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Neurological disorders, including Alzheimer's disease, Parkinson's disease, and stroke, remain major causes of global disability and mortality, with limited neuroprotective therapies available. Traditional Chinese medicine (TCM) offers multi-target therapeutic potential, but its mechanistic complexity requires systematic investigation using appropriate model systems. Zebrafish (Danio rerio) has emerged as a valuable vertebrate platform for TCM neuroprotection research due to its genetic homology with humans, optical transparency, and high-throughput screening compatibility. This review summarizes the application of zebrafish models in studying TCM for Alzheimer's disease, Parkinson's disease, cerebral ischemia, epilepsy, insomnia, depression, and spinal cord injury. Key findings indicate that TCM metabolites exert neuroprotective effects through multiple mechanisms, including anti-oxidative stress, anti-neuroinflammation, anti-apoptosis, neurotransmitter modulation, neurogenesis promotion, and vascular protection. Zebrafish models have proven particularly useful for high-throughput screening of active metabolites, real-time in vivo imaging of neurovascular processes, and rapid safety assessment. However, limitations such as the absence of a layered neocortex, differences in drug metabolism, and the predominantly acute nature of current models must be acknowledged. Addressing these challenges through model standardization, multi-omics integration, and cross-species validation will further enhance the translational relevance of zebrafish-based TCM research. This review provides a practical framework for leveraging zebrafish models to advance the mechanistic understanding and clinical development of neuroprotective TCM therapies.
The ability to inactivate gene function in an adult organism is essential for studies of biological processes such as regeneration and behavior. This is best achieved by engineering an allele that could be conditionally inactivated using drug-inducible Cre recombinase. Several recent studies clearly demonstrate the feasibility of engineering such conditional alleles in zebrafish. Meanwhile, achieving a sufficient degree of recombination to induce complete loss of function has remained a major limitation. Herein, we address this limitation by engineering a recombinant ubiquitin promoter ubbR consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carp beta-actin2 gene. Using phiC31-mediated targeted integration, we demonstrate that ubbR outperforms both parental promoters at all embryonic stages tested. Furthermore, the ubbR:CreERT2 driver line we generated ensures a high-level recombination of floxed alleles in adult zebrafish tissues. Finally, we demonstrate that our ubbR promoter-driven construct retains high activity when integrated at other genomic loci, making this promoter a promising new tool for robust expression of transgenes at all stages of zebrafish ontogenesis.
For eukaryotic organisms the maintenance and regulation of cellular homeostasis is essential. This process requires continuous elimination of cellular debris, including misfolded, oxidized, and aggregated proteins, and damaged/obsolete organelle. It also requires the remodelling of the cytoplasm compartments, and the provision of nutrients to support basic cellular functions. One of the central molecular mechanisms that maintain this precarious homeostasis is the recycling program of the cell: autophagy. Dysregulation of autophagy has a relationship with several diseases, including neurodegeneration, metabolic diseases, age-related disorders, heart dysfunction, cancer, and inflammatory diseases. The regeneration of some tissues and organs in humans is limited, which hinders our capacity to recover from diseases. Regenerative ability varies from species, organs, tissues, and development stages. Whereas mammals have a more limited regenerative capacity, restricted to few tissues or organs, such as skin or liver, other vertebrates, such as axolotls, salamanders, goldfish and zebrafish, possess a higher regenerative capacity. Zebrafish display exceptional regenerative abilities. Both embryo and adult zebrafish can regenerate organs like the fin, heart, liver, kidney, muscle, spinal cord, retina and optic nerves, and several brain regions. In recent years, several works remark the crucial role of autophagy in the repair of damaged tissues and the replacement of impaired organs or body parts after injury. Investigating the cellular function of autophagy in regenerative processes will allow us to develop new therapeutic strategies for human disorders. In this chapter, we discuss the current evidence supporting the important role of autophagy in mediating regenerative processes, emphasizing the relevance of zebrafish as a prominent vertebrate model to study autophagy.
