# Color markers

Aim

You need the information below when you track color-marked animals.

General information

▪Use a color scale (for example from a paint company) to find out which colors are most easily recognized by EthoVision XT in your setup and lighting conditions. Do this before applying color markers to your animals.

▪Use colors that have different hue values. For example, use red and green and not red and orange.

▪For birds and animals in general that perceive color in the red-green wavelength range, using red markers is not advisable as it looks like blood, and may influence the subjects’ behavior. Rodents have dichromatic vision, that is, they perceive wavelengths around the blue and the green, so probably red is seen as gray or a mix of blue and green. Albino animals have heavily impaired vision, both in acuity and color perception.

▪If you want to use red together with orange or pink, provide more light around (by means of additional lamps) and then close the aperture of the camera lens.

▪If the color of your marker is also present elsewhere in the arena, you can get rid of it by using a minimal marker size.

▪Marking your animals may stress them, and therefore affect their behavior. If necessary, ensure that you select a marking method that lasts for a longer period of time.

▪To improve color discrimination in EthoVision XT, you can alter the Saturation settings of the video image (this only applies for live tracking). In the Detection Settings window, under Video click Image Quality. Alter the Saturation value and check in the video window if the markers are more distinguishable. However, before altering the Image Quality always try to optimize the lighting and camera aperture settings.

Note that if you alter the Image Quality values you need to redefine the color detection (click Identification for each subject).

Marker characteristics

▪Make sure that the marker is visible as much as possible; preferably place it on the center of the animals backbone.

▪Make sure that the marker is 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. For color marker tracking it will help to prevent the jitter of the marker.

▪When you use marker assisted tracking, make sure the marker is not too big; the marker can 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.

Markers for rodents and other small mammals

There are several ways to color mark small mammals like rats and mice. Color mark as much of the head and back as possible. This way the mark is always visible, also when the animals rear or turn.

▪Livestock markers are intended for large animals but 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 they stay on for one or two days.

▪Fluorescent tubes can be used for color-tracking in the dark. It is possible to mark rats with fluorescent markers and illuminate the arena with UV-light (350-400 nm, 2 lamps of 1.2 W each). The fluorescent spots on the rats appear as patches of different color against a dark background. EthoVision XT can then effectively separate the two animals (source: J.P. Johansson and M.L. Carlsson, Institute of Clinical Neuroscience, Göteborg University, Göteborg, Sweden). See Tracking colors in the dark

▪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 (for example, Edding or Marks-A-Lot) 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.

▪Chalk ink markers can be used to mark rodents. They are available in a variety of bright colors. Their main advantages is that they are not toxic, are readily available and inexpensive, and, in contrast with other marker types, can be applied directly to dark fur without bleaching it first. Coloration may last several hours.

It is advised not to mark the animals just before you start a test, because the 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, you should habituate the animal to the marking procedure.

▪Colored ‘jackets’ can be used to color-track mammals with colored fur like cats. 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.

Markers for large mammals

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

Markers for birds

▪The feathers of birds can be marked with commercial products for dyeing human hair or permanent marker pens (see ‘Small mammals’ above).

▪Seabird ecologists often use picric acid (2,4,6-trinitrophenol, TNP) to mark their birds. You paint a dab of this yellow liquid on the feathers. It is permanent and very bright yellow, although it turns orange in the sun. To use it to mark animals you dissolve it in an excess of ethanol. If the acid (a powder) dries, it becomes explosive, so it is important to keep it dissolved (Source: J. Blount, Division of Environmental and Evolutionary Biology, University of Glasgow, UK).

Markers for fish

▪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) and trout (about 25 cm). Colored pearls can be bought from a toy store or a warehouse. Use bright colors that are as distinct as possible. For damsel fish pearls of 1 cm are used and for trout pears of 8 mm are used.

Damsel fish. 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).

Trout. The pearls can be attached at the base of the first ray of the dorsal fin. Use a curved needle and normal fishing-line to attach the pearls to the fin. X-ray images showed no injury after several weeks using this method. The fish can be anesthetized by placing them in a tank with 2-phenoxy-ethanol (0.3 ml/l), before attaching the pearl.

▪Visible implant fluorescent elastomer (VIE) tags are produced by North west Marine Technologies (NMT). 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 well 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 and Delcourt et al. (2012). Behavior Research Methods 43: 590–600).

Markers for 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).

