Zebrafish Danio Rerio Embryos Research Articles

Copper-Nanoparticle-Induced Neurotoxic Effect and Oxidative Stress in the Early Developmental Stage of Zebrafish (Danio rerio)

Copper nanoparticles (CuNPs) are extensively used in electronics, cosmetics, fungicides, and various other fields due to their distinctive qualities. However, this widespread usage can contribute to environmental contamination and heightened health risks for living organisms. Despite their prevalent use, the ecological impacts and biosafety of CuNPs remain inadequately understood. The present study aims to delve into the potential toxic effects of CuNPs on zebrafish (Danio rerio) embryos, focusing on multiple indexes such as embryonic development, neurotoxicity, oxidative stress, and inflammatory response. The results revealed a notable increase in the death rate and deformity rate, alongside varying degrees of decrease in hatching rate and heart rate following CuNPs exposure. Particularly, the frequency of spontaneous tail coiling significantly declined under exposure to CuNPs at concentrations of 500 µg/L. Furthermore, CuNPs exposure induced alterations in the transcriptional expression of GABA signaling pathway-related genes (gabra1, gad, abat, and gat1), indicating potential impacts on GABA synthesis, release, catabolism, recovery, and receptor binding. Additionally, CuNPs triggered oxidative stress, evidenced by disruption in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, along with elevated malondialdehyde (MDA) levels. This oxidative stress subsequently led to a proinflammatory cascade, as demonstrated by the increased transcriptional expression of inflammatory markers (il-1β, tnf-α, il-6, and il-8). Comparative analysis with copper ion (provided as CuCl2) exposure highlighted more significant changes in most indexes with CuCl2, indicating greater toxicity compared to CuNPs at equivalent concentrations. In conclusion, these findings provide valuable insights into the toxic effects of CuNPs on zebrafish embryo development and neurotransmitter conduction. Furthermore, they present technical methodologies for assessing environmental and health risks associated with CuNPs, contributing to a better understanding of their biosafety and ecological impact.

Evaluating the toxicity of estetrol, 17α-ethinylestradiol, and their combination with drospirenone on zebrafish larvae: A behavioural and proteomic study

ObjectiveTo characterise and compare the toxicity of estetrol (E4) and 17α-ethinylestradiol (EE2), and their respective mixture with the progestin drospirenone (DRSP) in zebrafish (Danio rerio) embryos. MethodsZebrafish embryos were exposed to E4, EE2, DRSP, E4+DRSP, and EE2+DRSP in a fish embryo acute toxicity (FET) test. A second test examined behavioural responses and, using label-free proteomics, identified changes in protein expression in response to hormonal treatments, across a range of concentrations, including those that are considered to be environmentally relevant. ResultsIn the FET test, no effects were found from E4 at concentrations ≤100 mg/L, while EE2 induced mortality and morphological abnormalities at concentrations of 1-2 mg/L. In the behavioural test, exposure to 30 ng/L EE2 (∼200 × predicted environmental concentration – PEC) resulted in hypoactivity in fish larvae and exposure to 0.3 ng/L EE2 (∼2 × PEC) led to quantitative changes in protein abundance, revealing potential impacts on RNA processing and protein synthesis machinery. Exposure to E4 did not alter behaviour, but several groups of proteins were modulated, mainly at 710 ng/L (∼200 × PEC), including proteins involved in oxidative phosphorylation. When combined with DRSP, EE2 induced reduced effects on behaviour and proteomic responses, suggesting an antagonistic effect of DRSP. E4+DRSP induced no significant effects on behaviour or proteomic profiles at tested concentrations. ConclusionsThese findings suggest that E4-based combined oral contraceptives present a more favourable environmental profile than EE2-based contraceptives, particularly during the early developmental stages of fish.

