Axis In Zebrafish Research Articles

Rnf111 has a pivotal role in regulating development of definitive hematopoietic stem and progenitor cells through the Smad2/3-Gcsfr/NO axis in zebrafish.

The ubiquitination or SUMOylation of hematopoietic related factors plays pivotal roles in hematopoiesis. RNF111, known as a ubiquitin ligase (Ubl), is a newly discovered SUMO-targeted ubiquitin ligase (STUbl) involved in multiple signaling pathways mediated by TGF-β family members. However, its role in hematopoiesis remains unclear. Herein, a heritable Rnf111 mutant zebrafish line was generated by CRISPR/Cas9-mediated genome editing. Impaired hematopoietic stem and progenitor cells (HSPC) of definitive hematopoiesis was found in Rnf111 deficient mutants. Ablation of Rnf111 resulted in decreased phosphorylation of Smad2/3 in HSPC. Definitive endoderm 2 inducer (IDE2), which specifically activates TGF-β signaling and downstream Smad2 phosphorylation, can restore the definitive hematopoiesis in Rnf111-deficient embryos. Further molecular mechanism studies revealed that Gcsfr/NO signaling was an important target pathway of Smad2/3 involved in Rnf111-mediated HSPC development. In conclusion, our study demonstrated that Rnf111 contributes to the development of HSPC by maintaining Smad2/3 phosphorylation and the Gcsfr/NO signaling pathway activation. Keywords: Rnf111, Ubiquitin ligase (UbL), HSPC, Smad2/3, Gcsfr/NO.

Brain Derived Neurotrophic Factor Stimulates Hypothalamic and Gonadal Reproductive Hormones and Oocyte Maturation in Zebrafish.

Brain-derived neurotrophic factor (BDNF) is a peptide widely known for its role in neurogenesis and synaptic plasticity. Its expression in non-neuronal tissues has been reported. In mammals, it is involved in ovarian development, follicle growth, oocyte maturation, and early embryonic development. In zebrafish, it was demonstrated that BDNF increases food intake and regulates metabolism. Reproduction and metabolism are tightly linked. We hypothesized that BDNF modulates reproductive hormones and reproductive functions in zebrafish. This study aimed to determine BDNF expression in the zebrafish reproductive axis and whether it modulates the reproductive endocrine milieu and oocyte biology in zebrafish. Our results show that bdnf and its receptor trkb, and BDNF-like immunoreactivity are detected in zebrafish gonads and liver cells. This suggests BDNF local production and possible actions within the gonads and liver. Intraperitoneal administration of 1, 10, or 100 ng/g bodyweight BDNF significantly (ANOVA, p<0.05) increased sgnrh/cgnrh-II, kiss1, and cyp19a1b mRNAs in the zebrafish brain; steroidogenic enzymes (star and cyp19a1a) and key receptors in the zebrafish gonads. In vitro incubation of zebrafish liver cells with BDNF significantly (ANOVA, p<0.05) increased estrogen receptor mRNAs and vitellogenin concentrations (ELISA) in the cells. BDNF (100 ng/mL) induced (ANOVA, p<0.05) oocyte maturation in vitro at 24 hours post-incubation and significantly upregulated cumulus-expansion related genes (ANOVA, p<0.05). Overall, our findings indicate a stimulatory role for BDNF in the reproductive axis of zebrafish. This provides impetus for future research on its mechanism of action and potential practical applications to enhance reproduction in aquaculture.

Effects of L-NAME and air exposure on mitochondrial energetic markers, thyroid hormone receptor/regulator system and stress/ease-responsive receptor expression in the brain/gut axis of zebrafish

