Male Zebrafish Research Articles

Analysis of oxygen consumption rates in zebrafish reveals differences based on sex, age and physical activity recovery.

Introduction: Mitochondrial dysfunction is linked to a variety of human diseases. Understanding the dynamic alterations in mitochondrial respiration at various stages of development is important to our understanding of disease progression. Zebrafish provide a system for investigating mitochondrial function and alterations during different life stages. The purpose of this study was to investigate our ability to measure mitochondrial oxygen consumption rates in zebrafish embryos, larvae, and adults as an indicator of mitochondrial function. Methods: Basal respiration of entire zebrafish embryos (5dpf), larvae (0.6-0.9cm), young adults (3-month-old), and old adults (12-month-old) was measured using an Oroboros Oxygraph, with a stirrer speed of 26rpm. For embryos and larvae, "leak" respiration (plus oligomycin), maximum respiration (plus uncoupler), non-mitochondrial respiration (plus inhibitors), and complex IV activity were also measured. To induce physical activity in adult fish, the stirrer speed was increased to 200rpm. Results and Discussion: We demonstrate the ability to accurately measure respiration rates in zebrafish at various ages using the Oroboros Oxygraph. When comparing zebrafish embryos to larvae, embryos have a higher maximum respiration. Three-month-old zebrafish males have higher basal respiration than females, while 12-month-old zebrafish females exhibit greater rates of respiration than males and younger females. When the stirrer speed was increased, respiration rates decrease, but with differences depending on sex. This study demonstrates a simple and accessible method to assess zebrafish physiology by mitochondrial oxygen consumption measurements in an unmodified Oroboros Oxygraph. The method should facilitate studies to understand the intricate interplay between mitochondrial function, development, and aging.

BDE-209-induced genotoxicity, intestinal damage and intestinal microbiota dysbiosis in zebrafish (Danio Rerio)

The environmental presence of polybrominated diphenyl ethers (PBDEs) is ubiquitous due to their wide use as brominated flame retardants in industrial products. As a common congener of PBDEs, decabromodiphenyl ether (BDE-209) can pose a health risk to animals as well as humans. However, to date, few studies have explored BDE-209's toxic effects on the intestinal tract, and its relevant mechanism of toxicity has not been elucidated. In this study, adult male zebrafish were exposed to BDE-209 at 6 μg/L, 60 μg/L and 600 μg/L for 28 days, and intestinal tissue and microbial samples were collected for analysis to reveal the underlying toxic mechanisms. Transcriptome sequencing results demonstrated a dose-dependent pattern of substantial gene differential expression in the group exposed to BDE-209, and the differentially expressed genes were mainly concentrated in pathways related to protein synthesis and processing, redox reaction, and steroid and lipid metabolism. In addition, BDE-209 exposure caused damage to intestinal structure and barrier function, and promoted intestinal oxidative stress, inflammatory response, apoptosis and steroid and lipid metabolism disorders. Mechanistically, BDE-209 induced intestinal inflammation by increasing the levels of TNF-α and IL-1β and activating the NFκB signaling pathway, and might induce apoptosis through the p53-Bax/Bcl2-Caspase3 pathway. BDE-209 also significantly inhibited the gene expression of rate-limiting enzymes such as Sqle and 3βhsd (p < 0.05) to inhibit cholesterol synthesis. In addition, BDE-209 induced lipid metabolism disorders through the mTOR/PPARγ/RXRα pathway. 16S rRNA sequencing results showed that BDE-209 stress reduced the richness and diversity of intestinal microbiota, and reduced the abundance of probiotics (e.g., Bifidobacterium and Faecalibacterium). Overall, the results of this study help to clarify the intestinal response mechanism of BDE-209 exposure, and provide a basis for evaluating the health risks of BDE-209 in animals.