Micro-and nano-plastics induce kidney damage and suppression of innate immune function in zebrafish (Danio rerio) larvae

Aquatic environments serve as critical repositories for pollutants and have significantly accumulated micro- and nanoplastics (MNPs) due to the extensive production and application of plastic products. While the disease resistance and immunity of fish are closely linked to the condition of their aquatic habitats, the specific effects of nanoplastics (NPs) and microplastics (MPs) within these environments on fish immune functions are still not fully understood. The present study utilized zebrafish (Danio rerio) embryos and larvae as model organisms to examine the impacts of polystyrene NPs (100 nm) and MPs (5 μm) on fish immune responses. Our findings reveal that NPs and MPs tend to accumulate on the surfaces of embryos and within the intestines of larvae, triggering oxidative stress and significantly increasing susceptibility to Edwardsiella piscicida infection in zebrafish larvae. Transmission electron microscopy examined that both NPs and MPs inflicted damage to the kidney, an essential immune organ, with NPs predominantly inducing endoplasmic reticulum stress and MPs causing lipid accumulation. Transcriptomic analysis further demonstrated that both NPs and MPs significantly suppress the expression of key innate immune pathways, notably the C-type lectin receptor signaling pathway and the cytosolic DNA-sensing pathway. Within these pathways, the immune factor interleukin-1 beta (il1b) was consistently downregulated in both exposure groups. Furthermore, exposure to E. piscicida resulted in restricted upregulation of il1b mRNA and protein levels, likely contributing to diminished disease resistance in zebrafish larvae exposed to MNPs. Our findings suggest that NPs and MPs similarly impair the innate immune function of zebrafish larvae and weaken their disease resistance, highlighting the significant environmental threat posed by these pollutants.

Abnormal eyes and spine development in zebrafish (Danio rerio) embryos and larvae induced by triphenyltin

Triphenyltin (TPT) is widely used in crop pest control and ship antifouling coatings, which leads to its entry into aquatic environment and poses a threat to aquatic organisms. However, the effects of TPT on the early life stages of wild fish in natural water environments remains unclear. The aim of this study was to assess the toxic effects of TPT on the early life stages of fish under two different environments: field investigation and laboratory experiment. The occurrence of deformities in wild fish embryos and larvae in the Three Gorges Reservoir (TGR) and the developmental toxicity of TPT at different concentrations (0, 0.15, 1.5 and 15 μg Sn/L) to zebrafish embryos and larvae were observed. The results showed that TPT content was higher in wild larvae, reaching 27.21 ng Sn/g w, and the malformation of wild fish larvae mainly occurred in the eyes and spine under natural water environment. Controlled experiment exposure of zebrafish larvae to TPT also resulted in eye and spinal deformities. Gene expression analysis showed that compared with the control group, the expression levels of genes related to eye development (sox2, otx2, stra6 and rx1) and spine development (sox9a and bmp2b) were significantly up-regulated in the 15 μg Sn/L exposure group, which may be the main cause of eye and spine deformity in the early development stage of fish. In addition, the molecular docking results further elucidate that the strong hydrophobic and electrostatic interactions between TPT and protein residues are the main mechanism of TPT induced abnormal gene expression. Based on these results, it can be inferred that TPT is one of the teratogenic factors of abnormal eye and spine development in the early life stage of fish in the TGR. These findings have important implications for understanding the toxicity of TPT on fish.

Sargassum spp. Ethanolic Extract Elicits Toxic Responses and Malformations in Zebrafish (Danio rerio) Embryos.

