Zebrafish Larvae Research Articles

Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish.

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Waterborne exposure to the antineoplastic 5-fluorouracil alters lipid composition in larval zebrafish (Danio rerio)

Antineoplastic medications are present in aquatic environments and are measured at relatively high concentrations in hospital sewage effluent. Thus, it is important to characterize risk associated with waterborne exposures to anticancer drugs. The drug 5-fluorouracil (5-FU) is used to treat several types of cancers, acting to inhibit cell division and cellular metabolism. The objectives of this study were to determine the effects of 5-FU on developmental endpoints and lipid composition in zebrafish. 5-FU did not negatively affect development nor survival in developing zebrafish at concentrations up to 1000 μg/L. However, 5-FU increased neutral lipid content in zebrafish larvae, indicating potential for lipid dysregulation. To further discern effects on lipids, lipidomics was conducted and a total of 164 lipids belonging to 14 lipid classes were identified. Significant changes (false discovery rate < 0.05) in abundance were detected for 19 lipids including some ceramides, ether-linked phosphatidylethanolamines, and sphingomyelins among others. We also measured the expression levels of 14 lipid-related enzymes and transporters (e.g., acox3, dgat1, fads2, fasn, elovl2) using real-time PCR; however, mRNA abundance levels were not affected, suggesting transcriptional changes may not be a primary mechanism underlying lipid dysregulation. Locomotor activity was measured in zebrafish as lipids are needed for swimming activity in larvae. Exposure to 5-FU did not affect locomotor activity up to 1000 μg/L. We conclude that lipids accumulate in larval zebrafish with exposure to 5-FU, which can subsequently affect lipid composition. These data reveal potential lipid signatures of 5-FU exposure and contribute to risk assessments for antineoplastic exposure in aquatic environments.

Toxicological and Sedative Effects of Chipilin (Crotalaria longirostrata) Leaf Extracts Obtained by Maceration and Supercritical Fluid Extraction.

Chipilin (Crotalaria longirostrata) is consumed as a vegetable in the preparation of traditional dishes. As a folk medicine, Chipilin extracts are used as a hypnotic and sedative agent; however, there are few reports that support these uses. This study aimed to characterize the compounds present in Chipilin leaf extracts and to investigate their sedative effect using zebrafish as an in vivo model. Extracts were obtained by maceration with water (H2O), ethanol (EtOH), and EtOH-H2O, while oleoresin was obtained by supercritical fluid extraction (SFE). Total phenolic and flavonoid contents were quantified by colorimetric methods. Phytochemical constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The chronic and acute toxicities of Chipilin extracts were tested in zebrafish embryos and larvae, respectively. Chipilin sedative effect was tested by the larvae response to dark-light-dark transitions. EtOH-H2O extracts had the highest value of total phenolics (5345 ± 5.1 μg GAE/g), followed by water and oleoresin (1815 ± 5.1 and 394 ± 5.1 μg GAE/g, respectively). In water extracts were identified the alkaloid trachelanthamidine, 1,2β-epoxy- and the alkyl ketone 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, while oleamide, α-monostearin, and erucamide were detected in all samples except in water extracts. Oleoresin extract had the lowest embryotoxicity (LC50 = 4.99 μg/mL) and the highest sedative effects. SFE is a green alternative to obtain Chipilin extracts rich in erucamide, an endocannabinoid analogue, which plays an important role in the development of the central nervous system and in modulating neurotransmitter release.

Anti-adipogenesis effect of indole-3-acrylic acid on human preadipocytes and HFD-induced zebrafish.