Acute toxicity of palm oil mill effluent on zebrafish (Danio rerio Hamilton-Buchanan, 1822): Growth performance, behavioral responses and histopathological lesions

Evaluating the toxicity of Palm Oil Mill Effluent (POME) is critical as part of the effort to develop waste management regulations for the palm oil industry. In this study, we investigated the acute toxicity of POME on growth performance, behavioral response, and histopathology of gill and liver tissues of zebrafish (Danio rerio). In total, 550 adult male zebrafish were used for the toxicity experiment including range finding test, acute toxicity test, growth performance and behaviour test. Static non-renewal acute toxicity bioassays were conducted by exposing fish to POME (1.584–9.968 mL/L) for 96 h. Growth performance, behavior response, and histopathological lesions in untreated and POME treated (96-h LC50: 5.156 mL/L) fish were measured at 24, 48, 72 and 96 h. Time-dependent significant decline in body length and body weight of POME-exposed zebrafish was observed. Furthermore, several behavioral changes were recorded, including hyperactivity, loss of balance, excessive mucus secretion, and depigmentation. Decreasing operculum movement and oxygen consumption rate as well as alterations in gill tissues (i.e. hyperplasia, hypertrophy, hemorrhage, and necrosis) of POME-exposed zebrafish were observed, suggesting a dysfunction in respiratory performance. On the other hand, liver tissue alterations (congestion, hemorrhage, hyperplasia, shrinkage of hepatocytes, hydrophilic degeneration, and necrosis) indicated a disruption in detoxification performance. We conclude that exposure to POME at acute concentration caused histopathological lesions both in gill and liver tissue along with changes in fish behaviors which disrupted respiratory and detoxification performance, resulting in mortality and reduced growth of zebrafish. These findings might provide valuable information for guiding POME management and regulation.

Multi- and transcriptomic changes of chronic exposure to bisphenol A reveals reproductive toxicity in male zebrafish.

Bisphenol A (BPA) is considered to be a threat to marine organisms owning to its widespread usage and potential aquatic toxicity. However, the reproductive toxicity of BPA to transgenerational inheritance in aquatic organisms is still unclarified. In this study, the morphological, histological, and transgenerational changes by BPA in zebrafish testis were investigated. Results showed that BPA caused abnormities in sperm number, activity, and fertility rate. Testicular transcriptional alterations detected by RNA-seq identified 1940 differentially expressed genes (DEGs) after BPA exposure, of which 392 were upregulated and 1548 were downregulated. Gene Ontology analysis showed that acrosin binding, binding of sperm to zona pellucida, and positive regulation of acrosome reaction were significantly enriched in BPA-induced DEGs. Pathway analysis indicated that cell adhesion molecules, steroid hormone biosynthesis and fatty acid biosynthesis, elongation, and metabolism were remarkably changed after BPA treatment. Thus, we deduce here that multi- and transcriptomic changes of chronic exposure to BPA reveals reproductive toxicity in male zebrafish.

ZIF-8 Nanoparticles Induce Behavior Abnormality and Brain Oxidative Stress in Adult Zebrafish (Danio rerio).

Zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs) are typical metal-organic framework (MOF) materials and have been intensively studied for their potential application in drug delivery and environmental remediation. However, knowledge of their potential risks to health and the environment is still limited. Therefore, this study exposed female and male zebrafish to ZIF-8 NPs (0, 9.0, and 90 mg L-1) for four days. Subsequently, variations in their behavioral traits and brain oxidative stress levels were investigated. The behavioral assay showed that ZIF-8 NPs at 90 mg/L could significantly decrease the locomotor activity (i.e., hypoactivity) of both genders. After a ball falling stimulation, zebrafish exposed to ZIF-8 NPs (9.0 and 90 mg L-1) exhibited more freezing states (i.e., temporary cessations of movement), and males were more sensitive than females. Regardless of gender, ZIF-8 NPs exposure significantly reduced the SOD, CAT, and GST activities in the brain of zebrafish. Correlation analysis revealed that the brain oxidative stress induced by ZIF-8 NPs exposure might play an important role in their behavioral toxicity to zebrafish. These findings highlight the necessity for further assessment of the potential risks of MOF nanoparticles to aquatic species and the environment.