Truncating variants in the DSP gene are associated with non-dilated left ventricular cardiomyopathy (ND-LVC), characterized by impaired ventricular function without dilation. The influence of physical activity in disease onset, severity, and progression in DSP variant carriers remains unclear. This study aimed to generate a zebrafish model carrying a human DSP truncating variant to characterize homozygous dspb-/- mutants in terms of cardiac function, structure, and gene expression. The impact of moderate and endurance exercise was additionally evaluated. Clinical data from human carriers were also analysed to assess the relationship between physical activity, age at diagnosis, disease severity and arrhythmic events. A CRISPR/Cas9-generated dspb-/- zebrafish model (p.T449fs*) was used to characterize structural, functional, and molecular phenotypes. Moderate exercise training protocols were applied to assess their effect on cardiac function, endurance capacity, and survival. Physically active human carriers were diagnosed at a younger age but did not show increased disease severity or events rates. Zebrafish mutants exhibited baseline systolic dysfunction, sarcomeric disorganization, and signaling pathway dysregulation. Importantly, moderate exercise partially restored cardiac function and improved endurance without increasing mortality. These findings indicate that exercise may serve as a phenotypic modifier in DSP-related ND-LVC, enhancing functional outcomes without accelerating disease progression. The dspb-/- zebrafish model provides a robust translational platform to investigate the interactions between lifestyle factors and the pathogenesis of inherited desmosomal cardiomyopathies.
The increasing environmental release of cerium oxide nanoparticles (CeO2 NPs) raised concerns about their potential risks to aquatic organisms, yet their chronic effects remained poorly understood. This study investigated the impact of CeO2 NPs on the glucose and lipid metabolic homeostasis of the gut-liver axis and their bioaccumulation patterns in adult zebrafish. Exposure to 100 μg/L and 1000 μg/L CeO2 NPs for 26 days inhibited the growth of zebrafish and caused intestinal damage. The analysis of inductively coupled plasma mass spectrometry (ICP-MS) indicated that Ce was concentrated in the intestinal tract and liver, with less bioaccumulation in muscle. In the groups treated with 100 and 1000 μg/L CeO2 NPs, the expression of glycolipid-related genes was downregulated in liver of zebrafish. Hepatic biochemical assays showed that exposure to CeO2 NPs (100 and 1000 μg/L) resulted in decreased levels of pyruvate and elevated levels of glucose. It also led to increased content of lipid in the liver, manifesting as increased levels of triglycerides (TG) and low-density lipoprotein (LDL). In addition, sequencing of intestinal 16S rRNA amplicons revealed that CeO2 NPs significantly altered the microbial community composition and induced intestinal injury. This study demonstrated that CeO2 NPs disrupted the gut-liver axis metabolic homeostasis and induced tissue-specific accumulation in zebrafish, illustrating its multifaceted toxicological impact.
RNA binding proteins have multiple diverse cellular functions and are often mis-regulated in disease. Despite their many cellular functions and implications in disease, very little is known about their physiological functions. Here we describe a novel zebrafish knockout model of the RNA binding proteins Hnrnpa1 and Hnrnpa3. Loss of Hnrnpa3 in zebrafish has no obvious morphological phenotype. Similarly, single mutants of the duplicated zebrafish hnrnpa1 genes, hnrnpa1a and hnrnpa1b, have no discernible phenotype, whereas the hnrnpa1a; hnrnpa1b double mutants are embryonic lethal. They display muscle, vascular and developmental defects with a reduced volume of the yolk extension. Metabolic profiling revealed severe changes in lipid metabolism in the hnrnpa1a; hnrnpa1b double mutants. Our analysis identified the involvement of Hnrnpa1 in many cellular pathways including the regulation of lipid metabolism and opens the door for future therapeutic studies in HNRNPA-associated diseases.
Adaptive flexibility is essential for organisms to survive and thrive in the face of environmental pressures. Throughout their life cycle, animals experience diverse stressful conditions. Early life stages are considered highly sensitive to environmental stressors, which may, during these stages, affect brain function, leading to persistent behavioural disruption. Notably, individuals often differ in their responses. Coping styles represent consistent, individual-specific behavioural responses to stressors, shaped by both genetic and environmental factors, and play a key modulating role in how animals respond to stress. Coping styles fall along a shy-bold behavioural continuum, reflecting differences in behavioural responses to stressors, with bold individuals being proactive and risk-prone, and shy ones reactive and cautious. This work aimed to study how zebrafish early life stress (ELS) influences later-life coping behaviours. To achieve this goal, zebrafish larvae were acutely exposed, at three specific developmental stages, 4-, 7-, and 15 days post-fertilisation (dpf), to a sequence of non-chemical environmental stressors and coping styles were assessed in the juvenile stage. Overall, the obtained results show that ELS modulates zebrafish later behavioural phenotype expression, altering the neurochemical profile and stress axis function. The developmental stage most sensitive to early-life stress was 15 dpf, with fish exhibiting lower activity than controls, regardless of their later-emerging coping style. Juveniles with a shy phenotype exhibited heightened susceptibility to ELS consequences over time, exhibiting a greater range of behavioural and physiological alterations than bold individuals, demonstrating that shy fish retain stronger biological imprints of ELS across development.