As a signal molecule, nitric oxide (NO) has several physiological actions in fish. However, the action of NO on the brain/gut axis, a classic inter-organal axis that bridges the gastrointestinal tract and the CNS, still requires more understanding. The short-term in vivo action of a NO inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), on mitochondrial energetic markers and the receptor expression of thyroid hormone (TH) and neuroendocrine hormones involved in stress/ease response was tested in the brain/gut axis of zebrafish exposed to either in non-stressed or air-exposed condition. L-NAME treatment decreased the NO content in brain and gut segments in non-stressed fish but rose upon L-NAME treatment in air-exposed fish that corresponded with the activation of inos, nnos, hif1a and hif1an transcript expressions. The brain/gut segments that showed spatial and differential sensitivity to L-NAME, modified the transcript expression patterns of stress (adra2da, adrb1, nr3c2)- and ease-responsive (htr2b, slc6a4a, mtnr1aa) hormone receptors. The expression pattern of the TH receptor/regulator system (thra, thrb, dio1, dio2, dio3) becomes more active in gut segments than brain segments upon L-NAME challenge in stressed zebrafish. The data provide evidence for a novel role of NO as an integrator of brain/gut axis segments in zebrafish, where the endogenously produced NO in mid-brain/posterior-gut axis aligns together upon air-exposure stress, providing a lead role to the posterior gut that activates and directs the neuroendocrine receptor expressions of stress/ease responsive genes. The data further invites studies exploring the therapeutic potential of L-NAME in this biomedical model to control the brain/gut axis segments.

Synergistic risk in the gut and liver: Insights into the toxic mechanisms and molecular interactions of combined exposure to triazophos and fenvalerate in zebrafish

The simultaneous or sequential application of pesticides such as triazophos (TRI) and fenvalerate (FEN) in agriculture results in their residues co-existing in the environments. However, the impact of co-exposure to TRI and FEN on the gut-liver axis, along with the underlying mechanisms, remains unclear. Our results showed that exposure to FEN (96 h-LC50 value of 0.096 mg a.i. L−1) was more toxic to adult zebrafish compared to TRI (96 h-LC50 value of 6.75 mg a.i. L−1). Furthermore, the study aimed to reveal the toxic potencies of individual and combined exposure to TRI and FEN on the liver-gut axis in zebrafish (Danio rerio). Our results also indicated that pesticide exposure decreased tight junction molecule expression and increased intestinal inflammatory molecule expression in D. rerio, with co-exposure demonstrating enhanced toxicity. Co-exposure altered gut flora structure and species abundance. RNA-Seq sequencing revealed changes in liver gene expressions, particularly enrichment of P53 signaling. Molecular docking demonstrated FEN's stronger binding to P53 and Caspase3, correlating with its higher toxicity. Liver pathology confirmed exacerbated liver damage by individual and co-exposures, with co-exposure inducing more severe liver injury. qPCR results showed increased pro-apoptotic gene expression and decreased anti-apoptotic gene expression, with co-exposure exhibiting an interactive effect. Overall, this study identifies specific targets and pathways influenced by these pesticides, revealing toxicity mechanisms involving the gut-liver axis, which is crucial for environmental risk assessment of pesticide mixtures.

Effects of penthiopyrad on the hypothalamic-pituitary-thyroid (HPT) axis in zebrafish

Exposure to specific pesticides has been demonstrated to alter normal thyroid function of aquatic vertebrates. This study aimed to investigate the impact of penthiopyrad (PO) on the thyroid function of zebrafish, further elucidating its toxic mechanisms on the early developmental stages of zebrafish. Exposure to sublethal doses of PO (0.3–1.2 mg/L) for 8 days from 2 h after fertilization resulted in a significant reduction in larval swim bladder size and body weight, accompanied by developmental abnormalities such as pigment deposition and abnormal abdominal development. Perturbations in the hypothalamic-pituitary-thyroid (HPT) axis in larvae manifested as a marked upregulation of crh, tg, ttr, and ugt1ab expression, alongside downregulation of trβ expression, culminating in elevated thyroxine (T4) and triiodothyronine (T3) levels. Additionally, molecular docking results suggest that PO and its metabolites may disrupt the binding of thyroid hormones to thyroid hormone receptor beta (TRβ), compromising the normal physiological function of TRβ. These findings highlight the PO-induced adverse effects on the HPT axis of larvae under sublethal doses, eventually leading to abnormal development and growth inhibition.

Reproductive and Developmental Effects of Sex-Specific Chronic Exposure to Dietary Arsenic in Zebrafish (Danio rerio).