The amount of Sargassum spp. arriving in the Caribbean Sea has increased steadily in the last few years, producing a profound environmental impact on the ecological dynamics of the coasts of the Yucatan Peninsula. We characterized the toxicological effects of an ethanolic extract of Sargassum spp. on zebrafish (Danio rerio) embryos (ZFEs) in a 96-h static bioassay using T1 (0.01 mg/L), T2 (0.1 mg/L), T3 (1 mg/L), T4 (10 mg/L), T5 (25 mg/L), T6 (50 mg/L), T7 (75 mg/L), T8 (100 mg/L), T9 (200 mg/L), and T10 (400 mg/L). In this extract, we detected 74 compounds by gas chromatography-mass spectrometry (GC-MS), of which hexadecanoic acid methyl ester, and 2-pentanone 4-hydroxy-4-methyl, were the most abundant. In ZFEs, a median lethal concentration of 251 mg/L was estimated. Exposed embryos exhibited extensive morphological changes, including edema in the yolk sac, scoliosis, and loss of pigmentation, as well as malformations of the head, tail, and eyes. By integrating these abnormalities using the Integrated Biological Response (IBRv2) and General Morphological Score (GMS) indices, we were able to determine that ZFEs exposed to 200 mg/L (T9) exhibited the most pronounced biological response in comparison with the other groups. In the comparative transcriptomic analysis, 66 genes were upregulated, and 246 genes were downregulated in the group exposed to 200 mg/L compared with the control group. In the upregulated genes, we identified several gene ontology-enriched terms, such as response to xenobiotic stimuli, cellular response to chemical stimulus, transcriptional regulation, pigment metabolic process, erythrocyte differentiation and embryonic hemopoiesis, extracellular matrix organization, and chondrocyte differentiation involved in endochondral bone morphogenesis, among others. In the down-regulated genes, we found many genes associated with nervous system processes, sensory and visual perception, response to abiotic stimulus, and the nucleoside phosphate biosynthetic process. The probable connections among the morphological changes observed in the transcriptome are thoroughly discussed. Our findings suggest that Sargassum spp. exposure can induce a wide negative impact on zebrafish embryos. Environ Toxicol Chem 2024;43:1075-1089. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Biological Effect of Microplastics with Different Functional Groups on the Bacterial Communities and Metabolic Functions of Zebrafish (Danio rerio) Embryos

To investigate the influences of functional groups on the biological effects caused by microplastics, the accumulation of three polystyrene microplastics (PS, PS-NH2, and PS-COOH) in zebrafish (Danio rerio) embryos were analyzed, and then the responses of metabolic functions and microbial communities in zebrafish larvae were revealed using the combination of the microbiome and metabolome methods. The results showed that all microplastics could accumulate in zebrafish with concentrations ranging from 143 to 175 μg·g-1, and there were no significant differences in the accumulation potentials among different PS treatments. Exposure to plain PS significantly affected the metabolic capacity of aminoglycosides in zebrafish larvae, whereas the metabolic processes of amino acids were affected by PS-NH2. In the PS-COOH treatment, the metabolic pathways of the tricarboxylic acid cycle, amino acids, and glycolysis in zebrafish were markedly altered. The metabolic functions of zebrafish larvae were changed by all PS microplastics, resulting in toxic effects on zebrafish, and the functional group modification of microplastics may have further enhanced these toxicities. Compared to that in the control, exposure to PS-NH2 significantly reduced the diversity of microbial communities in zebrafish larvae and increased the proportion of Proteobacteria in the composition, leading to an imbalance of the bacterial community in zebrafish and thus disrupting the metabolic functions in the fish. Therefore, the functional modifications of microplastics may significantly alter the related stresses on aquatic organisms, leading to unpredictable ecological risks.

Chemical Cues Released by Predators’ Consumption of Heterospecific Prey Alter the Embryogenesis of Zebrafish

