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ETHNOPHARMACOLOGICAL RELEVANCE: Indigenous use communities in the Western Cape (South Africa) where Aspalathus linearis (Brum.f) R.Dahlgren - or rooibos - grows naturally, has a long history of using rooibos for medicinal purposes. Apart from its well-known antioxidant effect, the Cederberg community in particular has been using rooibos as a treatment for high blood pressure. Given the detrimental effects of high blood pressure on endothelial cells, rooibos may either directly or indirectly affect vascular health. This, together with more recent reports of neuroprotective effects, may position rooibos as complementary medicine in related vascular conditions such as ischaemic stroke. AIMS OF THE STUDY: The study aimed to evaluate the potential benefit of acute administration of unfermented rooibos, on vascular health in a larval zebrafish model of stroke. MATERIALS AND METHODS: Stroke was induced via 24-h ponatinib exposure, in the presence or absence of an aqueous solution of an ethanolic extract of unfermented Rooibos (GreenOxithin™). The magnitude of stroke was assessed by monitoring larval locomotion and thrombus formation. In terms of specific mechanisms probed, changes in redox status (MDA and TEAC), neurological markers (TH and NeuroD1) and endothelial health (tight/adhesion junction protein expression) were assessed. RESULTS: Rooibos treatment limited thrombus formation and prevented stroke-induced deficits on larval motility. In terms of redox status, rooibos treatment prevented lipid peroxidation 3 days after initial stroke induction, reducing the need for significant upregulation of endogenous antioxidant mechanisms. Stroke-induced changes in neuronal (NeuroD1 and TH) protein expression were normalized in the presence of rooibos, suggesting a neuroprotective role. In terms of tight junction proteins, stroke-related decreases in ZO-1 expression were again prevented by rooibos treatment. In addition, rooibos treatment may beneficially modulate levels of claudin-5 and VE-cadherin, to indirectly limit stroke-associated vascular dysfunction. CONCLUSIONS: Taken together, activity data and physiological assessments suggest that unfermented rooibos may indeed have benefit in the context of stroke, via action at multiple targets. Thus, current data further our understanding of the mechanisms of actions of rooibos and warrant future research to confirm sufficient bioavailability of rooibos in target tissues, in mammalian systems.

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The cerebellum is a conserved structure of the vertebrate brain involved in the timing and calibration of movements. Its function is supported by the convergence of fibers from granule cells (GCs) and inferior olive neurons (IONs) onto Purkinje cells (PCs). Theories of cerebellar function postulate that IONs convey error signals to PCs that, paired with the contextual information provided by GCs, can instruct motor learning. Here, we use the larval zebrafish to investigate (1) how sensory representations of the same stimulus vary across GCs and IONs and (2) how PC activity reflects these two different input streams. We use population calcium imaging to measure ION and GC responses to flashes of diverse luminance and duration. First, we observe that GCs show tonic and graded responses, as opposed to IONs, whose activity peaks mostly at luminance transitions, consistently with the notion that GCs and IONs encode context and error information, respectively. Second, we show that GC activity is patterned over time: some neurons exhibit sustained responses for the entire duration of the stimulus, while in others activity ramps up with slow time constants. This activity could provide a substrate for time representation in the cerebellum. Together, our observations give support to the notion of an error signal coming from IONs and provide the first experimental evidence for a temporal patterning of GC activity over many seconds.

