Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Liver development in zebrafish （Danio rerio）

Liver is one of the largest internal organs in the body and its importance for metabolism, detoxification and homeostasis has been well established. In this review, we summarized recent progresses in studying liver initiation and development during embryogenesis using zebrafish as a model system. We mainly focused on topics related to the specification of hepatoblasts from endoderm, the formation and growth of liver bud, the differentiation of hepatocytes and bile duct cells from hepatoblasts, and finally the role of mesodermal signals in controlling liver development in zebrafish.     

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.     

Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio)

Gankyrin is a small ankyrin-repeat protein that previous research has confirmed to be overexpressed in hepatocellular carcinoma (HCC). Although relevant literature has reported on gankyrin functions in cellular proliferation and tumorigenesis, the exact role of gankyrin is poorly understood in animal model systems. This study analyzed hepatic lipid accumulation in gankyrin transgenic (GK) zebrafish. Bromodeoxyuridine (BrdU)-positive cells were predominantly increased in the liver bud of GK larvae, indicating that gankyrin functionally promoted cell proliferation at the larval stage in GK fish. However, over 90% of the viable GK adults showed an increased lipid content, leading in turn to liver steatosis. Liver histology and oil red O staining also indicated the accumulation of fatty droplets in GK fish, consistent with the specific pathological features of severe steatosis. Molecular analysis revealed that gankyrin overexpression induced hepatic steatosis and modulated the expression profiles of four hepatic microRNAs, miR-16, miR-27b, miR-122, and miR-126, and 22 genes involved in lipid metabolism. Moreover, significantly increased hepatic cell apoptosis resulted in liver damage in GK adults, leading to liver failure and death after approximately 10months. This study is the first to report gankyrin as a potential link between microRNAs and liver steatosis in zebrafish.     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Embryonic substances induce alarm response in adult zebrafish (Danio rerio)

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10⁻⁶ g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae

Activin A belongs to the superfamily of transforming growth factor-β and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml^–1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 μm² compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

Embryonic exposure to cypermethrin induces apoptosis and immunotoxicity in zebrafish (Danio rerio)

Cypermethrin (CYP) is widely used for control of indoor and field pests. As a result, CYP is one of the most common contaminants in freshwater aquatic systems. In the present study, we investigated the effects of CYP exposure on the induction of apoptosis and immunotoxicity in zebrafish during the embryo developmental stage. The mRNA levels of some key genes including P53, Puma, Bax, Apaf1, Cas9 and Cas3 on the mitochondrial pathway of cell apoptosis were significantly up-regulated at the concentration of 3 and 10 μg/l CYP. Correspondingly, the activities of Cas3 and Cas9 increased significantly after exposure to 3 or 10 μg/l CYP. In addition, the mRNA levels of iNOS and the total content of NO were also up-regulated significantly after CYP exposure. Moreover, it was also observed that the mRNA levels of IFN, CXCL-Clc, CC-chem and C3, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to 3 and 10 μg/l CYP, exhibiting CYP's prominent impacts on the innate immune system of zebrafish. Taken together, our results suggest that CYP has the potential to induce cell apoptosis and cause innate immune system disruption in zebrafish during the embryo stage. The information presented in this study will help elucidate the mechanism of CYP-induced toxicity in fish.     

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio)

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.     

Effect of silymarin on curcumin-induced mortality in zebrafish (Danio rerio) embryos and larvae

In presence of 7.5 microM of curcumin, no embryos or larva of zebrafish survived 3 days of incubation; however, coincubation with 144 microg/ml silymarin increased the survival rates of curcumin-treated embryos and larvae to about 70%. Moreover, in presence of 12.5 microM curcumin, all embryos died after 2 days of incubation; however, co-treatment with 144 microg/ml silymarin increased the survival rates of curcumin-treated embryos and larvae up to 60 and 50%, respectively. This protective effect was not found in the other phenolic compounds viz., ferulic acid, naringin, or crocin, tested. Finally, using a fluorescence microscope, accumulation of less curcumin has observed in the edema sac area of the larvae co-treated with curcumin and silymarin than in the larvae treated with curcumin only. The result suggests that the protective effects of silymarin may be due to a decreased accumulation of curcumin in the fish body.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Acridine mutagenesis of zebrafish (Danio rerio)

Mutagenesis screening, in which heritable traits are isolated following damage to the genome, is a powerful approach for investigating gene function. Among vertebrate model organisms, the zebrafish (Danio rerio) is ideally suited to mutagenesis screens. The success of large-scale screens is dependent on the way in which changes are identified. The type of damage induced is also pivotal. Single base coding region deletions and insertions are suited to abolition of gene function whilst inducing a small physical alteration to the genome. Such mutations are not commonly found following mutagenesis schemes reported to date. Here, we show that an acridine mutagen, ICR191, which in other model organisms frequently induces single base deletions and insertions, is mutagenic in zebrafish. ICR191 induces hallmark phenotypes associated with genetic damage in treated embryos. Alterations are heritable. Offspring of mutagenised fish had mutations in a marker gene and were found to produce offspring with abnormal development. Using an adaptation of a molecular mutation detection method, fluorescent arbitrary primed PCR, we identified an induced alteration directly. The estimated frequency of induced mutations was sufficiently high to make it feasible to employ this approach for mutagenesis screening.     

Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

Oxidative stress in zebrafish (Danio rerio) sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.     

Sex-specific recombination rates in zebrafish (Danio rerio)

In many organisms, the rate of genetic recombination is not uniform along the length of chromosomes or between sexes. To compare the relative recombination rates during meiosis in male and female zebrafish, we constructed a genetic map based on male meiosis. We developed a meiotic mapping panel of 94 androgenetic haploid embryos that were scored for genetic polymorphisms. The resulting male map was compared to female and sex-average maps. We found that the recombination rate in male meiosis is dramatically suppressed relative to that of female meiosis, especially near the centromere. These findings have practical applications for experimental design. The use of exclusively female meiosis in a positional cloning project maximizes the ratio of genetic map distance to physical distance. Alternatively, the use of exclusively male meiosis to localize a mutation initially to a linkage group or to maintain relationships of linked alleles minimizes recombination, thereby facilitating some types of analysis.     

Visual discrimination learning in zebrafish (Danio rerio)

Three experiments demonstrated visual discrimination learning in zebrafish (Danio rerio). In each experiment, zebrafish were given a choice between two visually distinct arms of a T-maze. Choice of one stimulus was always followed by a food reward, but choice of the other stimulus was not rewarded. Different colored sleeves fitted around the arms of the T-maze were used in Experiments 1 (green and purple) and 2 (red and blue). The stimuli used in Experiment 3 were white sleeves lined with horizontal or vertical black stripes. In all three experiments, zebrafish acquired a significant preference for the stimulus that led to a food reward. Experiments 1 and 2 also showed that zebrafish could learn a reversal of the discrimination. Finally, the effect of discontinuing food rewards was examined after reversal training in Experiment 2 and after initial discrimination training in Experiments 1 and 3. Non-reinforcement led to a decrease in correct responding in Experiments 2 and 3 independent of stimulus identity, but to an asymmetrical pattern of responding in Experiment 1. The median latency to make a choice response decreased over the course of acquisition in all three experiments; during extinction, median response times did not change at all in Experiment 1 and increased only very slightly in Experiment 2, but showed a substantial increase in Experiment 3. The implications of these results for the zebrafish as a model system for genetic studies of learning and memory are discussed.