Paclobutrazol (PBZ), a triazole-based plant growth retardant, is widely employed in agriculture and horticulture to control plant height and promote root growth. However, its persistence and mobility in the environment raise concerns regarding its bioavailability and toxicity in aquatic ecosystems. In this study, we investigated the reproductive toxicity of PBZ in zebrafish (Danio rerio), a well-established model for aquatic toxicology and vertebrate reproductive research. Adult zebrafish were exposed to PBZ (0, 0.1, 1 ppm) for 2, 4, 6, or 8 weeks. After exposure, breeding was conducted using exposed or unexposed pairs to assess maternal, paternal, and combined effects on reproduction. Fertilization and hatching rates were quantified, and gonads were processed for histological evaluation. Chronic PBZ exposure significantly reduced fertilization and hatching success in both sexes, especially at higher concentrations and longer exposure durations. Histological examination revealed a reduction in testicular cyst area without significant changes in gonadosomatic index (GSI). In ovaries, structural integrity remained largely intact, although a mild increase in atretic follicles was observed. These findings demonstrate that PBZ impairs zebrafish reproductive success through subtle but consequential gonadal disruptions and suggest potential risks to aquatic biodiversity and population sustainability.
Zebrafish larvae are increasingly used in behavioural pharmacology because of their translational relevance and suitability for anxiety- and stress-related studies. However, it remains unclear whether commonly used behavioural assays and molecular stress markers provide convergent evidence of anxiolytic-like drug activity. This study therefore aimed not to validate a single predictive screening platform, but to assess the concordance and interpretative value of behavioural and molecular endpoints in larval zebrafish anxiety-related drug screening. Reference compounds with established anxiolytic or antidepressant activity, including diazepam, amitriptyline, and fluoxetine, were used to anchor assay performance. TP003, a GABAergic modulator, and three serotonergic psychedelic compounds, DOI, 5-MeO-DMT, and psilocybin, were included to challenge the assays with pharmacologically diverse mechanisms relevant to stress- and anxiety-related responses. Diazepam showed the strongest cross-assay behavioural consistency compatible with an anxiolytic-like profile. However, this response was not accompanied by reduced cortisol levels or straightforward normalisation of stress-related gene expression. Amitriptyline reduced cortisol but produced only partial behavioural effects, whereas DOI affected selected behavioural preference endpoints and transcriptional markers without significantly reducing cortisol. Fluoxetine and 5-MeO-DMT altered locomotor activity in patterns requiring cautious interpretation, as reduced movement may reflect locomotor suppression or sedation rather than anxiolysis alone. These findings indicate that behavioural and molecular endpoints should not be treated as interchangeable measures of a single anxiety construct. Instead, combined behavioural and molecular assessment is most useful as a profiling framework to identify endpoint-dependent responses, detect sedative or nonspecific locomotor confounds, and support more cautious interpretation of putative anxiolytic-like effects in larval zebrafish.
Immunosuppression increases susceptibility to infections, highlighting the need for safe immunomodulatory strategies. This study investigated the immunomodulatory effects of wheat peptides (WP) and identified bioactive peptide sequences using zebrafish and mouse models. In chloramphenicol (CAP)-induced immunosuppressed zebrafish, WP (150 μg/mL) significantly restored macrophage counts (32.25% increase), neutrophil fluorescence intensity (28.87% increase), and IFN-γ levels (42.34% increase). In cyclophosphamide (CTX)-induced immunosuppressed mice, WP (0.5 g/kg) significantly increased spleen index, thymus index, serum IgA and IFN-γ levels, and fecal short-chain fatty acid concentrations. LC-MS/MS peptidomics identified peptide sequences; molecular docking targeting CHRM1 revealed that the octapeptide GFNDLGKR (GR8) exhibited the strongest binding affinity (CDOCKER energy = -161.15 kcal/mol). GR8 (10 μg/mL) validation in zebrafish significantly increased macrophage counts (60.62%), neutrophil fluorescence intensity (44.03%), and IFN-γ levels (60.84%). These findings demonstrate that WP exerts immunomodulatory effects through restoration of immune cells, organ indices, cytokines, and short-chain fatty acids (SCFA), and identify GR8 as a promising bioactive peptide for functional food development.
