What determines the coloration of transgenic (TG), (Night Pearl), and (GloFish) aquarium fish?
Genetically modified fish (transgenic *Danio rerio* and *Oryzias latipes*) first went on sale in 2003, initially in Taiwan and other Asian countries, and later in the USA. By 2005, there were already quite a few of them in Russia as well. The official producers of these fish are the Taiwanese corporation Taikong (which produces and sells fluorescent fish under the Taikong-1 “Diamond Series”, TK-2, TK-3 lines under the “Night Pearl” trademark) and the American company Yorktown Technologies LP (which sells fish in the USA under the “GloFish” trademark — derived from two English words: “glow” and “fish”). The University of Singapore announced the creation of luminescent goldfish that change color depending on water temperature.
The “Diamond Series” includes varieties named Emerald Night Pearl, Ruby Night Pearl, and Golden Night Pearl. They carry genes for green, red, or yellow fluorescent proteins and feature bright patterns on the dorsal fin and ray formations on the caudal fin. The “Platinum Series” includes Platinum Red Leopard and Platinum Green Leopard varieties. Demand creates supply, and rumors that all transgenic fish on sale are sterile have proven false; now they are successfully bred in Russia, Ukraine, and other countries as well.
How are TG fish of other colors (neither red nor green) produced? Orange fish were created by crossing TG carriers of the rfp gene and the gfp gene. In hybrids, expression of both genes occurs in muscle tissue (these are “double transgenic” fish), with more red protein synthesized, which is why we perceive their color as orange. The exact same TG fish look different under daylight versus ultraviolet lighting.
Under daylight, we perceive their color primarily through reflected rays. Under these conditions, an orange fish, whose color results from mixing two dyes (gfp and rfp), looks somewhat darker due to subtractive color mixing, as each dye absorbs its own part of the spectrum. Chromatophores located in the skin also influence the fish’s color. This is the same thing that happens on an artist’s palette when mixing paints.
The situation is entirely different when fish are illuminated with ultraviolet light. Here, the contribution of fluorescence to the visual image is very large, so the fish are perceived as self-luminous objects. An orange fish looks lighter than a red or green one because both rfp (the same amount as in a red fish) and gfp (adding about 15% more to the glow) fluoresce in its muscles. In this case, additive color mixing occurs. In daylight, gfp and rfp are more like simple paints and mix accordingly, whereas under ultraviolet light, gfp and rfp begin to fluoresce and mix like red and green light rays.
Fluorescent *Danio rerio* were specifically bred to detect environmental pollutants. For over a decade, fluorescent fish have been used by scientists worldwide to better understand crucial questions in genetics and molecular biology. Initially in the USA, glowing zebrafish were created to serve as living pollution indicators — in the presence of certain toxic substances in water, the fish were supposed to change color.
GloFish are a genetically modified form of the highly popular aquarium fish, the zebrafish. In nature, these fish are black or silver. Unlike them, GloFish are colored red and emit a faint reddish glow most noticeable under ultraviolet light. This unusual metamorphosis occurred thanks to the introduction of a red coloration gene, borrowed from a sea anemone, into the zebrafish DNA. It turned out that *Danio rerio* are ideally suited for experiments with GFP and RFP transgenes. Numerous pure genetic lines of these fish that strictly inherit specific traits have been established.
Regarding the topic at hand, zebrafish possess another advantageous feature: their embryos develop rapidly and remain nearly transparent for a long time. Adult fish with reduced pigmentation also retain this property to a large extent. Therefore, if a transgenic fish organism acquires the ability to produce a fluorescent protein, the glow will be easily observable even when the expression of the gene integrated into its genome occurs in tissues located beneath the skin and in internal organs.
The Japanese rice fish, or medaka (*Oryzias latipes*), possesses similar properties. Taiwanese and Japanese researchers experimented extensively with this species. As a result, today we have fluorescent medakas as well as zebrafish. But besides genetically modified *Danio rerio*, medakas, and Atlantic salmon, GM carp, GM trout, GM tilapia, GM channel catfish, and GM goldfish are also bred. Following zebrafish, new transgenic fish were created. This time, cichlids: the convict cichlid Amatitlania nigrofasciata and the angelfish *Pterophyllum scalare*. And surely someone else… In short, transgenic aquarium fish are merely the tip of the iceberg, most of which remains hidden from our eyes.
First, genes encoding green fluorescent proteins (GFP) and red fluorescent proteins (RFP) were isolated from marine jellyfish (*Aequorea victoria*) and anemones (*Discosoma* sp.), and then scientists learned to integrate these genes into the fish genome so that the synthesis of fluorescent proteins was triggered precisely when specific muscle groups began to form during the individual development of zebrafish embryos. It became possible to observe the formation of the muscular system in colors under a fluorescent microscope. The ability of proteins to glow under soft ultraviolet light made it possible to detect them, and consequently the muscle tissue in which they were synthesized, at very early stages of development.
It turned out that these fluorescent proteins continue to be synthesized in fish muscles throughout their lives. They accumulate in such quantities that they become visible to the naked eye. The fish takes on the color of the fluorescent protein it synthesizes, and as it grows, it becomes brighter and brighter! As they grow, genetically modified zebrafish turn intensely red or green. About a dozen such genes are currently known.