Environmentally cued hatching is prevalent, diverse, and crucial to many animals’ survival. Fish embryos use a variety of chemical cues to initiate hatching to avoid potential predators, yet the function of chemical cues released from the predatory consumption of heterospecific prey is largely unknown. Zebra cichlids (Metriaclima estherae) are ferocious predators that can feed on medaka (Oryzias latipes), though it is impossible for this to occur in their natural habitat. Zebrafish (Danio rerio) embryos have been employed as experimental subjects due to their sensitivity to a variety of chemical signals. In this study, zebrafish embryos were subjected to three types of chemical signals: predator cues (PCs, released from cichlids), heterospecific cues (HCs, released from medaka), and heterospecific dietary cues (HDCs, released from cichlids that have ingested medaka). As a result, the hatching times of zebrafish embryos were accelerated by 6.8% and 12.6% by PCs and HDCs, respectively. PCs and HDCs cause significantly reduced morphology in zebrafish embryos, including regarding total length, eye length, dorsal fin length, trunk height, caudal fin height, and body cavity, and increase yolk sac height. The PCs and HDCs diminished the larvae’s motion at 120 and 144 h post fertilization (hpf), which could be attributed to non-developmental embryogenesis. Overall, the impacts of HDCs on embryonic hatching, developmental morphology, and locomotor were more pronounced in comparison with PCs. Our findings demonstrate that predators’ dietary cues, even those released after predation on heterospecific prey, can modify embryogenesis, highlighting the critical functions of chemical signals in predation risk assessment using embryos.

Per- and Polyfluoroalkyl Substances: Impacts on Morphology, Behavior and Lipid Levels in Zebrafish Embryos.

The presence of per- and polyfluoroalkyl substances (PFASs) in aquatic environments is often persistent and widespread. Understanding the potential adverse effects from this group of chemicals on aquatic communities allows for better hazard characterization. This study examines impacts on zebrafish (Danio rerio) embryo physiology, behavior, and lipid levels from exposure to perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and heptadecafluorooctanesulfonic acid (PFOS). Embryos were exposed to lethal and sublethal levels of each chemical and monitored for alterations in physiological malformations, mortality, lipid levels, and behavior (only PFOA and PFHxS). The predicted 50% lethal concentrations for 120 hpf embryos were 528.6 ppm PFOA, 14.28 ppm PFHxS, and 2.14 ppm PFOS. Spine curvature and the inability of the 120 hpf embryos to maintain a dorsal-up orientation was significantly increased at 10.2 ppm PFHxS and 1.9 ppm PFOS exposure. All measured 120 hpf embryo behaviors were significantly altered starting at the lowest levels tested, 188 ppm PFOA and 6.4 ppm PFHxS. Lipid levels decreased at the highest PFAS levels tested (375 PFOA ppm, 14.4 PFHxS ppm, 2.42 ppm PFOS). In general, the PFAS chemicals, at the levels examined in this study, increased morphological deformities, embryo activity, and startle response time, as well as decreased lipid levels in 120 hpf zebrafish embryos.

An Integrated Metabolomics-Based Model, and Identification of Potential Biomarkers, of Perfluorooctane Sulfonic Acid Toxicity in Zebrafish Embryos.

Known for their high stability and surfactant properties, per- and polyfluoroalkyl substances (PFAS) have been widely used in a range of manufactured products. Despite being largely phased out due to concerns regarding their persistence, bioaccumulation, and toxicity, legacy PFAS such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid continue to persist at high levels in the environment, posing risks to aquatic organisms. We used high-resolution magic angle spinning nuclear magnetic resonance spectroscopy in intact zebrafish (Danio rerio) embryos to investigate the metabolic pathways altered by PFOS both before and after hatching (i.e., 24 and 72 h post fertilization [hpf], respectively). Assessment of embryotoxicity found embryo lethality in the parts-per-million range with no significant difference in mortality between the 24- and 72-hpf exposure groups. Metabolic profiling revealed mostly consistent changes between the two exposure groups, with altered metabolites generally associated with oxidative stress, lipid metabolism, energy production, and mitochondrial function, as well as specific targeting of the liver and central nervous system as key systems. These metabolic changes were further supported by analyses of tissue-specific production of reactive oxygen species, as well as nontargeted mass spectrometric lipid profiling. Our findings suggest that PFOS-induced metabolic changes in zebrafish embryos may be mediated through previously described interactions with regulatory and transcription factors leading to disruption of mitochondrial function and energy metabolism. The present study proposes a systems-level model of PFOS toxicity in early life stages of zebrafish, and also identifies potential biomarkers of effect and exposure for improved environmental biomonitoring. Environ Toxicol Chem 2024;43:896-914. © 2024 SETAC.