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Per- and polyfluoroalkyl substances (PFAS) are found in many consumer and industrial products. While some PFAS, notably perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are developmentally toxic in mammals, the vast majority of PFAS have not been evaluated for developmental toxicity potential. A concentration-response study of 182 unique PFAS chemicals using the zebrafish medium-throughput, developmental vertebrate toxicity assay was conducted to investigate chemical structural identifiers for toxicity. Embryos were exposed to each PFAS compound (≤100 µM) beginning on the day of fertilization. At 6 days post-fertilization (dpf), two independent observers graded developmental landmarks for each larva (e.g., mortality, hatching, swim bladder inflation, edema, abnormal spine/tail, or craniofacial structure). Thirty percent of the PFAS were developmentally toxic, but there was no enrichment of any OECD structural category. PFOS was developmentally toxic (benchmark concentration [BMC] = 7.48 µM); however, other chemicals were more potent: perfluorooctanesulfonamide (PFOSA), N-methylperfluorooctane sulfonamide (N-MeFOSA), ((perfluorooctyl)ethyl)phosphonic acid, perfluoro-3,6,9-trioxatridecanoic acid, and perfluorohexane sulfonamide. The developmental toxicity profile for these more potent PFAS is largely unexplored in mammals and other species. Based on these zebrafish developmental toxicity results, additional screening may be warranted to understand the toxicity profile of these chemicals in other species.

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Mapping brain activities is necessary for understanding brain physiology and discovering new treatments for neurological disorders. Such efforts have greatly benefited from the advancement in technologies for analyzing neural activity with improving temporal or spatial resolution. Here, we constructed a multielectrode array based brain activity mapping (BAM) system capable of stabilizing and orienting zebrafish larvae for recording electroencephalogram (EEG) like local field potential (LFP) signals and brain-wide calcium dynamics in awake zebrafish. Particularly, we designed a zebrafish trap chip that integrates with an eight-by-eight surface electrode array, so that brain electrophysiology can be noninvasively recorded in an agarose-free and anesthetic-free format with a high temporal resolution of 40 µs, matching the capability typically achieved by invasive LFP recording. Benefiting from the specially designed hybrid system, we can also conduct calcium imaging directly on immobilized awake larval zebrafish, which further supplies us with high spatial resolution brain-wide activity data. All of these innovations reconcile the limitations of sole LFP recording or calcium imaging, emphasizing a synergy of combining electrical and optical modalities within one unified device for activity mapping across a whole vertebrate brain with both improved spatial and temporal resolutions. The compatibility with in vivo drug treatment further makes it suitable for pharmacology studies based on multimodal measurement of brain-wide physiology.

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Plants used in traditional medicine in the management of epilepsy could potentially yield novel drug compounds with antiepileptic properties. The medicinal plant Securidaca longepedunculata is widely used in traditional medicine in the African continent, and epilepsy is among several indications. Limited knowledge is available on its toxicity and medicinal effects, such as anticonvulsant activities. This study explores the potential in vivo inhibition of seizure-like paroxysms and toxicity effects of dichloromethane (DCM) and ethanol (EtOH) extracts, as well as isolated xanthones and benzoates of S. longepedunculata. Ten phenolic compounds were isolated from the DCM extract. All of the substances were identified by nuclear magnetic resonance spectroscopy. Assays for toxicity and inhibition of pentylenetetrazole (PTZ)-induced seizure-like paroxysms were performed in zebrafish larvae. Among the compounds assessed in the assay for maximum tolerated concentration (MTC), benzyl-2-hydroxy-6-methoxy-benzoate (MTC 12.5 µM), 4,8-dihydroxy-1,2,3,5,6-pentamethoxyxanthone (MTC 25 µM), and 1,7-dihydroxy-4-methoxyxanthone (MTC 6.25 µM) were the most toxic. The DCM extract, 1,7-dihydroxy-4-methoxyxanthone and 2-hydroxy-1,7-dimethoxyxanthone displayed the most significant inhibition of paroxysms by altering the locomotor behavior in GABAA receptor antagonist, PTZ, which induced seizures in larval zebrafish. The EtOH extract, benzyl benzoate, and benzyl-2-hydroxy-6-methoxy-benzoate unexpectedly increased locomotor activity in treated larval zebrafish and decreased locomotor activity in nontreated larval zebrafish, seemingly due to paradoxical excitation. The results reveal promising medicinal activities of this plant, contributing to our understanding of its use as an antiepileptic drug. It also shows us the presence of potentially new lead compounds for future drug development.

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The innate immune system can remember previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed "trained immunity." Trained innate immune cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where ß-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of ß-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to ß-glucan stimulation.

