Novel insights on acetylcholinesterase inhibition by Convolvulus pluricaulis,scopolamine and their combination in zebrafish

Acetylcholinesterase(AChE)inhibitors increase the retention of acetylcholine(ACh)in synapses.Although they allevi-ate cognitive deficits in Alzheimer’s disease,their limited benefits warrant investigations of plant extracts with similar properties.We studied the anti-AChE activity of Convolvulus pluricaulis(CP)in a zebrafish model of cognitive impair-ment induced by scopolamine(SCOP).CP is a perennial herb with anti-amnesiac and anxiolytic properties.It contains alkaloid,anthocyanin,coumarin,flavonoid,phytosterol and triterpenoid components.Isoxazole(ISOX)was used as a positive control for AChE inhibition.CP-treated 168 hpf larvae showed a similar pattern of AChE inhibition(in the myelencephalon and somites)as that of ISOX-treated larvae.CP was superior to ISOX as evidenced by the retention of avoidance response behavior in adult zebrafish.Molecular docking studies indicated that ISOX binds Ser203 of the catalytic triad on the human AChE.The active components of CP-scopoletin and kaempferol-were bound by His447 of the catalytic triad,the anionic subsite of the catalytic center,and the peripheral anionic site.This suggested the ability of CP to mediate both competitive and non-competitive modes of inhibition.Surprisingly,SCOP showed AChE inhibition in larvae,possibly mediated via the choline-binding sites.CP+SCOP induced a concentration-dependent increase in AChE inhibition and ACh depletion.Abnormal motor responses were observed with ISOX,CP,ISOX+SCOP,and CP+SCOP,indicative of undesirable effects on the peripheral cholinergic system.Our study proposes the examination of CP,SCOP,and CP+SCOP as potential AChE inhibitors for their ability to modulate cognitive deficits.

Mechanism by which Rab5 promotes regeneration and functional recovery of zebrafish Mauthner axons

Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles and their fusion with the cellular membrane. Rab5 has been reported to play an important role in the development of the zebrafish embryo;however, its role in axonal regeneration in the central nervous system remains unclear. In this study, we established a zebrafish Mauthner cell model of axonal injury using single-cell electroporation and two-photon axotomy techniques. We found that overexpression of Rab5 in single Mauthner cells promoted marked axonal regeneration and increased the number of intra-axonal transport vesicles. In contrast, treatment of zebrafish larvae with the Rab kinase inhibitor CID-1067700markedly inhibited axonal regeneration in Mauthner cells. We also found that Rab5 activated phosphatidylinositol 3-kinase(PI3K) during axonal repair of Mauthner cells and promoted the recovery of zebrafish locomotor function. Additionally, rapamycin, an inhibitor of the mechanistic target of rapamycin downstream of PI3K, markedly hindered axonal regeneration. These findings suggest that Rab5 promotes the axonal regeneration of injured zebrafish Mauthner cells by activating the PI3K signaling pathway.

Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish:unveiling the role of vascular endothelial growth factor signaling and microglia

After brain damage,regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals,suggesting a close link between these processes.However,the mechanisms by which these processes interact are not well understood.In this work,we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury.To this end,we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms.First,using the Tg(fli1:EGFP×mpeg1.1:mCherry)zebrafish line,which enables visualization of blood vessels and microglia respectively,we analyzed regenerative angiogenesis from 1 to 21 days post-lesion.In parallel,we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry.We found that after brain damage,the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor(vegfaa and vegfbb)were increased.At the same time,neural stem cell proliferation was also increased,peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis,along with the recruitment of microglia.Then,through pharmacological manipulation by injecting an anti-angiogenic drug(Tivozanib)or Vegf at the lesion site,we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes,as well as microglial recruitment.Finally,we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis,as previously described,as well as injury-induced angiogenesis.In conclusion,we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process.In addition,we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes.This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

Potential therapeutic role of spermine via Rac1 in osteoporosis:Insights from zebrafish and mice