Obesity, defined as excessive or abnormal body fat accumulation, which could significantly increase the risk of cardiovascular disease, type 2 diabetes mellitus (T2DM) diseases and seriously affect people's quality of life. More than 2 billion people are overweight, and the incidence of obesity is increasing rapidly worldwide, it has become a widely concerned public health issue in the world. Diverse evidence show that active metabolites are involved in the pathophysiological processes of obesity. However, whether the downstream catabolite of tryptophan, 3-indole acrylic acid (IA), is involved in obesity remains unclear. We collected the samples of serum from peripheral blood of obesity and health controls, and liquid chromatography-mass spectrometry (LC-MS) was performed to identify the plasma levels of IA. Additionally, we verified the potential benefits of IA on human preadipocytes and HFD- induced zebrafish by cell viability assay, flow cytometry assay, Oil red O staining, total cholesterol (T-CHO), triglyceride (TG) and nonesterified free fatty acids (NEFA) measurements and Nile Red staining. RNA-Seq, functional analysis and western blot revealed the mechanisms underlying the function of IA. We found that the content of IA in peripheral blood serum of overweight people was significantly lower than that of normal people. In addition, supplementation with IA in zebrafish larvae induced by a high fat diet (HFD) dramatically reduced HFD induced lipid accumulation. IA had no effect on proliferation and apoptosis of preadipocytes, but significantly inhibited adipogenesis of preadipocytes by down-regulate CEBPα and PPARγ. RNA-Seq and functional analysis revealed that IA regulated the adipogenesis of preadipocytes through stimulate the phosphorylation of STAT1. Taken together, IA has been identified as a potent metabolite for the prevention or treatment of obesity.

Diosgenin-Conjugated Zinc Oxide Nanoparticles: A Sustainable Approach to Counter Antibiotic-Induced Oxidative Stress in the Aquatic Environment Using the in vivo Zebrafish Larvae Model (Danio rerio)

Diosgenin-Conjugated Zinc Oxide Nanoparticles: A Sustainable Approach to Counter Antibiotic-Induced Oxidative Stress in the Aquatic Environment Using the in vivo Zebrafish Larvae Model (Danio rerio)

Berberine and hesperidin prevent the memory consolidation impairment induced by pentylenetetrazole in zebrafish

This study verified the effects of the natural compounds berberine and hesperidin on seizure development and cognitive impairment triggered by pentylenetetrazole (PTZ) in zebrafish. Adult animals were submitted to a training session in the inhibitory avoidance test and, after 10 minutes, they received an intraperitoneal injection of 25, 50, or 100 mg/kg berberine or 100 or 200 mg/kg hesperidin. After 30 minutes, the animals were exposed to 7.5 mM PTZ for 10 minutes. Animals were submitted to the test session 24 h after the training session to verify their cognitive performance. Zebrafish larvae were exposed to 100 µM or 500 µM berberine or 10 µM or 50 µM hesperidin for 30 minutes. After, larvae were exposed to PTZ and had the seizure development evaluated by latency to reach the seizure stages I, II, and III. Adult zebrafish pretreated with 50 mg/kg berberine showed a longer latency to reach stage III. Zebrafish larvae pretreated with 500 µM berberine showed a longer latency to reach stages II and III. Hesperidin did not show any effect on seizure development both in larvae and adult zebrafish. Berberine and hesperidin pretreatments prevented the memory consolidation impairment provoked by PTZ-induced seizures. There were no changes in the distance traveled in adult zebrafish pretreated with berberine or hesperidin. In larval stage, berberine caused no changes in the distance traveled; however, hesperidin increased the locomotion. Our results reinforce the need for investigating new therapeutic alternatives for epilepsy and its comorbidities.

Graphene oxide/ε-poly-L-lysine self-assembled functionalized coatings improve the biocompatibility and antibacterial properties of titanium implants.

The construction of an antibacterial biological coating on titanium surface plays an important role in the long-term stability of oral implant restoration. Graphene oxide (GO) has been widely studied because of its excellent antibacterial properties and osteogenic activity. However, striking a balance between its biological toxicity and antibacterial properties remains a significant challenge with GO. ε-poly-L-lysine (PLL) has broad-spectrum antibacterial activity and ultra-high safety performance. Using Layer-by-layer self-assembly technology (LBL), different layers of PLL/GO coatings and GO self-assembly coatings were assembled on the surface of titanium sheet. The materials were characterized using scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle test. The antibacterial properties of Porphyromonas gingivalis (P.g.) were analyzed through SEM, coated plate experiment, and inhibition zone experiment. CCK-8 was used to determine the cytotoxicity of the material to MC3T3 cells, and zebrafish larvae and embryos were used to determine the developmental toxicity and inflammatory effects of the material. The results show that the combined assembly of 20 layers of GO and PLL exhibits good antibacterial properties and no biological toxicity, suggesting a potential application for a titanium-based implant modification scheme.

