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A Glofish Selection Experiment
June 9, 2015
6:11 pm
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BillT
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Wild Type fish chase off red Glofish from mating:

http://hatcheryinternational.c.....fish-from/

Bill Trevarrow [email protected]
June 11, 2015
3:31 am
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BallAquatics
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Cool article Bill.  Thanks for posting it.

Dennis

June 11, 2015
7:20 pm
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mikev
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Very cool indeed and thanks for posting... but there is a question here:

Do the wild type success caused by the glo- modifications at all?

Glofish has been developed from lab strains that are inbred to some degree and likely less vibrant... so one would want to check if the same effect is perhaps present in the wild vs normal lab competition.

June 11, 2015
10:57 pm
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BillT
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Yes! This leaves a lot unanswered.

Here are some possibilities:

The original glofish were made many years ago and were probably done in a way where many copies of a gene are inserted end-to end at a single site in the genome, or in several different sites. This can change in the first few generations of breeding as some of the end-to-end duplicates get deleted and genes at separate sites could become separated as chromosomes sort out differently among an animal's progeny.

This can lead to a reduction of brightness. much of this should have happened many years ago. Fish from each generation should be selected for brightness before being bred. Not likely to happen in a big fishfarm population (hundreds of thousands of fish). This short blurb did not mention the source of the fish or details of their genetics.

There are several colors of glofish, only one color was tested. Effects might vary with color.

GM fish (in labs at least)  are often crossed to other lines and then crossed together to recover the homozygous state (2 copies) for the flourescent gene. Each out cross replaces half the genes not being selected for with those to the line being outcrossed to. This will tend to replace genes from the first line with some of those from the second line. The genes removed and replaced could have either a "good" or "bad" effect on the first line.

To do a more perfect experiment of this type (which is done in mouse genetics labs) you would do backcrosses for about 20 generations. This would reduce the differences between the two strains to some small fraction of a percent, leaving only a relative few genes different between the strains. It takes years to go through these breeding steps before such a experiment could be done. The comparison could then be made between the two different strains with their genetics close to identical.

 

Common assumptions (among scientists) of why these fish would lose out is that:

1) Randomly throwing a gene into the genome could mess something up by inserting somewhere where it could break an gene or its controllers could have some negative effect.

2) There will be an energetic burden on the animal because it has to produce an extra protein which provides no offsetting adaptive advantage.

These hypotheses were not really addressed by the experiment. Each answer leads to more questions.

Bill Trevarrow [email protected]
June 11, 2015
11:53 pm
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mikev
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Yes, all true!

But I'd be sufficiently happy to just see a wild caught vs lab strain competition, just to answer if there is any glo- relevance in this work.

In fact to make it easier, one can perhaps take a lab strain with a visible mutation.... heck, in fact, how about running just a wild caught / leopard competition?

June 12, 2015
11:56 pm
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BillT
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Actually, once you make a hybrid however, the background genetics of the different lines will be blended together. Neither will win out totally. There are something like 20-30,000 genes. No set (from one parent or the other) is going to fully will out in competition, especially in new (to the fish) environments like living in an aquarium. Furthermore, you will only be able to assess and score visible mutations (unless you use molecular biology). You will not be able to determine the presence or absence of most of the background genes.

 

The way I think of lab line genetis, there are three kinds of zebrafish lines: lab lines, fish farm lines, and lines recently started from fish collected from the wild.

Lab lines are generally more inbred and are more difficult to handle (husbandry-wise) presumably for reasons typical of inbreeding problems, fixation of may slightly deleterious mutations in their genetic background. Most lab lines have breeding population sizes 20-200 fish/generation, with smaller numbers during selections. In addition, two of the most popular lines (AB and Tübengen) underwent very strong selections when they were established and have recently been shown to lack a significant part of their sex determination system.

These fish have been selected to be breedable by squeezing out eggs and sperm. They were also selected for a reproducible look to their embryos and being raised in certain conditions. Some of these lines were made homozygous in two generations. This allows more deleterious mutations to be fixed because they are not being selected out of the line over many generations. Visible mutations however, (including those affecting embryo development) have often been selected out.

