Olfactorily-mediated cortisol response to chemical alarm cues in zebrafish Danio rerio

Chemical cues released as a by-product of predation mediate antipredator behaviour, but little is known about the physiological responses to olfactory detection of predation risk. In this study, zebrafish Danio rerio were exposed to either chemical alarm cues from conspecifics, or water (control). Compared with water controls, D. rerio exposed to alarm cues responded behaviourally with antipredator behaviours such as erratic dashing and an increase in time spent near the bottom of the test aquarium. Danio rerio were sacrificed 5 min after exposure to test cues (alarm cues or water). Enzyme-linked immunosorbent assay (ELISA) revealed whole-body levels of cortisol that were significantly higher for fish exposed to alarm cues (mean ± SE, 11.9 ± 3.4 ng g⁻¹) than control fish (1.5 ± 0.7 ng g⁻¹). These data provide a benchmark for future studies of the proximate mechanisms of olfactorily mediated antipredator responses, modelling effects on aquatic life in a changing climate and, as a model organism, Danio rerio can further our understanding of anxiety in humans.     

Daily shoaling patterns in the zebrafish Danio rerio

Shoaling intensity in zebrafish Danio rerio is believed to vary throughout subjective day and night hours. This experiment examines long term variations in shoaling behavior. Adult zebrafish Dan io rerio were maintained under a 12：12 LD cycle （with dim red light serving as reduced visibility during subjective dark hours）, and their shoaling behavior was monitored every hour for a three-day period of time. Our results show that zebrafish perform shoaling behavior throughout subjective day and under reduced visibility conditions, although mean shoaling times during the light phase were significantly higher than mean shoaling times during the dark phase. However, on the 3^rd day of the experiment, mean shoaling times during the subjective night had increased and mean shoaling times during the subjective day had decreased. This shift in intensity was not seen on the first two days of the study, and may represent the influence of experience on the behavior of the test fish. We believe this study shows that shoaling behavior changes with light/dark cycles and that fish shoal even during reduced visibility conditions.     

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.     

The behaviour and ecology of the zebrafish, Danio rerio

The zebrafish Danio rerio, is an important model organism in developmental genetics, neurophysiology and biomedicine, but little is known about its natural ecology and behaviour. It is a small, shoaling cyprinid, native to the flood-plains of the Indian subcontinent, where it is found in shallow, slow-flowing waters. Zebrafish are group spawners and egg scatterers, although females are choosy with respect to sites for oviposition and males defend territories around such sites. Laboratory studies of zebrafish behaviour have encompassed shoaling, foraging, reproduction, sensory perception and learning. These studies are reviewed in relation to the suitability of the zebrafish as a model for studies on cognition and learning, development, behavioural and evolutionary ecology, and behavioural genetics.     

Distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

Mitochondria play a vital role during oocyte maturation, fertilization, and embryo development. In this study, confocal microscopy with the mitochondrial membrane potential-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) was used to investigate mitochondria distribution and activity of stage III zebrafish ovarian follicles. To support the mitochondrial origin of the fluorescence obtained by JC-1, a second mitochondrial probe, MitoTracker Green FM, was used. Cryo-scanning and transmission electron microscopy were also used to validate the distribution and localization of mitochondria obtained by mitochondrial staining. The mitochondrial probes were unable to penetrate the oocyte, and as a result it was not possible to observe stained mitochondria in the oocyte cytoplasm. However, mitochondrial staining of the granulosa cell layer surrounding the stage III zebrafish oocyte exhibited a contiguous aggregation pattern of mitochondria. Cryo-scanning electron microscopy studies also showed the oocyte surface to be covered by polygonal patterns of ridges of the same dimensions as the distributional arrangement of mitochondria in the granulosa cells. Though the results suggested the need for defolliculation to assess mitochondrial distribution and activity in the stage III zebrafish oocyte cytoplasm, the findings of this study will contribute to our understanding of oogenesis and folliculogenesis processes in fish.     

Parent-of-origin specific effects on the methylation of a transgene in the zebrafish,Danio rerio

We have examined the inheritance of a transgene locus in the zebrafish, Daniorerio and demonstrated that its methylation is af fected by the sex of the parent contributing the allele. This parent-of-origin effect on the zebrafish transgene appears to be identical to imprinting as seen in mammals except that in zebrafish, passage of the locus through a female tended to decreased its methylation, whereas passage through a male increased it. Methylation of the transgene in gametic tissues differed from somatic tissue with the locus being hypomethylated in sperm and hypermethylated in the unfertilized egg. The potential identification of imprinting in the zebrafish has important ramifications with respect to the evolution of the process as well as for understanding the role of imprinting in mammals. © 1995 Wiley-Liss, Inc.     

斑马鱼对无氧运动训练的适应性变化

该研究以斑马鱼为(Danio rerio)对象,研究了四周无氧运动训练对斑马鱼行为、形态、生长、肌肉生化组分及代谢酶活性的影响。旨在探索斑马鱼对无氧运动训练的适应性变化,为进一步了解鱼类适应无氧运动训练的分子机制提供基础数据。结果发现:斑马鱼的日常活跃程度经四周无氧运动训练后显著降低,群聚程度增加;训练组个体体重和体长增长减缓,更利于运动;肌糖原含量显著增加,运动持久能力加强;肌肉乳酸脱氢酶(LDH)活性显著增高,柠檬酸合成酶(CS)活性显著降低,无氧代谢能力加强。即,斑马鱼无氧运动能力和无氧代谢能力在训练后得以明显提升。     

斑马鱼繁殖内分泌学研究进展

作为四大模式动物之一,斑马鱼(Danio rerio)广泛应用于胚胎学、发育生物学、毒理学、分子生物学等研究。但关于斑马鱼繁殖内分泌生理和环境毒理方面的研究少见报道,本文综述了近年来,在内分泌学方面以斑马鱼作为实验动物的研究概况。     

Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.     

