|
|
Zebrafish FAQs |
|
|
Zebrafish FAQs |
Home Page
FAQs Streisinger
Zebrafish
ABZs Lab
Protocols Stages
Methods
Resources Contacts
THE "WHYs", "WHATs", AND "HOWs" OF ZEBRAFISH RESEARCH
IDENTIFICATION / APPEARANCE
AVAILABILITY
FEEDING
MAINTENANCE
BREEDING
HEALTH
* Where did the zebrafish originate?
Zebrafish, Danio rerio, are freshwater fish that were originally found in slow streams and rice paddies and in the Ganges River in East India and Burma.
* Where is the zebrafish in the food chain?
Zebrafish eat smaller living organisms than themselves and in the wild, they are eaten by bigger fish, small amphibians, mammals, or birds.
* Why are zebrafish used in research?
In the early 1970's, a scientist at the University of Oregon by the name of Dr. George Streisinger determined that the zebrafish is a wonderful model for studying vertebrate development and genetics. Since he began using them in his research, zebrafish embryos have become very popular worldwide as a means of understanding how not only fish, but all vertebrates including people, develop from the moment that sperm fertilizes an egg. The eggs are clear and develop outside of the mother's body, allowing scientists to watch a zebrafish egg grow into a newly formed fish under a microscope. The scientists watch while the cells divide and form different parts of the baby fish's body. In the development span of 2-4 days, some form to make the eyes; others, the heart, the liver, the stomach, the skin, the fins, etc. until the fish is complete and ready to begin it's new life. Scientists will occasionally move a cell to another spot to see if it will still go to form the same part of the body as it is known to do in other embryos or if it will do something different. Occasionally a cell is removed or destroyed to see what the result is to the fish once it has developed. This is how scientists are discovering the causes of birth defects in human children and it's how they are trying to find a way to prevent these birth defects by understanding why they happen and what original cells are involved. So, this little obscure fish of the Ganges is helping us to learn about how all vertebrates develop and why sometimes things go wrong in that development to cause birth defects and other health problems. It is serving a very important role in our understanding and some day it may play a huge role in overcoming these things.
* Why are zebrafish ideal models for development and disease research?
*Why are zebrafish a good choice for high school science labs?
Zebrafish are:
(1) inexpensive
(2) hardy, tolerating a reasonable amount of stress from fluctating classroom temperatures, lighting that is on Monday through Friday and dark Saturday and Sunday
(3) small enough to easily raise in inexpensive aquariums (school budgets are always small!)
(4) produce enough eggs that biology students all get some to examine
(5) examination of fish eggs pose no ethical problems (obviously this cannot be said of other vertebrate embryos)
(6) fish embryo development is similar to other species, so it provides a basis for comparative embryological studies and a spring board to discussion of evolution, if desired
(7) zebrafish projects are easily student-managed, thus saving the teacher's time, and generating many creative science research projects from the students
(8) egg production can be stimulated by controlling the periods of darkness (simply cover the aquarium).
Students enjoy watching these flashy little fish!
* How does zebrafish research help humans?
We can't do research on humans so we find models that mimic the human, do the research using zebrafish, and then try to figure out how to extrapolate the data to humans... not an easy task. We are really never sure how exactly the data will match what we would have found if we use humans. Now that we know the genome of the human and are working on many other organisms, that process is getting easier. For example, if we find some gene altered in the zebrafish due to exposure to a toxicant or from a disease, then we can use the database to search for a similar gene in humans. We also now know that there is considerable conservation of pathways across species. Zebrafish are vertebrates. This means that they are more closely related to humans than invertebrates. By virtue of their being more closely related to humans, they are more likely to be similar in any biological trait than is a more distantly related organism.
Some zebrafish mutants are known to develop and duplicate certain conditions and diseases common to humans. In studying their more simplified make up, it is frequently possible to determine what genes are involved and then compare them to the equivalent genes within the human genome.
Although zebrafish and humans are obviously very different, their embryonic development is remarkably similar. Furthermore, it is becoming more and more clear that all vertebrates follow an evolutionarily-conserved developmental program. This conservation extends even to the molecular level--where similar genes perform similar functions in many different species. You cannot do the kind of experiments in humans that are outlined above, and which are necessary to understand a given biological process, and how genes act to make that process work.