Petrosamine isolated from marine sponge Petrosia sp. demonstrates protection against neurotoxicity in vitro and in vivo

According to The World Alzheimer Report 2023 by Alzheimer’s Disease International (ADI) estimates that 33 to 38.5 million people worldwide suffer from Alzheimer’s Disease (AD). A crucial hallmark associated with this disease is associated with the deficiency of the brain neurotransmitter acetylcholine, due to an affected acetylcholinesterase (AChE) activity. Marine organisms synthesize several classes of compounds, some of which exhibit significant AChE inhibition, such as petrosamine, a coloured pyridoacridine alkaloid. The aim of this work was to characterize the activity of petrosamine isolated for the first time from a Brazilian marine sponge, using two neurotoxicity models with aluminium chloride, as exposure to aluminium is associated with the development of neurodegenerative diseases. The in vitro model was based in a neuroblastoma cell line and the in vivo model exploited the potential of zebrafish (Danio rerio) embryos in mimicking hallmarks of AD. To our knowledge, this is the first report on petrosamine’s activity over these parameters, either in vitro or in vivo, in order to characterize its full potential for tackling neurotoxicity.Graphical

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.

Long arm octopus (Octopus minor) extract prevents eye injury caused by particulate matter exposure in zebrafish (Danio rerio) embryos

Long arm octopus (Octopus minor) extract prevents eye injury caused by particulate matter exposure in zebrafish (Danio rerio) embryos

Ecological assessment of stream water polluted by phosphorus chemical plant: Physiological, biochemical, and molecular effects on zebrafish (Danio rerio) embryos

The rapid development of the phosphorus chemical industry has caused serious pollution problems in the regional eco-environment. However, understanding of their ecotoxic effects remains limited. This study aimed to investigate the developmental toxicity of a stream polluted by a phosphorus chemical plant (PCP) on zebrafish embryos. For this, zebrafish embryos were exposed to stream water (0, 25, 50, and 100% v/v) for 96 h, and developmental toxicity, oxidative stress, apoptosis, and DNA damage were assessed. Stream water-treated embryos exhibited decreased hatching rates, heart rates, and body lengths, as well as increased mortality and malformation rates. The general morphology score system indicated that the swim bladder and pigmentation were the main abnormal morphological endpoints. Stream water promoted antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and glutathione peroxidase (GPx)), lipid peroxidation, and DNA damage. It also triggered apoptosis in the embryos' heads, hearts, and spines by activating apoptotic enzymes (Caspase-3 and Caspase-9). Additionally, stream water influenced growth, oxidative stress, and apoptosis-related 19 gene expression. Notably, tyr, sod (Mn), and caspase9 were the most sensitive indicators of growth, oxidative stress, and apoptosis, respectively. The current trial concluded that PCP-polluted stream water exhibited significant developmental toxicity to zebrafish embryos, which was regulated by the oxidative stress-mediated activation of endogenous apoptotic signaling pathways.

Developmental effects of zebrafish (Danio rerio) embryos after exposure to glyphosate and lead mixtures

Natural aquatic environments have a heterogeneous composition; therefore, simultaneous exposure to multiple contaminants is relevant and more realistic when assessing exposure and toxicity. This study examines the combinatorial effects of two compounds found ubiquitously in drinking water across the United States: glyphosate and lead acetate. Zebrafish (Danio rerio) embryos were used as a model for investigating developmental delays following controlled exposures. Six different environmentally relevant exposure concentrations of glyphosate, ranging from 0.001 to 10 ppm, and lead acetate, ranging from 0.5 to 4 ppm, were applied first as single exposures and then as co-exposures. The sublethal endpoints of hatching and coagulation were quantified to determine potencies. Results indicate that higher concentrations of the individual chemicals correlate with later hatching with correlation coefficients of 0.71 and 0.40 for glyphosate and lead acetate respectively, while the co-exposure at lower concentrations induced earlier hatching with a correlation coefficient 0.74. In addition, increased levels of coagulation and glutathione reductase activity were observed following co-exposure, as compared to the individual exposures, suggesting potential toxicological interactions. These results support the need for further work assessing the combined potencies of aquatic contaminants rather than individual exposures.

