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.     

Inhibitory control in zebrafish,Danio rerio

We assessed whether zebrafish, Danio rerio, display inhibitory control using a simple and rapid behavioural test. Zebrafish were exposed to a prey stimulus placed inside a transparent tube, which initially elicited attack behaviour. However, zebrafish showed a rapid reduction in the number of attacks towards the prey, which indicated the ability to inhibit their foraging behaviour. Zebrafish also exhibited mnemonic retention of foraging inhibition, as indicated by a reduced number of attacks in a subsequent exposure to the unreachable prey. The ability to inhibit the foraging behaviour varied across three genetically separated wild-type strains and across different individuals within strains, suggesting that zebrafish show heritable within-species differences in inhibitory control. Our behavioural test might be suitable for screening large zebrafish populations in mutational studies and assessing the effects of pharmacologically active substances on inhibitory control.     

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.     

Population genomics of wild and laboratory zebrafish (Danio rerio)

Understanding a wider range of genotype–phenotype associations can be achieved through ecological and evolutionary studies of traditional laboratory models. Here, we conducted the first large‐scale geographic analysis of genetic variation within and among wild zebrafish (Danio rerio) populations occurring in Nepal, India, and Bangladesh, and we genetically compared wild populations to several commonly used lab strains. We examined genetic variation at 1832 polymorphic EST‐based single nucleotide polymorphisms (SNPs) and the cytb mitochondrial gene in 13 wild populations and three lab strains. Natural populations were subdivided into three major mitochondrial DNA clades with an average among‐clade sequence divergence of 5.8%. SNPs revealed five major evolutionarily and genetically distinct groups with an overall F_ST of 0.170 (95% CI 0.105–0.254). These genetic groups corresponded to discrete geographic regions and appear to reflect isolation in refugia during past climate cycles. We detected 71 significantly divergent outlier loci (3.4%) and nine loci (0.5%) with significantly low F_ST values. Valleys of reduced heterozygosity, consistent with selective sweeps, surrounded six of the 71 outliers (8.5%). The lab strains formed two additional groups that were genetically distinct from all wild populations. An additional subset of outlier loci was consistent with domestication selection within lab strains. Substantial genetic variation that exists in zebrafish as a whole is missing from lab strains that we analysed. A combination of laboratory and field studies that incorporates genetic variation from divergent wild populations along with the wealth of molecular information available for this model organism provides an opportunity to advance our understanding of genetic influences on phenotypic variation for a vertebrate species.     

Shade as enrichment: testing preferences for shelter in two model fish species

We compared preferences shown by zebrafish Danio rerio and three-spined stickleback Gasterosteus aculeatus for shelter provided by above-tank shade and artificial plants. Zebrafish showed no preference for either shelter, whereas sticklebacks showed a preference for both shelter types over open areas and for shade over plants. Our results suggest shade may be used as enrichment for captive fish and re-emphasise the importance of species-specific welfare considerations.     

International survey on the use and welfare of zebrafish Danio rerio in research

A survey was conducted regarding zebrafish Danio rerio use for scientific research with a focus on: anaesthesia and euthanasia; housing and husbandry; breeding and production; refinement opportunities. A total of 98 survey responses were received from laboratories in 22 countries in Europe, North America, South America, Asia and Australia. There appears a clear and urgent need to identify the most humane methods of anaesthesia and euthanasia. Aversive responses to MS‐222 were widely observed raising concerns about the use of this anaesthetic for D. rerio. The use of anaesthesia in fin clipping for genetic identification is widely practised and there appears to be an opportunity to further develop less invasive methods and refine this process. Optimization (and potentially standardization) of feeding is an area for further investigation. Given that diet and body condition can have such profound effects on results of experiments, differences in practice could have significant scientific implications. Further research into transition between dark and light phases in the laboratory appears to represent an opportunity to establish best practice. Plants and gravel were not considered practical by many laboratories. The true value and benefits need to be established and communicated. Overproduction is a concern both from ethical and financial viewpoints. There is an opportunity to further reduce wastage of D. rerio. There are clear concerns and opportunities for the scientific community to work together to further improve the welfare of these important laboratory models.     

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.     