Farm lines are often selected for fecundity (labs line were not until recently), can be larger than wild caught fish or lab lines. Often raised outside, but protected from large predators they tend to lose predator avoidance behaviors of wild fish.

The way they breed their fish selects for fecundity (which until recently was not done in lab lines) and therefore large size. They have large breeding populations (probably 1,000 - 100,000) and therefore can maintain their genetic diversity better. They frequently carry background mutations. Fish farm fish can have diseases but are robust.

Wild caught fish generated lines are smaller than fish farm lines. They come from populations that are presumably immense, but are then maintained with lab techniques using much smaller population sizes. Their genetic diversity will be undergoing reduction. Selection should be applied to maintain desired traits during this period, lest something undesirable get fixed.

These lines (at least initially) have some obvious predator avoidance behaviors that are lost in the aquarium environment (because no predators makes them a waste of energy). These include:

1) Feeding Behavior: The will mostly stay 4-8 inches below the surface, dash up to the water surface to grab a piece of food and then and then immediately back down to cruising depth. Reducing exposure to predators. More "domesticated" lines will "lawn mow" the surface, sucking in the food way more efficiently.

2) Flee or Approach: In 30 G tanks with the ends towards the sides, wild caught fish lines will flee to the back 20% of the tank when some one approaches one side (such as when feeding). Fish from the more "domesticated" lines will often crowd toward the front in expectation of being fed.

Living in an aquarium will put the fish from these different backgrounds, under different selections. Wild caught fish, for example, are often more difficult for the first few generations living in aquariums.

Bill Trevarrow [email protected]
June 13, 2015
5:23 am
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Anders247
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Interesting, though I'm not really surprised.

June 13, 2015
4:45 pm
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mikev
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Bill, very interesting, thanks.

Two points: do we actually know if Glofish is a lab-type or farm-type? (I do not think we know this for a fact!)

secondly: are you certain that the behavior of the fish is a function of line rather than individuals? (sure, wild caught fish would be more skittish and predator-aware than tank raised, but do we have an evidence this is present in F1? The question is interesting not just for danios, do we have any solid papers at all to answer this?)

June 13, 2015
8:44 pm
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BillT
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The glofish were first made in a lab in Singapore. They were intended to glow when exposed to some environment toxin as a bioassay. This did not work out well (they glowed without the toxin), so they eventually got re-purposed as pets.

At least for the last few years glofish have been raised in one or more fish farms in Florida. It seems likely to me that they regenerate their large breeding populations periodically in order to maintain their high intensity glow. This would allow genetic changes to be made before the new group gets expanded to a production breeding population.

It is likely they have been outcrossed to improve their husbandry traits etc. However, it is neither clear if this actually occurred nor how many times it might have been repeated.

Outcrossing would make for big genetic changes (somewhhat random with respect to the environmental conditions). Selection in the fish farm environment would be much slower and would depend on pre-existing genetic diversity in the population or on the creation of new mutations which is usually slow. Selection would adapt the population to its current conditions.

 

At least some of the traits are genetic. Here's a paper on the Nadia line of WT fish which I got started sometime around 2000 claiming most are:

A potential model system for studying the genetics of domestication: behavioral variation among wild and domesticated strains of zebra danio (Danio rerio). Barrie D. Robison and William Rowland Can. J. Fish. Aquat. Sci. 62: 2046–2054 (2005). This is behind a paywall, but I have a copy of this if you are interested.