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Animals influence how they disperse in the environment by sensing local cues and adapting how they move. However, controlling dispersal can present a particular challenge early in life when animals tend to be more limited in their capacities to sense and move. To what extent and by what mechanisms can newly hatched fish control how they disperse? Here, we reveal hatchling sensorimotor mechanisms for controlling dispersal by combining swim tracking and precise sensory manipulations of a model species, zebrafish. In controlled laboratory experiments, if we physically constrained hatchlings or blocked sensations of motion through vision and the lateral line, hatchlings responded by elevating their buoyancy and passively moving with faster surface currents. Complementarily, in stagnant water, hatchlings covered more ground using hyperstable swimming, strongly orienting based on graviception. Using experimentally calibrated hydrodynamic simulations, we show that these hatchling behaviors nearly tripled diffusivity and made dispersal robust to local conditions, suggesting this multisensory strategy may provide important advantages for early life in a variable environment.

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Purpose: To explore the pharmacological effects and mechanisms of Qinghao Biejia decoction (QBD) against non-small-cell lung cancer (NSCLC) based on network pharmacology and to verify the anticancer effect of artemisinin B (ART B), the active ingredient of QBD, on H1299 cells. Methods: Ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was applied to explore the chemoprofile of QBD. A zebrafish xenograft model was used to determine the anti-cancer efficacy of QBD. Cell counting kit-8 assay, terminal deoxyribonucleotide transferase-mediated-dUTP nick-end labeling assay; immunofluorescence, and flow cytometry were used to evaluate the in vitro anti-proliferative and pro-apoptotic effects of QBD and ART B on H1299 cells. Subsequently, the related targets and action mechanisms of both QBD and ART B predicted by network pharmacological analyses were experimentally validated by real-time PCR and Western blot assays on H1299 cells. Results: UPLC-QTOF-MS/MS identified a total of 69 compounds (such as ART B, mangiferin, and artemisinic acid) in QBD. The in vivo data showed that QBD significantly inhibited the growth of H1299 cells in xenograft larval zebrafish from 125 to 500 µg/mL. The in vitro data showed that QBD induced apoptosis of H1299 cells, accompanied by down-regulating the expression of BCL-2 and up-regulating the expression of BIM, PUMA, BAX, c-PARP, γ-H2A.X, c-CASP3, and c-CASP8. Alike QBD, ART B exerted similar anti-proliferative and pro-apoptotic effects on H1299 cells. Moreover, ART B inhibited expressions of BCL2L1, AKT1, AKT2, MMP-2, and EGFR, and up-regulated ALB expression. Mechanistically, ART B promoted apoptosis of H1299 cells by inhibiting PI3K/Akt signaling pathway. Conclusion: This study revealed the anti-NSCLC efficacy of QBD. ART B, the effective component of QBD, plays an anti-NSCLC role by down-regulating the PI3K-Akt signaling pathway. It suggests that QBD and ART B are promising drug candidates for NSCLC treatment.

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Azithromycin (AZM) is a widely used antibiotic in both human and veterinary medicine, and its use has significantly increased during the COVID-19 pandemic. However, potential adverse effects of AZM on aquatic organisms have not been well studied. In this study, we explored the neurotoxicity of AZM in zebrafish and delved into its underlying mechanisms. Our results showed that AZM exposure resulted in a spectrum of detrimental effects in zebrafish, encompassing abnormal behaviors, damaged neuronal development, aberrant lateral line nervous system development, vascular malformations and perturbed expression of genes related to neural development. Moreover, we observed a concentration-dependent exacerbation of these neurotoxic manifestations with increasing AZM concentrations. Notably, AZM induced excessive cell apoptosis and oxidative stress damage. In addition, alterations in the expression levels of the genes involved in the VEGF/Notch signaling pathway were evident in AZM-exposed zebrafish. Consequently, we hypothesize that AZM may induce neurotoxicity by influencing the VEGF/Notch signaling pathway. To validate this hypothesis, we introduced a VEGF signaling inhibitor, axitinib, and a Notch signaling agonist, valproic acid, alongside AZM exposure. Remarkably, the administration of these rescue compounds significantly mitigated the neurotoxic effects induced by AZM. This dual verification provides compelling evidence that AZM indeed induces neurotoxicity during the early developmental stages of zebrafish, primarily through its interference with the VEGF/Notch pathway. Innovatively, our study reveals the molecular mechanism of AZM-induced neurotoxicity from the perspective of the close connection between blood vessels and nervous system. These findings provide new insights into the potential mechanisms underlying the neurotoxic effect of antibiotics and highlight the need for further investigation into the ecotoxicological effects of antibiotics on aquatic organisms and the potential risks to human health.