Examples:
There are many zebrafish mutants with defective blood (studied primarily by Len Zon's lab - Massachusetts General Hospital) or defects in heart development (Mark Fishman's Lab - Massachusetts General Hospital, Didier Stainier's Lab - University of California, SF, or Debbie Yelon's lab - Skirball Institute, NYU School of Medicine). It is likely that blood function and heart development in zebrafish are similar to blood function and heart development in people. Some of these mutations might mimic human syndromes, and understanding them will provide us with valuable insight as to the underlying problem. Such an understanding could lead to new treatments. In addition, many of the proteins involved in early development are critical in later life. Defects in these proteins, or in the regulation of their expression, can lead to tumors later in life.
* What
types of research are commonly done with zebrafish?
* What are some possible future innovations in zebrafish research?
* What are the advantages and disadvantages of using zebrafish in labs? Advantages:
Disadvantages:
* How
can you tell a male from a female? Males are slender and torpedo-shaped,
usually with gold on their belly, ventral fin, pelvic fins, and pectoral fins, although
this isn't always true... especially in the Long Fin variety. Males can also be identified
by the fact that they tend to chase the females early in the morning before they breed.
Females are fat when filled with eggs and do not usually have gold on their undersides (or
very little of it, if any) . These are barbels, a
whiskery-like sensory organ, and are found on both males and females. The
function of the barbels is 1) chemosensory and 2) for disturbing bottom debris
in order to locate food. Barbels are common to fish
living at the bottom of eutrophic waterways where visual location of food is not
easy. The catfish are perhaps the best known example of a fish group having
barbels and they live at the bottom of deepish, frequently dirty, rivers and
water impoundment's. * How
big are these fish and would it be practical to raise them at home in a tropical fish
tank? The zebrafish is a small, hardy fish,
about 1" to 1½" long that many people have in their small aquariums
at home. The standard "wildtype" variety is somewhat clear-colored with black
stripes that run lengthwise down its body. For many years they have been bred by fish
hobbyists for home aquariums.
* Why do some zebrafish have long fins and others don't?
There are several different varieties of zebrafish... long finned, short finned (the natural wild type), striped (natural) and speckled, as well as albino and "pink" and "blue." All are merely genetic variants of the same species. They can all interbreed and you can do genetic experiments to determine dominance of the various traits. (i.e., are long fins dominant over short fins? etc.)
* How can we obtain mutant stocks of zebrafish for our high school lab?
The easiest way to obtain your stock of fish would be to try the local pet stores. They should have normal looking wildtype fish and they may have some variants that you could use although it is unlikely they would have a true albino (no dark pigment and pink eyes). Your most likely choices would be: a) leopard, a recessive with spots instead of stripes; b) long fin, a dominant with long flowing fins (a common type of mutant in cultured tropical fish); and c) brass, a dark pigment-lacking fish except for its eyes which are dark. It is a recessive. You can often get long fin in combination with one of the other two mutants. There are also a bunch of other mutants that are infrequently seen in the pet stores. There are hundreds of them in labs, but most of them are embryonic lethals and several pigmentation mutants. I would not recommend using lab strains if you can avoid it because they are very inbred and less robust and viable.
* What should I feed my fish to get them ready to breed?
First of all, zebras lay eggs every day! They eat most of their own eggs as a way of recycling the protein that is lost from producing the eggs. Feed your zebrafish LOTS of high protein foods. The best is LIVE brine shrimp but frozen will also work well. Cichlid food is higher in protein than other foods for fish, too. READ the nutrition information and pick foods high in protein. Do not feed more than they can consume, though, since excess food contaminates the water. Feed them well for at least one week or until the females get fat with extra eggs.
* What's the best food for newly hatched zebrafish?
