Bacterial lipopolysaccharides induce genes involved in the innate immune response in embryos of the zebrafish (Danio rerio)

The innate immune response in fish represents an early, rapid defence against pathogens. Environmental contaminants could disturb this defence and negatively influence the ability to protect against infection. However, analysis of immune-modulation has not yet been included in testing strategies for environmental risk assessment of chemicals. In order to establish an efficient, small scale test system, the ability to induce the innate immune response by bacterial lipopolysaccharides in zebrafish embryos was investigated. The level of expression of various genes involved in inflammation was used as the endpoint. We could show that immersion of embryos in LPS induced the gene expression of two key pro-inflammatory cytokines, tumor necrosis factor alpha and interleukin 1 beta in 32 h old zebrafish embryos. The gene induction required the removal of the chorion prior to lipopolysaccharide exposure.     

Impairment of the immune system in GH-overexpressing transgenic zebrafish (Danio rerio)

Growth hormone (GH) is an important regulator of immune functions in vertebrates, and it has been intensively reported a series of stimulatory actions of this hormone over on the immune system. Within aquaculture, overexpression of GH has been considered a promising alternative for promoting higher growth rates in organisms of commercial interest. Considering the various pleiotropic effects of GH, there are still few studies that aim to understand the consequences of the excess of GH on the physiological systems. In this context, our goal was to present the effects of the overexpression of GH on immune parameters using a model of zebrafish (Danio rerio) that overexpress this hormone. The results showed that GH transgenic zebrafish had 100% of mortality when immunosuppressed with dexamethasone, revealing a prior weakening of the immune system in this lineage. Morphometric analysis of thymus and head kidney revealed a reduction in the area of these structures in transgenic zebrafish. Moreover, the phenotypic expression of CD3 and CD4 thymocytes was also depreciated in transgenic zebrafish. Furthermore, a decrease was noted in the expression of genes RAG-1 (60%), IKAROS (50%), IL-1β (55%), CD4 (60%) and CD247 (40%), indicating that development parameters, of innate and acquired immunity, are being harmed. Based on these results, it can be concluded that the excess of GH impairs the immune functions in GH transgenic zebrafish, indicating that the maintenance of normal levels of this hormone is essential for the functioning of immunological activities.     

Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio)

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.     

Embryonic exposure to cis-bifenthrin enantioselectively induces the transcription of genes related to oxidative stress,apoptosis and immunotoxicity in zebrafish (Danio rerio)

Cis-bifenthrin (cis-BF) is used widely for agricultural and non-agricultural purpose. Thus, cis-BF is one of the most frequently detected insecticides in the aquatic ecosystem. As a chiral pesticide, the commercial cis-BF contained two enantiomers including 1R-cis-BF and 1S-cis-BF. However, the difference in inducing oxidative stress, apoptosis and immunotoxicity by the two enantiomers in zebrafish still remains unclear. In the present study, the zebrafish were exposed to environmental concentrations of cis-BF, 1R-cis-BF and 1S-cis-BF during the embryos developmental stage. We observed that the mRNA levels of the most genes related to oxidative stress, apoptosis and immunotoxicity including Cu/Zn-superoxide dismutase (Cu/Zn-Sod), catalase (Cat), P53, murine double minute 2 (Mdm2), B-cell lymphoma/leukaemia-2 gene (Bcl2), Bcl2 associated X protein (Bax), apoptotic protease activating factor-1 (Apaf1), Caspase 9 (Cas9), Caspase 3 (Cas3), interleukin-1 beta (IL-1β) and interleukin-8(Il-8) were much higher in 1S-cis-BF treated group than those in cis-BF or 1R-cis-BF treated ones, suggesting that 1S-cis-BF has higher risk to induced oxidative stress, apoptosis and immunotoxicity than 1R-cis-BF in zebrafish. The information presented in this study will help with elucidating the differences and environmental risk of the two enantiomers of cis-BF-induced toxicity in aquatic organisms.     

Distribution of GABA-immunolabeling in the early zebrafish (Danio rerio) brain.

