Manipulation of Color Patterns in Jumping Spiders for Use in Behavioral Experiments

To understand how and why colors evolve, we need experiments where we manipulate animal color patterns. Tiny animals are often understudied because their colors are difficult to manipulate. The main advantage of directly manipulating color patches is that this method does not affect the appearance of the rest of the animal, or alter their natural lighting environment.

We show how researches can apply enamel paints and makeup to jumping spiders. These same methods also work on a variety of other small, colorful, and understudied arthropods. I would advise first attempting to paint a dead specimen so that there is no rush to obtain the perfect paint consistency and quantity on the micro brush.

Visual demonstration of this method is essential because it is hard to explain what the right paint consistency is, how to apply the paint, what angle the brush would have, for instance. To begin, connect a dissecting microscope to a camera and computer, to more easily take pictures of the outcome. Turn on all three devices, and start the software that processes the camera input.

Next, select the relevant zoom at which the final picture will be taken. Stick an insect mounting pin or a small nail into a ball of non-hardening modeling clay that is approximately the size of a grape. Place the modeling clay and pin setup under the microscope and adjust the objectives so that they roughly focus on the head of the pin where the spider will be mounted.

Then, place an extra thin insect mounting pin into a new ball of non-hardening modeling clay, and place this beside the microscope. Set out the following materials in an organized fashion beside the microscope. A small piece of absorbent paper, A piece of white printer paper, the paints being applied, separate containers of paint thinner for each color being applied, plus one to be kept transparent and clean, individual micro brushes for each color, and one microbrush to be used only with clean thinner.

Using a toothpick, add a drop of paint to an open plastic dish. Add paint thinner and use the toothpick to mix the two to the right consistency, where the paint is completely homogenized, but not too runny. Use a microbrush to test the mixed paint on the white printer paper.

Then, put a pea sized drop of water based glue onto a corner of the printer paper. First, add a very small amount of glue onto the head of the mounting pin or nail that was previously prepared and placed under the microscope. Gently slide the anesthetized spider from its vial onto the table with its ventral side up, making sure to not tap or drop the spider onto the table.

Hold the modeling clay with both hands, making sure that both hands are steadied firmly against the table. Then, gently press the pinhead with glue on the spider's sternum, such that the spider will slightly bounce and extend its legs under the small pressure applied. Reposition the mounting setup underneath the microscope, so that the area to be painted is facing up and in focus.

First, we test the paint consistency, and adjust it again if necessary. Always first try applying the paint on the printer paper, to control the paint quality contained in the brush hairs. Before touching the spider, it is critical to make sure the paint has the right consistency, and that there is only the right amount of paint on the microbrush.

While looking through the microscope, use the dominant hand to bring the tip of the brush into the field of vision, and ensure, once again, that the brush hairs do not contain too much paint. If there is too much paint on the brush, wipe some of it onto the printer paper. Then, touch the brush to the spider on the largest area that will need to be painted over.

This will reveal if the consistency and quantity of the paint is right, which is when the paint slightly and slowly soaks into the hair or scales of the spider. If no paint gets applied, soak the brush into the paint and repeat this process, beginning with testing the paint on the printer paper. If the paint soaks in rapidly, and spills over into an area that should not be covered by paint, wipe the brush onto the absorbent paper and repeat the process from the beginning, by testing the paint on the printer paper.

If painting the face of the spider, use the extra thin pin in the non-dominant hand, to hold down the front legs and pedipalps. Depending on the brushes and paint, consider painting both sides of the face before attempting to paint the central area between the eyes, as the painting on the side areas can be joined by holding the brush parallel to the spider's face and inducing capillary action. When painting the pedipalps or legs, make sure to not touch any joins with a hardening paint, and make sure to not apply paint to the male sperm delivery organs on the underside of the distal segment of the pedipalps.

After painting one side of the spider's face, it is helpful to rotate the spider under the microscope, so that the other side of the face can be painted with the same dominant hand. Painting must be done both carefully and quickly, before the spider awakes from anesthesia. Finally, zoom out with the microscope to inspect the overall color manipulation and prepare to take a photograph.

First, switch the objective to the camera mode. Using the computer software, take a picture while making sure that the zoom selected is the one selected on the microscope, so that a scale bar is added. When the spider starts moving, hold the pin so that the spider's front legs are touching the spider's vial.

If needed, gently incline the pin to help the spider pull itself away from the dried glue and let the spider release itself. In this study, the tiny faces of Habronattus pyrrithrix males are color manipulated. Using the techniques demonstrated here, various degrees of color manipulations are effective.

Including completely concealing colors, or reducing or enhancing their intensity. For the males shown here, the aim is to increase or decrease the number of red patches displayed by males during courtship. Some males have their natural red faces concealed with gray enamel paint, while others have their colors maintained by painting over the natural red with red enamel paint as a control.

Likewise, since some spiders have red patches added to the pedipalps to increase the amount of red color patches displayed, gray paint is used to cover the pedipalp of the other males so that all males have paint applied to the sensitive area. However, having spiders from different groups painted on the exact same areas, to control for any possible negative effect of the paint, is not always feasible. In a different study, the red coloration is removed using a black eyeliner that has the same spectral properties as the underlying cuticle of the male, while the other male colors are left intact and natural.

For the natural looking males, the same amount of eyeliner is applied to the area on top of the carapace, just behind the anterior median eyes, to control for any potential odor or overall toxicity of the product. The behavior of both types of males is compared in a given context to assess any subtle differences that might suggest toxicity or unintended effects of the paints. Sham treated males and males whose faces are painted with eyeliner, are put two by two in the presence of a female, and specific male behaviors are observed.

In this case, all comparisons reveal no differences between treatments. Therefore, it can be concluded that no bias is introduced for either of the treatment categories. Likewise, when introduced singly to a female cage, males painted with gray or red enamel paint and non manipulated males, present no differences in behavior.

Therefore, it can be concluded that the painted males acted naturally. To not handicap the spider in any way, it is important to completely avoid adding paint onto the mouth part or the sperm delivery organs. One should also not add or leave any paint on the spider's eyes or joints.

Color manipulated spiders or other arthropods can be used in behavioral experiments to understand the role of color in a variety of context, ranging from courtship, to male, male interactions, to avoiding predators. The field of animal coloration is driven mostly by work on larger vertebrates, such as birds. With scaled down color manipulation techniques, we can now ask more questions about color in tiny animals.