Expression patterns of the creatine metabolism‐related molecules AGAT,GAMT and CT1 in adult zebrafish Danio rerio

AGAT, GAMT and CT1, three creatine synthesis and transport‐related molecules, have been widely studied in mammals. To explore their homologous genes in adult zebrafish Danio rerio, the gene expression patterns of these three genes in D. rerio were investigated. The results reveal that AGAT, GAMT and CT1 are expressed widely in diverse tissues of D. rerio where the homologous genes in mammals are also expressed.     

Detection of female mating status using chemical signals and cues

Males of many species choose their mate according to the female's reproductive status, and there is now increasing evidence that male fitness can depend on this discrimination. However, females will also aim to regulate their mating activity so as to maximize their own fitness. As such, both sexes may attempt to dictate the frequency and timing of female mating, reflecting the potentially different costs of female signaling to both sexes. Here, I review evidence that chemical cues and signals are used widely by males to discriminate between mated and unmated females, and explore the mechanisms by which female odour changes post‐mating. There is substantial empirical evidence that mated and unmated females differ in their chemical profile, and that this variation provides males with information on a female's mating status. Although there appears to be large variation among species regarding the mechanisms by which female odour is altered post‐mating, the transfer of male substances to females during or subsequent to copulation appear to play a major role. This transfer of substances by males may be part of their strategy to suppress reproduction by competing males, particularly in species where females mate more than once.     

Membrane and calcium clock mechanisms contribute variably as a function of temperature to setting cardiac pacemaker rate in zebrafish Danio rerio

Here, we show that heart rate in zebrafish Danio rerio is dependent upon two pacemaking mechanisms and it possesses a limited ability to reset the cardiac pacemaker with temperature acclimation. Electrocardiogram recordings, taken from individual, anaesthetised zebrafish that had been acclimated to 18, 23 or 28°C were used to follow the response of maximum heart rate (f_Hmax) to acute warming from 18°C until signs of cardiac failure appeared (up to c. 40°C). Because f_Hmax was similar across the acclimation groups at almost all equivalent test temperatures, warm acclimation was limited to one significant effect, the 23°C acclimated zebrafish had a significantly higher (21%) peak f_Hmax and reached a higher (3°C) test temperature than the 18°C acclimated zebrafish. Using zatebradine to block the membrane hyperpolarisation-activated cyclic nucleotide–gated channels (HCN) and examine the contribution of the membrane clock mechanisms to cardiac pacemaking, f _Hmax was significantly reduced (by at least 40%) at all acute test temperatures and significantly more so at most test temperatures for zebrafish acclimated to 28°C vs. 23°C. Thus, HCN channels and the membrane clock were not only important, but could be modified by thermal acclimation. Using a combination of ryanodine (to block sarcoplasmic calcium release) and thapsigargin (to block sarcoplasmic calcium reuptake) to examine the contribution of sarcoplasmic reticular handling of calcium and the calcium clock, f _Hmax was again consistently reduced independent of the test temperature and acclimation temperature, but to a significantly lesser degree than zatebradine for zebrafish acclimated to both 28 and 18°C. Thus, the calcium clock mechanism plays an additional role in setting pacemaker activity that was independent of temperature. In conclusion, the zebrafish cardiac pacemaker has a limited temperature acclimation ability compared with known effects for other fishes and involves two pacemaking mechanisms, one of which was independent of temperature.     

The effect of sex and shoal size on shoaling behaviour in Danio rerio

Male and female zebra fish Danio rerio were given choices of shoals that differed in sex and size. Male zebra fish preferred to associate with female shoals over male shoals, but had no preference when given a choice between a mixed‐sex shoal and either a male or female shoal. Female zebra fish showed no significant preference when given a choice between male and females shoals, nor between mixed‐sex shoals and either male or female shoals. When given choices between shoals of differing size, females preferred to associate with the larger shoal, whether or not they were composed of males or females. Males, however, had no preference for larger shoals over smaller shoals, whether or not they were composed of males or females. These results showed that male zebra fish were capable of distinguishing between males and females solely on the basis of visual cues. Furthermore, these results demonstrated a significant difference between the shoaling choices of male and female zebra fish, which may indicate a difference in the function of shoaling for the two sexes.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

The role of male visual and chemical cues on the activation of female courtship behaviour in the sex-role reversed peacock blenny

Chemical cues during courtship in peacock blenny Salaria pavo did not add significantly to the response of visual stimuli and females did not court when a male was not visible. The results showed that female S. pavo courtship behaviour was elicited exclusively by male visual features and not by a multicomponent signal in different sensory modalities. The time spent by females courting the male and exhibiting the nuptial colouration during visual trials correlated with the size of the male's crest, suggesting a signalling function for this trait. Alternative hypothesis for the function of the putative pheromones released by the male's anal gland are discussed. Evidence that female sexual motivation decreases towards the end of the breeding season is also provided, which should be considered in future mate choice experiments.