Osteoporosis(OP)is a prevalent metabolic bone disease.While drug therapy is essential to prevent bone loss in osteoporotic patients,current treatments are limited by side effects and high costs,necessitating the development of more effective and safer targeted therapies.Utilizing a zebrafish(Danio rerio)larval model of osteoporosis,we explored the influence of the metabolite spermine on bone homeostasis.Results showed that spermine exhibited dual activity in osteoporotic zebrafish larvae by increasing bone formation and decreasing bone resorption.Spermine not only demonstrated excellent biosafety but also mitigated prednisolone-induced embryonic neurotoxicity and cardiotoxicity.Notably,spermine showcased protective attributes in the nervous systems of both zebrafish embryos and larvae.At the molecular level,Rac1 was identified as playing a pivotal role in mediating the antiosteoporotic effects of spermine,with P53 potentially acting downstream of Rac1.These findings were confirmed using mouse(Mus musculus)models,in which spermine not only ameliorated osteoporosis but also promoted bone formation and mineralization under healthy conditions,suggesting strong potential as a bonestrengthening agent.This study underscores the beneficial role of spermine in osteoporotic bone homeostasis and skeletal system development,highlighting pivotal molecular mediators.Given their efficacy and safety,human endogenous metabolites like spermine are promising candidates for new anti-osteoporotic drug development and daily bone-fortifying agents.

Ethyl acetate fraction of Sargassum pallidum extract attenuates particulate matter-induced oxidative stress and inflammation in keratinocytes and zebrafish

Objective:To evaluate the effect of the ethyl acetate fraction derived from Sargassum pallidum extract against particulate matter(PM)-induced oxidative stress and inflammation in HaCaT cells and zebrafish.Methods:HaCaT cells and zebrafish were used to evaluate the protective effects of the ethyl acetate fraction of Sargassum pallidum extract against PM-induced oxidative stress and inflammation.The production of nitric oxide(NO),intracellular ROS,prostaglandin E_(2)(PGE_(2)),and pro-inflammatory cytokines,and the expression levels of COX-2,iNOS,and NF-κB were evaluated in PM-induced HaCaT cells.Furthermore,the levels of ROS,NO,and lipid peroxidation were assessed in the PM-exposed zebrafish model.Results:The ethyl acetate fraction of Sargassum pallidum extract significantly decreased the production of NO,intracellular ROS,and PGE_(2) in PM-induced HaCaT cells.In addition,the fraction markedly suppressed the levels of pro-inflammatory cytokines and inhibited the expression levels of COX-2,iNOS,and NF-κB.Furthermore,it displayed remarkable protective effects against PM-induced inflammatory response and oxidative stress,represented by the reduction of NO,ROS,and lipid peroxidation in zebrafish.Conclusions:The ethyl acetate fraction of Sargassum pallidum extract exhibits a protective effect against PM-induced oxidative stress and inflammation both in vitro and in vivo and has the potential as a candidate for the development of pharmaceutical and cosmeceutical products.

P-aminobenzoic acid promotes retinal regeneration through activation of Ascl1a in zebrafish

The retina of zebrafish can regenerate completely after injury.M ultiple studies have demonstrated that metabolic alte rations occur during retinal damage;however to date no study has identified a link between metabolites and retinal regeneration of zebrafish.Here,we performed an unbiased metabolome sequencing in the N-methyl-D-aspartic acid-damaged retinas of zebrafish to demonstrate the metabolomic mechanism of retinal regeneration.Among the differentially-ex pressed metabolites,we found a significant decrease in p-aminobenzoic acid in the N-methyl-D-aspartic acid-damaged retinas of zebrafish.Then,we investigated the role of p-aminobenzoic acid in retinal regeneration in adult zebrafish.Impo rtantly,p-aminobenzoic acid activated Achaetescute complex-like 1a expression,thereby promoting Müller glia reprogramming and division,as well as Müller glia-derived progenitor cell proliferation.Finally,we eliminated folic acid and inflammation as downstream effectors of PABA and demonstrated that PABA had little effect on Müller glia distribution.Taken together,these findings show that PABA contributes to retinal regeneration through activation of Achaetescute complex-like 1a expression in the N-methyl-Daspartic acid-damaged retinas of zebrafish.

Three-dimensional morphological and fluorescent imaging of zebrafish with a continuous-rotational light-sheet microscope

Light-sheet fluorescence microscopy(LSFM)has been widely used to image the three-dimensional(3D)structures and functions of various millimeter-size bio-specimen such as zebrafish.However,the sample adsorption and scattering cause shading of the light-sheet illumination,preventing the even 3D image of thick samples.Herein,we report a continuous-rotational light-sheet microscope(CR-LSM)that enables simultaneous 3D bright-field and fluorescence imaging.With a high-accuracy rotational stage,CR-LSM records the outline projections and the fluorescent images of the sample at multiple rotation angles.Then,3D morphology and fluorescent structure were reconstructed with a developed algorithm.Using CR-LSM,zebrafish’s whole-fish contour and blood vessel structures were obtained simultaneously.