Abstract: The process of domestication in fish is fundamentally important to conservation efforts because of the extensive
use of hatcheries to mitigate population declines. Research into the genetic changes associated with the domestication
process in many endangered species is impeded by a lack of genomic tools, long generation times, and large space
requirements. The study of the genetics of fish domestication could therefore benefit from the introduction of a model
system. In this paper, we document behavioral and growth rate differences observed between a domesticated laboratory
strain of zebra danio (Danio rerio) and a strain newly introduced into the laboratory from its native habitat in India.
Domesticated zebra danio showed a higher degree of surface orientation, a reduced startle response, and higher growth
rate compared with wild zebra danio. Wild–domesticated interstrain hybrids were intermediate in phenotype for all
traits. When strains were reared together, most interstrain behavioral differences were maintained, indicating a genetic
basis underlying the interstrain phenotypic variation. Phenotypic differences observed in this study are consistent with
the effects of domestication in other fish species, indicating that the zebra danio can be used as a model system for
studying the genetics of the domestication process in fish.

Bill Trevarrow [email protected]
June 14, 2015
5:42 am
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mikev
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That they came from a lab does not prove that the lab used a lab-line. The application was meant to be industrial, they had no reasons to worry about purity of research..... basically we don't know what it, not entirely impossible they even used wild caught!
But what we can be reasonably sure of is that subsequent versions of glofish came from the same line as the first (even those designed in Florida).

I do not think there is a possibility of fading color, the line is likely the same as on day 1. (I bred them a few gens and did not see any color deterioration)

Sure, I would love to see that paper. TBH, I'm skeptical it is possible to reprogram the personality part of genome in a small number of generations.... otoh the difference between tank and wild raising has a huge impact. (As one current example on my end : Sew03 (loach) juveniles are way more accepting of me than the w/c adults... and one generation with no selection cannot possibly alter the genome. Well, this particular situation will become definite when the juveniles reach the full size... maybe by that time they switch to seeing me as a danger too...)

June 14, 2015
8:56 pm
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BillT
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OK, let me know your e-mail and I can send a copy of the article to you (or anyone else interested).

 

I do not think there is a possibility of fading color, the line is likely the same as on day 1. (I bred them a few gens and did not see any color deterioration)

There are at least two ways the expression of a gene inserted into a genome using techniques from around the turn of the century could get reduced soon after they are made. Genes were then added to ZF by injecting DNA encoding the genes into fertilized eggs. Enzymes in the egg recognize the cut DNA ends and "fix" them by sticking them to other cut ends or making a cut in the some chromosomal DNA and sticking the inserted DNA on to those cut ends. This can lead to multiple site in insertion on the same or different chromosomes.

Fluorescence is thought to go up and down with gene copy number in a fish. Subsequent breeding and separate the different insertion sites such that they go to different sperm or eggs and pass on fewer copies of the genes to offspring. Strings of an inserted gene lined up at one site (is called concatenation) are subject to either increases or losses on the number of genes in the group by inexact crossing over among chromosome pairs which would add gene copies to one chromosome but remove from the other. These two mechanisms can remove gene copies and therefore reduce expression and therefore fluorescence early in the life of the transgenic line (first few generations). Usually these losses stabilize, perhaps because all the easy losses have occurred. There could still be differences between heterozygous (fluorescence is dominant) and homozygous fish.

Modern transformation mechanisms for ZF use molecular techniques like Crisp3r to do more controlled inserts. So these problems may not be as significant now.

 

I'm skeptical it is possible to reprogram the personality part of genome in a small number of generations.... otoh the difference between tank and wild raising has a huge impact. (As one current example on my end : Sew03 (loach) juveniles are way more accepting of me than the w/c adults... and one generation with no selection cannot possibly alter the genome.

The paper (A potential model system for studying the genetics of domestication: behavioral variation among wild and domesticated strains of zebra danio (Danio rerio). Barrie D. Robison and William Rowland Can. J. Fish. Aquat. Sci. 62: 2046–2054 (2005).) is about the genetics basis of behavior as seen inn hybrids of the two lines they used. They crossed them together and mixed the genetics 50-50. So there is a lot of genetic change, but not, at that point, due to selection.

One possible way selection could have an effect in one generation is if you got very few survivors in the first generation or two. Any survivors might be surviving because of some genetic advantage (perhaps a combination of low frequency alleles in several different genes) they have unbeknownst to you. They then pass this on to the next generation where the likelihood of these combinations being reproduced are increased because the alleles are now dominant in your breeding population. Suddenly the fish do better the next generation.

