Color-marking animals for EthoVision

In EthoVision XT there are two methods for tracking animals with color markers:

• Color marker tracking (treat marker as center point) — Only the colored marker is tracked, not the whole animal, so nose-point and tail-base tracking is not possible. It is suitable for any species.
• Marker assisted tracking — Tracks the animals’ body using the usual contrast methods, and then in addition use the marker to determine the animal’s identity. You can use this method in social interaction tests where you need to track the nose point and tail.

In each case, you'll need some kind of colored mark on the animal which can be tracked.

Lighting conditions

Lighting conditions play an important role in ensuring different colors can be distinguished.

• It must be bright enough — Dark or dim conditions can result in low contrast between the animal and the background and therefore tracking might not be optimal. Dim light also makes it difficult to distinguish colors. When it is not possible to use strong illumination in your setup, try using fluorescent marker colors with UV lighting.
• It must be even — Lighting should be diffuse, e.g., coming from multiple directions, through a diffuser, or bounced off the ceiling. This keeps colors and contrast consistent throughout the arena.
• For optimal color separation, illuminate your setup with lamps that approximate to day-light in color temperature, that is, have a wide spectrum range.

General Tips for Markers

EthoVision distinguishes markers on the basis of Hue, Saturation, and Brightness. Hue is the color (e.g. red vs blue), saturation is how colorful it is (high saturation is colorful, low saturation is muddy and grayish), and brightness is light vs dark. Some general tips:

• Use hues that are widely separated. For example, use red and green, not red and orange.
• It may be wise to avoid using red for marking, since it looks like blood.
• If the color of your marker is also present elsewhere in the arena, you can get rid of it by setting a minimal marker size in EthoVision, excluding areas with that color from the arena, or use a different colored marker.
• Marking animals may stress them, and therefore affect their behavior. A longer-lasting method requires less handling and can be used farther in advance.
• If you need to record in dark conditions, using UV illumination with fluorescent markers can provide enough visibility. This is most suitable when you are only tracking the colored marker and not the outline of the animal (i.e., no nose-point and tail-base tracking).
• Place the marker where it can be seen by the camera, preferably on the center of the animal's back. A marker on the head of an animal may go out of view when the animal looks down.
• Make the mark as round as possible, this will ensure that the relative movement of the center of gravity of the marker is the same in all directions when the edges of the marker change due to posture changes or otherwise. 
• Don't make the marker too big if using marker-assisted tracking; the marker may interfere with proper detection of the body contour. For example, make sure that a dark marker on a white animal does not cover the complete width of the animal because it can cause the body to be split in two.

Small mammals (and birds)

These methods work with many small animals, e.g., mice and rats. Many also work with birds.

• Chalk markers ( https://www.chalkink.com/ ) and terracotta markers are each types of non-toxic opaque paint markers which work well even on dark-colored animals.
• Livestock markers intended for large animals can also be used on smaller animals. These markers come as paint sticks and spray cans, and are available in many colors including fluorescent ones. The markers are weather-resistant, non-toxic and stay on for one or two days. See links under Large Mammals below.
• The fur of rats and mice can be marked with commercial products for dyeing human hair. Dark fur can be bleached (like dark human hair, to make it blonde) before coloring. Hair dyes are available in many bright colors, and provide long-lasting coloration. Schwarzkopf PolyColor Hair color Cream, for example, can be applied to rat’s fur with a cotton swab.
• Permanent marker pens present a cheap and easy way to mark small mammals. Take care to make a large and uniform blob. This method may only work for a short period of time as many species may remove the mark by grooming.
• It is advised not to mark the animals immediately before starting a test, because handling of the animals might affect their subsequent behavior. If you use a marker that stays on the animal for only a short period of time, habituate the animal to the marking procedure.
• Colored ‘jackets’ can be used to color-track mammals with colored fur. Jackets cover most of the cat’s back, and can be provided with velcro strips, so one can place pieces of cloth of different colors to test optimal color combinations. Orange vests may give problems when animals are red-haired, so it should be replaced by either (bright) green, yellow or pink.

Large mammals

You can use livestock markers to mark large animals such as cows, sheep and pigs. Examples include:

https://carmelindustries.com/collections/livestock-markers

https://markal.com/pages/all-weather

Fish (and amphibians)

Fish can be color marked by attaching plastic pearls. This technique has been used successfully in Mediterranean damsel fish (Chromis chromis, size: about 9 cm long). Colored pearls with a diameter of 1 cm can be bought from a toy store. The pearls can be attached under the dorsal fin with blue non-resorbable surgery polyamide mono filament (for example, from B/Braun), using a surgical needle under anaesthesia. The fish can be anesthetized by placing them in a small tank with 2-phenoxy-ethanol (0.3 ml/l for the damsel fish) for five minutes, before attaching the pearl. The bottom and sides of the arena have to be very dark. The pearls do not significantly impair swimming in damsel fish (Source: M. Ylieff, Institut de Zoologie, Université de Liège, Belgium).

Visible implant fluorescent elastomer (VIE) tags are produced by North west Marine Technologies (NMT). https://www.nmt.us/visible-implant-elastomer/ VIE material is implanted beneath transparent or translucent tissue (it may become difficult to detect it beneath pigmented tissue). The material is injected as a liquid and soon turns into a pliable, bio-compatible solid. VIE colors are visible under normal lighting conditions and have greatly enhanced visibility under ultra-violet light or with other fluorescence enhancing techniques. You can test which colors work best in your own setup with a color test patch from NMT. (Source: J. Merilä, Evolutionary Biology Centre, Uppsala University, Sweden). VIE tags also work with tadpoles.

Reptiles

Nail polish (available in many colors) can be used to mark lizards. The mark usually lasts 10 days in small (up to 10 cm) lizards in the field. (Source: M. Massot, Laboratoire d’Ecologie, Université Pierre et Marie Curie, Paris, France).

Insects

Acrylic paint (for example, Talens Decorfin) can be used to mark insects. The paint can be applied with a fine brush or thread. Hold the insect with forceps until the paint is dry (Source: G. Driessen, Institute of Evolutionary and Ecological Sciences, Leiden University, The Netherlands; S. Belmain, Natural Resources Institute, Chatham Maritime, UK).

Some species of parasitic wasp develop a colored abdomen when you feed them honey with non-toxic food coloring. The advantage of this method over marking with paint is that you do not have to anesthetize the insects (which may influence their behavior and survival). The method works best in insects with unpigmented abdomens (Source: G. Driessen, Institute of Evolutionary and Ecological Sciences, Leiden University, The Netherlands).

You can also use visible implant fluorescent elastomer (VIE) tags (see ‘Fish’ above).