Metabolomic-based analysis reveals bile acid-mediated ovarian failure induced by low temperature in zebrafish

As ectotherms, fish are highly sensitive to temperature fluctuations, which can profoundly impact their reproductive cycles. In this study, we investigated the fertility and histological characteristics of zebrafish(Danio rerio) ovaries exposed to a temperature gradient ranging from the thermopreferendum temperature of the species,27℃, to lower temperatures of 22℃, 20℃, and 13℃ over a period of two weeks. Comparative metabolomic(six biological replicates for each temperature) and transcriptomic(four biological replicates for each temperature) analyses were conducted under the four temperature conditions. Results indicated that lower temperatures inhibited oocyte development and differential metabolites were involved in steroid hormone production,antioxidant function, and lipid and protein catabolism.Disrupted reproductive hormones, increased proteolysis,and lipid degradation significantly impeded oocyte development and egg maturation. Notably, a significant increase in bile acid content was noted in the ovaries of the cold-treated fish, indicating that bile acids play a critical role in ovarian failure. Overall, these findings provide valuable insights into the mechanisms governing the reproductive response of fish to cold stress.

c-myb is involved in CML progression and is a therapeutic target in the zebrafish CML model

Background:Despite the success of tyrosine kinase inhibitors in chronic myeloid leukemia(CML)therapy,CML still faces the challenges of drug resistance and progression to blast crisis.Twenty-five percent of patients have imatinib resistance and treatment difficulties due to heterogeneity after progression,but little is known about the mechanism.A key transcription factor in hematopoiesis,MYB,has been reported to increase abnormally in several types of aggressive blood disorders including CML.Methods:This study used a zebrafish model to explore the relationship between BCR/ABL1 and c-myb in CML progression.A CML zebrafish model was crossed with a c-myb hyperactivity transgenic line.Results:It was found that both exogenous BCR/ABL1 and c-myb could up-regulate the expression of neutrophil-related genes.More seriously,neutrophil accumulation was observed when BCR/ABL1 was combined with c-myb overexpression.Further studies showed that c-myb may be one of the downstream targets of BCR/ABL1 and the effect of BCR/ABL1 on neutrophils was c-myb dependent.Taking advantage of this inheritable in vivo model,it was shown that a combination of imatinib and flavopiridol,a cyclin-dependent kinase inhibitor targeting MYB,could more effectively alleviate the aggressive phenotype of the double transgene line.Conclusion:In summary,this study suggests that c-myb acts downstream of BCR/ABL1 and is involved in CML progression and is therefore a risk factor and a valuable target for the treatment of CML progression.The model used in the study could be helpful in high-throughput drug screening in CML transformation.

Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish(Danio rerio)

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Effects of Imazethapyr-Based Herbicide Formulation in the Zebrafish (Danio rerio) Hepatocyte Cell Line (ZF-L): Cytotoxicity and Oxidative Stress

Seizures of agrochemical formulations have increased in Brazil and Rio Grande do Sul is among the Brazilian states with the highest number of seizures of these products obtained illicitly. The use of illicit formulations can cause significant harm to agricultural production, the environment, and non-target species. This study evaluated the cytotoxicity and oxidative stress of a seized formulation containing the herbicide imazethapyr (IMZT). Characterization of the herbicide included gas chromatography-mass spectrometry (GC-MS) and thermal analyses (thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)). Hemolytic and cytotoxicity assays in ZF-L hepatic cells showed IC50 values of 12.75 µg/mL, 3.01 µg/mL, 2.67 µg/mL, and 1.61 µg/mL for erythrocytes, [3(4,5-dimethyl)-2 bromide-5 diphenyl tetrazolium] (MTT), neutral red (NR), and lactate dehydrogenase (LDH) assays, respectively. The median IC50 of 2.84 µg/mL was used in oxidative stress assays, revealing increased reactive oxygen species (ROS) production, reduced total sulfhydryl content, and decreased superoxide dismutase (SOD) and catalase (CAT) activity. This study is the first to report in vitro oxidative stress induced by IMZT in the ZF-L cell line, emphasizing the importance of in vitro assays for assessing the toxic effects of seized agrochemicals on human health and the environment.