Results of a preliminary experiment conducted by Prof. Gong to determine what colors can theoretically be obtained using green and red fluorescent proteins. These proteins were extracted from the muscles of TG fish and mixed in various proportions. Depending on the proportion of each protein in the mixture, different colors are produced.
However, the general regulation principle is known: the required amount of fluorescent proteins in muscle tissue is achieved by altering the strength of promoters, i.e., their ability to ensure reporter gene expression. As a result, more of one fish-coloring protein is synthesized and less of another, yielding a specific resulting color. The color of fish capable of synthesizing red fluorescent protein in their muscles can actually vary greatly.
This mainly depends on the color of their skin, which partially shields the light rays reflected by the colored musculature of the fish or emitted by it during fluorescence under blue and ultraviolet light. Currently, there are many color forms of ordinary unmodified zebrafish, so-called background lines.
These lines were used to produce transgenic zebrafish of various color shades using classical breeding methods rather than genetic engineering (genes responsible for colored protein synthesis behave as dominant, and their inheritance follows classical Mendelian laws).
For example, there are albino fish completely devoid of the ability to synthesize black pigment (melanin) in their bodies. Their skin is very light and transparent, so if their genome contains a gene responsible for synthesizing red fluorescent protein, the pinkish-red color of the muscles will be clearly visible. And these fish truly look as bright as possible. Conversely, fish with skin coloration close to the wild type are noticeably darker, as melanin granules shield the muscle color.
It should be noted that when creating stable lines of TG fish of various colors and shades that reliably pass their traits to offspring, classical breeding work comes to the forefront. Transgene-carrying fish are crossed with representatives of specific pure genetic lines (background lines), and then offspring with the desired traits are painstakingly selected. The inheritance of genes responsible for gfp and rfp synthesis obeys Mendelian laws, with these genes behaving as dominant. The color of fish capable of synthesizing red fluorescent protein in their muscles can actually be very diverse.
However, there are also special TG aquarium fish whose production method is kept secret, leaving us to guess how they were made. These are creations of the Taikong corporation known as TK-3 (Candycane). The manufacturer merely indicates that the fish synthesize gfp and rfp under the control of an “actin” promoter, but how was such an unnatural distribution of fluorescent proteins achieved?
According to one hypothesis, these fish are made from two halved TG embryos, one possessing gfp genes and the other rfp genes. The front half of one was grafted onto the rear half of the other. If the operation is performed at early embryonic stages, successful fusion of the halves is entirely possible. In short, this is the result of virtuoso manual work. No wonder these TG fish are not yet widely available for sale.
Taiwanese researchers were not satisfied with the “natural” appeal of zebrafish and decided to enhance it. Scientists from the Taiwanese biotech firm Taikong Corporation inserted a specific gene into the hereditary apparatus of the world-famous decorative fish *Danio rerio*, a gene nature bestowed only upon certain Pacific jellyfish. This gene is responsible for synthesizing a fluorophore protein that emits pleasant greenish rays in the dark. Initially, the genetic engineers aimed to facilitate observation of the internal organs of these translucent fish.
However, a representative of a company engaged in breeding and selling aquarium fish saw a photograph of the fish glowing with a greenish ghostly light at a scientific conference. At the company’s request, a red glow gene isolated from a sea coral was added to the zebrafish genome. As a result, they delighted aquarists with a new “novelty” — a fish with fluorescent green light. Transgenic fish with the transplanted jellyfish gene were given the poetic name “Night Pearl”.
There is some concern about what might happen if these genetically modified fish escape into natural rivers and streams. GloFish creators assert that they pose no ecological threat because they cannot survive in the wild, being tropical fish that require water temperatures around 24–26°C.
Let’s compare how the natural coloration given by nature differs from that of genetically modified zebrafish. Answering this question makes it easy to understand whether environmentalists’ concerns regarding the possibility of transgenic zebrafish entering natural biotopes were justified. It is easy to notice that the natural coloration of fish depends on how light falls on them.
These fish look bright only when illuminated frontally and seen in reflected light. In transmitted light, they become quite pale, and if light falls on them at various angles, the fish produce bright flashes. Furthermore, natural coloration effectively breaks up the fish’s body, dividing it into separate fragments similar to environmental elements and surface glare. Such coloration evolved for a reason: it functions to camouflage the fish from predators.
Transgenic fish, however, always remain bright. They are clearly visible from the bottom against the blue sky and foliage—which is precisely from below that most aquatic predators attack their prey. Yet they are also clearly visible from above and will be easy prey for predatory birds. When lighting levels drop, normally colored fish turn gray and become almost invisible, whereas transgenic fish are completely betrayed by the fluorescent component of their coloration. Moreover, the bright pink-orange color provokes attacks even from a full predator and irritates fish that are not strictly predatory but simply quite large.
In short, transgenic zebrafish have not a single chance of surviving in nature — there is no need to worry about that. It should be noted that upon eating a transgenic fish, a predator will easily digest it just like any other prey. Fluorescent proteins are non-toxic and fully degraded by digestive enzymes. Thus, no trace of a genetically modified zebrafish will long remain in a natural water body.
Keeping transgenic fluorescent fish.
Which fish are artificially colored for commercial purposes. 
