This educational resource is part of the Cephalopod Week spotlight

Cephalopods (octopuses, squids, and cuttlefish) are considered to be masters of camouflage, blending in with their environment effortlessly. In another Science Friday Educate resource , we learned about how the chromatophore layer allows cephalopods to instantly generate one of three types of patterns ( uniform, mottled, or disruptive) using only three colors: red, yellow, and brown. In this activity, we’ll investigate the two deeper layers of cephalopod skin, the iridophore layer, and the leucophore layer, and how they contribute to the remarkable camouflage capabilities of cephalopods.

Take a look at this squid skin:

In this gif, you can see some red chromatophores pulsing. You can also see a shimmering area that starts off looking like pale gold, then shifts to bright blue, and finally ends with emerald green. That shimmering area is an iridophore (“iridophore” means “bearer of rainbows”), a reflective type of tissue found in the second layer of cephalopod skin, known as the iridophore layer.

Let’s take a closer look at the structure of an iridophore:

Each iridophore organ is surrounded by several stacks of plates of chitin. Chitin is a translucent, somewhat tough material found in many organisms, including the cell walls of fungi, the scales of fish, and the shells of crustaceans (like crabs). When a ray of light strikes the top surface of chitin, some of it reflects, while the rest of it passes through. The light that passes through strikes the bottom surface of chitin, and again, some of that light reflects while the rest of it passes through. This process repeats itself through all of the plates in a stack.

The many rays of light that reflect off of the plates of chitin combine through a process called thin-film interference. In thin-film interference , some colors get canceled out, resulting in the visibility of other colors. You can see thin film interference in soap bubbles…

Each has a thin semi-transparent film of some substance overlying an object and in the iridophore, it is chitin.

When light strikes the top surface of a transparent or semi-transparent film, some of the light reflects off of the surface of the film and bounces off, while the rest of the light penetrates into the film itself. The light that manages to penetrate through the top surface refracts or bends slightly because it travels a bit slower through the film. Of the light that penetrates into the film, when it reaches the bottom side of the film, some of it will reflects off of the bottom while the rest of the light passes through the bottom surface and exits the film. In the image below, follow the different paths that light can take when it hits a semi-transparent film.

When a light ray encounters the top surface of a semi-transparent film, some of it penetrates through to the bottom surface, where some of it reflects. If the distance traveled by the ray at (1) is an odd number (1, 3, 5, …) multiplied by half the wavelength of a given color (350 nm for red), then when it combines with another light ray at (2), there is destructive interference of that color, resulting in the visibility of the complementary color (in this case, green) at (3).

The rays of light that reflect off the bottom surface (let’s call these bottom rays) travel a bit further than the rays of light that reflect off the top surface (let’s call these top rays), because they must pass through the thickness of the film twice: once going in, and once going out. That extra distance is important. It offsets the wavelengths of the bottom rays from the wavelengths of the top rays when they combine. If that offset is equal to a distance that is half of the wavelength of a given color, then that color is effectively canceled out , removing that wavelength (color) of light.

This is called destructive interference. When particular colors are canceled out, then the complementary colors (their opposites) predominate. So when red is canceled out, then the complementary color green becomes visible.

Scientists are still trying to figure out how cephalopods manipulate iridophores to control their color changes, but a current hypothesis is that cephalopods might be able to change the thickness or angle of the chitin plates in their iridophores to change their color. Changing either the angle or the thickness of the chitin plates would change the distance traveled by rays of light that pass through the chitin, causing destructive interference, and thus altering the colors displayed by the iridophore.

Iridescent Cellophane , cut into at least three 3” x 3” sheets (Iridescent Cellophane can usually be found in Craft Supply Stores)

Scotch Tape or Masking Tape (both, if possible)

A digital camera (preferably on a phone or other handheld device)

9 plastic cups (we used red Great Value 9 Fluid Ounce Party Cups, but any cup that you can easily cut with scissors or a box cutter would work)

An Exacto knife, box cutter, or sharp scissors

A desk lamp with an adjustable head

A dark room, to cut down on ambient light

A table-top that is not reflective, or colored black or white

A printout of the Activity 1 Worksheet

A set of color markers

Step 1) How To Start A Hole In The Bottom Of The Cup *With the help of an adult

Step 2) Removing The Bottoms Of Your Cups

Step 3) How To Tape Your Cups Together

Step 4) How To Assemble The “Rainbow” Viewer

Step 5) Label The Angles Of Your Viewer

Step 6) Putting Your Viewer In Place

Step 7) Lining Up Your Viewer

Step 2) Taking Pictures Of Each Angle

After you’ve finished, complete the Reflections section of the Activity 1 Worksheet.