Skin Toxicity of Wufang Babu Ointment to Zebrafish

[Objectives]Using wild-type AB strain of zebrafish as experimental animal,this study investigated the damaging effect of Wufang Babu Ointment on skin cells,in order to evaluate the skin toxicity of Wufang Babu Ointment.[Methods]Wild-type AB strain of zebrafish with an age of 2 d were taken and fed in different concentrations of Wufang Babu Ointment solution for 24 h.The number of deaths in each group of zebrafish was recorded,and the mortality rate was calculated.Using Origin 8.0 software,the maximum non lethal concentration(MNLC)was simulated.Zebrafish raised in different concentrations of Wufang Babu Ointment solution for 24 h were placed under an anatomical microscope for taking photos,to analyze and calculate the incidence of skin damage in zebrafish.Based on the statistical analysis results of this indicator,the skin toxicity of Wufang Babu Ointment was evaluated.[Results]The MNLC of Wufang Babu Ointment on zebrafish was 671μg/mL;Wufang Babu Ointment can induce skin damage at the concentrations of 224μg/mL(1/3 MNLC)and 671μg/mL(MNLC).[Conclusions]Wufang Babu Ointment had certain skin toxicity to zebrafish.

Acute Single and Joint Toxicity Effects of Deltamethrin and Lambda-cyhalothrin on Zebrafish(Danio rerio)

In this research,the single and combined toxicity effects of two commonly used pesticides,lambda-cyhalothrin and deltamethrin,were investigated on zebrafish at 20℃,with a weight of(1±0.1)g and a length of(3.5±0.35)cm.The study revealed that lambda-cyhalothrin exhibited higher toxicity compared to deltamethrin.Additionally,when used together,these pesticides showed significantly increased toxic effects on zebrafish.The 96-h LC 50 values were determined to be 3.059μg/L(confidence limits 0.077-0.351μg/L)for lambda-cyhalothrin and 1.304μg/L(confidence limits;0.046-0.228μg/L)for deltamethrin,both demonstrating a significant positive correlation(P<0.05).These results underscore the importance of regulating and managing pesticide use to safeguard aquatic organisms and uphold environmental sustainability.

Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin

Enantioselectivity of chiral pollutants is receiving growing concern due to the difference in toxicology and environment fate between enantiomers.In this study,enantiomers of insecticide beta-cypermethrin （beta-CP） were separated on selected chiral column by HPLC,and the toxicity of enantiomers was evaluated using the zebrafish embryo-larval assays.The enantiomers of beta-CP were baseline separated on Chiralcel OD and Chiralpak AD columns and detected by circular dichroism （CD） at 236 nm.Better separation could be achieved at lower temperature （e.g.,20°C） and with lower levels of polar modifiers.Pure enantiomers were obtained on Chiralcel OD.The CD spectra of enantiomers were recorded.By comparing the elution order with a previous similar study,the absolute configuration of beta-CP enantiomers was determined.The individual enantiomers were used in zebrafish embryo test,and the results showed that beta-CP enantioselectively induced yolk sac edema,pericardial edema and crooked body.The 1R-cis-αS and 1R-trans-αS enantiomers showed strong developmental toxicities at concentration of 0.1 mg/L,while the 1S-cis-αR and 1S-trans-αR induced no malformations at higher concentration （e.g.,0.3 mg/L）.The results suggest that the enantioselective toxicological effects of beta-CP should be considered when evaluating its ecotoxicological effects.

Kai Xin San ameliorates scopolamine-induced cognitive dysfunction

Kai Xin San(KXS, containing ginseng, hoelen, polygala, and acorus), a traditional Chinese herbal compound, has been found to regulate cognitive dysfunction; however, its mechanism of action is still unclear. In this study, 72 specific-pathogen-free male Kunming mice aged 8 weeks were randomly divided into a vehicle control group, scopolamine group, low-dose KXS group, moderate-dose KXS group, high-dose KXS group, and positive control group. Except for the vehicle control group and scopolamine groups(which received physiological saline), the doses of KXS(0.7, 1.4 and 2.8 g/kg per day) and donepezil(3 mg/kg per day) were gastrointestinally administered once daily for 2 weeks. On day 8 after intragastric treatment, the behavioral tests were carried out. Scopolamine group and intervention groups received scopolamine 3 mg/kg per day through intraperitoneal injection. The effects of KXS on spatial learning and memory, pathological changes of brain tissue, expression of apoptosis factors, oxidative stress injury factors, synapse-associated protein, and cholinergic neurotransmitter were measured. The results confirmed the following.(1) KXS shortened the escape latency and increased residence time in the target quadrant and the number of platform crossings in the Morris water maze.(2) KXS increased the percentage of alternations between the labyrinth arms in the mice of KXS groups in the Y-maze.(3) Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that KXS promoted the production of Nissl bodies and inhibited the formation of apoptotic bodies.(4) Western blot assay showed that KXS up-regulated the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic protein Bax. KXS up-regulated the expression of postsynaptic density 95, synaptophysin, and brain-derived neurotrophic factor in the cerebral cortex and hippocampus.(5) KXS increased the level and activity of choline acetyltransferase, acetylcholine, superoxide dismutase, and glutathione peroxidase, and reduced the level and activity of acetyl cholinesterase, reactive oxygen species, and malondialdehyde through acting on the cholinergic system and reducing oxidative stress damage. These results indicate that KXS plays a neuroprotective role and improves cognitive function through reducing apoptosis and oxidative stress, and regulating synapse-associated protein and cholinergic neurotransmitters.

