Zebrafish

Characteristics, Life cycle, Development, Genome Research


Overview


Zebrafish (Danio rerio) are small, freshwater fish commonly found in the tropics. Although they are native to South Asia (Nepal, India, etc), Zebrafish are among the most common aquarium fish.

With regards to habitat, Zebrafish are typically found in shallow ponds, canals and streams, etc (stagnant or slow-flowing waters of between 18 and 24 degrees Celsius). As omnivorous organisms, they feed on a range of organisms such as insects, worms and zooplankton as well as various plant matter in their environment.

Although Zebrafish are not of economic importance as a source of food, they have become increasingly important in research studies as model systems through which researchers are trying to understand the genetic and neuronal basis for behavior. As such, they are particularly important in developmental biology, biomedicine and neurophysiology are model organisms.

 

* The name Zebrafish comes from the blue stripes that run horizontally on each side of their bodies.

 



Zebrafish Classification 


·         Kingdom: Animalia

·         Phylum: Chordata

·         Class: Teleostei

·         Subclass: Actinopterygii

·         Order: Cypriniformes

·         Family: Cyprinidae

·         Genus: Danio


Zebrafish by Oregon State University [CC BY-SA 2.0 (https://creativecommons.org/licenses/by-sa/2.0)]Zebrafish by Oregon State University [CC BY-SA 2.0 (https://creativecommons.org/licenses/by-sa/2.0)]


Zebrafish Characteristics (Appearance and Morphology)


As already mentioned, Zebrafish are small fish that measure about 6 cm in length on average. In addition to a moderately elongated body that gives them the classic fish shape, Zebrafish have a mouth that forms slightly upwards and a dorsally compressed head. They lack oral teeth (on their jaws) but Zebrafish have teeth attached to their fifth brachial arch consisting of a dentine layer, an enamel coating as well as a pulp core.

Zebrafish also have two pairs of skin sensory appendages known as barbels. These include the rostal (nasal) barbels that extend to the orbit's anterior margin and the long maxillary barbels on either side of the mouth that act as taste buds and are also used to search for food.

Although Zebrafish are generally silvery white in color, they have about 5 horizontal lines (blue in color) on either side of their body extending to the caudal fin. Moreover, the upper (dorsal) part and belly part may appear pale yellow in color causing some Zebrafish to appear more golden.


Some of the pigments that give Zebrafish their color include:

 

  • The dark blue melanophores - Responsible for the blue stripes
  • Iridescent iridophores
  • Gold xanthophores - Gives the pale yellow color of the dorsal and belly part of the fish

 


The difference between male and female Zebrafish:

 

  • Whereas the female Zebrafish tend to be larger in size with a rounded belly (whitish in color) male Zebrafish are more slender with a golden appearance on their belly
  • Males also have a golden appearance on their pelvic and ventral fin
  • Females (adult) have a small genital papilla located in front of the anal fin

 

Some of the other anatomical features of Zebrafish include:

 

  • A striped anal fin with between 10 and 12 branched rays
  • A soft dorsal fin with between 30 and 32 branched rays


Life Cycle



On average Zebrafish have a lifespan of three and a half years (3.5years). However, some live up to 5.5 years. As they age, Zebrafish have been shown to portray a declining exercise capacity similar to mammals. This has been shown to be the case with performance and trainability of the organism as they age.


The following are some of the main stages of the lifecycle of a Zebrafish:

 

  • Unfertilized egg
  • Embryo
  • Larval stage
  • Juvenile
  • Adult

 

A female Zebrafish can produce as many as several hundreds of eggs per spawning. Given that eggs are released to be fertilized externally, the production of this many eggs allows for many offspring to survive through the development process before they reach adulthood even as some die in the process.

The eggs, about 0.7mm in diameter, are released on a substrate where they become activated and start undergoing developmental steps. Although they are non-adhesive and tend to be released on un-prepared substrate, the eggs of Zebrafish have been shown to be activated by water even in cases where they are not fertilized by male sperm. The unfertilized eggs do not develop beyond the first few cleavage phases.

Zygotes are formed once the eggs are fertilized. This may occur immediately as the eggs are released (and fertilized) or within a period of 72 hours. Fertilization is followed by egg division in the cleavage phase. This may take between 40 minutes and 2 hours.