Particulate Matter Induced Adverse Effects on Eye Development in Zebrafish (Danio rerio) Embryos.

Particulate matter (PM) can cause human diseases, particularly respiratory diseases. Since eyes are directly exposed to the air, they might be directly adversely affected by PM. Therefore, we determined the toxicity caused to eye development by PM using zebrafish (Danio rerio) embryos. The PM-induced embryo toxicity was dependent on dose and time and caused significant morphological defects, reducing the total body length and the total eye area. Reactive oxygen species (ROS) overproduction was confirmed in the PM treatment group, and antioxidant genes (cat and sod2), photoreceptor cell development, pigmentation genes (atoh8, vsx1, and rho), eye-embryogenesis genes (pax6a and pax6b), and eye-lens-development genes (cryaa) were downregulated, while eye-development genes (crybb1) were upregulated. In conclusion, PM had a direct adverse effect on the eyes, and zebrafish embryos can be used as a model to evaluate PM-induced eye toxicity in vivo.

Morphological, teratogenic and behavioral evaluations of Gelidium spinosum methanol extract on zebrafish embryos

Gelidium spinosum is an edible red seaweed from the family Gelidiaceae with possibility to be developed. The potential medical benefits of G. spinosum have been established, yet adequate empirical research on its toxicity is still lacking. Hence, the present work was aimed to examine the toxicity of G. spinosum methanol extract (GsME) on zebrafish (Danio rerio) embryos and to identify the phytoconstituents using gas chromatography-mass spectrometry (GC-MS) analysis. Results of this study showed that GsME induced morphological defects in zebrafish embryos, including reduction in eye size and body length. Moreover, yolk sac size and mortality were increased in zebrafish embryos exposed to GsME in a dose-dependent manner. GsME at high concentrations triggered teratogenic effects in zebrafish embryos such as decrease in heartbeat/minute, lack of pigmentation, lack of somite, structural deformity, pericardial oedema, and yolk oedema. The LC50 and EC50 of GsME were < 100 mg/L, which classified as a harmful category. The teratogenic index of GsME was found to be > 1, indicating its teratogenic attribute. Additionally, GsME exerted behavioral effects i.e. significantly lower total distance of movement and slower swimming speed of zebrafish embryos. GC-MS analysis of GsME was confirmed the presence of amino acid, phenolics, carboxylic acids, reducing sugars, saturated fatty acids and brominated saturated fatty acid in the extract. It suggesting that compounds of 13-bromotetradecanoic acid, palmitic acid, and stearic acid containing in GsME were contributed to the toxic effects.

Assessment of thimerosal effects in sublethal concentrations on zebrafish (Danio rerio) embryos exploring NMR-based metabolomics profile

Thimerosal, a preservative commonly used in the pharmaceutical and cosmetic industry, has raised concerns regarding its potentially toxic effects as an organic mercury compound. Within this context, using an NMR-based metabolomics profile and chemometric analysis, zebrafish embryos were used as an in vivo model to study the effects of thimerosal in metabolic profiles after exposure to sublethal concentrations of the mercury compound. The thimerosal concentrations of 40 and 80 nM were employed, corresponding to 40% and 80% of the LC50, respectively, for zebrafish embryos. The most significant alterations in the metabolic profile included changes in carbohydrates, amino acids, nucleotides, trimethylamine-N-oxide, ethanolamine, betaine, and ethanol. Furthermore, thimerosal exposure affects various metabolic pathways, impairing the nervous system, disrupting protein metabolism, and potentially causing oxidative damage. Therefore, adopting a metabolomics approach in this investigation provided insights into the potentially implicated metabolic pathways contributing to the deleterious effects of thimerosal in biological systems.