Metabolomic changes following GenX and PFBS exposure in developing zebrafish

Short chain per- and polyfluoroalkyl substances (PFAS), including hexafluoropropylene oxide dimer acid (GenX) and perfluorobutane sulfonate (PFBS), are replacement chemicals for environmentally persistent, long-chain PFAS. Although GenX and PFBS have been detected in surface and ground water worldwide, few studies provide information on the metabolic alterations or risks associated with their exposures. In this study, larval zebrafish were used to investigate the toxicity of early-life exposure to GenX or PFBS. Zebrafish were chronically exposed from 4 h post-fertilization (hpf) to 6 days post-fertilization (dpf) to 150 µM GenX or 95.0 µM PFBS. Ultra-high-performance liquid chromatography paired with high-resolution mass spectrometry was used to quantify uptake of GenX and PFBS into zebrafish larvae and perform targeted and untargeted metabolomics. Our results indicate that PFBS was 20.4 % more readily absorbed into the zebrafish larvae compared to GenX. Additionally, PFBS exposure significantly altered 13 targeted metabolites and 21 metabolic pathways, while GenX exposure significantly altered 1 targeted metabolite and 17 metabolic pathways. Exposure to GenX, and to an even greater extent PFBS, resulted in a number of altered metabolic pathways in the amino acid metabolism, with other significant alterations in the carbohydrate, lipid, cofactors and vitamins, nucleotide, and xenobiotics metabolisms. Our results indicate that GenX and PFBS impact the zebrafish metabolome, with implications of global metabolic dysregulation, particularly in metabolic pathways relating to growth and development.

Ketamine induces insomnia-like symptom of zebrafish at environmentally relevant concentrations by mediating GABAergic synapse.

Although the stimulative effects on the normal behaviors of fish posed by ketamine (KET) were well-studied, the adverse effects on the behavioral functions induced by KET at nighttime were unknown. Here, we used zebrafish larvae as a model exposed to KET (10, 50, 100, and 250 ng/L) at environmental levels for 21 days. The behavioral functions at nighttime, morphological changes during exposure stage, and alterations on the associated genes transcriptional levels of fish were determined. The difficultly initiating sleep was found in the fish exposed to KET, while the sleep duration of the animals was at the normal levels in exposure groups. The significant suppressions of the developmentally relevant genes, including bmp2, bmp4, and pth2ra were consistent with the developmental abnormalities of fish found in exposure groups. Moreover, the expression of γ-aminobutyric acid (GABA) receptor increased and melatonin (MTN) receptor decreased while the levels of GABA and MTN remained unchanged after exposure, by gene expression analysis and molecular docking. In addition, the transcriptional expression of apoptotic genes, including tp53, aifm1, and casp6, was significantly upregulated by KET. After a 7-day recovery, the insomnia-like behaviors (shorter sleep duration) were observed in zebrafish from the 250 ng/L-KET group. Accordingly, the adverse outcome pathway framework of KET was constructed by prognostic assessment of zebrafish larvae. This study suggested that the adverse outcomes of KET on the sleep health of organisms at environmentally relevant concentrations should be concerned.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorateaminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5days' post fertilization after neomycin (20μM) and gentamicin (20mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100μM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300μM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20μM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake ofneomycininto hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production andaminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish

The vertebrate stress response (SR) is mediated by the hypothalamic–pituitary–adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. Genetic abrogation of glucocorticoid receptor (nr3c1) decreased basal locomotor activity in light and darkness. Some key HPI axis receptors (mc2r [ACTH receptor], nr3c1), but not nr3c2 (mineralocorticoid receptor), were required to adapt to light more efficiently but became dispensable when longer illumination was provided. Such light adaptation was more efficient in dimmer light. Our findings show that the HPI axis contributes to the SR, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPI axis activity.