The present study investigated the reproductive and developmental effects of sex-specific chronic exposure to dietary arsenic in zebrafish. Adult zebrafish (Danio rerio) were exposed to environmentally realistic doses of arsenic via diet [0 (control; no added arsenic), 30 (low), 60 (medium), and 100 (high) μg/g dry weight, as arsenite] for 90 days. Following exposure, arsenic-exposed females from each dietary treatment were mated with control males, and similarly, arsenic-exposed males from each dietary treatment were mated with control females. In females, arsenic exposure resulted in a dose-dependent decrease in reproductive performance (fecundity, fertilization success, and hatching success). Moreover, a dose-dependent increase in developmental toxicity (larval deformities and larval mortality) was observed with maternal exposure to arsenic. In contrast, in males, arsenic exposure also induced similar reproductive and developmental toxicity; however, the adverse effects were mainly evident only in the medium and high dietary arsenic treatment groups. We also examined the sex-specific effects of dietary arsenic exposure on the expression of genes that regulate the hypothalamus-pituitary-gonadal-liver (HPG-L) axis in fish. The gene expression results indicated the downregulation of HPG-L axis genes in females irrespective of the arsenic treatment dose; however, the reduced expression of HPG-L axis genes in males was recorded only in the medium and high arsenic treatment groups. These observations suggest that chronic arsenic exposure in either females or males causes reproductive and developmental toxicity in zebrafish. However, these toxic effects are markedly higher in females than in males. Our results also suggest that arsenic can act as an endocrine disruptor and mediate reproductive and developmental toxicity by disrupting the HPG-L axis in zebrafish.

Enrofloxacin exposure undermines gut health and disrupts neurotransmitters along the microbiota-gut-brain axis in zebrafish

The environmental prevalence of antibiotic residues poses a potential threat to gut health and may thereby disrupt brain function through the microbiota-gut-brain axis. However, little is currently known about the impacts of antibiotics on gut health and neurotransmitters along the microbiota-gut-brain axis in fish species. Taking enrofloxacin (ENR) as a representative, the impacts of antibiotic exposure on the gut structural integrity, intestinal microenvironment, and neurotransmitters along the microbiota-gut-brain axis were evaluated in zebrafish in this study. Data obtained demonstrated that exposure of zebrafish to 28-day environmentally realistic levels of ENR (6 and 60 μg/L) generally resulted in marked elevation of two intestinal integrity biomarkers (diamine oxidase (DAO) and malondialdehyde (MDA), upregulation of genes that encode inter-epithelial tight junction proteins, and histological alterations in gut as well as increase of lipopolysaccharide (LPS) in plasma, indicating an evident impairment of the structural integrity of gut. Moreover, in addition to significantly altered neurotransmitters, markedly higher levels of LPS while less amount of two short-chain fatty acids (SCFAs), namely acetic acid and valeric acid, were detected in the gut of ENR-exposed zebrafish, suggesting a disruption of gut microenvironment upon ENR exposure. Along with corresponding changes detected in gut, significant disruption of neurotransmitters in brain indicated by marked alterations in the contents of neurotransmitters, the activity of acetylcholin esterase (AChE), and the expression of neurotransmitter-related genes were also observed. These findings suggest exposure to environmental antibiotic residues may impair gut health and disrupt neurotransmitters along the microbiota-gut-brain axis in zebrafish. Considering the prevalence of antibiotic residues in environments and the high homology of zebrafish to other vertebrates including human, the risk of antibiotic exposure to the health of wild animals as well as human deserves more attention.

Synergistic detoxification efficiency and mechanism of triclocarban degradation by a bacterial consortium in the liver-gut-microbiota axis of zebrafish (Danio rerio)

Triclocarban (TCC), an emerging organic contaminant, poses a potential threat to human health with long-term exposure. Here, Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 were utilized to degrade TCC at environmental related concentrations for enhancing TCC biodegradation and investigating whether the toxicity of intermediate metabolites is lower than that of the parent compound. The results demonstrated that the bacterial consortium could degrade TCC by 82.0% within 7 days. The calculated 96 h LC50 for TCC, as well as its main degradation product 3,4-Dichloroaniline (DCA) were 0.134 mg/L and 1.318 mg/L respectively. Biodegradation also alleviated histopathological lesions induced by TCC in zebrafish liver and gut tissues. Liver transcriptome analysis revealed that biodegradation weakened differential expression of genes involved in disrupted immune regulation and lipid metabolism caused by TCC, verified through RT-qPCR analysis and measurement of related enzyme activities and protein contents. 16 S rRNA sequencing indicated that exposure to TCC led to gut microbial dysbiosis, which was efficiently improved through TCC biodegradation, resulting in decreased relative abundances of major pathogens. Overall, this study evaluated potential environmental risks associated with biodegradation of TCC and explored possible biodetoxification mechanisms, providing a theoretical foundation for efficient and harmless bioremediation of environmental pollutants.