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To optimize performance during vital tasks, animals are capable of tuning rhythmic neural signals that drive repetitive behaviors, such as motor reflexes under constant sensory stimuli. In the oculomotor system, animals track the moving image during slow phases while repetitively resetting the eye position from the eccentricity during quick phases. During optokinetic response (OKR), larval zebrafish occasionally show a delayed quick phase; thus, the eyes remain tonically deviated from the center. In this study, we scrutinized OKR in larval zebrafish under a broad range of stimulus velocities to determine the parametric property of the quick-phase delay. A prolonged stimulation revealed that the slow-phase (SP) duration-the interval between two quick phases-was tuned increasingly over time toward a homeostatic range, regardless of stimulus velocity. Attributed to this rhythm control, larval zebrafish exhibited a tonic eye deviation following slow phases, which was especially pronounced when tracking a fast stimulus over an extended time period. In addition to the SP duration, the fixation duration between spontaneous saccades in darkness also revealed a similar adaptive property after the prolonged optokinetic stimulation. Our results provide a quantitative description of the adaptation of rhythmic eye movements in developing animals and pave the way for potential animal models for eye movement disorders.

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Zebrafish have a remarkable capacity for spontaneously regenerating their central nervous system. Larval zebrafish are optically transparent and therefore are widely used to dynamically visualize cellular processes in vivo, such as nerve regeneration. Regeneration of retinal ganglion cell (RGC) axons within the optic nerve has been previously studied in adult zebrafish. In contrast, assays of optic nerve regeneration have previously not been established in larval zebrafish. In order to take advantage of the imaging capabilities in the larval zebrafish model, we recently developed an assay to physically transect RGC axons and monitor optic nerve regeneration in larval zebrafish. We found that RGC axons rapidly and robustly regrow to the optic tectum. Here, we describe the methods for performing the optic nerve transections, as well as methods for visualizing RGC regeneration in larval zebrafish.

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Emerging contaminants, such as neonicotinoid pesticide acetamiprid (Ace), are frequently detected in the water environment, which can interact with existing heavy metal cadmium (Cd) to produce unpredicted influence. Limited studies have evaluated the effects of multiple pollutant exposures on aquatic animals. Here, we characterized the joint toxicity of Ace and Cd exposure to zebrafish (Danio rerio). The results revealed that Cd and its combined exposure with Ace had an inhibitory effect on the growth of larval zebrafish and induced morphological defects. Combined exposure to high doses of Ace and Cd could significantly reduce the levels of TG, glucose, and pyruvate in larval zebrafish. Untargeted metabolomics revealed that Cd treatment (285) produced more differentially expressed metabolites (DEMs) than Ace treatment (115), and combined treatment produced the most DEMs (294). The KEGG pathway enrichment analysis showed that they could disrupt riboflavin metabolism, amino acid metabolism, and glycolipid metabolism in the larvae of D. rerio. ELISA showed that VB2, FMN, and FAD levels were significantly increased. In addition, gene expression analysis exhibited that the mRNA levels of essential genes related to glycolipid metabolism were substantially affected, such as PK, PEPckc, PPAR-α, and FABP6. Furthermore, targeted amino acid metabolomics confirmed that both single exposure to Cd and combined exposure to Ace and Cd altered the levels of amino acids in larvae, including ALA, ARG, MET, PRO, TYR, VAL, GLY, ORN, and PHE. Taken together, exposure to Ace and Cd, alone or in combination, exerted harmful effects on the individual development, riboflavin metabolism, glycolipid metabolism, and amino acid metabolism disorder of D. rerio. These findings highlighted that more attention should be paid to the compound toxicity of chemical mixtures to aquatic organisms.