Very tiny baby zebrafish in large facilities eat even tinier living organisms called paramecia that live in water. These paramecia are grown in jars and are fed to the fish by squirting hundreds of them from an eye dropper into their water. You can't see them very well. It just looks like cloudy water in the eye dropper. In general, live food is best for baby fish. It is easier for them to digest. Your fish will not have to eat until they are floating up off the bottom, though. Hobbyists traditionally use infusoria, a mix of ciliated microscopic organisms that can be grown in cultures of boiled grass or hay. For details see a hobbyist book for those instructions. You can buy an infusoria culture from the pet store in either tablet or powder form. The directions for cultivating and feeding the fry will be on the package. Be sure to explain to the person at the pet store that you want to feed live micro-organisms to baby egg-layers. Another possible food for both the fish and a ciliate culture is the yolk of a boiled egg after it has been pushed through a small size mesh (usually an old nylon stocking). People who have zebrafish at home frequently buy and feed other packaged foods bought from the pet store instead of raising their own. Try to stay away from artificial substitutes since they tend to pollute the water too easily. As the fish get bigger and become adults, they eat live baby brine shrimp which are a little larger than paramecia, but are still hard to see because of their small size. You should be able to buy frozen baby brine shrimp or eggs to hatch from a pet store. Be sure not to over feed this and siphon out any left over after 30 minutes. Otherwise it will decay and the bacteria will make your baby fish sick.
* We are having trouble raising zebrafish. We have had quite a bit of success getting the eggs to hatch, but we are unable to keep them alive for more than 24 hours afterwards. We think it's because there isn't any food for them to eat. We've tried feeding them crushed flake food and have even experimented with vinegar eels, but still no success. One student who had a huge amount of success raising the fish used just crushed flake food. What do you think is happening?
If your fish are hatching but die soon afterward there are a number of possibilities. Some of them might be best explored with the use of a microscope with a high power magnifying lens. The baby fish do not really eat much until they develop their gas bladders and can float up into the water and swim around easily. It is at this time they chase down small prey items (like paramecia) and eat them. Even if they do not eat, it will take several days for them to die of starvation. It sounds more likely to me that something else is going on. We have found that vinegar eels (which we also feed our older fish) do not work well as a first food. We use paramecia. It may be that the baby fish may not able to deal with eating the vinegar eels, but some people here at the UO think that the problem is that there is too much transfer of the vinegar (a weak acid) causing their water to become too acid. The details of this issue are unresolved, but they have not worked well for us as a first food.Another problem we have had is the presence of a small ciliate (coleps) that will kill and eat the baby zebrafish. You can usually tell if you have this problem if the number of baby fish decreases very rapidly. The bodies would be eaten up rapidly and completely. It would appear as if someone had removed the baby fish.
There is also the possibility that the water you are raising the fish in may be contaminated with some chemical that is causing their death. Alternatively there may be either fungus or bacteria growing on dead fish or uneaten food that could cause the fish to die. The water should always be as clear as possible and everything dead should be removed. If you suspect a chemical problem with the water, try a different source of water.
The success with the crushed flake food can result in two ways:
By observing your baby fish, the water they live in, and what, if anything, they are eating, you should be able to tell which of these possible scenarios are occurring in your particular case.
The easiest thing you could try would be an infusoria culture. Details can be found in many aquarium books, but it is basically made by boiling some clean grass in water letting it cool down and set with the top open for several days to weeks until a culture of bacteria and protozoans develops. You could probably speed this process up by adding some old aquarium or pond water.
* Should I feed frozen brine shrimp to my baby zebrafish?
No. Frozen brine shrimp are considered a non-optimal food for a number of reasons. The freezing process damages the shell of the shrimp resulting in the loss of nutrients. The shrimp will become food for bacteria or fungus, both bad. The frozen brine shrimp are dead so they will start rotting immediately and sink to the bottom where they will be a less attractive food item for the fish. Instead, you should use live baby brine shrimp that you hatch yourself from eggs (cysts actually). These will swim around a bit, eliciting interest from the fish, and will provide a fresh source of food.
* How big should my baby zebrafish be before I begin feeding brine shrimp to them?
If a brine shrimp can't fit down the baby fish's throat they won't be able to eat it. What we usually do is to continue to feed the baby fish paramecia in addition to the live brine shrimp at least for a while. One way you can tell if the baby fish are eating the brine shrimp is that their bellies will turn red from the brine shrimp in their stomachs. Ideally, you should not feed more brine shrimp than the fish can eat in a short period of time... maybe a half hour. Several very small feedings would be preferable to a few large ones.
* Is anyone using plankton to feed zebrafish larvae? I am specifically interested in the frozen blocks used in fish farming.