Keluwih (Artocarpus camansi) extract effects in zebrafish models of Parkinson’s disease

Parkinson’s disease is a condition that affects the central nervous system in the brain and is brought on by a lack of dopamine. Uncontrollable tremors, uncoordinated movement, and stiffness characterize Parkinson’s disease. Until now, the medication for Parkinson’s disease is limited to relieve the symptoms and maintain the quality of life; thus, the progression of the disease can be delayed. In order to search for alternative therapy from herbs, Kluwih (Artocarpus camansi) has been used traditionally to relieve convulsants. This research aims to observe 96% ethanol extract of A. camansi leaves in dopamine and locomotor activity in adult male and female zebrafish (Dario rerio). The A. camansi extract concentration was 2.5; 5; 7.5; and 10 mg/ml for 28 days. Zebrafish locomotion was observed for 5 minutes on days 0; 7; 14; 21; and 28. ELISA measured the observations of dopamine after 28 days. The 96% ethanol extract of A. camansi leaves at 5 mg/ml can increase dopamine levels after induced with rotenone, but the dopamine level decreased at 7,5 and 10 mg/ml. The maximal concentration to increase locomotor activity is also at 5 mg/ml, along with dopamine concentration. Our findings revealed that 5 mg/ml of 96% ethanol extract of A. camansi leaves was the optimal dosage to stimulate dopamine release and enhance locomotor activity.

Tris(1,3-dichloro-2-propyl) phosphate causes female-biased growth inhibition in zebrafish: Linked with gut microbiota dysbiosis

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is ubiquitous in aquatic environment, but its effect on intestinal health of fish has yet not been investigated. In the present study, the AB strain zebrafish embryos were exposed to environmentally realistic concentrations (0, 30, 300, and 3000 ng·L−1) of TDCIPP for 90 days, after which the fish growth and physiological activities were evaluated, and the intestinal microbes were analyzed by 16S rRNA gene high-throughput sequencing. Our results manifested that the body length and body weight were significantly reduced in the female zebrafish but not in males. Further analyses revealed that TDCIPP resulted in notable histological injury of intestine, which was accompanied by impairment of epithelial barrier integrity (decreased tight junction protein 2), inflammation responses (increased interleukin 1β), and disruption of neurotransmission (increased serotonin) in female intestine. Male intestines maintained intact intestinal structure, and the remarkably increased activity of glutathione peroxidase (GPx) might protect the male zebrafish from inflammation and intestinal damage. Furthermore, 16S rRNA sequencing analysis showed that TDCIPP significantly altered the microbial communities in the intestine in a gender-specific manner, with a remarkable increase in alpha diversity of the gut microbiome in male zebrafish, which might be another mechanism for male fish to protect their intestines from damage by TDCIPP. Correlation analysis revealed that abnormal abundances of pathogenic bacteria (Chryseobacterium, Enterococcus, and Legionella) might be partially responsible for the impaired epithelial barrier integrity and inhibition in female zebrafish growth. Taken together, our study for the first time demonstrates the high susceptibility of intestinal health and gut microbiota of zebrafish to TDCIPP, especially for female zebrafish, which could be partially responsible for the female-biased growth inhibition.

Additive effects of microplastics on accumulation and toxicity of cadmium in male zebrafish

Microplastics (MPs) have emerged as contaminants of concern because of their ubiquitous presence in almost all aquatic environments. The ecological effects of MPs are complex and depend on multiple factors including their age, size and the ecological matrix. There is an urgent need for multifactorial studies to elucidate their impacts. We measured the effects of virgin and naturally aged MPs, alone, pretreated with cadmium (Cd), or in combination with ionic Cd, on the bioaccumulation of Cd, metallothionein expression, behavior, and histopathology of adult zebrafish (Danio rerio). Zebrafish were exposed to virgin or aged polyethylene MPs (0.1% MPs enriched diets, w/w) or waterborne Cd (50 μg/L) or a combination of the two for 21 days. There was an additive interaction between water-borne Cd and MPs on bioaccumulation in males but not in females. Cd accumulation increased by twofold when water-borne Cd and MPs were combined. Water-borne Cd induced significantly higher levels of metallothionein compared to MPs pre-exposed to Cd. However, Cd-treated MPs caused greater damage to the intestine and liver compared to untreated MPs suggesting that bound Cd could be released or modulate MPs toxicity. We also showed that co-exposure to water-borne Cd and MPs increased anxiety in the zebrafish, compared with water-borne Cd alone, suggesting using microplastics as a vector may increase toxicity. This study demonstrates that MPs can enhance the toxicity of Cd, but further study is needed to elucidate the mechanism.

Short-term exposure of Cannabidiol on Zebrafish (Danio Rerio): Reproductive Toxicity.