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Although their production was banned in the United States in 1977, polychlorinated biphenyls (PCBs) continue to pose significant risks to the developing nervous system. Perinatal exposure to PCBs is associated with increased risk of neuropsychiatric disorders, perhaps due to altered patterns of dendritic arborization of central neurons. Non-dioxin-like (NDL) PCB congeners enhance dendritic arborization of developing mammalian neurons via sensitization of ryanodine receptors (RYR). Structure-activity relationships (SAR) of RYR sensitization by PCBs have been demonstrated using mammalian and rainbow trout (Oncorhynchus mykiss) tissue homogenates. The purpose of this study is to determine whether this SAR translates to developmental neurotoxicity (DNT) of PCBs in vivo, a question that has yet to be tested. To address this gap, we leveraged a zebrafish model to evaluate the developmental neurotoxicity potential of PCBs 28, 66, 84, 95, 138, and 153, congeners previously shown to have broadly different potencies towards sensitizing RYR. We first confirmed that these PCB congeners exhibited differing potency in sensitizing RYR in zebrafish muscle ranging from negligible (PCB 66) to moderate (PCB 153) to high (PCB 95) RYR activity. Next, enzymatically dechorionated embryos were statically exposed to varying concentrations (0.1-10 µM) of each PCB congener from 6 h post-fertilization to 5 days post-fertilization (dpf). Embryos were observed daily using stereomicroscopy to assess mortality and gross malformations and photomotor behavior was assessed in larval zebrafish at 3, 4, and 5 dpf. The body burden of each PCB was measured by gas chromatography. The key findings are: 1) None of these PCBs caused death or overt teratology at the concentrations tested; 2) A subset of these PCB congeners altered photomotor behavior in larval zebrafish and the SAR for PCB behavioral effects mirrored the SAR for RYR sensitization; and 3) Quantification of PCB levels in larval zebrafish ruled out the possibility that congener-specific effects on behavior were due to differential uptake of PCB congeners. Collectively, the findings from this study provide in vivo evidence in support of the hypothesis that RYR sensitization contributes to the DNT of PCBs.

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Early-life seizures (ELS) are often associated with the development of cognitive deficits. However, methods to predict and prevent these deficits are lacking. To increase the range of research models available to study cognitive consequences of ELS, we investigated whether seizures in larval zebrafish (Danio rerio) lead to behavioral deficits later in life. We thus modified the existing pentylenetetrazole (PTZ)-induced seizure model in larval zebrafish, exposing zebrafish to PTZ daily from 5 to 7 days post-fertilization (dpf). We then compared later-life learning, social behavior (shoaling), and behavioral and chemical measures of anxiety in the PTZ-exposed zebrafish (PTZ group) to that of naïve clutchmates (untouched controls, UC) and to a second control group (handling control, HC) that experienced the same handling as the PTZ group, but without PTZ exposure. We observed that only the PTZ group displayed a significant deficit in a y-maze learning task, while only the HC group displayed a social deficit of decreased shoaling. HC fish also showed an increased frequency of behavioral freezing and elevated cortisol responses to netting, heightened stress responses not seen in the PTZ fish. Since mild stressors, such as the handling the HC fish experienced, can lead to learned, advantageous responses to stress later in life, we tested escape response in the HC fish using an acoustic startle stimulus. The HC group showed an enhanced startle response, swimming significantly farther than either the PTZ or UC group immediately after being startled. Taken together, these results indicate that seizures in larval zebrafish impair learning and the development of an adaptive, heightened stress response after early-life stress. These findings expand the behavioral characterization of the larval zebrafish seizure model, strengthening the power of this model for ELS research.

