024 (202418860001), the Global Joint Research Program funded by the Pukyong National University (202506320001), and a grant from the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea (Project No.: RS-2023-00232749).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: JJ, BK; Methodology: JEK, AHP, JJ, BK; Investigation (Animal maintenance \u0026amp; Experiments): JEK, AHP, BK; Formal Analysis (Metabolomics): HHJ, JJ, BK; Formal Analysis (Behavioral/Locomotor Activity): JEK, AHP, KBH; Writing \u0026ndash; Original Draft: JJ, BK; Writing \u0026ndash; Review \u0026amp; Editing: JJ, BK, HHJ; Funding Acquisition: JJ, BK. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheng, L. J. et al. Record High Temperatures in the Ocean in 2024. \u003cem\u003eAdv. Atmos. Sci.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00376-025-4541-3\u003c/span\u003e\u003cspan address=\"10.1007/s00376-025-4541-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerry, A. L., Low, P. J., Ellis, J. R. \u0026amp; Reynolds, J. D. 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Sci.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms26072843\u003c/span\u003e\u003cspan address=\"10.3390/ijms26072843\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Zebrafish, Thermal stress, Metabolomics, Lipidome, Climate change, Embryogenesis","lastPublishedDoi":"10.21203/rs.3.rs-7397518/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7397518/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlobal warming has led to continuous increases in marine surface temperatures, posing significant challenges to aquatic ecosystems and the aquaculture industry. Thermal stress during early development can profoundly alter physiological, morphological, and metabolic process in fish, with potential long-term consequences for growth, behavior, and survival. In this study, we investigated the effects of elevated temperature during zebrafish embryogenesis on developmental timing, morphological phenotypes, larval behavior, and metabolic regulation. Embryos were exposed to control and thermal stress conditions, and subsequent phenotypic and biochemical analyses were conducted from hatching through the larval stage. Thermal stress accelerated hatching to within 48 hpf, but this was accompanied by morphological abnormalities. At the late larval stage, larvae exposed to thermal stress exhibited significantly increased swimming velocity and distance with altered spatial occupancy patterns. Untargeted metabolomic profiling via UPLC-QTOF-MS elucidated alterations in purine metabolism, amino acid turnover, nucleotide metabolism, and phospholipid composition. Notably, phosphatidylethanolamines and phosphatidylserines were significantly depleted and phosphatidylinositols was elevated, implicating disruptions in pathways involved in membrane integrity, autophagy regulation, and ferroptosis. These findings demonstrate that thermal stress during embryogenesis elicits coordinated physiological, behavioral, and molecular responses in zebrafish, providing mechanistic insights into the vulnerability of aquatic organisms to climate change-driven temperature fluctuations.\u003c/p\u003e","manuscriptTitle":"Thermal Stress During Embryogenesis Alters the Metabolome of Zebrafish Larvae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 13:09:09","doi":"10.21203/rs.3.rs-7397518/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d017dbed-1ce9-41d6-9866-34b7bf402da7","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55481417,"name":"Biological sciences/Developmental biology"},{"id":55481418,"name":"Biological sciences/Ecology"},{"id":55481419,"name":"Earth and environmental sciences/Ecology"},{"id":55481420,"name":"Biological sciences/Physiology"},{"id":55481421,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-04-18T00:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 13:09:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7397518","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7397518","identity":"rs-7397518","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}