Developmental Toxicity of C10 Massoia Lactone, the Main Constituent of Cryptocarya massoia, on Zebrafish (Danio rerio) Embryos

C10 massoia lactone (C10) is the main component of massoia essential oil derived from Cryptocarya massoia plant bark, which is used as natural flavoring agent of “generally recognized as safe” status. In this study, the developmental toxicity of C10 was evaluated on zebrafish (Danio rerio) embryos at an exposure level of 0–2000 µg·L−1, and acute toxicity was determined with respect to lethal effects, hatching rates, and morphological changes. Additionally, morphological changes were determined for the endpoints as the occurrence of yolk edema, pericardial edema, spine curvature, and shortened body length after treatment until 96 h post-fertilization (hpf). The complete lethality of C10 was achieved with embryos treated at 2000 µg·L−1, and most embryos treated at 1000 µg·L−1 developed pericardial edemas with some spine curvature. Some embryos exhibited delayed development with shortened body length when compared with the control. Hatchability was completely accomplished at the tested dose of 1000 µg·L−1, and cardiac malformation was observed using a transgenic zebrafish line Tg(cmlc:EGFP), with a lower heartbeat rate in embryos treated with C10 for 72 hpf. After 96 hpf, heartbeat rates were normalized when compared with the control group, and two cardiac development-related genes such as nppa and canca1 were differently expressed in C10-treated embryos by 2.3-fold and 0.4-fold, respectively. Therefore, C10 must be studied further in other higher organisms for its risk.

Realistic microplastics harness bacterial presence and promote impairments in early zebrafish embryos: Behavioral, developmental, and transcriptomic approaches.

The plastisphere is a newly recognized ecosystem. However, its interaction with early life stages of aquatic vertebrates is a multifaceted issue that requires further research. This study investigated the involvement of bacteria in shaping realistic microplastics hazards in zebrafish Danio rerio embryos. Fish were exposed to bottle micro-fragments (FR) and textile micro-fibers (FI) of polyethylene terephthalate (5–15 μm), concomitant with Aeromonas salmonicida achromogenes challenge from 2h post-fertilization for 3 days. Egg chorion showed affinity for FR and FI, inducing earlier embryo hatching. However, this effect was masked by biofilm invasion. Fragments were more detrimental than fibers on developmental parameters, while bacterial presence compromised body length, eye, and yolk sac surface area. In a further finding, MPs alone increased locomotor activity in zebrafish larvae, without synergistic effect when combined with bacteria. Data showed that realistic MPs had no significant effects except for downregulated sod and cyp1a gene expression, whereas bacterial challenge inhibited larval potency for most of the evaluated mRNA levels (mpx (immune system), apoeb (lipid metabolism), nfkb and tfa (inflammation), cyp and sod (oxidative stress)). This study provides new insights into realistic microplastic effects under relevant conditions when combined with environmental pathogen within the first life stages of aquatic vertebrates.

Confocal Microscopy Technique in Teratogenicity Testing Using Zebrafish (Danio rerio) Embryos as Model.

Teratogenicity refers to the ability to cause adverse effects on the normal development of embryos resulting in retardation of growth as well as structural and functional abnormalities in the developing embryos. Zebrafish (Danio rerio) is one of the prime model organisms for teratogenicity testing, owing to the many advantages it offers, particularly its relatively large and initially transparent embryos, which allow real-time imaging of the various developmental stages. Confocal microscopy provides the best technique for imaging cellular dynamics within zebrafish embryos as it gives high-resolution imaging of thick tissues. This chapter focuses on major teratogenicity testing techniques using confocal microscopy. Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay, immunohistochemistry assay, and reactive oxygen species (ROS) detection are important methods for studying the teratogenicity of drugs or compounds using 6h post-fertilization Zebrafish embryos.