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ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory and practice of traditional Chinese medicine (TCM), the pathogenesis of lung carcinoma is associated with many syndromes, such as "sputum stasis", "cough", "lung fever", "lung toxin", and "hemoptysis", which should be removed for therapeutic purpose. Tea is not only a world-wide beverage, but also a TCM herb, possessing activities against the above syndromes. Recently, green tea extract exerted inhibitory effects on a variety of tumor cells. As a pigment active substance of green tea, theabrownin (TB) has been found to inhibit many cancer cells. AIM OF THE STUDY: This study focused on the efficacy and mechanism of TB on non-small cell lung cancer (NSCLC) cell lines. The in vivo efficacy of TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells NSCLC cells were determined, and its mechanism of action was explored. MATERIALS AND METHODS: In vivo, two lung cancer cell lines, H1299 (p53-deficient) and A549 (p53-wild type) were selected to establish xenograft models of larval zebrafish, respectively. For in vitro experiments, wound healing assay, DAPI staining, TUNEL assay, immunofluorescence assay, and flow cytometry were conducted in these two cell lines. RNA sequencing (RNAseq), real time PCR (qPCR) and Western blot (WB) were performed for the mechanism study. RESULTS: The in vivo results showed that TB significantly inhibited the H1299 and the A549 xenograft tumor growth in larval zebrafish (dosage ranged from 2.13 to 21.3 µg/ml). Wound healing assay results showed that TB suppressed the migration of H1299 cells. DAPI staining, TUNEL assay, and immunofluorescence assay results showed that TB inhibited the growth of H1299 cells by inducing apoptosis. RNAseq, qPCR and WB data showed that TB significantly up-regulated the MAPK/JNK pathway-related proteins (ASK-1, JNK and c-JUN) through phosphorylation activation, accompanying with down-regulation of the epithelial-mesenchymal transition (EMT)-associated genes (N-CADHERIN, SLUG, FIBROWNECTIN and ZEB1) and anti-apoptotic molecules (BCL-2), and up-regulation of the metastasis-related gene HSPA6 and the pro-apoptotic molecules (BIM, BAX, PARP, c-PARP, γ-H2A.X, c-CASP3, c-CASP8, c-CASP9, DDIT3 and DUSP8). CONCLUSION: This study determined the in vivo efficacy of green tea-derived TB on p53-deficient NSCLC (H1299) cells and p53-wild type NSCLC (A549) cells and clarified its p53-independent mechanism mediated by the activation of MAPK/JNK signaling pathway.

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Prochloraz (PCZ), an imidazole fungicide, has been extensively used in horticulture and agriculture to protect against pests and diseases. To investigate the potential toxicity of PCZ on aquatic organisms, larval zebrafish, as a model, were exposed to a series of concentrations (0, 20, 100, and 500 µg/L) of PCZ for 7 days. With transcriptomic analysis, we found that exposure to high dose PCZ could produce 76 downregulated and 345 upregulated differential expression genes (DEGs). Bioinformatics analysis revealed that most of the DEGs were characterized in the pathways of glycolipid metabolism, amino acid metabolism and oxidative stress in larval zebrafish. Targeted metabolomic analysis was conducted to verify the effects of PCZ on the levels of acyl-carnitines and some amino acids in larval zebrafish. In addition, biochemical indicators related to glycolipid metabolism were affected obviously, manifested as elevated triglyceride (TG) levels and decreased glucose (Glu) levels in whole larvae. The expression levels of genes associated with glycolipid metabolism were affected in larvae after exposure to PCZ (PK, GK, PEPckc, SREBP, ACO). Interestingly, we further confirmed that PCZ could induce oxidative stress by the changing enzyme activities (T-GSH, GSSG) and upregulating several related genes levels in larval zebrafish. Generally, our results revealed that the endpoints related to glycolipid metabolism, amino acid metabolism and oxidative stress were influenced by PCZ in larval zebrafish.