Most of the phytoplankton is too small.
* How do I maintain their tank?
Zebrafish like clean old water. Be sure to remove any uneaten food, dead eggs and fry, and detritus from the bottom of the tank. Do this daily if necessary to keep the dirt from accumulating on the bottom of the tank. Zebrafish originate from fast moving rivers and streams in northeastern India and the water quality in that area is very good.
* How often should I change the water for my babies?
If the water looks hazy or if you see coleps swimming around, it is a good idea to increase the amount of water you exchange. The haziness is probably bacteria. You should get rid of the bacteria and coleps as much as possible. I have often changed 90% or more of the water and then if I was not satisfied, repeated it.
* Do I use egg water for my baby zebrafish until they go into the adult tank?
After the fish hatch, use clean tank water.
* Does egg water need to be refrigerated? How long does it remain good?
It does not have to be refrigerated. Of course, the colder temperature would reduce the growth rate of any bacteria or other organisms that would be in it. If it is clean and nothing is growing in it, it should last forever.
* What kind of light do you use for your zebrafish? Do you use fluorescent light, or natural light, or...?
Here is our understanding of lighting and zebrafish breeding.
* I have a couple of beakers filled with zebrafish embryos. When will they need some kind of aeration before they are big enough for the adult tank?
No, your baby fish in beakers do not need to be aerated. They should not be crowded enough for oxygen depletion to become a problem. We usually keep 20 to 30 baby fish in a beaker. I believe that the main consideration here is not the oxygen levels, but having enough food per fish so that they are not competing for it. A possible drawback of aerating small fish is that a too vigorous stream of bubbles may damage the fragile babies.
* Is it absolutely imperative to keep the number of eggs in a 250 ml beaker under 20?
This factor seems to be more of an effect on the size of the fish rather than their outright survival, so the answer depends on what you are wanting.
* How do I get my zebrafish to breed?
Different breeders use different methods. We are providing 2 methods below. Method #1 is the preferred method of the people at the University of Oregon's zebrafish facility. Method #2 is the one recommended by a high school science teacher who has successfully bred zebrafish for his science classes' breeding program.
Method #1: One afternoon, put several egg-bearing females and a larger number of male zebrafish into a bare tank... no gravel. The size of the tank would depend on how many fish you are using, but a 5- or 10-gallon tank is acceptable. That evening, put 1 or more very clean glass bowls (i.e. fingerbowls) into a corner of the tank. These bowls should contain 2 layers of very clean marbles. Place a skirting of black plastic around the tank and allow the fish to spend a full night in the dark. Expose the fish to normal light the next morning and after an hour or two, remove the dishes into which the fish will have laid their eggs. During spawning, the eggs fall among the marbles where the adults cannot eat them. Remove the eggs from the dishes with an eyedropper or small straw and wash them well with distilled water. Healthy fertilized eggs will appear clear for the first day or so. Bad eggs will appear white instead of clear. This will be apparent either immediately or after a day or so. They develop in their egg shell for a few days before they hatch out. You may be able to see small black spots as their eyes develop. If you have access to a dissecting microscope or a good magnifying lens you maybe able to make out more details. They should hatch in 2-4 days. They will lay on the bottom for a day until their swim bladders develop and then they should be swimming around and looking for food. Raise the eggs to babies in beakers or small glass containers (100 mls per 15 eggs). Feed the babies after they are 4 days old. At 10 days, they can be moved to larger containers with more water.
Method #2: Use a 10 gallon tank with at least one layer of marbles covering the bottom. The water should be at least one to two inches above the marbles. Put a submersible heater in the tank... be sure it is completely covered with water so it does not have hot spots exposed to the air which can damage the heater. Adjust the temperature in the tank to between 78o and 82o F. Use NO gravel or filter in the tank. Put an air stone or bubble wand in for aeration and water movement. One afternoon, put several gravid (heavy with eggs) females and several more males than females in the tank. (More males help to assure that the eggs are fertilized.) Be sure they have at least 8 to 10 hours of darkness at night since the sunrise will stimulate them to spawn. The males will chase the females when they are mating. Mating will usually be within 1-2 hours of when the lights turn on. Do NOT feed them now since the food will fall beneath the marbles and decay among the eggs. Leave the adults in the tank for about 2 days. The eggs will begin to hatch after two days and the young will begin swimming after four days. Remove the parents by the 2nd day or they will eat the first swimmers. Once the fry are swimming you can carefully remove the marbles and VERY carefully siphon any dirt from the bottom of the tank. Replace any water removed from the tank with OLD aquarium water. After several weeks when your fry are large enough, you can begin feeding them live baby brine shrimp but be careful to wash the baby shrimp in fresh water before feeding and make sure that the fry are large enough to eat the shrimp. Once they are feeding well, you can transfer the babies to another larger tank with under-gravel filtration.