Cannabidiol (CBD), a medically active component of hemp, is a popular ingredient in healthcare and personal-care products. The increasing demand for CBD and the legalization of hemp growth may promote chronic exposure of non-target organisms to CBD. In this study, the reproductive toxicity of CBD was investigated on adult zebrafish. With CBD treatment, female zebrafish spawned less with higher natural mortality and malformation rates. Both female and male zebrafish showed a decreased gonadosomatic index with an increased percentage of pre-mature oocytes and sperm and had an increased hepatosomatic index with decreased content of vitellogenin. The value of estrogen/testosterone (E2/T) decreased in female zebrafish and increased in male zebrafish. Sex hormone synthesis genes were downregulated in ovaries and upregulated in testicles, except for cyp11a, in contrast to the other genes. Apoptosis-related genes were upregulated in the zebrafish brain, gonad, and liver. These results show that CBD might damage the reproductive function by inducing an apoptotic response, further inhibiting zebrafish reproductive ability.

Single-cell transcriptomic analysis reveals heterogeneity of the patterns of responsive genes and cell communications in liver cell populations of zebrafish exposed to polystyrene nanoplastics

Nanoplastics (NPs) are a group of emerging environmental pollutants with potential toxicity and health risk on biosystem and ecosystem. Great efforts have been devoted to describing the uptake, distribution, accumulation, and toxicity of NPs at various aquatic organisms; however, the heterogeneous response patterns in zebrafish (Danio rerio) liver cell populations caused by NP exposure have not yet been clarified. Investigation of the heterogeneous response patterns in zebrafish liver cell populations after NPs exposure provides us significances to explore the NP cytotoxicity. In this article, the heterogeneous response patterns in zebrafish liver cell populations after polystyrene (PS)-NPs exposure were studied. Significantly increased content of malondialdehyde and decreased levels of catalase and glutathione were observed, indicating the oxidative damage of zebrafish liver induced by PS-NPs exposure. Afterwards, the liver tissues were enzymatically dissociated and used for single-cell transcriptomic (scRNA-seq) analysis. Nine cell types were identified based on unsupervised cell cluster analysis followed by their marker genes. Hepatocytes were the cell type most impacted by PS-NP exposure, and heterogeneous response patterns of male and female hepatocytes were observed. The PPAR signaling pathway was up-regulated in hepatocytes from both male and female zebrafish. Lipid metabolism-related functions were altered more notably in male-derived hepatocytes, while female-derived hepatocytes were more sensitive to estrogen stimulus and mitochondria. Macrophages and lymphocytes were also highly responsive cell types, with specific immune pathways activated to suggest immune disruption after exposure. Oxidation-reduction process and immune response were significantly altered in macrophages, and oxidation-reduction process, ATP synthesis, and DNA binding were most altered in lymphocytes. Our study not only integrates scRNA-seq with toxicology effects to identify highly sensitive and specific populations of responding cells, revealing highly specialized interactions between parenchymal and non-parenchymal cells and expanding our current understanding of PS-NPs toxicity, but also highlights the importance of cellular heterogeneity in environmental toxicology.

Environmental stressors differentially modulate anxiety-like behaviour in male and female zebrafish

How differently male and female responds in a stressful situation is a matter of curiosity. Apart from curiosity, this opens a new arena to the synthesis of personalized/individualized medications. Here, we used zebrafish, a suitable experimental animal model to study stress and anxiety. We evaluated the differential responses in adult male and female zebrafish on the acute exposure of three different stressors: Caffeine (100 mg/L), Conspecific alarm substance (3.5 ml/L), and sight of sympatric predators (Leaf fish and Snakehead) with the help of two different behavioural paradigms (Novel tank test & Predator exposure). Behavioural responses were captured over 6 minutes and quantified using Smart 3.0. Male zebrafish were found to be more responsive to caffeine treatment. Conspecific alarm substance-challenged males and females showed robust alarm reactions whereas females were found to be more prone to it. Female zebrafish showed statistically significant aversion to the visual representation of sympatric predators. Taken together, each stressor induced differential responses in male and female zebrafish.

Kidney transcriptome and cystic kidney disease genes in zebrafish.