The spatial and temporal pattern of GABA-expression in the brains of zebrafish (Danio rerio) embryos was studied by means of immunohistochemical techniques. GABA is said to exert neurotrophic actions in the early regulation of the differentiation of the central nervous system. In early stages GABAergic cells form distinct clusters throughout the CNS. As development progresses, more GABAergic clusters appear, and a pattern of GABAergic axonal projections is well defined. Although there is a corresponding pattern of distribution and appearance of GABA-expression in the brain of different teleosts, further studies are needed to establish its role during early morphogenesis of the CNS of vertebrates.     

Effect of aging on male reproduction in zebrafish (Danio rerio)

The study was designed to test the hypothesis that male aging is associated with a change in reproductive function in the zebrafish. Young (290 ± 37 d) and older (911 ± 48 d) males were combined with females (604 ± 24 d) to test the effect of male age on the number and fertility of eggs laid by their mates. 48% of breeding trials with young males and 25% of the trails with older males resulted in egg deposition. Although young males were associated with significantly more successful breeding attempts than older males, number of eggs laid per clutch, number and percent of fertilized eggs and the number and percent living embryos were not statistically different between young and older males. These data suggest that male aging is associated with altered reproductive behavior and/or female response but not in sperm quality per se. Consistent with this interpretation were the findings that percent motility and sperm motility characteristics did not differ between sperm from young and older males as assessed by computer-assisted sperm analysis. However, older males contained higher quantities of extractable sperm than did young males, perhaps associated with fewer successful breeding attempts. Age-related effects on male reproductive in the zebrafish may therefore be a consequence of behavioral or morphological features that play a role in female mate choice and/ or male sexual response.     

Live imaging early immune cell ontogeny and function in zebrafish Danio rerio

The success of a robust vertebrate inflammatory response is in part because of the migratory potential of its haematopoietic components. Once these cells converge at an inflammatory site, they interact with each other as well as non‐immune tissues and infectious agents to help manage both the scale and the duration of any ensuing response. Exactly how these blood cells, that constitute the innate and adaptive immune systems, contribute to such immune responses remain largely unknown. Traditionally, assessing these contributions relied upon histological analysis of fixed tissue sections complemented with in vitro dynamic data. Although informative, translating results from these studies into the multicellular whole‐animal setting remain difficult. Recently, non‐invasive live imaging of the immune system in animal models is providing significant insights into how immune cells function within their intact natural environment. Although the majority of these studies have been conducted within mice, another vertebrate, the zebrafish Danio rerio is being recognized as an ideal platform for non‐invasive live imaging applications. The optical transparency, rapid development, genetic tractability and highly conserved innate and adaptive immune systems of this well‐established developmental model have been exploited in a number of recent studies evaluating the immunocompetence of fluorescently tagged blood cells. In addition, similar live imaging studies are helping to dissect the ontogeny of blood‐cell development by tracking various haematopoietic precursor cells to assess their contribution to different blood lineages. This review will examine some recent advances that have helped D. rerio emerge as a live imaging platform as well as its potential to offer valuable insights into the genetics behind diseases associated with immune cell dysfunction.     

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.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

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.     

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.     

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.     

Ontogeny of cardiovascular control in zebrafish (Danio rerio): effects of developmental environment

The goal of this symposium paper was to identify and quantify developmental plasticity in the onset of cardiovascular responses in the zebrafish. Developmental plasticity was induced by altering the developmental environment in one of three ways: (1) by developing zebrafish in a constant current of 5 body lengths per second, (2) by developing zebrafish at a colder temperature (20 degrees C), and (3) by developing zebrafish in severe hypoxia (DO=0.8 mg/L). Early morphological development was significantly affected by each of the treatment environments with hypoxia slowing development the most and producing the highest variation in measurements. Development in constant water current did not significantly affect the timing onset of cardiovascular responses to the pharmacological agents applied. Development at 20 degrees C significantly delayed the onset of all cardiovascular responses measured by 2-3 days. Development in hypoxia, however, not only delayed onset of all cardiovascular responses, but also shifted the onset relative to the developmental program. Hypoxia clearly has a profound affect on the onset of cardiovascular regulation and it will take many more studies to elucidate the mechanisms by which hypoxia is having its effect. Furthermore, long term studies are also needed to assess whether the plasticity measured in this study is adaptive in the evolutionary sense.     

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.     

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.     

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.