Voltage imaging has emerged as a powerful tool for recording membrane potential changes in living cells, offering a direct measurement of rapid neuronal events with high temporal precision. Since the brain is a three-dimensional circuit, it is essential to record signals across a volume. However, achieving effective three-dimensional voltage imaging over large neuronal populations remains challenging due to the need for high imaging speed, high signal-to-noise ratio, and extensive volume coverage. In this study, we demonstrate in vivo three-dimensional voltage imaging in larval zebrafish using oblique plane microscopy and QFDBD-QUAS-driven expression of the genetically encoded voltage indicator Ace-mNeon2-Kv2.1, achieving volumetric imaging rates of up to 200 volumes per second (VPS). This approach enables dye-free voltage imaging, simplifying experimental workflows and improving the reproducibility of in vivo voltage imaging experiments for investigating neuronal circuit dynamics in the living zebrafish animal model.
Animals undergo major behavioral adjustments during ontogeny, but how the underlying cognitive algorithms change during this process remains elusive. Here, we describe that zebrafish shift from light-seeking to dark-seeking as they grow from larval to juvenile stage. Combining complementary phototaxis assays in virtual reality and modeling, we dissect the computational basis of this transition. We identify three parallel pathways, one analyzing ambient whole-field luminance levels, one spatially comparing light levels across the eyes, and one computing luminance change in each eye. Larvae mostly use the latter two computations, whereas juveniles largely employ the first one. Using a library of agent-based models, we predict behavior in more complex environments. Model-based extraction of latent cognitive variables suggests potential neural correlates of this behavioral inversion. We suggest that zebrafish phototaxis is regulated via parallel processing streams, which could be a universal mechanism for adjusting behavior depending on developmental stage, context, or internal state.
Human-induced environmental change increases unpredictability, disrupting habitats and social structures in ways that many animals are poorly adapted to. This may also reduce the reliability of information faster as contingencies shift. While independently acquired information is often more accurate, increased unpredictability may make recency the key to reliability, increasing the value of social information, which is cheaper and faster to update. Unpredictability might thus shift the balance between the values of these types of information, increasing reliance on more recent social information. Thus, independent of information quality, the predictability of background conditions may affect how animals make decisions. To test whether living in an unstable environment changes the inherent value of information, zebrafish (Danio rerio) were housed for 3 mo under either highly unpredictable Dynamic (water temperature, feeding times, habitat complexity, group size and membership fluctuated) or Stable conditions. Across 6 behavioral assays, Dynamic condition fish showed more information-seeking, greater attention to social stimuli, more sensitivity to social cues, and were less coordinated but swam closer together in shoals. When personal and social information conflicted, they were also more likely to prioritize recent social information over previously learned personal information. Together, these results indicate that long-term housing in an unpredictable environment diminishes the value of information faster, raises the value of social information to allow for faster updating, shifts decision-making strategies independent of the quality of information itself, and disrupts coordinated schooling in zebrafish. This sensitivity may fundamentally alter how animal collectives navigate an increasingly unpredictable world.
Larval zebrafish are often mounted laterally to ensure consistent anatomical positioning and to standardize imaging of body axes across early development. However, this conventional approach often tethers sample orientation to a single microscope configuration and limits optical accessibility. We present a mounting protocol for larval zebrafish that enables optical access from both dorsal and ventral orientations while preserving lateral sample position. This approach uses common laboratory consumables to establish a mounting platform that eliminates any need to remount samples between the use of upright and inverted microscopes. By establishing a hydrophobic seal, mounted embryos can be inverted with ease to access the sample from either orientation. A seamless transition here facilitates reliable identification and longitudinal tracking of the same biological region of interest across microscope configurations. This protocol is broadly applicable to live imaging experiments requiring flexibility in imaging geometry, minimal sample handling, and high reproducibility. Key features • Dual-configuration mounting setup can be established from standard, low-cost consumables in any laboratory setting. • Preserves lateral mounting in agarose for consistent anatomical positioning and experimental continuity. • Enables seamless transition between upright and inverted microscopes without the need for specimen remounting.
