The goal of this protocol is to manipulate the color patterns of jumping spiders and other very small arthropods with paint in order to study questions related to sexual selection, sexual cannibalism, predation, aposematism, or any other field of animal coloration.
In the field of behavioral ecology, many experiments are designed to investigate the evolutionary purposes of colorful traits in the context of sexual selection and predation. Methods are various but mostly consist of modifying the color patterns of individuals with diverse colorants. Such techniques have been used across many vertebrate taxa, particularly in birds, but have remained underdeveloped for invertebrates because of the difficulty of effectively manipulating color in small organisms. Instead, to manipulate the appearance of invertebrates, scientists have usually modified the lighting environment to filter out certain wavelengths. However, such a method affects not only the phenotypic trait of interest but the entire appearance of the individual and its surrounding. Here, scaling down the techniques previously used on colorful birds, we present ways of manipulating the colors of small arthropods, using equally emblematic but understudied species: the colorful jumping spiders.
Animals often have elaborate color patterns that they display during sexual encounters, agonistic encounters, or to deter predation. These traits may convey information to receivers such as the signaler's individual quality as a mate1, fighting ability as a competitor2, or palatability as a prey item3. To understand the adaptive purposes of colorful traits, researchers have designed experiments that involve manipulating colors in various ways. Some researchers have used colored decoy stimuli such as models4,5,6,7,8, photographs9, or videos10,11,12 that are presented to receivers in behavioral experiments. Others, especially when using invertebrates, have manipulated the lighting environment to affect the appearance of colors of live individuals13,14,15,16,17. All these manipulations, while ingenious, have the disadvantage of removing potentially important natural behavior and/or affecting much more than the trait of interest. In large vertebrates, such as birds, researchers very often manipulate color directly on live animals (reviewed in Hill and McGraw, 200618). Individual feathers or beaks have been directly colored with markers2,19,20,21,22,23,24, dyes containing hydrogen peroxide often used in hair lighteners25,26,27, or various paints including nail polish28. In invertebrates, such studies that manipulate color patterns directly on live animals are comparatively rare but have still provided immense insight into the function and evolution of color29,30,31,32,33,34,35,36,37,38,39. Even arthropod studies seem to be biased towards larger taxa that can be more easily handled and painted, leaving color patterns in very small species relatively understudied.
Here, we describe a delicate color manipulation technique that was developed for very small animal taxa. Specifically, this method involves manipulating the facial coloration of male jumping spiders under a microscope in order to investigate the importance of such colorful traits in the context of mate choice and sexual cannibalism. In this case, we used Habronattus pyrrithrix (collected from Phoenix, AZ, USA) as a model species (Figure 1). We have published the results of experimental work using some of these techniques elsewhere38,39, but here we describe the methods in more detail than has been done previously, in a way that should make them accessible to others attempting to replicate them or adapt them for use on other very small taxa. Such protocols should open up experimental opportunities on animals that can be as colorful as the most emblematic birds but that are usually understudied.
1. Equipment preparation
2. Anesthetizing the spider
3. Mounting the spider under the microscope
4. Painting the spider
5. Taking the spider's picture
6. Releasing the spider from the pin or nail
7. Analysis of the spiders' behavior
8. Measuring the reflectance properties of the color manipulation on the painted subject
Effectiveness of color-manipulation
Using these techniques, various degrees of color manipulation are effective, including concealing colors completely or reducing or enhancing their intensity. This is evident from both photographs and measurements of spectral reflectance (Figure 2, Figure 3, and Figure 4). Here we show color-manipulated male Habronattus pyrrithrix compared to natural red-faced males. Spectral properties were measured using a UV-VIS spectrophotometer (see Table of Materials) that can precisely measure colored areas as small as 1mm in diameter. Measurements were taken relative to a diffuse reflectance white standard (see Table of Materials).
On rare occasions (5 out of 108 males painted with black eyeliner 1 (see Table of Materials) on their face), the water-soluble eyeliner began to wear off the spiders' faces after a week or two. This was not observed for the other brand of eyeliner (eyeliner 2; see Table of Materials). In both cases, spider's cages were sprayed with water three to five times per week. Different conditions of maintenance may affect the wear of water-based paint. The enamel paint was still intact for all manipulated males (n=221), even for those still alive after 6 months.
