Danio rerio
An adult female zebrafish
Conservation status
Least Concern (IUCN 3.1)[1]
Scientific classification
Kingdom:	Animalia
Phylum:	Chordata
Class:	Actinopterygii
Order:	Cypriniformes
Family:	Cyprinidae
Subfamily:	Danioninae
Genus:	Danio
Species:	D. rerio
Binomial name
Danio rerio (F. Hamilton, 1822)
Synonyms
Barilius rerio Brachydanio frankei Brachydanio rerio Cyprinus chapalio Cyprinus rerio Danio frankei Danio lineatus Nuria rerio Perilamopus striatus

The zebrafish (Danio rerio) is a freshwater fish belonging to the minnow family (Cyprinidae) of the order Cypriniformes. Native to South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio^[2] (and thus often called a "tropical fish" although both tropical and subtropical). It is also found in private ponds.

The zebrafish is an important and widely used vertebrate model organism in scientific research, for example in drug development, in particular pre-clinical development.^[3] It is also notable for its regenerative abilities,^[4] and has been modified by researchers to produce many transgenic strains.^[5][6][7]

Taxonomy

The zebrafish is a derived member of the genus Brachydanio, of the family Cyprinidae. It has a sister-group relationship with Danio aesculapii.^[8] Zebrafish are also closely related to the genus Devario, as demonstrated by a phylogenetic tree of close species.^[9]

Distribution

Range

The zebrafish is native to fresh water habitats in South Asia where it is found in India, Pakistan, Bangladesh, Nepal and Bhutan.^{[1][10][11][12]} The northern limit is in the South Himalayas, ranging from the Sutlej river basin in the Bangladesh–India border region to the state of Arunachal Pradesh in northeast Indian.^[1][11] Its range is concentrated in the Ganges and Brahmaputra River basins, and the species was first described from Kosi River (lower Ganges basin) of India. Its range further south is more local, with scattered records from the Western and Eastern Ghats regions.^[12][13][14] It has frequently been said to occur in Myanmar (Burma), but this is entirely based on pre-1930 records and likely refers to close relatives only described later, notably Danio kyathit.^{[12][15][16][17]} Likewise, old^{[clarification needed]} records from Sri Lanka are highly questionable and remain unconfirmed.^[15]

Zebrafish have been introduced to California, Connecticut, Florida and New Mexico in the United States, presumably by deliberate release by aquarists or by escape from fish farms. The New Mexico population had been extirpated by 2003 and it is unclear if the others survive, as the last published records were decades ago.^[18] Elsewhere the species has been introduced to Colombia and Malaysia.^[11][19]

Habitats

Zebrafish typically inhabit moderately flowing to stagnant clear water of quite shallow depth in streams, canals, ditches, oxbow lakes, ponds and rice paddies.^{[12][13][19][20]} There is usually some vegetation, either submerged or overhanging from the banks, and the bottom is sandy, muddy or silty, often mixed with pebbles or gravel. In surveys of zebrafish locations throughout much of its Bangladeshi and Indian distribution, the water had a near-neutral to somewhat basic pH and mostly ranged from 16.5 to 34 °C (61.7–93.2 °F) in temperature.^[12][13][21] One unusually cold site was only 12.3 °C (54.1 °F) and another unusually warm site was 38.6 °C (101.5 °F), but the zebrafish still appeared healthy. The unusually cold temperature was at one of the highest known zebrafish locations at 1,576 m (5,171 ft) above sea level, although the species has been recorded to 1,795 m (5,889 ft).^[12][13]

Description

The zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a zebra's stripes, and which extend to the end of the caudal fin. Its shape is fusiform and laterally compressed, with its mouth directed upwards. The male is torpedo-shaped, with gold stripes between the blue stripes; the female has a larger, whitish belly and silver stripes instead of gold. Adult females exhibit a small genital papilla in front of the anal fin origin. The zebrafish can reach up to 4–5 cm (1.6–2.0 in) in length,^[16] although they typically are 1.8–3.7 cm (0.7–1.5 in) in the wild with some variations depending on location.^[13] Its lifespan in captivity is around two to three years, although in ideal conditions, this may be extended to over five years.^[20][22] In the wild it is typically an annual species.^[1]

Psychology

In 2015, a study was published about zebrafishes' capacity for episodic memory. The individuals showed a capacity to remember context with respect to objects, locations and occasions (what, when, where). Episodic memory is a capacity of explicit memory systems, typically associated with conscious experience.^[23]

The Mauthner cells integrate a wide array of sensory stimuli to produce the escape reflex. Those stimuli are found to include the lateral line signals by McHenry et al. 2009 and visual signals consistent with looming objects by Temizer et al. 2015, Dunn et al. 2016, and Yao et al. 2016.^[24]

Reproduction

Stages of zebrafish development. Photos to scale except adult, which is about 2.5 cm (1 in) long.