Rays of light that manage to pass through the chromatophore and iridophore layers are either absorbed by the relative darkness of the cephalopod’s body or (if they are in the right place) are reflected back by leucophores in the leucophore layer.

Leucophores are cells covered with rice-shaped reflective granules called leucosomes. Like disco-balls, leucophores reflect light back in many different directions without changing the color of that light. Oftentimes, leucophores appear as white spots (“leuco” means “white”). However, leucophores actually take on the color of whatever light shines upon it. Under blue light, leucophores would make the cephalopod look blue; under white light, leucophores would make the cephalopod look white. Leucophores help cephalopods to camouflage by reflecting whatever color of light is already in the environment. Leucophores can’t be directly controlled by cephalopods. However, by controlling the chromatophore and iridophore layers above, leucophores can alter the amount of light that reaches them.

Step 1) Place a piece of black construction paper under the iridescent foil.

Take pictures as before. Also, draw your observations on the Activity 2 Worksheet.

Step 2) Replace the construction paper with a sheet of crumpled aluminum foil. This will act as a simulated leucophore, since it reflects and scatters light back through the iridophore layer.

Take pictures, and draw your observations, focusing particularly on how the aluminum foil changes the colors that you see.

Step 3) Cut a square of colored cellophane. Place it over the opening of your desk lamp, and wrap a rubber band around it to hold it in place. Turn on the lamp.

Replace the aluminum foil with the black construction paper. Take pictures and draw your observations.

Step 4) Replace the construction paper with aluminum foil.

Take pictures and draw your observations.

Step 5) Complete the questions in Reflections section of the Activity 2 Worksheet, and listed down below.

Complete the Reflections section of the Activity 2 Worksheet.

Step 2) Set Up Your Background

Step 4) Look For Specific Colors

Step 6) Resizing The Mask For Your Image

Step 8) Select The Rest Of The Colors For Your Palette

Step 9) Using Real Underwater Backgrounds

Step 10) Select A Location For Your Cephalopod

Cephalopods don’t only use one layer at a time; they utilize all three layers simultaneously to create a composite image. This is possible because each layer is at least partially translucent, allowing light to pass through it, even as each layer makes its contribution to “staining” the light a certain color. Here are some real examples of the three layers of cephalopod skin working together:

In this picture, the iridophore layer displays a light blue color, while the chromatophore layer displays larger red spots, with smaller brown and yellow spots.

Credit: Lydia Mathger, Eric J. Denton, N. Justin Marshall, and Roger Hanlon

In this picture, the iridophore layer contributes a predominantly orange background to the red, brown, and yellow chromatophores above. Notice how the iridophore layer brightens the chromatophores (or conversely, how the chromatophores stain the light from the iridophores).

Credit: Lydia Mathger, Eric J. Denton, N. Justin Marshall, and Roger Hanlon

Here, you can see the influence of a leucophore as it creates a stark white background for the chromatophore layer above it. By contracting the chromatophores, a cephalopod can emphasize the whiteness of the leucophore layer. Note the shimmering orange spots around the edges of the leucophore, which are caused by iridophores.

Credit: Lydia Mathger, Eric J. Denton, N. Justin Marshall, and Roger Hanlon

The iridophore and leucophore layers are the second and third layers of cephalopod skin. To learn about the first layer, the chromatophore layer, try this activity. When finished with the chromatophore layer activity, try to repeat the camouflage activity found in that lesson using the full palette of a cephalopod! It is recommended that you either do the entire camouflage activity online (using drawing software like Google Draw) or that you print out photos of iridophores/leucophores for use in a hard-copy version.

Next Generation Science Standards Addressed by this Activity:

4-PS4-1. Develop a model of waves to describe patterns in terms of amplitude and wavelength and that waves can cause objects to move.

4-PS4-2. Develop a model to describe that light reflecting from objects and entering the eye allows objects to be seen.

4-LS1-1 : Construct an argument that plants and animals have internal and external structures that function to support survival, growth, behavior, and reproduction.

MS-PS4-2 : Develop and use a model to describe that waves are reflected, absorbed, or transmitted through various materials.

HS-PS4-1 : Use mathematical representations to support a claim regarding relationships among the frequency, wavelength, and speed of waves traveling in various media.

Resource by Randy Otaka Editing by Brian Soash, Shirley Campbell, Ariel Zych, and Lauren Young Digital Production by Lauren Young

Randy Otaka is an elementary school special education teacher and robotics coach for Wahiawa Elementary School in Wahiawa, Hawaii. He has a passion for STEM education and implements interactive and engaging lessons, such as using Minecraft to model the cardiovascular system to creating scale model solar system orbital paths.

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