Effects of environmental enrichment on activity and lameness in commercial broiler production

The aim of this pilot study was to investigate the effects of commercially applied environmental enrichments on behavior and lameness in broilers. Two consecutive flocks of broilers were observed at 16 days and 30 days of age to investigate differences between enriched (peat, bales of lucerne hay, and elevated platforms) and control birds with regards to behavioral activities and lameness. More running (p < .001), worm running (p = .006), play fighting (p = .015), dust bathing (p = .009), and ground pecking while standing (p < .001) were observed at 16 days than at 30 days. Across both ages, enriched birds showed more wing flapping (p = .016), wing stretching (p = .002), body shaking (p = .002), ground scratching (p < .001), and ground pecking while standing (p < .001) and lying (p = .003) compared with control birds. Even when no enrichments were close, enriched birds showed more body shaking (p = .008) and ground pecking while standing (p < 0.001) and lying (p = .010) than birds in comparable locations in control pens. There was a tendency for a lower gait score (i.e., reduced lameness) with the enriched treatment (p = .077). In conclusion, enriched birds showed higher levels of several activities compared with control birds, and demonstrated higher levels in areas where no enrichments were present.     

Environmental enrichment improves behaviors rather than the growth and physiology of rock bream Oplegnathus fasciatus

Environmental enrichment has the potential to improve the welfare and post-release survival of hatchery fish stocked for conservation purposes. However, the effectiveness of environmental enrichment is partly dependent on the fish species, life stage, and specific enrichment structure used. To enhance the effectiveness of environmental enrichment, it is crucial to focus on characteristic differences in enrichment structures, such as type and level. This study investigated how differences in enrichment type and level affected physiological and behavioral aspects of the welfare of pre-release juvenile rock bream Oplegnathus fasciatus by evaluating growth performance, basal and stressed cortisol levels, antioxidant enzyme activities, and exploratory behaviors regarding anxiety and flexibility. Fish were reared for 4 weeks in different enrichment treatments: barren, low-level cover structure, high-level cover structure, low-level interference structure (LI), and high-level interference structure (HI). The results revealed that fish reared with the LI treatment showed less anxiety and greater flexibility with respect to exploratory behaviors, without oxidative damage being detected. Despite exhibiting less anxiety as well, fish reared in the HI treatment had oxidative damage, indicated by lower superoxide dismutase activity, compared to those in the barren treatment. In addition, none of these enrichment structures enhanced growth performance or mitigate chronic and acute stress responses. Overall, the low-level interference structure may be more favorable in promoting the behavioral welfare of the fish. Application of this type and level of enrichment may increase the survival of the hatchery fish after release, which is critical to stocking success.     

Ontogeny of odorant receptor gene expression in zebrafish,Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26°C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ. © 1996 John Wiley & Sons, Inc.     

Dose-dependent effects of developmental mercury exposure on C-start escape responses of larval zebrafish Danio rerio

Zebrafish Danio rerio embryos were exposed to 0, 25, 50 or 75 ppb Hg²⁺ from 0 to 24 h post‐fertilization (hpf) then placed into Hg²⁺‐free water. Inductively coupled plasma‐mass spectrophotometer analysis of whole embryo Hg²⁺ content at 24 hpf showed a positive correlation with exposure regime (Pearson's one‐tailed, r²= 0·698, P < 0·01); at 5 days post‐hatch (dph), whole larval Hg²⁺ content was not detectable. Hg²⁺‐induced behavioural deficits in larvae were, therefore, due to changes during embryogenesis and not to residual Hg²⁺ in the larvae. At 5 dph, larvae were tested for responses to different frequencies but equal intensities of vibrational stimuli generated by a remotely controlled plastic hammer. Data were recorded by high‐speed videography and computer‐analysed for latency of response (ms), amplitude of the response as measured by maximum initial velocity [normalized as body (standard) lengths s^?1; V_max] and duration of behaviour from initial head movement to cessation of caudal tail movement (ms). A single mechanical stimulus resulted in behavioural outcomes that were related to embryonic Hg²⁺ uptake. Response latency increased with exposure level and displayed an increase of ×1·5–2·5 over control values (ANOVA, P < 0·01). The V_max decreased with exposure level to a low of 71% of control at the highest Hg²⁺ concentration (ANOVA, P < 0·01). Duration of behaviour displayed a biphasic response pattern in which exposure to 0, 50 or 75 ppb Hg²⁺ did not result in a significantly different response yet exposure to 25 ppb Hg²⁺ caused a significantly longer time of active response (ANOVA, P < 0·01). Repeated stimulation (1, 2 or 4 hits s^?1) resulted in a concentration‐dependent increase in response failures. Regardless of stimulation frequency, larvae exposed to 0 or 25 ppb Hg²⁺ as embryos maintained higher V_max levels for longer intervals during the testing period than those exposed as embryos to either 50 or 75 ppb Hg²⁺.     