* How should I condition my zebrafish to prepare them for breeding?
For beginners it is important to be aware of the value of seasons. You should allow your fish to rest at 78oF with 6-8 hours of light a day for a few weeks, then increase the temperature to 80oF with 8 hours illumination per day for a week, then 82oF for 10 hours/day; finally 84oF for 12-14 hours/day, while at the same time, increasing the number of water changes, the quality of feed, and the quantity of live food. All of these conditions help to ensure that your zebrafish will be in prime breeding condition. Be sure to keep sexes separated. When you are ready for them to breed, place them together in tank at night. Fish will loose their prime condition in a few weeks and they will need to be put back into the resting mode for about a week.
For large batches I use 7 styrofoam shipping boxes available free at most tropical outlets. Green colored nylon netting from fabric stores is draped inside the box and pinned to the top. It helps to sew the corners together to prevent fish from getting stuck. Hang this net a few inches above the bottom. Simply move the net (with the fish in it) to another box every 3 days. An 18" x 18" box will hold several hundred eggs from a dozen or more zebrafish. By the time you get back to the 1st box (21 days), fry in that box are large enough to move easily. Feed live blackworms (not tubifex or bloodworms) to the breeders to keep them in prime condition. The blackworms that fall to the bottom and get through the net will clean the water of the excess fry feed. Recommended fry feed consists of dried peas with infusoria (available as a prepared mix commercially) which I feel works best, followed by live brine shrimp that have been fed Selcon (obtainable from Artemia), or at least boiled egg yolk. The live worms do not appear to harm any fry. They do collect all the loose shrimp eggs and filter the water well. The largest fry eat these small worms which reproduce by splitting, instead of going after their smaller kin. Seven boxes like this, 1 net, and 24 adult zebrafish, can easily produce a couple hundred fry per day. To keep your breeders in top quality condition, rest them after 21 days and replace them with fish in prime condition. A small closet with shelves and a single space heater is all you need to great results for very little expense.
(The above protocol was written by Roger Hawthorne of Albany Aquarium who was the original supplier of George Streisinger's first zebrafish models.)
* Which would be better for mating, wildtype male with female mutant, or vice versa?
Either way should be fine, although trying both ways would be better, since they do not always cooperate by mating when you want them to.
* My zebrafish has gotten extremely fat. I assume she is pregnant. I have separated her from the rest of the fish. It has been 3 weeks now, and I haven't seen any change in her. She is still "chubby." How much longer do I wait? How long will she be pregnant for? Should I place her with the other fish?
Zebrafish lay eggs which are fertilized by the male after they are laid. Your fish is probably full of eggs and waiting for a male (usually skinnier and slightly yellow on the bottom side) to mate with. She won't lay the eggs without a male. After they lay the eggs they will tend to eat them so if you want to keep the eggs you should either have something like marbles, rods, plants, or a mesh in the tank that the eggs will sink into and that will keep the fish away from the eggs. The eggs are not sticky so you can remove them with an eyedropper or maybe a straw to a clean container in which they can hatch (in 3-5 days). It is also a good idea to get them as clean as possible without damaging them. The fish will usually mate early in the morning when they first see the light (sunrise or when you turn the lights on). Typically the male fish will chase the female around. The eggs are about a millimeter in diameter (between 1/16" and 1/32") and almost clear.
* I have a breeder colony of about 60 zebrafish who have been giving me nice clutches of eggs pretty much whenever I needed them. Suddenly yesterday and today, no eggs! Is this common?