Combined Effects of Polystyrene Nanosphere and Homosolate Exposures on Estrogenic End Points in MCF-7 Cells and Zebrafish.

Micro- and nanoplastics (MNPs) and homosalate (HMS) are ubiquitous emerging environmental contaminants detected in human samples. Despite the well-established endocrine-disrupting effects (EDEs) of HMS, the interaction between MNPs and HMS and its impact on HMS-induced EDEs remain unclear. This study aimed to investigate the influence of MNPs on HMS-induced estrogenic effects and elucidate the underlying mechanisms in vitro and invivo. We assessed the impact of polystyrene nanospheres (PNSs; , ) on HMS-induced MCF-7 cell proliferation (HMS: , equivalent to ) using the E-SCREEN assay and explored potential mechanisms through transcriptomics. Adult zebrafish were exposed to HMS () with or without PNSs (, ) for 21 d. EDEs were evaluated through gonadal histopathology, fertility tests, steroid hormone synthesis, and gene expression changes in the hypothalamus-pituitary-gonad-liver (HPGL) axis. Coexposure of HMS and PNSs resulted in higher expression of estrogen receptor (ESR1) and the mRNAs of target genes (pS2, AREG, and PGR), a greater estrogen-responsive element transactivation activity, and synergistic stimulation on MCF-7 cell proliferation. Knockdown of serum and glucocorticoid-regulated kinase 1 (SGK1) rescued the MCF-7 cell proliferation induced by PNSs alone or in combination with HMS. In zebrafish, coexposure showed higher expression of SGK1 and promoted ovary development but inhibited spermatogenesis. In addition, coexposure led to lower egg hatchability, higher embryonic mortality, and greater larval malformation. Coexposure also modulated steroid hormone synthesis genes (cyp17a2, hsd17[Formula: see text]1, esr2b, vtg1, and vtg2), and resulted in higher () release in females. Conversely, males showed lower testosterone, , and gene expressions of cyp11a1, cyp11a2, cyp17a1, cyp17a2, and hsd17[Formula: see text]1. PNS exposure exacerbated HMS-induced estrogenic effects via SGK1 up-regulation in MCF-7 cells and disrupting the HPGL axis in zebrafish, with gender-specific patterns. This offers new mechanistic insights and health implications of MNP and contaminant coexposure. https://doi.org/10.1289/EHP13696.

Foxp2 deficiency impairs reproduction by modulating the hypothalamic-pituitary-gonadal axis in zebrafish†.

FOXP2 was initially characterized as a transcription factor linked to speech and language disorders. Single-cell RNA sequencing reveals that Foxp2 is enriched in the gonadotrope cluster of the pituitary gland and colocalized with the hormones LHB and FSHB in chickens and mice, implying that FOXP2 might be associated with reproduction in vertebrates. Herein, we investigated the roles of foxp2 in reproduction in a Foxp2-deficient zebrafish model. The results indicated that the loss of Foxp2 inhibits courtship behavior in adult male zebrafish. Notably, Foxp2 deficiency disrupts gonad development, leading to retardation of follicle development and a decrease in oocytes in females at the full-growth stage, among other phenotypes. The transcriptome analysis (RNA-seq) also revealed that differentially expressed genes clustered into the estrogen signaling and ovarian steroidogenesis-related signaling pathways. In addition, we found that Foxp2 deficiency could modulate the hypothalamic-pituitary-gonadal axis, especially the regulation of lhb and fshb expression, in zebrafish. In contrast, the injection of human chorionic gonadotropin, a specific LH agonist, partially rescues Foxp2-impaired reproduction in zebrafish, suggesting that Foxp2 plays an important role in the regulation of reproduction via the hypothalamic-pituitary-gonadal axis in zebrafish. Thus, our findings reveal a new role for Foxp2 in the regulation of reproduction in vertebrates.

Enantioselective Toxicity and Potential Endocrine-Disruptive Effects of the Insecticides Flufiprole and Ethiprole on Danio rerio.