Allozyme and microsatellite genetic variation in natural samples of zebrafish,Danio rerio

In this paper, we report the results from allozyme and microsatellite markers in natural populations of the zebrafish, a species of great significance in biological studies. Four zebrafish wild samples from West Bengal, India, were analysed in a preliminary survey of levels and patterns of genetic variation. Results indicate high levels of genetic variability and weak genetic structure, although the latter is consistent with the geographical features of the area under study, sampling sites being located in the Ganges and Brahmaputra delta region, which is characterized by high waterways connectivity.     

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.     

Circadian rhythms in zebrafish (Danio rerio) behaviour and the sources of their variability

Over recent decades, changes in zebrafish (Danio rerio) behaviour have become popular quantitative indicators in biomedical studies. The circadian rhythms of behavioural processes in zebrafish are known to enable effective utilization of energy and resources, therefore attracting interest in zebrafish as a research model. This review covers a variety of circadian behaviours in this species, including diurnal rhythms of spawning, feeding, locomotor activity, shoaling, light/dark preference, and vertical position preference. Changes in circadian activity during zebrafish ontogeny are reviewed, including ageing-related alterations and chemically induced variations in rhythmicity patterns. Both exogenous and endogenous sources of inter-individual variability in zebrafish circadian behaviour are detailed. Additionally, we focus on different environmental factors with the potential to entrain circadian processes in zebrafish. This review describes two principal ways whereby diurnal behavioural rhythms can be entrained: (i) modulation of organismal physiological state, which can have masking or enhancing effects on behavioural endpoints related to endogenous circadian rhythms, and (ii) modulation of period and amplitude of the endogenous circadian rhythm due to competitive relationships between the primary and secondary zeitgebers. In addition, different peripheral oscillators in zebrafish can be entrained by diverse zeitgebers. This complicated orchestra of divergent influences may cause variability in zebrafish circadian behaviours, which should be given attention when planning behavioural studies.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Philosophy of environmental enrichment: Past,present, and future

The brief tenure of environmental enrichment has been influenced both directly and indirectly by the field of psychology, from the work of B.F. Skinner to that of Hal Markowitz. Research on enrichment supports the supposition that an enriched environment does indeed contribute to a captive animal’s well‐being. Critical elements of effective environmental enrichment are 1) assessing the animal’s natural history, individual history, and exhibit constraints and 2) providing species‐appropriate opportunities, i.e., the animal should have some choices within its environment. This paper presents a historic perspective of environmental enrichment, proposes a broader, more holistic approach to the enrichment of animals in captive environments, and describes a framework or process that will ensure a consistent and self‐sustaining enrichment program. Zoo Biol 20:211–226, 2001. © 2001 Wiley‐Liss, Inc.     

Size‐dependent reproductive success of wild zebrafish Danio rerio in the laboratory