N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a rubber antioxidant commonly used to manufacture automobile tires. The tire-road wear particles (TRWP) generated during the driving of automobiles, carried by rainwater and surface runoff, will bring the 6PPD and polyethylene microplastics contained in TRWP into the water body, posing a threat to the health and safety of aquatic life. However, the cardiotoxic effects and underlying mechanisms of their combined exposure remain unclear. In this study, zebrafish were exposed to PE-NPs (50 nm in diameter), 6PPD, and their mixtures to investigate cardiac developmental toxicity and molecular mechanisms. The results showed that 6PPD alone caused cardiac phenotypic abnormalities, including shortened SV-BA distance, reduced stroke volume, and decreased heart rate. Co-exposure with PE-NPs further aggravated these toxic phenotypes and significantly disrupted the expression of genes related to myocardial contraction, cardiac development, and heart failure. Mechanistically, PE-NPs enhanced the pericardial accumulation of nanoplastics, triggered more severe ROS overproduction, and induced oxidative stress by upregulating transcription of gpx, cat, and CuZn-SOD while reducing SOD, CAT, and GSH activities. Meanwhile, co-exposure activated the p53-mediated apoptotic pathway, increased the bax/bcl2 ratio, stimulated caspase8 expression, and ultimately aggravated pericardial cell apoptosis in zebrafish larvae. Collectively, PE-NPs exacerbate 6PPD-induced cardiotoxicity in zebrafish early life stages by enhancing pericardial accumulation, amplifying oxidative damage, and promoting apoptosis. This study provides novel mechanistic insights for ecological risk assessment of combined pollution caused by tire-wear-derived 6PPD and nanoplastics in aquatic environments.
Cis-regulatory modules (CRMs) are central to gene regulation, yet in vivo authentication of candidate CRMs remains a major bottleneck. Here, we introduce a parallel reporter assay for zebrafish that enables simultaneous, quantitative analysis of multiple transcriptional reporters using standard Tol2-mediated transgenesis. Activity of individual reporters is established through detection of a molecular barcode, allowing direct comparison of CRM activity within the same biological sample. We find that barcoded reporters function independently and that variability arising from mosaicism and genomic position effects is effectively mitigated by analyzing modest numbers of embryos routinely obtained in transient transgenesis experiments. Using whole embryos, we demonstrate robust detection of reporter activity across CRMs with distinct levels and spatial domains of activity. We further show that pre-enrichment enhances sensitivity. This enables cell type-specific comparisons of CRM activity and reveals context-dependent differences in amplitude. Together, these findings demonstrate that conventional zebrafish transgenesis supports reliable, multiplexed analysis of transcriptional reporters. This approach provides a practical framework for higher-throughput authentication of candidate CRMs in vivo and complements existing strategies for detailed spatial characterization.
Long-term preservation of genetic lines is crucial for animal research. Current methods heavily rely on cryopreservation of haploid sperm, whereas diploid germline stem cells (GSCs) may provide a superior alternative. Here, using zebrafish, we establish an integrated platform combining in vivo expansion of GSCs (eGSCs), cryopreservation and direct genetic recovery. The eGSCs are amplified in cyp11a2-/- mutants and exhibit a total number of about 300-fold higher than conventional GSCs after cryopreservation and thawing. The eGSCs achieve a success rate of 82% after transplantation, compared with less than 1% for conventional GSCs. Furthermore, we develop the GSC-deficient nanos2-/- mutant as an ideal host for eGSC transplantation, enabling efficient production of both sperm and oocytes within a single maturation period. This allows rapid recovery of maternal-zygotic mutants directly in the F1 generation. Overall, our strategy provides a robust platform for preserving and restoring zebrafish homozygous mutants, opening new opportunities for zebrafish research.