Potential toxicity of the color manipulation
One should avoid getting paint on the spiders' eyes so as not to obstruct their vision, nor on their chelicerae, mouth parts and other orifices, and possibly other soft body parts to prevent possible ingestion and poisoning. One should also be careful with painting joints or parts that contain sensory hairs (such as the legs and pedipalps) so as not to restrict their mobility or sensory system. However, if such color manipulations on these body regions are necessary, or if there is any doubt about the possibility of subtle negative effects, it is then best to apply paints to individuals in all treatment categories. This way, one would avoid unintentionally manipulating the sensory systems of individuals in ways that might be biased against one of the treatments only. For instance, in an experiment using males manipulated shown in Figure 4, the aim was to increase and decrease the number of red patches displayed by males during courtship. Since some males would get their natural red faces concealed with gray enamel paint (to decrease the amount of red displayed), the other males for which we wanted to maintain a red face were painted red over their naturally red face with the same product as the gray-faced males. Similarly, since we wanted to add red patches to the pedipalp on certain males to increase the amount of red color patches displayed, gray paint was used to cover the pedipalp of other males so that all males would be painted on this sensitive area (see Figure 4). Although preferable, this strategy may not always be feasible. For instance, in another experiment, the red coloration was removed by using a black eyeliner giving the same spectral property as the underlying cuticle of the male, while leaving the other male colors intact and natural (Figure 2). In this case, for natural looking males, the same amount of eyeliner was applied to the area on the top of their carapace just behind their anterior median eyes (an area that is not clearly visible to females), to control for potential odor or overall toxicity of the product. However, the location where the paint is applied may affect spiders differently. Therefore, to assess subtle differences in the way or the location where the paint was applied may have on the integrity of the spider, the behavior of both types of males in a context that was relevant to our hypotheses (relative to mate choice and sexual cannibalism) was compared. Males were put two-by-two in the presence of a female, and we compared their delay to become active, their delay to courting, and the total duration they spent courting with general linear mixed effect models (using the function lmer with the R package lme443 in R version 3.5.244 with the female identity as a random effect, and using the maximum likelihood criterion to obtain p-values). In this case, all comparisons reveal no differences between treatments (see Table 1) and it was therefore concluded that we did not introduce a bias in favor of one or the other treatment category.
In either case, when having very similar treatment categories (Figure 4), or only sham treated individuals (Figure 2 and Figure 3), researchers should assess how their model species are affected by the paint they use and ensure that they still behave in a similar and ecologically relevant manner. One could record data to assess the possible effects of toxicity as much as possible, for instance by comparing activity rates between treated and unmanipulated individuals. Our spiders painted with enamel paint like in Figure 4 were compared to unmanipulated males in an otherwise identical context. Specifically, males were introduced singly to a female cage and their delay to leave the vial, delay to courting and courting rate (prior to copulation, and prior to being attacked or cannibalized) were compared. No differences were found (when using similar linear mixed effect models as above) and we therefore concluded that our painted males behaved naturally (Table 2).
Finally, it is important to note that any spiders in the experiments (usually females) that cannibalized color-manipulated males never appeared to suffer from negative effects. Spiders digest their prey externally, usually leaving the painted areas of the cuticle behind. However, if adapting this method for other systems where color-manipulated animals will be consumed, one should assess the potential risks of toxicity.
Figure 1. Adult male Habronattus pyrrithrix illustrating how tiny their colored body regions are. Photographed by Lyle Buss. Please click here to view a larger version of this figure.
Figure 2. Experimental color manipulation used to conceal red facial coloration in Habronattus pyrrithrix. (A) The intact red facial coloration before color manipulation. (B) The facial coloration of the same male after concealing the natural red coloration with black eyeliner 1. (C) Representative reflectance spectra for the natural red face, the natural underlying black cuticle, and the red face painted with black eyeliner 2. Modified from Taylor and McGraw 201339. Please click here to view a larger version of this figure.
Figure 3. Experimental color manipulation used to reduce the size and redness of the red facial patch of male Habronattus pyrrithrix. (A) The intact red facial coloration before color manipulation. (B) The facial coloration of the same male after applying diluted black eyeliner (Urban Decay) to the front part of the face, and non-diluted black eyeliner along the edges of the facial patch to reduce the size of the red area. (C) Mean spectral curves of sham-treated control males (n = 21) and color-manipulated males (n = 21), compared with the population mean (n=57) and the 10 drabbest males from a previous study41. Figure reproduced from Taylor et al. 201438. Please click here to view a larger version of this figure.
Figure 4. Experimental color manipulation used to modify the color of the red facial patch of male Habronattus pyrrithrix. Habronattus pyrrithrix males painted with (A) red, (B) red and gray, and (C) gray enamel paint over their natural red face and naturally cream-colored pedipalps. (D) Mean spectral curves for unmanipulated males (n = 9), and males with their face covered with red enamel paint (n = 9). By applying a brighter red over the spider's face, we effectively enhanced its red facial coloration. Because enamel paint fully covers the underlying scales, color could also be changed entirely, as is the case with the gray enamel. (E) In this experiment, red and gray enamel paints were chosen to be matched for total brightness (total reflectance over the range of wavelengths visible to these spiders). Differences in the scale of the Y-axes in D and E are due to different techniques (such as distance to the sample and size of areas measured) for measuring color samples on paper (E) vs. direct measurements of colors on the face of the spider (D). Please click here to view a larger version of this figure.