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Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.

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To thrive, organisms must maintain physiological and environmental variables in suitable ranges. Given that these variables undergo constant fluctuations over varying time scales, how do biological control systems maintain control over these values? We explored this question in the context of phototactic behavior in larval zebrafish. We demonstrate that larval zebrafish use phototaxis to maintain environmental luminance at a set point, that the value of this set point fluctuates on a time scale of seconds when environmental luminance changes, and that it is determined by calculating the mean input across both sides of the visual field. These results expand on previous studies of flexible phototaxis in larval zebrafish; they suggest that larval zebrafish exert homeostatic control over the luminance of their surroundings, and that feedback from the surroundings drives allostatic changes to the luminance set point. As such, we describe a novel behavioral algorithm with which larval zebrafish exert control over a sensory variable.

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Epoxiconazole (EPX), as a broad-spectrum triazole fungicide, is widely used in agriculture to resist pests and diseases, while it may have potential toxicity to non-target organisms. In the present study, early developmental stage zebrafish were used as the subject organisms to assess the toxicity of EPX, and the possible mechanism of toxicity was also discussed by biochemical and transcriptomic analysis. Through embryo toxicity test, we had made it clear that the 96 h LC50 of embryo was 7.204 mg/L, and acute exposure to EPX effected hatching rate, heartbeats, body length and even morphological defects. Then, by being exposed to EPX for 7 days at concentrations of 175 (1/40 LC50), 350 (1/20 LC50) and 700 (1/10 LC50), biochemical parameters were affected, mainly manifested as increase of the triglyceride (TG) level and decrease of glucose content. Correspondingly, the transcription of genes related of glucose metabolism, lipid metabolism and cholesterol metabolism were also affected significantly in larval zebrafish. Moreover, some pathways, including lipid metabolism, glucose metabolism and amino acid metabolism were affected through transcriptome sequencing analysis in the larval zebrafish. Further data analysis based on the sequencing, EPX exposure also affected the expression of genes related to cell apoptosis. We further conformed that the bright fluorescence on the liver and bright spots near the liver by acridine orange staining. In addition, the mRNA levels of apoptosis related genes were also significantly affected in the EPX exposed larval zebrafish. Taken together, the work could provide an insight into toxic effects of EPX on the zebrafish larvae at embryo toxicity and transcriptional levels, providing some evidences for the toxic effects of triazole fungicides on non-target organisms.

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CPI-613 is a mitochondrial metabolism disrupter that inhibits tricarboxylic acid (TCA) cycle activity. The consequences of TCA cycle disruption on various metabolic pathways and overall organismal physiology are not fully known. The present study integrates in vivo experimental data with an in silico stoichiometric metabolism model of zebrafish to study the metabolic pathways perturbed under CPI-613 exposure. Embryo-larval life stages of zebrafish (Danio rerio) were exposed to 1 µM CPI-613 for 20 days. Whole-organism respirometry measurements showed an initial suppression of O2 consumption at Day 5 of exposure, followed by recovery comparable to the solvent control (0.01% DMSO) by Day 20. Comparison of whole-transcriptome RNA-sequencing at Day 5 vs. 20 of exposure showed functional categories related to O2 binding and transport, antioxidant activity, FAD binding, and hemoglobin complexes, to be commonly represented. Metabolic enzyme gene expression changes and O2 consumption rate was used to parametrize two in silico stoichiometric metabolic models representative of Day 5 or 20 of exposure. Computational simulations predicted impaired ATP synthesis, α-ketoglutarate dehydrogenase (KGDH) activity, and fatty acid ß-oxidation at Day 5 vs. 20 of exposure. These results show that the targeted disruption of KGDH may also impact oxidative phosphorylation (ATP synthesis) and fatty acid metabolism (ß-oxidation), in turn influencing cellular bioenergetics and the observed reduction in whole-organism O2 consumption rate. The results of this study provide an integrated in vivo and in silico framework to study the impacts of metabolic disruption on organismal physiology.

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