Introduction: Polycystic kidney disease (PKD) is a condition where fluid filled cysts form on the kidney which leads to overall renal failure. Zebrafish has been recently adapted to study polycystic kidney disease, because of its powerful embryology and genetics. However, there are concerns on the conservation of this lower vertebrate in modeling polycystic kidney disease. Methods: Here, we aim to assess the molecular conservation of zebrafish by searching homologues polycystic kidney disease genes and carrying transcriptome studies in this animal. Results and Discussion: We found that out of 82 human cystic kidney disease genes, 81 have corresponding zebrafish homologs. While 75 of the genes have a single homologue, only 6 of these genes have two homologs. Comparison of the expression level of the transcripts enabled us to identify one homolog over the other homolog with >70% predominance, which would be prioritized for future experimental studies. Prompted by sexual dimorphism in human and rodent kidneys, we studied transcriptome between different sexes and noted significant differences in male vs. female zebrafish, indicating that sex dimorphism also occurs in zebrafish. Comparison between zebrafish and mouse identified 10% shared genes and 38% shared signaling pathways. String analysis revealed a cluster of genes differentially expressed in male vs. female zebrafish kidneys. In summary, this report demonstrated remarkable molecular conservation, supporting zebrafish as a useful animal model for cystic kidney disease.

The gut-microbiome and anxiety in male and female zebrafish

Recent research from the CDC indicates that 41.5% of adults in the US classify themselves as anxious or depressed. Interestingly, anxiety disorders are disproportionately more prevalent in women than men. It is well established that the microbiome greatly influences stress responses, mood states, and other aspects of brain health including anxiety disorders. It is also known that the microbial composition differs in males and females. This study aims to determine the similarities and differences between the male and female zebrafish microbiome and identify if these differences impact anxiety. Zebrafish were treated with amoxicillin to decrease the total intestinal bacterial abundance. The zebrafish were then subjected to an acute stress protocol or a chronic stress protocol. For acute stress, fish were net stressed for three minutes, followed by 3 minutes of acclimation. Chronic stress was induced over 8 days with fish being stressed daily prior to the three minute net stress. Fish behavior and anxiety were assessed using the EthoVision tracking system and software. We observed a 48% and 67% decrease in bacterial abundance in males and females respectively treated with amoxicillin (p&lt;0.05, n= 5). Interestingly both males and females treated with antibiotics showed significantly fewer right and left crosses following acute stress exposure- no significant differences were observed between male and female fish (p&lt;0.05, n=10). In chronically stressed fish, no differences were seen in anxiety-like behavior in control or antibiotic-treated fish regardless of sex. These results support current research indicating the overall role of the microbiome in anxiety and depression however, the differences between intestinal bacteria in males and females behavior remains unclear. Elon University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Developmental Toxicity Assessment of Drymaria Cordata (Linn.) Willd using Zebrafish Embryo.

Drymaria cordata (Linn.) Willd is a creeping herb belonging to the Caryophyllaceae family, widely used as a traditional medicine in Africa and North-east India for various ailments. Many therapeutic applications of D. cordata have been reported in various scientific studies, but the teratogenicity study of this herb has not been documented till now. The present study aimed to assess the developmental toxic effect and median lethal concentration (LC50) of methanol extract of Drymaria cordata leaf (DCME) using zebrafish embryos. After spawning of male and female zebrafish, healthy zebrafish embryos were selected by microscopic screening and transferred into 96-well plate for the study. Embryos were exposed to DCME at concentrations ranging from 50-400 μg/ml in 2% DMSO from 24 hpf to 72 hpf. Developmental and morphological abnormalities were microscopically evaluated. Fifty percent lethal concentration (LC50) of DCME was determined by observation from 24 hpf to 72 hpf. The concentration-dependent toxic effects of DCME on developing embryos of zebrafish were found in the study in a time-dependent manner. At 72 hpf, the median lethal concentration (LC50) of DCME was found with visible developmental defects, such as heartbeat rate, less pigmentation, oedema, spinal curvature, immature yolk sac as well as reduced hatching rate and a slow growth. The median lethal dose was found to be 448 μg/ml at 72 hpf for zebra fish embryos, meriting further studies on toxicological profiling of the plants.