Phenylpyrazole insecticides are widely used as chiral pesticides. However, the enantioselective toxicity and potential endocrine-disrupting effects of these insecticides on aquatic organisms remain unclear. Herein, the enantioselective toxicity and potential endocrine-disrupting effects of flufiprole and ethiprole were investigated by using zebrafish embryos/larvae as a model. The acute toxicity of R-flufiprole and R-ethiprole toward zebrafish embryos and larvae was 1.8-3.1-fold higher than that of the S-configuration. Additionally, R-flufiprole and R-ethiprole had a greater effect on the expression of genes related to the hypothalamus-pituitary-gonad axis in zebrafish compared with the S-configuration. Nevertheless, both S-flufiprole and S-ethiprole exhibited a greater interference effect on the expression of genes related to the hypothalamus-pituitary-thyroid axis and a greater teratogenic effect on zebrafish than the R-configuration. Thus, this study demonstrates that both flufiprole and ethiprole exhibit enantioselective acute toxicity and developmental toxicity toward zebrafish. Furthermore, those pesticides potentially possess enantioselective endocrine-disrupting effects.

Co-exposure of nanoplastics and arsenic causes neurotoxicity in zebrafish (Danio rerio) through disrupting homeostasis of microbiota–intestine–brain axis

Nanoplastics (NPs) and arsenic (As) are toxic pollutants prevalent on the earth and have gained considerable attention in recent decades. Although numerous studies reported NPs and As can cause neurotoxicity there are still significant knowledge gaps in illustrating their combined toxicity and its mechanism. In this study, the co-exposure of environmentally relevant concentrations of NPs and As caused neurobehavioral toxicity in zebrafish, as evidenced by reduced swimming ability, anxiety and impaired short-term learning memory. Potentially, its toxicity mechanism is through disrupting the homeostasis of microbiota-intestine-brain axis in zebrafish. Specifically, the co-exposure reduced the 5-hydroxytryptamine (5-HT) production in intestine, which led to lower levels of 5-HT transported by the blood circulation to the brain. Ultimately, neurobehavior was adversely affected by the reduced binding of 5-HT to its receptors. Intestine, the primary source of 5-HT, its impaired health (aggravation in oxidative stress, mitochondrial damage and histopathological alterations) induced the dysregulation in the 5-HT system, which may be induced by the increased accumulation of As in the intestine by the co-exposure. Besides, the reduced 5-HT levels were correlated with decreased Firmicutes and Protecbacteria and increased Actinobacteriota and Chloroflexi in intestines. Potentially, intestinal microbiota adversely regulates the intestine-brain axis by reducing SCFAs levels. Thus, the alteration of intestinal microbiota structure may be the other reason for the dysregulation of intestine-brain axis. In summary, co-exposure of NPs and As induced neurobehavior toxicity probably through disrupting the homeostasis of microbiota-intestine-brain axis. This study provides insights into assessing the environmental health risks of the pollution of NPs and As to aquatic organisms.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

Probiotic Lactobacillus rhamnosus alleviates the neurotoxicity of microcystin-LR in zebrafish (Danio rerio) through the gut-brain axis

Microcystin-LR (MCLR) is one of the most toxic cyanobacterial toxins and is harmful to the central nervous system of fish. Probiotic additives can improve neuroendocrine function in fish. Although both MCLR and probiotics aim at the nervous system, whether they interact with each other and the mechanisms remain unexplored. In the present study, 4-month-old zebrafish were exposed to 0, 2.2, and 22 μg/L of MCLR for 28 days with or without the probiotic L. rhamnosus. We found that MCLR exposure could inhibit the swimming speed of zebrafish, while the presence of L. rhamnosus mitigated this abnormality. To elucidate the mechanism of how L. rhamnosus alleviates MCLR-induced neurotoxicity, we examined the bioaccumulation of MCLR, changes in neurotransmitters, immune biochemical indicators, and hormone content of the hypothalamic-pituitary-interrenal (HPI) axis in zebrafish along the gut-brain axis. Our results showed L. rhamnosus could reverse the abnormal swimming behavior and eventually alleviate neurotoxicity in zebrafish by modulating intestinal and brain neural signaling, neuroinflammation, and HPI axis responses. This study provides implications for the application of probiotics in the aquaculture industry.