Antinociceptive Analysis of Natural Monoterpenes Eugenol, Menthol, Carvacrol and Thymol in a Zebrafish Larval Model.

In the last decade, a considerable number of studies have broadened our knowledge of the nociceptive mechanisms of pain, a global health problem in both humans and animals. The use of herbal compounds such as eugenol, menthol, thymol, and carvacrol as analgesic agents has accompanied the growing interest in this area, offering a possible solution for this complex problem. Here, we aimed to explore how these natural substances-at three different concentrations (2, 5 and 10 mg/L)-affect the pain responses in zebrafish (Danio rerio) larvae exposed to 0.05% acetic acid (AA) for 1 min. By analysing the activity of acetylcholinesterase (AChE), 5'-ectonucleotidase and NTPDases, as well as aversion and exploratory behaviours, it was observed that that although all substances were effective in counteracting the pain stimulus, the concentration range within which they do so might be very limited. Eugenol, despite its acknowledged properties in fish anaesthesia, failed to alleviate the pain stimulus at low concentrations. Contrastingly, menthol exhibited the most promising results at the lowest concentrations tested. Overall, it is concluded that menthol might be a good analgesic for this species, qualifying it as a substance of interest for prospective studies.

Characterization of the zebrafish as a model of ATP-sensitive potassium channel hyperinsulinism

IntroductionCongenital hyperinsulinism (HI) is the leading cause of persistent hypoglycemia in infants. Current models to study the most common and severe form of HI resulting from inactivating mutations in the...

Nanoplastics and nano-ZnO facilitate Cd accumulation in zebrafish larvae via a distinct pathway: Revelation by LA-ICP-MS imaging

Nanoplastics and nano-ZnO facilitate Cd accumulation in zebrafish larvae via a distinct pathway: Revelation by LA-ICP-MS imaging

Burn pit-related smoke causes developmental and behavioral toxicity in zebrafish: Influence of material type and emissions chemistry

Combustion of mixed materials during open air burning of refuse or structural fires in the wildland urban interface produces emissions that worsen air quality, contaminate rivers and streams, and cause poor health outcomes including developmental effects. The zebrafish, a freshwater fish, is a useful model for quickly screening the toxicological and developmental effects of agents in such species and elicits biological responses that are often analogous and predictive of responses in mammals. The purpose of this study was to compare the developmental toxicity of smoke derived from the burning of 5 different burn pit-related material types (plywood, cardboard, plastic, a mixture of the three, and the mixture plus diesel fuel as an accelerant) in zebrafish larvae. Larvae were exposed to organic extracts of increasing concentrations of each smoke 6-to-8-hr post fertilization and assessed for morphological and behavioral toxicity at 5 days post fertilization. To examine chemical and biological determinants of toxicity, responses were related to emissions concentrations of polycyclic hydrocarbons (PAH). Emissions from plastic and the mixture containing plastic caused the most pronounced developmental effects, including mortality, impaired swim bladder inflation, pericardial edema, spinal curvature, tail kinks, and/or craniofacial deformities, although all extracts caused concentration-dependent effects. Plywood, by contrast, altered locomotor responsiveness to light changes to the greatest extent. Some morphological and behavioral responses correlated strongly with smoke extract levels of PAHs including 9-fluorenone. Overall, the findings suggest that material type and emissions chemistry impact the severity of zebrafish developmental toxicity responses to burn pit-related smoke.

Chronotropic and vasoactive properties of the gut bacterial short chain fatty acids in larval zebrafish.