N | Dependent variable | p | t | Red-faced | ±SE | Black-faced | ±SE | nFID |
202b | Male delay to leave dish | 0.35 | -0.93 | 140.0 | 23.9 | 109.8 | 23.9 | 102 |
179c | Male delay to court | 0.74 | 0.33 | 983.4 | 127.1 | 1031.0 | 126.5 | 95 |
204a | Male courtship effort | 0.52 | 0.63 | 181.2 | 24.4 | 203.0 | 24.4 | 102 |
204a | Male courtship effort prior to any attack | 0.41 | 0.68 | 89.0 | 15.7 | 97.5 | 15.7 | 102 |
Table 1. Effect of male face color manipulation on behavior, when painted with black eyeliner vs. sham treated (Figure 2). The structure of each model is given, as well as the mean estimates in seconds (±SE) for each treatment group. N = number of males, p and t = p-value and t-value for the male treatment, nFID = the number of levels in the random effect female identity. aOut of the 104 male tests performed, 102 were successfully recorded, leading to 204 unique males observed. b2 males were cannibalized by the female prior to ever exiting the petri dish. c25 males were cannibalized by the female prior to ever courting the female.
N | Dependent variable | p | t | Unmanipulated | ±SE | Painted | ±SE |
32a | Male delay to leave dish | 0.87 | -0.17 | 380.8 | 143.1 | 345.4 | 152.4 |
31b | Male delay to court | 0.93 | -0.09 | 502.6 | 105.8 | 488.1 | 116.6 |
31b | Male courtship effort | 0.74 | -0.33 | 2324.3 | 455.0 | 2102.1 | 484.4 |
31b | Male courtship effort prior to any attack | 0.68 | 0.42 | 1495.1 | 450.8 | 1770.1 | 479.9 |
Table 2. Effect of male face color manipulation on behavior, when painted with red or gray enamel paint (n = 15, Figure 4) vs. unmanipulated males (n = 17). The structure of each model is given, as well as the mean estimates in seconds (±SE) for each treatment group. N = number of males, p and t = p-value and t-value for the male treatment. a17 unmanipulated males were compared to a subset of all the painted males in our experiment (n = 221). Specifically, they were compared to 15 painted males (5 red (Figure 2A), 5 red and gray (Figure 2B), and 5 gray (Figure 2C)) tested in the same context (in presence of a female) and in the same specific time period. This is important because unmanipulated males were tested towards the end of the experiment (in August and September 2018), which corresponds to the end of their natural breeding season and where males are generally less active. Keeping all these other variables equal allows us to compare the painting treatment without introducing other biases. bOne male (all gray) was cannibalized prior to ever courting the female.
Here, we show that the colors of tiny body parts of arthropods can effectively be manipulated using colorants such as makeup and enamel paints.
The first critical step to achieve such delicate manipulation is to be able to immobilize small animals that usually cannot be restrained in one's hand. Here, to be able to paint sensitive areas such as jumping spider's face, we anesthetized individuals with CO2 and mounted them on the head of a pin. This allows work close to the spider's eyes with less stress than the spider would likely experience if it were awake (with light from the microscope shining into their faces during the painting process).
The method also requires getting good quality micro brushes, and, most critically, appropriate coloring substances. The most difficult step in applying paint without spillage but with good coverage is to get the right consistency. Therefore, the coloring substances need to be easily diluted with a thinner, and easily dried out for thickening. Different type of paints could be used; here, the results are presented with water-soluble (non-waterproof) eyeliners and enamel paints. Non-waterproof eyeliners have the advantage of being easily liquefied when mixed with water. However, this trades off with the dilution of the pigmentation (which may not or may be desirable (see for instance Figure 3)). Enamel paints have a consistency that can easily be controlled by adding enamel thinner, while still providing full coverage. However, this characteristic trades off with the possibility of maintaining the hair or scale structure of the body part painted. In addition, enamel paints are long lasting. The downside to this is that enamel paint and thinner emit strong odors during application and before drying. One additional difficulty regarding the coloring substances may be to find the right shade, with the right spectral properties. It is for instance hard to get red eyeliner to use in parallel with black eyeliner, as eyeliners are often more pink than red. It is also hard to get makeup powder (or pigments) that do not contain any glitter (which can sometimes be only visible under the microscope). Many makeup products also reflect UV light which, while invisible to the experimenters, might be conspicuous to the animals studied.
