The array of colors seen in the skin of guppies is due to the presence of pigment cells called chromatophores. These cells include the light-absorbing melanophores, xanthophores and erythrophores, and the light-reflecting leucophores and iridophores. Classified by color, xanthophores and erythrophores are yellow to red, leucophores are opaque white, iridophores are iridescent or silvery, and melanophores are brown to black. Xanthophores and erythrophores contain carotenoids and pteridines, which act as colored filters, preferentially absorbing short wavelengths. Carotenoids absorb visible light primarily in the 400–500 nm range, producing yellow to red colors. Pteridines primarily absorb light between 340 and 500 nm and may appear yellow (xanthopterin and sepiapterin), red (drosopterin or erythropterin), or colorless to the human eye.

Iridophores and leucophores produce structural colors through some combination of reflection, constructive interference and scattering. Iridophores contain organized stacks of reflecting crystalline platelets of guanine. Light is reflected at the planar surfaces and the color of the reflected light is primarily determined by the thickness, spacing and refractive index of the platelets. Leucophores are dendritic cells containing globular, membranous vesicles filled with cytoplasm, purines or colorless pteridines. Leucophores could potentially produce a wide range of colors through, depending on the diameter of the vesicles and refractive indices of the substances within them. However, most cells identified as leucophores appear white, probably because the vesicles they contain fall within the size range in which wavelength-independent scattering.

Melanophores contain melanins – highly polymerized compounds synthesized from tyrosine – which absorb light throughout the human visible spectrum and into the ultraviolet range, and thus cause darkening of the integument.

Chromatophores are typically arranged in the guppy skin in three or four contiguous cell layers. Carotenoids and pteridines in the xanthophore layer (if present) differentially absorb shorter wavelengths (violet to blue). A fraction of the light that passes through the xanthophores is reflected back out into the environment by platelets in the iridophores. The remaining light is either absorbed by the melanophores or reflected back through the skin by the reflective shield in the innermost layer (if present). The internal structure of the iridophores affects the color as well as the brightness of the color patch. If the reflecting platelets are dispersed and oriented randomly within the cytoplasm, all wavelengths of light will be reflected and the color patch will appear silver, yellow, orange or red, depending on the amounts and types of pigments present in the xanthophores. If the reflecting platelets are stacked to produce constructive interference in the short-wavelength region, the result will be a structural blue in the absence of xanthophores, or various hues of yellow-green, green or blue-green if xanthophores are present (see Fig. 1). The three functional elements are: 1) a filtering layer (carotenoid), 2) a reflecting layer (guanine) and 3) an absorbing layer (melanin).

Figure1
Wavy lines depict the paths of light of differing wavelengths through the cell layers.

In this article I want to show the typical coloration of a wildtype guppy. The reason for this is that I want to understand the "normal" coloration of guppies and have it as a reference when studying the phenotypes of modern guppies.

	This wildtype specimen will allow us to illustrate several important concepts about coloration in guppies. Here you can observe the colors blue, green, red, yellow, purple, silver and black. These colors result by the presence of a particular set of chromatophores colored areas
	Here we can observe a section of the body at a 60X magnification, which exemplifies some of the "typical" characteristics of guppies. You can see a "net-like" pattern on top of the red spot, which is the result of the presence of melanophores in the edges of scales. It is this "reticulated" pattern that gives the guppy its name..Poecilia reticulata.

Blue is produced by light reflected by iridophores. These cells are deep underneath the scales. The scales themselves, in guppies, are transparent. Green is produced when blue light travels through cells with yellow pigment (xanthophores - with Pteridines). Red is produces by carotenoid-containing erythrophores, and like green, purple is the result of blue light traveling through red cells (acting as a filter).

As you can see in Figures 2 and 3, the structure of guppy skin is a s follows: Epidermis (outermost), scales, dermis (in between and underneath the scales), and subcutaneous layer (lowermost layer between skin and muscle tissue).