Toxicity of Graphene Quantum Dots in Zebrafish Embryo

Objective To evaluate the bio-safety of graphene quantum dots （GQDs）, we studied its effects on the embryonic development of zebrafish. Methods In vivo, biodistribution and the developmental toxicity of GQDs were investigated in embryonic zebrafish at exposure concentrations ranging from 12.5-200μg/mL for 4-96 h post-fertilization （hpf）. The mortality, hatch rate, malformation, heart rate, GQDs uptake, spontaneous movement, and larval behavior were examined. Results The fluorescence of GQDs was mainly localized in the intestines and heart. As the exposure concentration increased, the hatch and heart rate decreased, accompanied by an increase in mortality. Exposure to a high level of GQDs （200μg/mL） resulted in various embryonic malformations including pericardial edema, vitelline cyst, bent spine, and bent tail. The spontaneous movement significantly decreased after exposure to GQDs at concentrations of 50, 100, and 200μg/mL. The larval behavior testing （visible light test） showed that the total swimming distance and speed decreased dose-dependently. Embryos exposed to 12.5 μg/mL showed hyperactivity while exposure to higher concentrations （25, 50, 100, and 200μg/mL） caused remarkable hypoactivity in the light-dark test. Conclusion Low concentrations of GODs were relatively non-toxic. However, GQDs disrupt the progression of embryonic development at concentrations exceeding 50 μg/mL.

Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver

AIM To evaluate the effects of chronic exposure to ethanol in the liver and the expression of inflammatory genesin zebrafish.METHODS Zebrafish(n = 104),wild type,adult,male and female,were divided into two groups:Control and ethanol(0.05 v/v).The ethanol was directly added into water;tanks water were changed every two days and the ethanol replaced.The animals were fed twice a day with fish food until satiety.After two and four weeks of trial,livers were dissected,histological analysis(hematoxilineosin and Oil Red staining) and gene expression assessment of adiponectin,adiponectin receptor 2(adipor2),sirtuin-1(sirt-1),tumor necrosis factor-alpha(tnf-a),interleukin-1b(il-1b) and interleukin-10(il-10) were performed.Ultrastructural evaluations were conducted at fourth week.RESULTS Exposing zebrafish to 0.5% ethanol developed intense liver steatosis after four weeks,as demonstrated by oil red staining.In ethanol-treated animals,the main ultrastructural changes were related to cytoplasmic lipid particles and droplets,increased number of rough endoplasmic reticulum cisterns and glycogen particles.Between two and four weeks,hepatic mR NA expression of il-1b,sirt-1 and adipor2 were upregulated,indicating that ethanol triggered signaling molecules which are key elements in both hepatic inflammatory and protective responses.Adiponectin was not detected in the liver of animals exposed and not exposed to ethanol,and il-10 did not show significant difference.CONCLUSION Data suggest that inflammatory signaling and ultrastructural alterations play a significant role during hepatic steatosis in zebrafish chronically exposed to ethanol.

Toxicity of Multi-Walled Carbon Nanotubes,Graphene Oxide,and Reduced Graphene Oxide to Zebrafish Embryos

Objective This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes （MWCNTs）, graphene oxide （GO）, and reduced graphene oxide （RGO）, on zebrafish embryos. Methods The 2-h post-fertilization （hpf） zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations （1, 5, 10, 50, 100 mg/L） for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations Is were evaluated. Results Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. Conclusion MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed.

Identification of zRAP55,a gene preponderantly expressed in StagesⅠandⅡ oocytes of zebrafish

In an in silico search for gonand specific expressed genes, we have identified zRAP55 which is enriched in the ovary of zebrafish . zRAP55 encodes a protein of 382 amino acids with a highly conserved Lsm domain. zRAP55 protein shares more than 56% identities with that of other vertebrate species. RT-PCR results show that it is predominantly expressed in the ovary. In situ hybridization and immunohistochemistry studies reveal that zRAP55 is ubiquitously dispersed throughout the cytoplasm of stages Ⅰ and Ⅱ oocytes, whereas no expression is observed in stages Ⅲ and Ⅳ oocytes. As an RNA associated protein, zRAP55 might function in the control of protein translation at the early stages of oogenesis in zebrafish.