Markers for amphibians

For amphibians you can use visible implant fluorescent elastomer (VIE) tags (see ‘Fish’ above). They work fine with small tadpoles.

Markers for insects

▪Acrylic paint (for example, Royal Talens Amsterdam deco) can be used to mark insects. The paint can be applied with a fine brush or thread. You have to 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 a non-toxic dye, as used for coloring food. The dyes can be obtained from a normal bakery. 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 will work best in insects with unpigmented abdomens (Source: G. Driessen, Institute of Evolutionary and Ecological Sciences, Leiden University, The Netherlands).

▪Mosquitoes can be fed with a sugar solution in water containing fluorescent dyes. Blue-black light is used to illuminate the subjects. See the paper by Sarkar et al. 2017, International Journal of Mosquito Research 4(6): 5-9 (animals were not video-tracked though). Other techniques using fluorescent powders or dyes are described by Verhulst et al. (2013), Parasites & Vectors 6: 200-206.

▪For insects you can also use visible implant fluorescent elastomer (VIE) tags (see ‘Fish’ above).

▪Aphids have successfully been color marked with fluorescent powders. The powders that are fluorescent under ultraviolet light can be obtained from Day Glo, Cleveland, Ohio. For optimal detection, a high intensity UV long wave UV·366 nm lamp can be used. To apply the powder to the aphids, gently dust the powder with a powder insufflator. Make sure, each aphid gets at least 10 grains of powder. For more information see Thomas et al. (1997). Journal of Agricultural Entomology 14(2): 187-198.

▪Ants can be color marked with a small drop of paint on the thorax or the abdomen. Anesthetize the ant with CO2. To restrain the ant during painting, use a sponge with a single strand of hair attached to it. Place the ant under the hair and apply the color mark. Transfer the ant to a plastic tube and allow the paint to dry for approximately 20 minutes. After 24 hours, the ant can be used in experiments.

▪For a review of the marking techniques see Hagler & Jackson (2001). Annual Reviews of Entomology 46: 511–543.

Markers for zooplankton

▪Nanoparticles known as Quantum dots can have been applied to the carapace of crustaceans Daphnia sp. Like any other fluorescent molecule, they must be excited with light of one wavelength in order to emit fluorescence at a longer wavelength. Among the advantages of these markers, the great photostability (animals can be tracked for up to 24 hours after labeling, without loss of signal due to photo-bleaching. Note that some quantum dots may be toxic to animals.

For more information see Lard et al. (2010). PLoS ONE 5(10): e13516. doi:10.1371/journal.pone.0013516.

Tracking colors in the dark

If you want to apply color markers and track the animals in the dark, you need lighting conditions that allow EthoVision XT to detect the marker and detect the contour of the animals.

To track colors in the dark, combine fluorescent markers with ultraviolet (UV) light. UV light makes the marker emit visible light, which is then detected by the camera. EthoVision will track the marker like it would normally do in daylight. See Markers for rodents and other small mammals

To track the contour of the animal, you must also use light sources other than UV. If you only use UV light, the contour of the animal won’t be seen. A couple of solution that may work:

▪Use visible light. Make sure that the contrast between animal and background is maximized.

▪Use infrared (IR) light. Some good color cameras are also sensitive to IR light. You can then use IR light to enhance the contour of the animal, provided that the camera lens does not have a IR-block filter.

Note that fine-tuning this setup may take considerable effort and time.

Useful web sites

▪Carmel livestock markers: http://www.livestockmarkers.com.

▪La-Co livestock markers: http://www.allweathermarker.com.

▪Net-Tex livestock markers: http://www.net-tex.co.uk and search for “marker”.

▪Edding markers: http://www.edding.com.

▪Marks-A-Lot markers: http://www.avery.com.

▪MS Schippers (livestock or rodent markers): https://www.schippers.nl. See the product line “MS Merkstift” (in Dutch).

▪Chalk Ink markers: http://www.chalkink.com. Choose a 15-mm broad tip marker.

▪Schwarzkopf hair dyes: https://www.schwarzkopf.com

▪Royal Talens acrylic paint: https://www.royaltalens.com/en/products/acrylic-colours/.

▪B/Braun surgical sutures and needles for attaching pearls to fish: http://www.bbraun.com.

▪Northwest Marine Technology fish markers: http://www.nmt.us/.

▪Invitrogen quantum dots http://www.thermofisher.com/nl/en/home/brands/invitrogen.html.

▪Fluorescent powders: https://www.dayglo.com/