Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.     

Effects of pollution on reproductive behaviour of fishes

This review attempts to integrate pollution research with behavioural ecology by focusing on reproductive behaviour of fishes. A search of Aquatic Sciences and Fisheries Abstractsand other sources showed that only 0.1% of 19 199 studies of aquatic pollution and fishes during the past 20 years have made this link. Effects on parental care and courtship have been investigated using a variety of pollutants (e.g. acidification, herbicide, thermal effluent) in several fish families (e.g. Cichlidae, Poeciliidae, Gasterosteidae, Cyprinidae).Eleven of the 19 studies found a change in behaviour from the norm. Effects on courtship included decreases or increases in frequency of displays, increased courtship duration, or performance of male-like behaviour by masculinized females. Studies of parental care have found decreased nest-building activity, decreased offspring defence, or changes in division of parental care between the sexes. Few studies have measured reproductive success or extrapolated their results to effects on populations.We develop a framework for exploring links between pollution and behavioural ecology which suggests potential impacts on life history trade-offs in reproduction, genetic changes in populations, and population sizes. Many reproductive behaviours of fish species are readily quantifiable and behaviours such as courtship by male guppies and other members of the Poeciliidae show some promise for pollution monitoring and behavioural toxicity tests. Choice of such assays would have to compete with the sensitivity and practicality of more traditional methods but may serve as useful complements. There is considerable scope for further research into conservation. A synthesis between behavioural ecology and toxicology should thus provide useful insights for both fields     

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.     

Can cost–benefit analysis explain fish distribution patterns?

This review describes some recent theoretical and empirical research into the way fish respond to spatial heterogeneity in their environment, and particularly to patchily distributed resources. The ways in which the need to acquire food interacts with other important requirements such as thermoregulation and predator avoidance to determine space use are considered, as are interactions with other members of the same species, viewed both as shoaling companions and as competitors. Recent developments in ideal free theory are discussed and the extent to which this formulation can explain how fish distribute themselves among feeding patches is examined. The implications of overtly aggressive interactions, in the form of dominance hierarchies and territorial systems, for distribution patterns in the laboratory and in the field are also reviewed. Finally the extent to which cost–benefit analysis can help to explain fish distribution patterns and the relative importance of behavioural processes compared to other determinants of the distribution of fish are considered.     

Otolith Shape Analysis as a Tool for Species Identification and Studying the Population Structure of Different Fish Species

An analysis and a comparison of the methods of geometric morphometrics as applied to fish species identification and to studies on the population structure of fish stocks based on peculiarities of the otolith shape are performed. A review of the geometric morphometric methods used in studies on fish otoliths is provided. The results of our own research on possible utilization of elliptical Fourier analysis for species identification are also described.     

Preference for structured environment in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis)

Information about the welfare and husbandry of pet and laboratory fish is scarce although millions of fish are sold in pet shops and used in laboratory research every year. Inadequate housing conditions can cause behavioural problems also in fish since they are complex animals with sophisticated behaviour. In this study, we investigated the influence of environmental complexity on compartment preference and behaviour in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis). For the preference test, large aquaria were divided by two semi-transparent walls of Plexiglas into an empty compartment, a structured compartment enriched with plants and clay pots, and a smaller compartment in-between, where food was provided. For observation, the empty and structured compartments were divided into six zones of similar size by defining three vertical layers and two horizontal areas (back vs. front area). Seven groups of six to nine zebrafish and seven groups of seven or eight checker barbs were observed on four days each (within a time period of ten days) to assess compartment use and activity, and to assess behavioural diversity and use of zones within compartments. Both zebrafish and checker barbs showed a significant preference for the structured compartment. Nevertheless, in neither species did behavioural diversity differ between the empty and structured compartment. Zebrafish used all zones in both compartments to the same extent. Checker barbs, however, used the structured compartment more evenly than the empty compartment, where they mainly used the lower and middle zones. These results suggest that zebrafish and checker barbs have a preference for complex environments. Furthermore, they indicate that the behavioural and ecological needs of fish may vary depending on species, and recommendations for husbandry should be specified at species level.     

Estimating the probability of identity among genotypes in natural populations: cautions and guidelines

Individual identification using DNA fingerprinting methods is emerging as a critical tool in conservation genetics and molecular ecology. Statistical methods that estimate the probability of sampling identical genotypes using theoretical equations generally assume random associations between alleles within and among loci. These calculations are probably inaccurate for many animal and plant populations due to population substructure. We evaluated the accuracy of a probability of identity (P(ID)) estimation by comparing the observed and expected P(ID), using large nuclear DNA microsatellite data sets from three endangered species: the grey wolf (Canis lupus), the brown bear (Ursus arctos), and the Australian northern hairy-nosed wombat (Lasiorinyus krefftii). The theoretical estimates of P(ID) were consistently lower than the observed P(ID), and can differ by as much as three orders of magnitude. To help researchers and managers avoid potential problems associated with this bias, we introduce an equation for P(ID) between sibs. This equation provides an estimator that can be used as a conservative upper bound for the probability of observing identical multilocus genotypes between two individuals sampled from a population. We suggest computing the actual observed P(ID) when possible and give general guidelines for the number of codominant and dominant marker loci required to achieve a reasonably low P(ID) (e.g. 0.01-0.0001).     