Are you collecting eggs from all your tanks, all the time? The fish can get exhausted if you've got them on marbles all the time -- it's a good idea to have them on a rotating schedule. The fish can be finicky about the light cycle. Flashing a light on them in the middle of their night can mess up their schedules pretty thoroughly, but I wouldn't think a half-hour delay in their dawn would be that traumatic. The most probable solution is to pull the marbles from the low fecundity tanks to give them a rest, and to swap around males and females.
* I'm a high school senior doing a experiment concerning zebrafish breeding. We've gotten many eggs but it is taking a lot time for us to see free-swimming fry and we don't have a very large survival rate. Also, our eggs seem to take much longer than usual to hatch. Could there be a reason for this?
There are at least a handful of possible causes I can think of:
1) The temperature is too low and the eggs are either slowed down in their development or not developing correctly due to the cold. If your eggs are being raised in too low a temperature, they may not be developing properly. To see this would require a dissecting scope. Fixing the problem is relatively easy, though. Keep the eggs in a warmer place. We normally raise our eggs at 28.5O C. (about 83O F). They can develop at lower temperatures but they should be warmer than just normal room temperature. I would not recommend temperatures below 24O C. Floating a dish of eggs in your aquarium might be a way to do this for you.
2) The eggs may be contaminated and are being killed by bacteria or fungus before they hatch. If this is the case, you should be able to see either the bacteria or the fungus without the use of microscope. Bacteria will make the water cloudy and smell bad. Fungus will produce a network of fine filaments that will reach all over the dish. The solution for these problems is to better clean your eggs. This can be done by hand with a pipette under a dissecting microscope, or with extensive, but gentle washing in a tea strainer using clean water. A fairly good chemical method is to lightly bleach the eggs when they are first collected. This requires a good quality bleach (i.e. Clorox® or Purex®) rather than a cheap brand. I have used 10 microliters of bleach in 100 mls of clean fish water for one minute followed by three rinses in clean fish water to clean the eggs. This is a 1:10,000 dilution of the bleach, which might be easier to do than measuring 10 microliters.
3) The eggs may not be getting properly fertilized. You could tell this by a visual inspection of the eggs. Using a dissecting microscope, the eggs should look relatively clear (not opaque) and should be shaped like those in a standard developmental series (such as is shown in the
Zebrafish K-12 website). If the eggs are opaque they are dead. It may take a day for this to become apparent. This may also be apparent without a dissecting microscope. If illuminated from below, opaque eggs will look dark since they will block out the light. Illuminated from above, the eggs will look white since they will reflect the light.*I've collected some embryos and some have little worms on them. Will they hurt the embryos? Do you know what they are?
It is unlikely that the little worms you describe will hurt your eggs. We sometimes notice little worms on our eggs, and they have always proven to be one of two things: either vinegar eels (which are not eels but actually little worms that we occasionally feed to the baby fish) or the larvae of some harmless midges that live in the fish water. These are also a food source if the fish find them. Neither of these worms will eat the eggs or be a health-hazard to your fish. You won't see the vinegar eels unless you are feeding them to your fish, but the midges seem to be able to colonize fish tanks all over the country. The actual flies they turn into are extremely small. The larval worms are usually found on the sides or bottoms of the tank in little tubes covered with whatever dirt-like substance is lying in the tank.
Of course, there’s always the chance that yours could be some strange organism that we haven’t encountered, but generally, the kinds of worms that would be a problem for your fish would live and remain on the inside of your fish, not the outside.
Coleps are small rugby ball-shaped ciliates (protozoans). They move in a helical (spiral) path and are covered with a series of small plates. These creatures will sometimes contaminate your paramecia cultures. In large numbers they can attack, kill, and completely eat you baby fish in about two hours. (See Mazanec, A. and Trevarrow, B. (1998). Coleps, Scourge of the Baby Zebrafish. The Zebrafish Science Monitor, University of Oregon Press, Eugene, Oregon http://zfin.org/zf_info/monitor/vol5.1/vol5.1.html#Coleps, Scourge of the Baby Zebrafish).
If your baby fish are disappearing "without a trace," it is likely that this is your problem.
* What percentage of hatchlings generally survive in controlled conditions?
Because our fish are inbred, we generally have a 60-85% survival rate which is fairly good. We consider 90% to be very good. Others who use less inbred fish claim 95% or more.
|
Thanks to the following people for providing information
for this page |