The use of in silico extreme pathway (ExPa) analysis to identify conserved reproductive transcriptional-regulatory networks in humans, mice, and zebrafish

Vertebrate sex determination and differentiation are coordinated by the activations and maintenance of reproductive transcriptional-regulatory networks (TRNs). There is considerable interest in studying the conserved design principles and functions of reproductive TRNs given that their intricate regulation is susceptible to disruption by gene mutations or exposures to exogenous endocrine disrupting chemicals (or EDCs). In this manuscript, the Boolean rules describing reproductive TRNs in humans, mice, and zebrafish, were represented as a pseudo-stoichiometric matrix model. This model mathematically described the interactions of 35 transcription factors with 21 sex determination and differentiation genes across the three species. The in silico approach of Extreme Pathway (ExPa) analysis was used to predict the extent of TRN gene activations subject to the species-specific transcriptomics data, from across various developmental life-stages. A goal of this work was to identify conserved and functional reproductive TRNs across the three species. ExPa analyses predicted the sex differentiation genes, DHH, DMRT1, and AR, to be highly active in male humans, mice, and zebrafish. Whereas FOXL2 was the most active gene in female humans and mice; and CYP19A1A in female zebrafish. These results agree with the expectation that regardless of a lack of sex determination genes in zebrafish, the TRNs responsible for canalizing male vs. female sexual differentiation are conserved with mammalian taxa. ExPa analysis therefore provides a framework with which to study the TRNs that influence the development of sexual phenotypes. And the in silico predicted conservation of sex differentiation TRNs between mammals and zebrafish identifies the piscine species as an effective in vivo model to study mammalian reproductive systems under normal or perturbed pathologies.

Sex differences in the expression of aggressive behavior and influences on social choice in zebrafish (Danio rerio)

Aggregation behavior (grouping) provides many animals with the ecological benefits of protection from predators, increased access to food, and greater mating opportunities, despite possible costs. Many factors likely contribute to the social choices animals make, and we sought to determine if the individual expression of aggression would correlate with shoalmate choices. We quantified aggressive or submissive tendencies of individual male and female zebrafish and shoalmate preferences within dichotomous choice assays. We hypothesized that fish, regardless of their individual aggressive behaviors, would benefit most and therefore choose to spend time near larger shoals and shoals of the opposite sex. Both sexes spent significantly more time near shoals rather than be alone. Males spent significantly more time with the largest shoal, and females exhibited a similar trend. Both sexes spent more time with shoals of females, rather than males. Males were generally more consistent in their aggressive behaviors across multiple assays and females exhibited more individual-variability. More aggressive male zebrafish were more likely to choose shoals of males over females and were more likely to swim alone, but female zebrafish did not exhibit a correlation between aggressive behaviors and social choices. Our results demonstrate clear sex differences in the expression of individual behavior and influences on shoaling.

Bisphenol A has a sex-dependent disruptive effect on hepatic lipid metabolism in zebrafish

Bisphenol A (BPA) is an endocrine disruptor that has adverse effects on lipid metabolism. However, most of the current studies on the effects of BPA on lipid metabolism in fish have focused on middle- and short-term exposure tests. The aim of this study was to investigate the effects of long-term BPA exposure on liver lipid metabolism in zebrafish. Post-fertilization embryos were exposed to environmentally relevant concentrations of BPA for 120 days, and the changes in triglyceride (TG), total cholesterol (TC) levels, and gene expression related to liver lipid metabolism were investigated in both male and female fish. The results showed that long-term exposure to BPA led to lipid deposition in liver, and there was a sex difference. In the liver of female fish, there was higher lipid transport and synthesis at low concentration of BPA, while overall metabolic levels were increased at high concentration of BPA. In contrast, BPA showed a dose-dependent effect on the lipid deposition in male fish. The expression of mRNA of TG transport-related and lipid synthesis-related genes was significantly up-regulated and the expression of genes related to lipid catabolism, was significantly down-regulated with increasing BPA dose. Taken together, our results indicate that long-term exposure to BPA can increase lipid deposition in a gender-specific manner. This may be due to the different responses of lipid metabolism related genes to BPA in male and female zebrafish. These results will provide a new reference for a deeper understanding of the ecotoxicological effects of BPA on aquatic animals.