Flunitrazepam and its metabolites exposure disturb the zebrafish gut-liver axis: Combined microbiome and metabolomic analysis

Due to clinical treatment and illegal use, psychoactive substances have been widely detected in the aquatic environment. In this study, we investigated the effects of the benzodiazepine drug flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ) on the gut-liver axis of zebrafish. Zebrafish were exposed to two concentrations of FLZ and 7-FLZ (0.05 and 1 μg/L) for 30 days. Results showed that both FLZ and 7-FLZ exposure altered the relative abundance of Proteobacteria at the phylum level, with significant differences observed at the genus level for pathogenic bacteria such as Paracoccus, Shewanella, and Aeromonas. Metabolomics results showed both exposures significantly interfered with nucleotide and amino acid metabolism. The imbalance of gut microbiota and metabolic disorder increased the level of malondialdehyde, which in turn heightened the permeability of the gut mucosal barrier. FLZ and 7-FLZ induced oxidative stress in the liver via the gut-liver axis, leading to decreased levels of glucose, total cholesterol, and triglyceride, as well as the down-regulation of glycolipid metabolism-related genes (PPARα, PPARγ, FABP2, Fabp11, PFKFB3, and LDHA). Metabolomics results revealed that FLZ and 7-FLZ significantly affected the biosynthesis of amino acids and arginine, and other metabolic pathways such as nucleotide, nicotinate and nicotinamide, and purine in the liver. Our results unveiled the mechanisms behind the toxicological effects of psychoactive substances on the gut-liver axis, providing valuable data for ecological and environmental risk assessments.

C5a drives the inflammatory response with bacterial dose effect by binding to C5aR1 in zebrafish infected with Aeromonas hydrophila.

The complement system is essential to host defense, but its excessive activation caused by severe pathogen invasion is a driving force in adverse inflammatory. The binding of complement component 5a (C5a) and complement component 5a receptor 1 (C5aR1) is the key to trigger complement-mediated inflammatory response in mammals. However, the role of C5a-C5aR1 axis in fish immune response remains obscure. In this study, the role of C5a-C5aR1 axis of zebrafish (Danio rerio) after serious infection with Aeromonas hydrophila was investigated. C5a and C5aR1 of zebrafish were cloned, with CDS sequences of 228 and 1041 bp, respectively, and they were widely expressed in various tissues with the highest expression in the liver and spleen, respectively. The survival of zebrafish was closely correlated to the dose of A. hydrophila. The cytokine storm occurred at high concentrations of A. hydrophila infection. At 24h post infection (hpi), the expression of C5a and C5aR1 in the spleen increased 26.8-fold and 9.9-fold in treatment group 1 (TG1, 3.0×107CFU/mL) (P<0.01), and 4.7-fold and 3.4-fold in treatment group 2 (TG2, 1.0×107CFU/mL) (P<0.05), respectively. Correspondingly, proinflammatory cytokines interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-17 (IL-17) were positively correlated to C5a and C5aR1at mRNA and protein expression levels. The expression of IL-1β was significantly increased in the spleen at 6 hpi, with a 599.2-fold and 203.2-fold upregulation in TG1 and TG2 (P<0.001), respectively. Moreover, after inhibition of C5a-C5aR1 binding treated with C5aR1 antagonist (W-54011), zebrafish showed lower expression of C5a, C5aR1, and cytokines, less intestinal damage, and significantly enhancement of survival (P<0.05) after A. hydrophila challenge. This study revealed that the inflammatory effect of C5a was achieved by binding to C5aR1 in zebrafish, providing novel insights into using C5a-C5aR1 axis as an effective target to reduce bacterial inflammation and disease in fish.

Ccdc57 is required for straightening the body axis by regulating ciliary motility in the brain ventricle of zebrafish

Recently, cilia defects have been proposed to contribute to scoliosis. Here, we demonstrate that coiled-coil domain-containing 57 (Ccdc57) plays an essential role in straightening the body axis of zebrafish by regulating ciliary beating in the brain ventricle (BV). Zygotic ccdc57 (Zccdc57) mutant zebrafish developes scoliosis without significant changes in their bone density and calcification, and the maternal-zygotic ccdc57 (MZccdc57) mutant embryos display curved bodies since the long-pec stage. The expression of ccdc57 is enriched in ciliated tissues and immunofluorescence analysis reveals colocalization of Ccdc57-HA with acetylated α-tubulin, implicating it in having a role in ciliary function. Further examination reveals that it is the coordinated cilia beating of multiple cilia bundles (MCB) in the MZccdc57 mutant embryos that is affected at 48 hours post fertilization, when the compromised cerebrospinal fluid flow and curved body axis have already occurred. Either ccdc57 mRNA injection or epinephrine treatment reverses the spinal curvature in MZccdc57 mutant larvae from ventrally curly to straight or even dorsally curly and significantly upregulates urotensin signaling. This study reveals the role of ccdc57 in maintaining coordinated cilia beating of MCB in the BV.