Short-chain fatty acids (SCFAs) by the gut bacteria have been associated with cardiovascular dysfunction in humans and rodents. However, studies exploring effects of SCFAs on cardiovascular parameters in the zebrafish, an increasingly popular model in cardiovascular research, remain limited. Here, we performed fecal bacterial 16S sequencing and gas chromatography/mass spectrometry (GC-MS) to determine the composition and abundances of gut microbiota and SCFAs in adult zebrafish. Following this, the acute effects of major SCFAs on heart rate and vascular tone were measured in anaesthetized zebrafish larvae using fecal concentrations of butyrate, acetate, and propionate. Lastly, we investigated if co-incubation with butyrate may lessen the effects of angiotensin II (Ang II) and phenylephrine (PE) on vascular tone in anaesthetized zebrafish larvae. We found that the abundance in Proteobacteria, Firmicutes, and Fusobacteria phyla in the adult zebrafish resembled those reported in rodents and humans. SCFA levels with highest concentration of acetate (27.43µM), followed by butyrate (2.19µM) and propionate (1.65µM) were observed in the fecal samples of adult zebrafish. Immersion in butyrate and acetate produced a ~20% decrease in heart rate (HR), respectively, with no observed effects of propionate. Butyrate alone also produced a ~25% decrease in the cross-sectional width of the dorsal aorta (DA) at 60 min (*P<0.05), suggesting compensatory vasoconstriction, with no effects of either acetate or propionate. In addition, butyrate significantly alleviated the decrease in DA cross-sectional width produced by both Ang II and PE. We demonstrate the potential for zebrafish in investigation of host-microbiota interactions in cardiovascular health.

Effects of developmental exposure to individual and combined PFAS on development and behavioral stress responses in larval zebrafish

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals known for their widespread use and persistence in the environment. Laboratory and epidemiological studies investigating these compounds have signaled their neurotoxic and endocrine-disrupting propensities, prompting further research into their effects on behavioral stress responses and their potential role as risk factors for stress-related disorders such as anxiety and depression. This study elucidates the ramifications of early developmental exposures to individual and combined PFAS on the development and behavioral stress responses of larval zebrafish (Danio rerio), an established model in toxicological research. Wild-type zebrafish embryos were enzymatically dechorionated and exposed to PFOS, PFOA, PFHxS, and PFHxA between 6 and 120 h post-fertilization (hpf). We targeted environmentally relevant concentrations stemming from the USEPA 2016 Hazard Advisory Limit (HAL, 0.07 μg/L) and folds higher (0.35, 0.7, 1.75, and 3.5 μg/L). Evaluations at 120 hpf encompassed mortality, overall development, developmental defects, and larval activity both at baseline stress levels and following exposure to acute stressors (acoustic and visual). Larval exposure to PFOA, PFOS, or PFHxS (0.07 μg/L or higher) elicited significant increases in mortality rates, which capped at 23.1%. Exposure to individual chemicals resulted in limited effects on overall development but increased the prevalence of developmental defects in the body axis, swim bladder, pigmentation, and eyes, as well as the prevalence of yolk sac and pericardial edemas. Larval activity at baseline stress levels and following exposure to acute stimuli was significantly altered. Combined exposure to all four chemicals intensified the breadth of developmental and behavioral alterations, suggesting possible additive or synergistic effects. Our findings shed light on the developmental and neurobehavioral disturbances associated with developmental exposure to PFAS at environmentally relevant concentrations, the added risks of combined exposures to these chemicals, and their possible role as environmental risk factors for stress-related disorders.

Differential Roles of Diet on Development and Spinal Cord Regeneration in Larval Zebrafish.

The zebrafish is a powerful model organism for studying development and regeneration. However, there is a lack of a standardized reference diet for developmental and regeneration experiments. Most studies evaluate the rate of growth, survival, and fecundity. In this study, we compare three diets and their effects on growth and regeneration after a spinal cord injury (SCI). Fish were fed daily for 1 week with daily measurements of overall length and width of spinal injury. Fish fed a live rotifer diet grew 32%, whereas a commercially available diet only led to a 4% increase in body length. Similarly, differences in rate of regeneration were observed with over 80% of rotifer-fed larvae forming a glial bridge after injury compared to <10% of zebrafish fed with the commercial diet. Our data highlight the need for establishing a standardized diet for regeneration studies to improve research reproducibility.