During this phase, cell division may result in between 16 and 64 cells as the embryo moves to the next phase. The cleavage phase in which cell division is experienced produces the blastula. This is the form of the embryo resembling a hollow ball and contains many layers of cells that surround a cavity known as the blastocele.

The next stage of development is known as gastrulation and results in the production of three germ layers and thus the body plan of the organism. Whereas blastulation occurs between 2 and 5 hours after fertilization, gastrulation occurs between 5 and 10 hours following fertilization.

This is followed by two other phases namely, segmentation and the pharyngula stage before hatching. In these two stages, structures of the organism develop further with the head and tail becoming more pronounced and visible. This prepares the offspring for hatching.

Hatching occurs between 48 and 72 hours after fertilization. This, however, has been shown to largely depend on the thickness of the chorion (in which the embryo resides) as well as the muscular activity of the embryo.

Depending on these factors, hatching of some embryo may be delayed. Once it is hatched, the larvae measures about 3mm in length. Using secretory cells located on the surface of its head, the larvae attach to hard surfaces from where they grow using nutrients from the yolk.

In a period of about 3 days, the larval undergoes morphogenesis which is characterized by the development of various anatomical structures. By the late larval stage (about 7 days after fertilization) the organism is capable of swimming, moving its jaws and even feeding on various food material.

At the juvenile phase (between 2 and 10 weeks old) the organism is capable of eating small organisms such as small worms and shrimp. The juvenile can be kept with adult fish when they are in the late part of their juvenile phase and they reach maturity at about 10 weeks of age. Once they mature, they are ready to reproduce and the life cycle continues.

 

* Although there are male and female Zebrafish, all first develop into ovaries before differentiating into male gonads a few weeks after hatching.


* During courtship, the male Zebrafish uses a number of strategies to attract the female and lead her to the spawning site. These may include using his snout to nudge her, swimming around her with his fins raised or swimming back and forth between the female and the spawning site.


In the event that the female follows to the spawning site, the male spreads his dorsal and caudal fins around her so that their genital pores align. This is followed by the female releasing the eggs as the male releases the sperm cells to fertilize them.


Breeding


Essentially, breeding refers to the mating and consequently the production of offspring by given organisms. With Zebrafish becoming increasingly important models for research studies, breeding of these organisms has also become  vital in order to study the various characteristics of the organism as it develops to adulthood.

While it may sound simple, successful breeding of Zebrafish is a complex process that is influenced by such components as abiotic factors of the environment, courtship and various behavioural aspects among others. In order to achieve successful breeding, ideal housing conditions are essential.


Some of the characteristics of the ideal housing conditions include:

 

·      Mechanical filters that remove large debris

·      Activated carbon filters - remove contaminants and small elements

·      Biological filters - remove organic waste

·      Ultraviolet sterilizing unit

·      14-hour lighting and 10-hour darkness cycle - allows the fish to breed at dawn

·      Water between 23 and 28 degrees Celsius

·      pH maintained between 6.2 and 7.5

 

For between 5 and 10 fish, a 5 to 10-gallon tank is recommended. This allows sufficient space for the fish to be social and not too small to become aggressive. The same number of males to that of the female is also recommended.

Although Zebrafish have been shown to be particularly hardy, breeding requires that they are fed well in order to keep them healthy. For the most part, live foods such as Brine shrimp larva are recommended in place of foods purchased from the pet store. However, crumbs of hard-boiled egg yolk have also been shown to be excellent food for the fish.

 

When ready to collect the eggs, marbles should be laid out at the bottom of the tank the night before. This helps prevent the fish from consuming the eggs given that they settle between the marbles. To collect eggs, a net can be used to take out the marbles or water strained through a tea strainer. Eggs can then be kept in a Petri dish (about 100 eggs per Petri dish) with a pipette being used to clean the eggs (to remove dead embryo). For optimal development, the embryo should be maintained at between 24 and 29 degrees Celsius. 

 

* When the Zebrafish are removed from the tank in order to collect the eggs (30 minutes after daylight) keeping them separated is recommended for the period it will take to collect the eggs to boost mating and the release of more eggs.


* New types of tanks have been introduced with a sloped interior. This removable insert creates an environment that resembles the natural environment in which the Zebrafish spawn and thus promotes the spawning process. In addition, it eliminates the need to slant the tank thus making the breeding process easier for researchers and enthusiasts.



Use of Zebrafish in Research

Zebrafish have a number of characteristics that have made them some of the most ideal organisms for research purposes in fields of genetics, developmental biology, and neurophysiology among others.