Leptin modulates oocyte maturation by a central and a direct pathway in zebrafish.

Loss of LEPR function (LOF) in mammals leads to diverse phenotypes including morbid obesity and infertility while zebrafish show relatively minor phenotypes. This however allows the study of LEPR LOF in the absence of the detrimental effects of hyperglycemia or obesity. Here, we show evidence that leptin plays a role in the central as well as peripheral regulation of the hypothalamic-pituitary-gonadal (HPG) axis in zebrafish. Animals with a Lepr LOF show dysregulated pituitary HPG genes as well as evidence that oocytes mature slower and/or exhibit an increased rate of atresia. In culture, Lepr LOF attenuates the effect of 17α-20β-dihydroxy-4 pregnen-3-one in promoting germinal vesicle breakdown (GVBD) and increases the rate of GVBD as well as attenuates the rate of oocyte atresia.

IPPD-induced growth inhibition and its mechanism in zebrafish

N-isopropyl-N-phenyl-1,4-phenylenediamine (IPPD) is used as a ubiquitous antioxidant worldwide, it is an additive in tire rubber easily discharged into the surrounding environment. At present, there is no study concerning the subacute toxicity of IPPD on fish. We used zebrafish embryos (2 h post-fertilization) exposed to IPPD for 5 days at concentrations of 0, 0.0012, 0.0120 and 0.1200 mg/L to investigate its toxic effects of embryonic development, disruption of growth hormone/insulin-like growth factor (GH/IGF) and hypothalamic-pituitary-thyroid (HPT) axis. The results showed that IPPD exposure decreased hatchability, weakened movement ability, reduced body length, and caused multiple types of deformities in zebrafish embryos. The expression of genes involved to GH/IGF and HPT axis were altered after exposure to IPPD in zebrafish larvae. Meanwhile, exposure to IPPD significantly decreased thyroxine (T4) and 3,5,3′-triiodothyronine (T3) contents in larvae, which indicated that HPT axis was in a disturbed state. Moreover, treatment of IPPD decreased the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) as well as levels of glutathione (GSH). While the contents of malondialdehyde (MDA) were elevated after exposure to IPPD. The present study thus demonstrated that IPPD induced oxidative stress, caused developmental toxicity and disrupted the GH/IGF and HPT axis of zebrafish, which could be responsible for developmental impairment and growth inhibition.

Silver nanoparticles modify the hypothalamic-pituitary-interrenal axis and block cortisol response to an acute stress in zebrafish, Danio rerio.

The present study aimed at assessing the effects of exposure to silver nanoparticle (AgNP) and a subsequent acute stress on the expression of various genes involved in the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish, Danio rerio. The fish were exposed to 0 (Control), 0.1 (LC), 0.4 (MC), and 1.2 (HC) mg Ag/L (as AgNP) over a 2-week period, followed by an acute air exposure stress. The whole body cortisol and the expression of selected genes in the fish brain and kidney were analyzed, before and after the acute stress. The results showed that AgNP increased basal cortisol levels and the expression of corticotropin releasing factor, prohormone convertase 1, pro-opiomelanocortin, and melanocortin 2 receptor; however, it suppressed/inhibited whole body cortisol, brain corticotropin releasing factor responses, pro-opiomelanocortin, and the kidney melanocortin 2 receptor responses to the acute stress. AgNP down-regulated the expression of the steroidogenic acute regulatory protein, but it intensified the gene expression in response to the acute stress. Before the acute stress, LC treatment exhibited an up-regulation in Cytochrome P450-11A-1 expression, but MC and HC treatments induced down-regulation. After the acute stress, the AgNP-exposed fish exhibited decreased Cytochrome P450-11A-1 expressions, compared with the Control. Exposure to AgNP significantly increased Cytochrome P450-11B expression. However, after the acute stress, LC treatment exhibited an up-regulation, but MC and HC treatments exhibited down-regulation in the Cytochrome P450-11B gene expression. In conclusion, AgNP suppressed cortisol response to stress, which appears to be a consequence of alterations in the HPI axis at the transcriptomic levels.