Estrone (E1) is one of the major natural estrogens influencing the reproductive system of vertebrates. However, it contaminates aquatic environments due to runoff from livestock waste and the discharge of domestic sewage. The aim of this study was to investigate the impact of exposure to environmentally relevant concentrations of E1 on oogenesis and spawning in zebrafish (Danio rerio). Fish were exposed, in duplicate, to 20, 200, and 2000 ng/L of E1 for 49 days for oogenesis analysis evaluating histology, sex steroids, morphometry, cell proliferation, apoptosis, immunohistochemistry for insulin-like growth factor (IGF) and inducible nitric oxide synthetase (iNOS). 17β-estradiol (E2) and 11-ketotestosterone (11-KT) were assessed by ELISA assay. Nitric oxide production was evaluated indirectly by quantifying nitrite. Results revealed that E1 exposure altered the proportions of ovarian follicles and somatic components, with significant increase of oogonia, follicular atresia and inflammatory infiltrate, and decrease of follicular cells. Levels of E2, and immunoreaction for IGF1 increased in E120 and 200 ng/L groups, but 11-KT, IGF2 and IGF1R were not altered. Nitrite concentrations in ovaries were significantly elevated in E1 200 and 2000 ng/L groups, but iNOS immunoreaction was not altered. These changes led to reduced egg production in all groups exposed to E1 and significatively decreased fertilization rates at 200 and 2000 ng/L. Overall, the findings demonstrate that E1, even at concentrations commonly found in surface waters, has detrimental effects on ovarian development, gametogenesis and reproductive success in zebrafish. Monitoring environmental estrogens levels in aquatic environments is therefore essential for fish conservation.
Epiboly is a crucial morphogenetic process during early animal embryogenesis that expands surface area of embryonic tissues while thinning them. During zebrafish development, epiboly spreads the superficial enveloping layer (EVL), germ layers, and yolk syncytial layer (YSL) to cover the yolk cell. Here we investigated functions of the three zebrafish dchs genes, dchs1a, dchs1b, and dchs2 that encode large atypical cadherins and report that they have partially overlapping functions in epiboly progression. By inserting GFP or mNeongreen fluorescent proteins at the C-terminal Dchs1b intracellular domain of the endogenous dchs1b locus using homologous recombination, we observed the resulting Dchs1b fluorescent fusion proteins localized in both the cell membrane and the cytoplasm of EVL, embryonic cells and YSL during gastrulation. The dynamic microtubule and actin cytoskeleton of the yolk cell are essential for epiboly. Our studies demonstrate that the yolk cell microtubules are more bundled and show faster polymerization during epiboly in dchs triple loss-of-function mutant embryos than in wild-type embryos, indicating that dchs genes are required for limiting microtubule polymerization and promoting dynamics during epiboly. The epiboly progression defects of dchs1b-deficient mutants were suppressed by mutations in the tetratricopeptide repeat protein 28 (ttc28) gene encoding a cytoplasmic protein previously shown to bind to Dchs1b intracellular domain and alter microtubule dynamics during early cleavages. We further demonstrate that MZdchs1b mutants exhibit abnormal organization and dynamics of yolk cell actin cytoskeleton during epiboly. Together, these lines of evidence as well as our transcriptomic analyses support the notion that, like during early embryonic cleavages, Dchs1b plays a major role, while Dchs1a and Dchs2 proteins have supporting roles regulating microtubule dynamics and organization of both microtubule and actin cytoskeleton to ensure normal epiboly.
The heavy metal lead (Pb) is widely accepted as a toxicant that impairs different physiological functions dependent on dose. The central nervous system (CNS) is highly sensitive to Pb especially when exposure occurs during early developmental periods. Moreover, even low Pb exposures during development can be detrimental and have long-lasting effects on the individual. Pb has also been implicated to exert changes across generations, impairing physiological traits as well as behavioral markers exerting an impairment in the health span. In this study, the developmental effect of environmental-relevant concentrations of Pb (0.01, 0.1, and 1 ppb; μg/L) were assessed in the F1 (multigenerational) and F2 (transgenerational) generations from an F0-exposed zebrafish (Danio rerio). Behavioral, oxidative, and lipid-profiling were assessed for these generations to identify changes inherited from developmental Pb contact. Behavioral assessments revealed an embryonic Pb exposure in the F0 generation led to significant hypolocomotion, anxiolytic- and anxiogenic-like behaviors, impaired social interaction, and reduced decision-making capacity in F1 larvae. On the other hand, minimal effects were observed in the F2 larvae. Lipidomic analysis indicated these behavioral phenotypes were associated with altered levels of sphingomyelins, phosphatidylcholines, and cholesterol esters, suggesting disrupted neuronal signaling and membrane homeostasis. Enzymatic alterations were related to glutathione metabolism and those might be driven by some mechanistic Pb-induced epigenetic modifications. Together, these findings provide evidence that low-level Pb exposure induces multigenerational neurobehavioral toxicity, mediated by lipidomic and redox pathway disruptions, with implications for long-term environmental health risk assessment.