Manipulating the coloration of arthropods by directly applying colorants onto their body parts comes with advantages and inconveniences when compared to other methods. Its main limitation is that one cannot absolutely dismiss the possibility of some subtle toxicity effects. However, one can ensure not to introduce biases against one treatment group by applying paint to all treatment categories, and/or one can test whether the paint application interferes with behaviors of interest. With the methods presented here, we collected enough evidence to suggest that the paint application led to negligible to no negative effect (Table 1 and Table 2). The main advantage of this method is that tiny patches of color can be targeted, their color 'removed' (see Figure 2), made duller (Figure 3) or brighter (Figure 4), in isolation from the rest of the body coloration and the individual's environment. This contrasts with the most common alternative method which consists of manipulating the lighting conditions, and thereby modifying the visual appearance of the whole individual and its surroundings. In fact, even when not specifically manipulating lighting conditions, one can successfully manipulate color and see limited or no effects of this manipulation if the lighting environment is not appropriate39. Therefore, it is important to measure and consider the light environment where any experiments will be conducted (i.e., measure irradiance) and make sure to closely match it to natural lighting conditions (for instance using full spectrum light bulbs that mimic natural light when in captivity). Overall, by using micro brushes and a microscope, this protocol allows for more precise manipulation of tiny color patches than most other direct coloring methods that have been used previously on invertebrates. Most previous studies have used animals with color patches that are relatively large compared to the faces of jumping spiders (e.g., manipulation of butterfly wing colors29,34,35, the bodies of adult hemipterans ('true bugs')30,36 and grasshoppers31, or the legs of relatively large wolf spiders32,33,37). The methods presented here open up opportunities to study the amazing diversity of color patches on taxa that are understudied due to their small size.
Similar techniques could be applied to other arthropods that can be immobilized or anesthetized and for areas where paint would not affect the mobility or health of the individual (i.e., excluding areas such as joints, structures such as hair or arolia that are needed for appropriate locomotion, mouth parts, or other orifices such as breathing structures). These techniques can also be extended to include a larger palette of dyes, paints, and makeups that are widely available.
Finally, these delicate techniques could be used not only to manipulate color on small organisms, but also to manipulate patterns (such as stripes) in relatively larger organisms. This should be beneficial to a wide variety of researchers that can adapt our methods to their own studies of sexual selection, communication, aposematic prey signals, and other contexts in which animals use color.
The authors have nothing to disclose.
This work was supported by funding from the National Science Foundation (IOS-1557867 to LAT), the Florida Museum of Natural History, and the Entomology and Nematology Department at the University of Florida. Publication fee of this article was funded in part by the University of Florida Open Access Publishing Fund.
CO2 tank | AirGas (Radnor, PA) | #CD 50 | to anesthesize spiders |
Enamel paint thinner | Testors (Vernon Hills, IL) | 75611792569 | to thin enamel paint |
Flat enamel paint | Testors (Vernon Hills, IL) | red: 075611115009, black: 075611114903, white: 075611116808 | can be thinned with enamel paint thinner |
Light microscope | Zeiss (Jena, Germany) | stemi 508 | to paint small areas with precision |
Light microscope camera | Zeiss (Jena, Germany) | Axiocam 105 color | to take picture before and after manipulation for documentation |
Light microscope camera software | Zeiss (Jena, Germany) | Zen 2 blue edition | to process pictures taken before and after manipulation |
Liquid liner eyeliner, shade “Perversion” | Urban Decay (Costa Mesa, CA) | (discontinued) | non-waterproof eyeliner which can be thinned with water; eyeliner 2 |
MegaLiner liquid eyeliner, black | WetnWild (Los Angeles, CA) | SKU# 871A | non-waterproof eyeliner which can be thinned with water; eyeliner 1 |
Micro brushes | MicroMark (Berkeley Heights, NJ) | #84648 | to allow precise painting of small areas |
Non-hardening modelling clay | Van Aken International Claytoon (North Charleston, SC) | 18165 | to stick small nail or insect pin in and flexily adjust their angles |
Small nail or insect mounting pins | BioQuip (Rancho Dominguez, CA) | #1208B7 | to glue spiders on as well as moving away spider’s appendages in front of the area to paint |
Small plastic containers such as the lids of snap-cap insect collection vials | BioQuip (Rancho Dominguez, CA) | #8912 | to mix paint and thinner to the right consistency |
Small syringe | Fisher Scientific | 1482910F | to transfer small amount of enamel thinner |
Spectralon white standard | Labsphere Inc. (North Sutton, NH) | WS-1-SL | to measure spectral properties of colors |
UV-VIS spectrophotometer | Ocean Optics (Dunedin, FL) | USB 2000 (spectrophotometer) with PX-2 (light source) | to measure spectral properties of colors |
Water soluble school glue | Elmer's (High Point, NC) | #E304 | to mount the spiders onto a nail/pin |
Wood toothpicks | Up&Up, Target Corporation (Minneapolis, MN) | #253-05-0125 | to transfer drops of enamel paint |