The approximate generation time for Danio rerio is three months. A male must be present for ovulation and spawning to occur. Zebrafish are asynchronous spawners^[25] and under optimal conditions (such as food availability and favorable water parameters) can spawn successfully frequently, even on a daily basis.^[26] Females are able to spawn at intervals of two to three days, laying hundreds of eggs in each clutch. Upon release, embryonic development begins; in absence of sperm, growth stops after the first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makes D. rerio a convenient research model species.^[20] Sex determination of common laboratory strains was shown to be a complex genetic trait, rather than to follow a simple ZW or XY system.^[27]

The zebrafish embryo develops rapidly, with precursors to all major organs appearing within 36 hours of fertilization. The embryo begins as a yolk with a single enormous cell on top (see image, 0 h panel), which divides into two (0.75 h panel) and continues dividing until there are thousands of small cells (3.25 h panel). The cells then migrate down the sides of the yolk (8 h panel) and begin forming a head and tail (16 h panel). The tail then grows and separates from the body (24 h panel). The yolk shrinks over time because the fish uses it for food as it matures during the first few days (72 h panel). After a few months, the adult fish reaches reproductive maturity (bottom panel).

To encourage the fish to spawn, some researchers use a fish tank with a sliding bottom insert, which reduces the depth of the pool to simulate the shore of a river. Zebrafish spawn best in the morning due to their Circadian rhythms. Researchers have been able to collect 10,000 embryos in 10 minutes using this method.^[28] In particular, one pair of adult fish is capable of laying 200–300 eggs in one morning in approximately 5 to 10 at time.^[29] Male zebrafish are furthermore known to respond to more pronounced markings on females, i.e., "good stripes", but in a group, males will mate with whichever females they can find. What attracts females is not currently understood. The presence of plants, even plastic plants, also apparently encourages spawning.^[28]

Exposure to environmentally relevant concentrations of diisononyl phthalate (DINP), commonly used in a large variety of plastic items, disrupt the endocannabinoid system and thereby affect reproduction in a sex-specific manner.^[30]

Feeding

Zebrafish are omnivorous, primarily eating zooplankton, phytoplankton, insects and insect larvae, although they can eat a variety of other foods, such as worms and small crustaceans, if their preferred food sources are not readily available.^[20]

In research, adult zebrafish are often fed with brine shrimp, or paramecia.^[31]

In the aquarium

Zebrafish are hardy fish and considered good for beginner aquarists. Their enduring popularity can be attributed to their playful disposition,^[32] as well as their rapid breeding, aesthetics, cheap price and broad availability. They also do well in schools or shoals of six or more, and interact well with other fish species in the aquarium. However, they are susceptible to Oodinium or velvet disease, microsporidia (Pseudoloma neurophilia), and Mycobacterium species. Given the opportunity, adults eat hatchlings, which may be protected by separating the two groups with a net, breeding box or separate tank. In captivity, zebrafish live approximately forty-two months. Some captive zebrafish can develop a curved spine.^[33]

The zebra danio was also used to make genetically modified fish and were the first species to be sold as GloFish (fluorescent colored fish).

Strains

In late 2003, transgenic zebrafish that express green, red, and yellow fluorescent proteins became commercially available in the United States. The fluorescent strains are tradenamed GloFish; other cultivated varieties include "golden", "sandy", "longfin" and "leopard".