环境DNA技术在鱼类生态学中的应用研究进展

全球渔业衰退是21世纪人类面临的重要挑战之一。为了有效地遏制鱼类资源的衰退,精确的鱼类生态调查是其首要任务。传统的鱼类监测以渔获物采集与形态学鉴定为主,往往耗时耗力且效果不佳,已无法满足现阶段大尺度上的精确调查。环境DNA （eDNA）技术作为一种近年来新兴的鱼类生态调查方法,其与传统方法相比具有灵敏度高、经济高效、采样受限小且对生态系统无干扰的优势,目前其已被广泛地应用于鱼类物种监测、多样性调查、生物量评估以及繁殖活动监测等方面的研究。然而,eDNA技术在鱼类生态学研究的具体应用中暴露出的一些问题将会影响其监测结果的精确性,诸如操作流程的不规范、基因数据库的不完善以及eDNA在环境中生态学过程的不明确等。鉴于上述原因,首先对eDNA技术的发展历程、分析流程以及eDNA技术在鱼类生态学研究领域中的研究进展进行了综述,而后着重分析了eDNA技术的发展当前所面临的困难与挑战,并提出了相应的解决方案,最后对eDNA技术未来在鱼类生态学研究领域中的发展趋势做出了展望。通过本研究,以期能够为eDNA技术在鱼类生态学领域中的准确应用提供理论基础。     

Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

Studying behavioural variation in salmonids from an ecological perspective: observations questions methodological considerations

Salmonid fish are an ecologically important and extensively studied group of fish which concern many interest groups in our society. The aim of this paper is to discuss and suggest solutions to the multifaceted problems associated with studying behavioural variation in salmonids, with focus on designing behavioural studies that are ecologically relevant. Many of the general problems and solutions discussed can be applied to other animals as well. First, the importance of asking clear questions when conceiving behavioural studies is addressed, using Tinbergen’s four questions and associated theories as stepping stones towards generating testable hypotheses about behavioural variation. We then address a range of methodological challenges encountered when attempting to study behavioural variation in salmonids and suggest solutions to overcome these problems. A range of approaches is discussed, from highly controllable laboratory experiments to monitoring studies of behaviour in the wild. The importance of combining lab- and field approaches to evaluate the ecological relevance of behavioural variation is highlighted. Finally, we suggest a general framework using a multi-faceted research approach to address questions about the behavioural ecology of salmonids (and other animals) so that knowledge can progress, and the ecological relevance of behavioural studies can be validated.     

A review of chemical communication in freshwater fish

Since communication between individuals of a species of fish by chemical agents (pheromones) was first demonstrated in 1932, such a process has been suggested in many aspects of fish behavidïo'ur and development. This review describes observations on such mechanisms in shoaling behaviour and beneficial conditioning of water, homing of migratory fish, communication of alarm, 'crowding factor' (which adversely affects growth, survival and fecundity in dense populations), pair formation and spawning, and a range of other social interactions. Some of the chemicals involved have been isolated and identified, but most are indicated by behavioural observations. Pheromones are of great significance in fish behaviour and ecology, and are likely to be an important factor in culture operations.     

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.     

Use of monoclonal antibodies in identification and characterization of fish viruses

Monoclonal antibodies (MAbs) for fish viruses have been used increasingly in fish health management programs and as tools in research. The initial and most widespread use has been in the development of improved diagnostic and virus identification assays. MAbs with broad reactivity that provide standardized reagents for use in general screening assays for particular groups of fish viruses have been developed, whereas MAbs with more restricted specificities permit the rapid serotyping of fish viruses and, in some cases, the identification of specific virus strains. The large number and variety of MAbs now available for some fish viruses, such as the aquatic birnaviruses and rhabdoviruses, present an ideal opportunity to select appropriate MAbs to construct standardized virus identification assays for use by all fish pathologists worldwide. Furthermore, these MAbs have been used to identify specific epitopes that stimulate a protective immune response to many fish viruses, and to investigate the mechanisms of antibody-mediated neutralization of fish viruses. They also have been used to study the epidemiology of specific viruses as well as antigenic variation and the mechanisms by which new strains arise. Such information is important for the development of reliable diagnostic assays for short-term control methods as well as the development of effective vaccines for long-term control of virus diseases in fish. This article presents a brief overview of MAb production and a review of some current applications of MAbs in the identification and characterization of viruses of fish.     