Benzo(a)pyrene exposure in early life suppresses spermatogenesis in adult male zebrafish and association with the methylation of germ cell-specific genes

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that are widely present in aquatic ecosystems. To assess the impact of early-life exposure to benzo[a]pyrene (BaP), a representative PAH, on reproductive ability in adult male zebrafish (Danio rerio), fertilized embryos were exposed to 0.05, 0.5, 5 and 50 nM of BaP for 96 h, and then the hatched larvae were raised to adulthood in clean water. In one-year-old male fish, the percentage of spermatozoa in testis was significantly reduced in the 0.5, 5 and 50 nM treatments. When the treated fish were mated with untreated fish, significantly decreased rate of egg fertilization and hatching success and significantly elevated malformation rate the F1 larvae were observed in the 0.5, 5 and 50 nM treatments. The transcriptional levels of genes along the brain-pituitary-gonadal axis, involving gnrh3, gnrhr3, fshβ, lhβ, lhγ, lhrγ and ar, were downregulated. In addition, embryonic BaP exposure upregulated the promotor methylation of germ cell-specific genes in the testis of adult fish. The upregulated methylation of ddx4, dnd1, nanos2 in the testis might be associated with the downregulated mRNA levels of these genes, which could be another reason for the inhibition of spermatogenesis. These results indicate that early-life exposure to BaP suppress the reproductive capability of adult male fish, which would cause a decrease in fish population.

Short daylight photoperiod alleviated alarm substance-stimulated fear response of zebrafish

Photoperiod has been well-documented to be involved in regulating many activities of animals. However, whether photoperiod takes part in mood control, such as fear response in fish and the underlying mode(s) of action remain unclear. In this study, adult zebrafish males and females (Danio rerio) were exposed to different photoperiods, Blank (12 h light: 12 h dark), Control (12 h light: 12 h dark), Short daylight (SD, 6 h light: 18 h dark) and Long daylight (LD, 18 h light: 6 h dark) for 28 days. After exposure, fear response of the fish was investigated using a novel tank diving test. After alarm substance administration, the onset to higher half, total duration in lower half and duration of freezing in SD-fish were significantly decreased, suggesting that short daylight photoperiod is capable of alleviating fear response in zebrafish. In contrast, comparing with the Control, LD didn’t show significant effect on fear response of the fish. Further investigation revealed that SD increased the levels of melatonin (MT), serotonin (5-HT) and dopamine (DA) in the brain while decreased the plasma level of cortisol comparing to the Control. Moreover, the expressions of genes in MT, 5-HT and DA pathways and HPI axis were also altered consistently. Our data indicated that short daylight photoperiod might alleviate fear response of zebrafish probably through interfering with MT/5-HT/DA pathways and HPI axis.

The kisspeptin-GnIH signaling pathway in the role of zebrafish courtship and aggressive behavior induced by azoxystrobin

Azoxystrobin, a strobilurin widely used to control rice diseases, has raised concerns about possible adverse effects on aquatic ecosystems. At present, very little is known about the effects of azoxystrobin on courtship and aggressive behavior and the potential underlying mechanisms. In the present study, after exposing adult male and female zebrafish to worst-case scenario concentrations of azoxystrobin (0, 2 μg/L, 20 μg/L, and 200 μg/L) for 42 d, we observed a decrease in courtship behavior and an increase in aggressive behavior in both male and female zebrafish. In addition, to elucidate the molecular mechanism of the behavioral effects of azoxystrobin, we quantified the changes in the concentrations of kisspeptin, 5-HT, GnIH, and their corresponding receptor mRNA expression in the brain. The results showed that 200 μg/L azoxystrobin decreased the concentrations of kisspeptin and increased the concentration of GnIH in both male and female zebrafish brain. In addition, azoxystrobin also significantly reduced 5-HT concentration in female zebrafish brain. Further investigation revealed that altered courtship and aggressive behavior were associated with the expression levels of genes (kiss1, kiss2, gnrh3, gnrhr3, 5ht1a, and 5ht2a) involved in kisspeptin-GnIH signaling pathway. In conclusion, our study suggested that azoxystrobin may impair courtship and aggressive behavior in zebrafish by interfering with the kisspeptin-GnIH signaling pathway, which may have more profound effects on natural zebrafish populations.