The leopard danio, previously known as Danio frankei, is a spotted colour morph of the zebrafish which arose due to a pigment mutation.^[34] Xanthistic forms of both the zebra and leopard pattern, along with long-finned strains, have been obtained via selective breeding programs for the aquarium trade.^[35]

Various transgenic and mutant strains of zebrafish were stored at the China Zebrafish Resource Center (CZRC), a non-profit organization, which was jointly supported by the Ministry of Science and Technology of China and the Chinese Academy of Sciences.^{[citation needed]}

Wild-type strains

The Zebrafish Information Network (ZFIN) provides up-to-date information about current known wild-type (WT) strains of D. rerio, some of which are listed below.^[36]

Hybrids

Hybrids between different Danio species may be fertile: for example, between D. rerio and D. nigrofasciatus.^[9]

Scientific research

Zebrafish chromatophores, shown here mediating background adaptation, are widely studied by scientists.

A zebrafish pigment mutant (bottom) produced by insertional mutagenesis.^[9] A wild-type embryo (top) is shown for comparison. The mutant lacks black pigment in its melanocytes because it is unable to synthesize melanin properly.

D. rerio is a common and useful scientific model organism for studies of vertebrate development and gene function. Its use as a laboratory animal was pioneered by the American molecular biologist George Streisinger and his colleagues at the University of Oregon in the 1970s and 1980s; Streisinger's zebrafish clones were among the earliest successful vertebrate clones created.^[37] Its importance has been consolidated by successful large-scale forward genetic screens (commonly referred to as the Tübingen/Boston screens). The fish has a dedicated online database of genetic, genomic, and developmental information, the Zebrafish Information Network (ZFIN). The Zebrafish International Resource Center (ZIRC) is a genetic resource repository with 29,250 alleles available for distribution to the research community. D. rerio is also one of the few fish species to have been sent into space.

Research with D. rerio has yielded advances in the fields of developmental biology, oncology,^[38] toxicology,^[29][39][40] reproductive studies, teratology, genetics, neurobiology, environmental sciences, stem cell research, regenerative medicine,^[41][42] muscular dystrophies^[43] and evolutionary theory.^[9]

Model characteristics

As a model biological system, the zebrafish possesses numerous advantages for scientists. Its genome has been fully sequenced, and it has well-understood, easily observable and testable developmental behaviors. Its embryonic development is very rapid, and its embryos are relatively large, robust, and transparent, and able to develop outside their mother.^[44] Furthermore, well-characterized mutant strains are readily available.

Other advantages include the species' nearly constant size during early development, which enables simple staining techniques to be used, and the fact that its two-celled embryo can be fused into a single cell to create a homozygous embryo. The zebrafish is also demonstrably similar to mammalian models and humans in toxicity testing, and exhibits a diurnal sleep cycle with similarities to mammalian sleep behavior.^[45] However, zebrafish are not a universally ideal research model; there are a number of disadvantages to their scientific use, such as the absence of a standard diet^[46] and the presence of small but important differences between zebrafish and mammals in the roles of some genes related to human disorders.^[47][48]

Regeneration

Zebrafish have the ability to regenerate their heart and lateral line hair cells during their larval stages.^[49][50] The cardiac regenerative process likely involves signaling pathways such as Notch and Wnt; hemodynamic changes in the damaged heart are sensed by ventricular endothelial cells and their associated cardiac cilia by way of the mechanosensitive ion channel TRPV4, subsequently facilitating the Notch signaling pathway via KLF2 and activating various downstream effectors such as BMP-2 and HER2/neu.^[51] In 2011, the British Heart Foundation ran an advertising campaign publicising its intention to study the applicability of this ability to humans, stating that it aimed to raise £50 million in research funding.^[52][53]

Zebrafish have also been found to regenerate photoreceptor cells and retinal neurons following injury, which has been shown to be mediated by the dedifferentiation and proliferation of Müller glia.^[54] Researchers frequently amputate the dorsal and ventral tail fins and analyze their regrowth to test for mutations. It has been found that histone demethylation occurs at the site of the amputation, switching the zebrafish's cells to an "active", regenerative, stem cell-like state.^[55][56] In 2012, Australian scientists published a study revealing that zebrafish use a specialised protein, known as fibroblast growth factor, to ensure their spinal cords heal without glial scarring after injury.^[4][57] In addition, hair cells of the posterior lateral line have also been found to regenerate following damage or developmental disruption.^[50][58] Study of gene expression during regeneration has allowed for the identification of several important signaling pathways involved in the process, such as Wnt signaling and Fibroblast growth factor.^[58][59]

In probing disorders of the nervous system, including neurodegenerative diseases, movement disorders, psychiatric disorders and deafness, researchers are using the zebrafish to understand how the genetic defects underlying these conditions cause functional abnormalities in the human brain, spinal cord and sensory organs.^{[60][61][62][63]} Researchers have also studied the zebrafish to gain new insights into the complexities of human musculoskeletal diseases, such as muscular dystrophy.^[64] Another focus of zebrafish research is to understand how a gene called Hedgehog, a biological signal that underlies a number of human cancers, controls cell growth.