Size‐dependent reproductive success of wild zebrafish Danio rerio was studied under controlled conditions in the laboratory to further understand the influence of spawner body size on reproductive output and egg and larval traits. Three different spawner size categories attained by size‐selective harvesting of the F₁‐offspring of wild D. rerio were established and their reproductive performance compared during a 5 day period. As to be expected, large females spawned more frequently and had significantly greater clutch sizes than small females. Contrary to expectations, small females produced larger eggs when measured as egg diameter with similar amounts of yolk compared to eggs spawned by large spawners. Eggs from small fish, however, suffered from higher egg mortality than the eggs of large individuals. Embryos from small‐sized spawners also hatched later than offspring from eggs laid by large females. Larval standard length (L_S)‐at‐hatch did not differ between the size categories, but the offspring of the large fish had significantly larger area‐at‐hatch and greater yolk‐sac volume indicating better condition. Offspring growth rates were generally similar between offspring from all size categories, but they were significantly higher for offspring spawned by small females in terms of L_S between days 60 and 90 post‐fertilization. Despite temporarily higher growth rates among the small fish offspring, the smaller energy reserves at hatching translated into lower condition later in ontogeny. It appeared that the influence of spawner body size on egg and larval traits was relatively pronounced early in development and seemed to remain in terms of condition, but not in growth, after the onset of exogenous feeding. Further studies are needed to explore the mechanisms behind the differences in offspring quality between large‐ and small‐sized spawners by disentangling size‐dependent maternal and paternal effects on reproductive variables in D. rerio.     

筑巢材料在实验大、小鼠环境丰荣中的应用

相对于标准鼠盒,实验大、小鼠更喜好有筑巢材料的“环境丰荣”鼠盒.向标准鼠盒中放入筑巢材料,可以为大、小鼠提供筑巢、躲避、攀爬、休息的环境.因此,在实验大、小鼠饲养过程中,提供筑巢材料,是一个简单有效的丰富实验动物环境,提高实验动物福利的方法.本文就筑巢材料应用背景、近年来在啮齿类实验动物环境丰荣中的应用、对实验动物福利的影响三方面做以综述,并对今后筑巢材料在改善实验动物福利的应用前景作以展望.     

Thermally induced plasticity of body shape in adult zebrafish Danio rerio (Hamilton, 1822)

We examined the effect of temperature during the early development on the phenotypic plasticity of Danio rerio. The effect of temperature was examined during two different early developmental periods of 280°d (the product of days × temperature) each, 28‐308°d or 280‐560°d, by subjecting the experimental populations to three different water temperatures (22°C, 28°C, and 32°C). Before and after the end of the 280°d period of the different thermal exposure, all populations were cultured in standard temperature (28°C). Five to 10 months after exposure to the different thermal regimes, the body shape of the adults was analyzed by geometric morphometrics. In both ontogenetic windows and experimental repetitions, the results showed that developmental temperature and sex significantly affected the body shape of adult zebrafish. Thermally induced shape variation discriminated the fish that developed at 22°C from those developed at 28°C–32°C. In the early developmental period (DP1, 28–308°d postfertilization), dorsal, anal, and caudal fin structures differed between the animals that developed at 22°C and 28°C–32°C. In the later developmental period (DP2, 280–560°d postfertilization), caudal, anal, pectoral, and pelvic fins, as well as the gill cover and lower jaw, were affected when animals developed at different temperatures. These results show that thermal history during a short period of embryonic and larval life affects the body form of adult zebrafish with potentially functional consequences. Based on previous data on the effects of temperature on fish development, we suggest thermally induced muscle and bone remodelling as possible mechanism underlying the observed plasticity. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Effects of the rearing environment on average behaviour and behavioural variation in steelhead

In the context of conservation hatcheries that seek to bolster wild populations by releasing captively-reared fishes into the wild, steelhead Oncorhynchus mykiss were used to test the hypothesis that naturalistic rearing environments promote adaptive behaviour that might otherwise not develop in typical hatchery environments. When comparisons were made among fish reared in barren, structured or structurally variable environments ( i.e. the location of the structure was repositioned every 2–3 days), structure in the rearing environment increased future exploratory behaviour, but only if the structure was stable. Under conditions of high perceived predation risk, the fish no longer exhibited increased exploratory behaviour, suggesting that it is expressed in an adaptive, context-dependant manner. Another concern with hatcheries is that relaxed selection over multiple generations in captivity can increase maladaptive behavioural variation. Compared to rearing in hatchery-typical barren environments, rearing in structured-stable environments decreased behavioural variation. This effect, which occurred during development and did not involve selection, demonstrates a different mechanism for change in behavioural variation in captivity. These experiments show that effects of structure and structural stability occur at the level of both average behaviour and behavioural variation, and suggest that these effects should be considered when fishes are reared in hatcheries for later release into the wild.