Figure 2

Figure 3

A couple of example of these cells in guppy skin at 200X magnification.

	This is the same wildtype specimen at 200X. This is the area immediately underneath the dorsal fin. Here you can see melanophores at the edges of the scales, and what appears to be xanthophores evenly distributed in between top and bottom scales (in the dermis - see arrows).
	In this photograph of the same wildtype specimen we can see a dense group pf erythrophores. But we cannot tell at what depth in the dermis these cells are located.
	Here you can see iridophores, or reflecting cells. Again, one cannot tell the depth or location of these cells.
The reflected light is either white (leucophores?), blue (iridophores most likely), yellow (white light reflected though a xanthophore), blue(iridophore), or green (blue light reflected through a xanthophore). These colors suggests that reflecting cells are underneath the pigment cells.

Removing scales from a portion of the skin of guppies can help us understand the location of cells involved in coloration of guppies. When we remove scales, we also remove the epidermis (on top of scales) and portion of the dermis (sandwiched in between the scales - see Figures 2 and 3). Note that only about 25% of the scale is "exposed" and he rest is overlapped with neighboring scales.

The following photograph series is of a different line of wildtype guppies. We removed scales to investigate the possibility of learning more about the position of cells responsible for coloration in guppies.

First of all, please not the symmetry between the right ad left side of this specimen.

Right Side	Left Side
Right Side	Left Side

	This is a 60X photograph of the left side of the guppy, where one can see the "typical" distribution of coloration in he skin. One can see red/orange, blue, purple and black.
	We remove scales, and with the scales we also removed many of the melanophores and some of the iridophores. The erythrophores/xanthophores remain.
	This is one of the scales, but unfortunately we cannot see much color detail. We can observe melanophores and (even if not clearly) xanthophores distributed throughout thet same area where the melanophores exist.
	This is a 60X magnification photograph of the area near the base of the caudal tail on the right side of the specimen, where you can clearly observe a metallic blue color reflected from iridophores.
	This is roughly the same area where scales have been removed. One can see how most of the iridophores have also been removed. The red/orange spot has a metallic look to it, and we suspect that leucophores may be underneath this patch of red/orange cells.
	Here you can see a 60x magnification photograph without scales. Here we made an effort to scrape the layer of dermis which remains after removing scales. You can see a flop of dermis (see arrow) with orange/red cells, which once scraped off, leave a clear section of subcutaneous tissue with little or none chromatophores.

Conclusion: Our method of removing scales is not very sophisticated and may be improved with some practice or as we learn better ways to do it. However, from this experiment (if you can call it that), we can tentatively conclude that, in wildtype guppies, most of the chromatophores are located in he dermis underneath the scales, with some located in the dermis between (sandwiched) scales, and only maybe a few in the epidermis.

We are going to continue to explore this method of removing scales with modern guppy strains, and perhaps we will write other articles about our findings. In the mean time, here are some examples of scales removed from modern guppy strains. In these photographs that follow, one can observe that scales can have a variety of types and density (amount of cells and "pigment" concentrations) of chromatophores associated with them in modern strains. For example, you can observe the presence of different types of melanophores, erythrophores, xanthophores, iridophores and (maybe) leucophores. We cannot tell for sure if these cells are located on top (epidermis) or in between (sandwiched) scales in the dermis. What is clear is that coloration in modern guppies depends to a large extent on the types and quantities of chromatophores that are located either on top or sandwiched between scales.

Investigations of these sort can help answer questions on whether certain mutations can "cover-up" certain alleles expressed deep underneath in the dermis. For example, on the bottom left photograph below one can see a very dense population of xanthophores (and iridophores) which I think are sandwiched between scales. If the individual guppy also is expressing another phenotype which is strictly expressed deep in the dermis, it could be covered up.

It is too early to tell the full utility of this approach or to make any sort of wide-reaching conclusion. But we will continue with this approach in conjunction with reciprocal modern-wildtype crosses and see where it takes us. We hope you find this article informative.