Genetics

Background genetics

Inbred strains and traditional outbred stocks have not been developed for laboratory zebrafish, and the genetic variability of wild-type lines among institutions may contribute to the replication crisis in biomedical research.^[65] Genetic differences in wild-type lines among populations maintained at different research institutions have been demonstrated using both Single-nucleotide polymorphisms^[66] and microsatellite analysis.^[67]

Gene expression

Due to their fast and short life cycles and relatively large clutch sizes, D. rerio or zebrafish are a useful model for genetic studies. A common reverse genetics technique is to reduce gene expression or modify splicing using Morpholino antisense technology. Morpholino oligonucleotides (MO) are stable, synthetic macromolecules that contain the same bases as DNA or RNA; by binding to complementary RNA sequences, they can reduce the expression of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after the 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are interpermeable to large molecules,^[68][69] allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies.^[70] Morpholinos are commonly microinjected in 500pL directly into 1-2 cell stage zebrafish embryos. The morpholino is able to integrate into most cells of the embryo.^[71]

A known problem with gene knockdowns is that, because the genome underwent a duplication after the divergence of ray-finned fishes and lobe-finned fishes, it is not always easy to silence the activity of one of the two gene paralogs reliably due to complementation by the other paralog.^[72] Despite the complications of the zebrafish genome, a number of commercially available global platforms exist for analysis of both gene expression by microarrays and promoter regulation using ChIP-on-chip.^[73]

Genome sequencing

The Wellcome Trust Sanger Institute started the zebrafish genome sequencing project in 2001, and the full genome sequence of the Tuebingen reference strain is publicly available at the National Center for Biotechnology Information (NCBI)'s Zebrafish Genome Page. The zebrafish reference genome sequence is annotated as part of the Ensembl project, and is maintained by the Genome Reference Consortium.^[74]

In 2009, researchers at the Institute of Genomics and Integrative Biology in Delhi, India, announced the sequencing of the genome of a wild zebrafish strain, containing an estimated 1.7 billion genetic letters.^[75][76] The genome of the wild zebrafish was sequenced at 39-fold coverage. Comparative analysis with the zebrafish reference genome revealed over 5 million single nucleotide variations and over 1.6 million insertion deletion variations. The zebrafish reference genome sequence of 1.4GB and over 26,000 protein coding genes was published by Kerstin Howe et al. in 2013.^[77]

Mitochondrial DNA

In October 2001, researchers from the University of Oklahoma published D. rerio's complete mitochondrial DNA sequence.^[78] Its length is 16,596 base pairs. This is within 100 base pairs of other related species of fish, and it is notably only 18 pairs longer than the goldfish (Carassius auratus) and 21 longer than the carp (Cyprinus carpio). Its gene order and content are identical to the common vertebrate form of mitochondrial DNA. It contains 13 protein-coding genes and a noncoding control region containing the origin of replication for the heavy strand. In between a grouping of five tRNA genes, a sequence resembling vertebrate origin of light strand replication is found. It is difficult to draw evolutionary conclusions because it is difficult to determine whether base pair changes have adaptive significance via comparisons with other vertebrates' nucleotide sequences.^[78]

Developmental genetics

T-boxes and homeoboxes are vital in Danio similarly to other vertebrates.^[79][80] The Bruce et al. team are known for this area, and in Bruce et al. 2003 & Bruce et al. 2005 uncover the role of two of these elements in oocytes of this species.^[79][80] By interfering via a dominant nonfunctional allele and a morpholino they find the T-box transcription activator Eomesodermin and its target mtx2 – a transcription factor – are vital to epiboly.^[79][80] (In Bruce et al. 2003 they failed to support the possibility that Eomesodermin behaves like Vegt.^[79] Neither they nor anyone else has been able to locate any mutation which – in the mother – will prevent initiation of the mesoderm or endoderm development processes in this species.)^[79]

Pigmentation genes