Boldness predicts social status in zebrafish (Danio rerio)

This study explored if boldness could be used to predict social status. First, boldness was assessed by monitoring individual zebrafish behaviour in (1) an unfamiliar barren environment with no shelter (open field), (2) the same environment when a roof was introduced as a shelter, and (3) when the roof was removed and an unfamiliar object (Lego® brick) was introduced. Next, after a resting period of minimum one week, social status of the fish was determined in a dyadic contest and dominant/subordinate individuals were determined as the winner/loser of two consecutive contests. Multivariate data analyses showed that males were bolder than females and that the behaviours expressed by the fish during the boldness tests could be used to predict which fish would later become dominant and subordinate in the ensuing dyadic contest. We conclude that bold behaviour is positively correlated to dominance in zebrafish and that boldness is not solely a consequence of social dominance.     

Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure

The aim of this study was to identify periods in zebrafish (Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE(2)) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE(2) were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE(2)effects was evaluated by exposing zebrafish during the gonad transition stage (43-71 dpf) to 1.67, 3 or 10 ng EE(2)/L. Significant effects of EE(2) on adult reproduction were found with 3 and 10 ng EE(2)/L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE(2) exposure (71 dpf), all individuals in the 3 and 10 ng EE(2)/L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE(2) exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities.     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

Development of the cranium and paired fins in the zebrafish Danio rerio (Ostariophysi,Cyprinidae)

Because of the genetic and developmental information available, Danio rerio stands out as a vertebrate model system in which significant progress in the areas of development and evolution can be made. Despite its increasing popularity, little research has been done on skeletal development. In this report, we provide developmental information on the structure and composition of the zebrafish skull, pectoral, and pelvic girdle. We describe the sequence of ossification of the skull and paired fins from a large series of cleared and Alizarin red-stained specimens at larval and adult stages. The most commonly followed developmental sequence in Danio rerio is described. Chondrocranial development is noted from Alcian blue-stained specimens. General trends in ossification patterns are examined from developmental, phylogenetic, and functional contexts. No clear pattern in ossification order of dermal versus cartilage bones is evident. Ossification sequence conforms to functional need in a general way, but there are inconsistencies in the details of order. Selected phylogenetic comparisons of ossification sequence within cranial regions are made among Danio rerio, Betta splendens, Oryzias latipes, and Barbus barbus. Greater sequence conservation is apparent between D. rerio and Barbus barbus, the ostariophysans, than among other taxon pairs. Intraspecific variation in ossification order is apparent, most of which involves small adjustments in timing. © 1996 Wiley-Liss, Inc.     

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.     

壬基酚暴露对斑马鱼求偶行为与繁殖成功率的影响

壬基酚(NP)是普遍存在于水生生态系统中的一种环境内分泌干扰物。研究了不同浓度下(0,0.1,1,10,50,100μg/L)NP暴露对斑马鱼(Daniorerio)求偶行为与繁殖成功率的影响。结果表明,NP暴露对斑马鱼求偶行为与繁殖成功率影响显著,100μg/LNP暴露显著减少斑马鱼求偶时间、降低产卵量与受精率(P(0.05)。斑马鱼产卵量与求偶总时间、平均每次求偶时间、长于5s的求偶时间显著正相关(P(0.05),与求偶频率不相关(P=0.951)。NP暴露可能通过影响斑马鱼求偶时间,进而影响产卵量。斑马鱼求偶时间有望作为评估水体NP污染有效的生物标记。     

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.     

双酚A对斑马鱼肝脏和性腺的作用

为了阐明内分泌干扰物双酚A(BPA)对水生动物的毒理作用机制,本文研究了BPA对斑马鱼急性毒性及组织学损伤作用。结果表明:BPA对斑马鱼具有急性毒性效应,96h半致死浓度为6.3mg·mL-1,96h暴露对斑马鱼的肝脏产生了浓度依赖性损伤;在高浓度组,精巢结构损伤,卵泡萎缩严重(闭锁率35%);在0.2mg·mL-1亚致死浓度BPA组中,暴露96h后,斑马鱼肝脏受到轻微损伤,性腺组织结构没有受到明显损伤,卵巢中处于第Ⅲ时相晚期和第Ⅳ时相配子数比率增加,单个卵母细胞直径增加(P0.05);在2mg·mL-1亚致死浓度BPA组中,暴露96h,斑马鱼的肝脏组织受到了一定损伤,细胞核萎缩变形,细胞肿胀,核固缩,性腺组织受损不明显,卵巢中第Ⅲ时相晚期和第Ⅳ时相配子数比率有所增加;在6.1mg·mL-1BPA组中,暴露96h,斑马鱼肝脏产生了极大的损伤,细胞空泡化,大面积的组织坏死,空洞产生,精巢组织结构受到一定损伤,卵巢中发现大量细胞萎缩现象;BPA对斑马鱼肝脏具有靶器官毒性,对性腺作用值得进一步研究。     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)

Some chemicals have the potential to adversely affect sexual development through multiple endocrine actions. Prochloraz is an imidazole fungicide that displays diverse mechanisms of action, including inhibition of aromatase activity, inhibition of androgen synthesis, and antagonism of the androgen receptor. The objective of this study was to assess the effects of prochloraz on the sexual development of zebrafish (Danio rerio) in the Fish Sexual Development Test (FSDT) proposed as an OECD test guideline for detection of endocrine disruptors. Zebrafish were exposed to prochloraz (0, 16, 64 or 202 microg/L) for 60 days from 24 h post fertilization. Fish exposed to 202 microg/L prochloraz showed an increased proportion of males. Furthermore, the incidence of intersex and the stages of the gonads were altered in the treated fish compared to the control fish. A significant vitellogenin decrease was observed in both female and male zebrafish at an exposure concentration of 202 microg/L prochloraz. However, in the male fish, significantly increased vitellogenin concentrations were observed in the groups exposed to 16 or 64 microg/L prochloraz. This study serves as a part of the validation of the FSDT and indicates that the FSDT is suitable in detecting compounds with multiple endocrine actions. This is of importance in the assessment of the potential risk of existing and new chemicals.     

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.     

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.     

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.     

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.     

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae)

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.