Zebrafish as a cancer model

The zebrafish has developed into an important model organism for biomedical research over the last decades. Although the main focus of zebrafish research has traditionally been on developmental biology, keeping and observing zebrafish in the lab led to the identification of diseases similar to humans, such as cancer, which subsequently became a subject for study. As a result, about 50 articles have been published since 2000 in which zebrafish were used as a cancer model. Strategies used include carcinogenic treatments, transplantation of mammalian cancer cells, forward genetic screens for proliferation or genomic instability, reverse genetic target-selected mutagenesis to inactivate known tumor suppressor genes, and the generation of transgenics to express human oncogenes. Zebrafish have been found to develop almost any tumor type known from human, with similar morphology and, according to gene expression array studies, comparable signaling pathways. However, tumor incidences are relatively low, albeit highly comparable between different mutants, and tumors develop late in life. In addition, tumor spectra are sometimes different when compared with mice and humans. Nevertheless, the zebrafish model has created its own niche in cancer research, complementing existing models with its specific experimental advantages and characteristics. Examples of these are imaging of tumor progression in living fish by fluorescence, treatment with chemical compounds, and screening possibilities not only for chemical modifiers but also for genetic enhancers and suppressors. This review aims to provide a comprehensive overview of the state of the art of zebrafish as a model in cancer research. (Mol Cancer Res 2008;6(5):685-94).     

A practical guide to amplicon and metagenomic analysis of microbiome data

Advances in high-throughput sequencing(HTS)have fostered rapid developments in the field of microbiome research,and massive microbiome datasets are now being generated.However,the diversity of software tools and the complexity of analysis pipelines make it difficult to access this field.Here,we systematically summarize the advantages and limitations of micro-biome methods.Then,we recommend specific pipelines for amplicon and metagenomic analyses,and describe commonly-used software and databases,to help researchers select the appropriate tools.Furthermore,we introduce statistical and visualization methods suit-able for microbiome analysis,including alpha-and beta-diversity,taxonomic composition,difference compar-isons,correlation,networks,machine learning,evolu-tion,source tracing,and common visualization styles to help researchers make informed choices.Finally,a step-by-step reproducible analysis guide is introduced.We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the bio-logical significance behind the data.     

Zebrafish Danio rerio shows behavioural cross-context consistency at larval and juvenile stages but no consistency between stages

Coping style is defined as a set of individual physiological and behavioural characteristics that are consistent across time and context. In the zebrafish Danio rerio, as well as in many other animals, several covariations have been established among behavioural, physiological and molecular responses. Nonetheless, not many studies have addressed the consistency in behavioural responses over time starting at the larval stage. Therefore, this study aimed to improve the understanding of behavioural consistency across contexts and over time in zebrafish from the larval to juvenile stages. Two distinct experiments were conducted: a larval stage experiment (from 8 to 21 days post fertilization, dpf) and a juvenile stage experiment (from 21 to 60 dpf). On one hand, the larval experiment allows to focus on the transition between 8 and 21 dpf, marked by significant morphological changes related to the end of larval stage and initiation of metamorphosis. On the other hand, the juvenile experiment allows to properly cover the period extending from the end of larval stage to the juvenile stage (60 dpf), including metamorphosis which is itself completed around 45 dpf. Within each experiment, boldness was determined using a group risk-taking test to identify bold and shy individuals. A novel environment test was then performed at the same age to evaluate consistency across contexts. Groups of fish (either bold or shy) were bathed in an alizarin red S solution for later identification of their initially determined coping style to evaluate behavioural consistency over time. Fish were then reared under common garden conditions and challenged again with the same behavioural tests at a later age (21 and 60 dpf in the larval and juvenile experiments, respectively). Behavioural consistency was observed across contexts, with bold fish being more active and expressing higher thigmotaxis regardless of age. There was, however, little behavioural consistency over age, suggesting behavioural plasticity during development. Moreover, the use of alizarin red S to conduct this experiment provides new perspectives for the further study of the longitudinal evolution of various traits, including behaviour, over life stages in fish.     

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.     

Transposable elements in fish functional genomics: technical challenges and perspectives

The recent introduction of several transposable elements in zebrafish opens new frontiers for genetic manipulation in this important vertebrate model. This review discusses transposable elements as mutagenesis tools for fish functional genomics. We review various mutagenesis strategies that were previously applied in other genetic models, such as Drosophila, Arabidopsis, and mouse, that may be beneficial if applied in fish. We also discuss the forthcoming challenges of high-throughput functional genomics in fish.     

Fish stem cell cultures

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.     

Thermal biology of zebrafish (Danio rerio)

Zebrafish has become one of the most important animal models in research. Most of the variables studied using zebrafish are influenced by water temperature. The objective of this review was to analyze the published data on the thermal biology of the zebrafish. The paper first provides a brief introduction to zebrafish ecology and thermal tolerance, and continues with a review of the influence of temperature on several physiological variables, including development, growth, metabolism, reproduction, behavior, circadian biology and toxicology. Although a number of papers have already studied the effects of temperature on the zebrafish biology, knowledge in this field is still scarce, especially compared with other model organisms such as the rat, and therefore further research should be encouraged.