 

Another tangentially relevant point about selection is that smaller selective differences will have a stronger effect in populations of larger size. Home aquarium population sizes are usually small, lab population sizes a bit larger, fish farms very much larger, and wild populations immense. Smaller selective advantages can have effects in large populations while they might be slower acting or be swamped out be drift in smaller populations. Selection in a small population would have to be very strong to act fast. Flat out survival would probably work.

Bill Trevarrow [email protected]
June 17, 2015
5:37 am
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mikev
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Bill, thanks for the paper, let me read it and get back on this, the subject is really interesting.

As for Glo-fish -- actually nothing to say here, I don't have the key information about what was done there.

Specifically:

We do not know if the original glo fish (OGF) had multiple copies of the gene or not. It may be that they inserted the gene into multiple eggs but then selected one that has exactly one copy of the gene. No info.
If the OGF had mutlple copies of the gene we do not know if more than one copy is active.
We actually do not know if the intensity depends on the number of the functional genes. The entire mechanism is not clear since unlike other coloration in the fish it does not go through chromatophores but likely comes from a pigment present in every(?) normal cell. (When I raised GF the color showed up in larva earlier than chromatophores could develop)
and to make it worse GF involved surgery on two genes... first there was an insertion of a recessive allele to suppress the normal coloration, this produced a (call this Base) form that is somewhat in the albino direction... and then one of the actual Glo genes was inserted (this is dominant). The Base form can be recovered by breeding GF's against normals in the 2nd generation....I did this.... and the only things that we do know are connected to it:
1. Since it would be insane to repeat the process of constructing the Base form for all the variations of Glo color, we can assume that all Glo versions are lab- or farm- or (unlikely!) recent w/c... basically they are all the same.
2. And the Base form may turn out to be a known lab line, which would prove that all GF's come from lab lines....

I think these 1. and 2. are really the only things we do know..... ?

June 18, 2015
8:32 am
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BillT
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The entire mechanism is not clear since unlike other coloration in the fish it does not go through chromatophores but likely comes from a pigment present in every(?) normal cell. (When I raised GF the color showed up in larva earlier than chromatophores could develop)

Glow fish glow because they have one or more gene(s) for a fluorescent protein inserted somewhere in their genome. Exactly where the gene goes in the genome affects where and at what age the gene turns on and off. If a cell has such a fluorescent protein in it, it will fluoresce.

A molecule is fluorescent when it can absorb a photon of light, and then release a photon of lower energy (which has a shorter wavelength). Not all molecules can do this. The protein in glofish was first discovered in a jellyfish and was adapted for research uses. It was also modified to change the wavelength of light it releases. The proteins releasing different colored proteins have cleaver names like GFP (green fluorescent protein), RFP (red fluorescent protein), BFP (blue fluorscent protein).

This has been done lots of times for research purposes. Here are pictures of GFP and some other colors in research zebrafish. One common research use is make a specific set of cells fluorescent (so they can easily bee seen) and then do something the affects them. This is most useful when very small numbers of cells are labeled. It often takes a microscope to see them. This could be something like change the shape of an axon of a neuron in the nervous system or where some group of cell.

There are some lines of GFP fish which lay glowing eggs. They stayed glowing in most tissues through development and into adults. Other lines might fluoresce only at certain stages of development. If you look closely at some glofish you can see differences in the details of which parts are glowing.

 

first there was an insertion of a recessive allele to suppress the normal coloration, this produced a (call this Base) form that is somewhat in the albino direction

What you are calling the base form is just normal (not transgenic) mutations that are combined to make the fish more clear by suppressing normal pigment formation. This lets more of the light that activates the fluorescence in and more of the emitted light can get out to be seen.

In labs these lines are fish are easy to get and would be used for this purpose. Here is an article on the current favorite line for doing this.

Bill Trevarrow [email protected]
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