Zebrafish have been shown to carry highly conserved orthologues in their genome resembling many of the genes associated with various human neurological disorders. As a result, research studies on these organisms may yield results that can help determine how to treat such disorders.


Apart from the fact that Zebrafish share much of their genome with human beings, some of the other facts that make them ideal for research studies in genetic manipulation include:

 

·      They lay many eggs which is ideal for research studies

·      They can be easily bred in the laboratory with all the necessary material and other equipment

·      They are not expensive to breed and maintain

·      The transparent larvae of Zebrafish allow for easier imaging of their neuronal structure and function (of live organisms)

·      Compared to other research subjects like mice, adult Zebrafish prefer to be housed together in groups and thus consume less space

·      Eggs are released and fertilized externally thus making it easier to study or manipulate from this early stage

 

* About 70 percent of human genes can be found in Zebrafish.

 

Natural history museum Naturalis,Leiden.Research Cell zoomer.Photo of cell of zebrafish by Henk Caspers/Naturalis Biodiversity Center[CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]Natural history museum Naturalis,Leiden.Research Cell zoomer.Photo of cell of zebrafish by Henk Caspers/Naturalis Biodiversity Center[CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)]


Zebrafish Genome compared to Human

As previously mentioned, Zebrafish share a significant percentage of their genes with human beings.

Based on this understanding, studying the genetic material of Zebrafish can better help understand various diseases and conditions in human beings and even possibly discover how to treat such diseases.

Genetic mutations in human beings have been associated with various conditions. However, a researcher may wish to determine whether any loss of function of the gene does indeed cause a condition observed in an individual.

To do this, a similar gene is mutated in the fish (Zebrafish) to observe the outcome. In some cases, the mutated gene in the human being with the condition may be introduced into the DNA of the fish. Through such genetic studies, it would be possible to learn more about the condition and thus how to proceed.

Here, once genetic studies on the Zebrafish have provided more information about the disease, it becomes possible for researchers to test new drugs on the fish and determine whether they would work on human beings.

Given that Zebrafish are able to produce many offspring, this presents a significant advantage in that many Zebrafish can be used in the study to determine the percentage of organisms that may have benefited from the treatment.

 

A number of human diseases have been successfully modeled in Zebrafish, these include:

 

  • Duchenne muscular dystrophy
  • Human melanoma
  • Human neurodegenerative diseases

 


Although Zebrafish have many advantages for modeling human diseases, some of the disadvantages include:

 

·      They are not mammals and thus lack mammalian organs (e.g. lungs and mammary gland etc)

·      Some of the diseases caused by orthologous genes vary significantly between human beings and fish

·      The genome of Zebrafish contains many gene duplications. This has been shown to result in subfunctionalization and neofunctionalization

 

With regards to regeneration, Zebrafish have been shown to be capable of regenerating lost fins as well as a number of lesioned organs such as the retina, spinal cord, and the heart among a number of other tissues. As a result, Zebrafish have also become excellent models for heart regeneration studies as well as animal aging research.

For Zebrafish that experience injuries to the heart as well as their fins, they have been shown to regenerate tissue that allows them to recover and regain normal functions in these organs. Through biological studies of these organisms, researchers hope to gain valuable information that may help deal with some human heart diseases and potentially prolong life.


See also a page on Cytopathology

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References

Allison D'Costa and Iain Shepherd. (2009). Zebrafish Development and Genetics: Introducing Undergraduates to Developmental Biology and Genetics in a Large Introductory Laboratory Class. ResearchGate. 

 

Aswin L. Menke, Jan M. Spitsbergen, Andre P. M. Wolterbeek And Ruud A. Woutersen. (2011). Normal Anatomy and Histology of the Adult Zebrafish. Toxicologic Pathology, 39: 759-775, 2011.

 

Benjamin Tsang, Hifsa Zahid, Rida Ansari, and Richard Chi Yeung Lee. (2017). Breeding Zebrafish: A Review of Different Methods and a Discussion on Standardization. ResearchGate, 

 

Cristina Santoriello and Leonard I. Zon. (2012). Hooked! Modeling human disease in zebrafish.

 

Links

 

https://www.biomedcentral.com/collections/AZBR

https://www.cell.com/trends/genetics/pdf/S0168-9525(13)00113-3.pdf



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