Int J Primatol DOI 10.1007/s10764-012-9638-8

Using the Hard, Randy, and Rittler Test to Evaluate Vision in Capuchins (Cebus libidinosus)

Tiago Siebert Altavini & Leonardo Dutra Henriques & Daniela Maria Oliveira Bonci & Balázs Vince Nagy & Dora Fix Ventura & Valdir Filgueiras Pessoa

Received: 11 May 2012 /Accepted: 28 August 2012 # Springer Science+Business Media, LLC 2012

Abstract The identification of types in primates is fundamental to understanding the evolution and biological function of color perception. The Hard, Randy, and Rittler (HRR) pseudoisochromatic test categorizes human color vision types successfully. Here we provide an experimental setup to employ HRR in a nonhuman primate, the capuchin (Cebus libidinosus), a platyrrhine with polymorphic color vision. The HRR test consists of plates with a matrix composed of gray circles that vary in size and brightness. Differently colored circles form a geometric shape (X, O, or Δ) that is discriminated visually from the gray background pattern. The ability to identify these shapes determines the type of dyschromatopsy (deficiency in color vision). We tested six capuchins in their own cages under natural sunlight. The subjects chose between two HRR plates in each trial: one with the gray pattern only and the other with a colored shape, presented on the left or right side at random. We presented the test 40 times and calculated the 95 % confidence limits for chance performance based on the binomial test. We also genotyped all subjects for exons 3 and 5 of the X-linked opsin genes. The HRR test diagnosed two subjects as protan dichromats (missing or defective L-cone), three as deutan dichromats (missing or defective M-cone), and one female as trichromat. Genetic analysis supported the behavioral data for all subjects. These findings show that the HRR test can be applied to diagnose color vision in nonhuman primates.

T. S. Altavini : L. D. Henriques : V. F. Pessoa (*) Laboratory of Neurosciences & Behavior and Primate Center, University of Brasília, 70910 Brasília, DF, Brazil e-mail: [email protected]

L. D. Henriques Department of Experimental Psychology, Institute of Psychology, and Center for Neuroscience and Behavior, University of São Paulo, 66281 São Paulo, Brazil

D. M. O. Bonci : B. V. Nagy : D. F. Ventura Department of Experimental Psychology, Institute of Psychology, and Center for Neuroscience and Behavior, University of São Paulo, 66281 São Paulo, Brazil T.S. Altavini et al.

Keywords Capuchin . Color vision . Hard, Randy, and Rittler pseudoisochromatic test . Platyrrhine

Introduction

Monkeys use color as an important cue for the perceptual identification of objects in their environment (Jacobs 2009). We can assess color vision using microspectropho- tometry, electroretinographic flicker photometry, genotype sequencing, or behavioral tasks (Jacobs 2007). In particular, researchers have used monochromatic stimuli widely to evaluate primate color vision in behavioral studies (Jacobs 2007; Kelber et al. 2003). However, this requires equiluminant monochromatic to prevent subjects from discriminating between lights based on their brightness rather than on wavelength (Jacobs 1981). Despite the contribution of these studies (Jacobs 2009), the reflectance functions of natural surfaces present relatively continuous variation across the spectrum (Lennie and D’Zmura 1988) and equiluminant edges are rare in nature. Indeed, color vision is important for the detection of targets against dappled or variegated backgrounds, where varies randomly (Mollon 1989). Pseudoisochromatic tests are effective in diagnosing humans with deficient color vision (Mollon 1989). These are usually composed of a figure formed by colored dots within a field of differently colored dots. The dots vary randomly in size, luminance, and saturation (Birch et al. 1979), so that the observer is unable to discriminate the figure from the background based on brightness or contour. Thus, if the observer can discriminate the figure, we can infer that the discrimination was based on . Several studies have applied pseudoisochromatic tests to assess color vision in nonhuman primates. These studies used the Ishihara plate test (Saito et al. 2003, 2005a, b) or an adaptation of the digital Cambridge Colour Test (Mancuso et al. 2006, 2009). The advantage of the Cambridge Colour Test over plate tests is that it allows quantitative measurement of color discrimination thresholds. However, it requires specialized equipment and is more expensive than plate tests. We developed a relatively inexpensive protocol that applies an alternative plate test, the Hardy, Randy, and Rittler (HRR) test, to nonhuman primates. Although the HRR test does not measure color discrimination thresholds, it does allow us to determine whether subjects are di- or trichromats, to classify dichromats as protan (missing or defective L-cone) or deutan (missing or defective M-cone), and to rank the severity of dyschromatopsia (deficiency in color vision) in anomalous trichromats as , medium, or strong, corresponding to different color contrasts in the HRR plates. The HRR test is considered to be more efficient than the because it provides detailed information regarding severity and reliably classifies protans and deutans (Cole et al. 2006; Crone 1961). HRR plates are also much simpler than Ishihara plates, and thus, we assumed, easier to use with nonhuman primates. The severity of dyschromatopsia diagnosed by this test correlates with the degree of photopigment abnormality in humans, diagnosed using an anomaloscope (Neitz et al. 1996). The anomaloscope consists of two different light sources that have to be matched by the subject. One is a monochromatic while the other is a yellow mixture of and . The diagnosis relies on the amount of green and red each subject will require on the mixture to match the monochromatic yellow. Hard, Randy, and Rittler Test in Capuchins

Our objective was to ascertain whether the HRR test is a viable tool of primate color vision evaluation. The genus Cebus can express four different alleles in the X chromosome that code four different opsins with absorption peaks around 535 nm, 545 nm, 552 nm, and 561 nm (Soares et al. 2010). We aimed to design a protocol that enabled us to classify nonhuman primates as expressing one or more (in the case of trichromats) of these opsins. To achieve this we tested the ability of Cebus libidinosus to discriminate the geometric shapes of the HRR test and genotyped the subjects to test whether the classification of the subjects was consistent with their color vision phenotype.

Methods

Subjects and Experimental Cage

We tested three male and three female adult capuchins in their home cages at the Primate Center of the University of Brasília. We gave all subjects food once a day and water ad libitum. There were six cages along a corridor, each with two compartments isolated by a door operated from the corridor. The cages were 2.5 m high; the smaller compartment was 2 m wide and 1 m long, while the larger compartment was 2 m wide and 3 m long. Three individuals (male 1, male 3, and female 3) had previously been tested in color discrimination experiments that used Munsell chips (Gomes et al. 2002). The other three subjects were naive.

Stimuli and Apparatus

We used the red/green subset of the HRR fourth edition color vision test. The HRR plates consist of geometric shaped targets (circle, cross, and triangle) composed of colored circles with random variation in size, saturation, and brightness in a matrix of gray circles varying in brightness and size. There are 17 plates in the red/green subset. Although some plates have two shapes, each has a different hue, and the set totals 27 shapes. All colored figures corresponded to a positive stimulus (SD+). We used plate 0 as a negative stimulus (SD–) because it has no colored shapes. Plates 2 and 3 have shapes that are easy to discriminate even for dichromats. Plates 7–10, termed screening plates, determine whether the subject is di- or trichromat. Plates 11–20 are used to diagnose the type of dischromatopsy in humans because some shapes are visible for protans, while others are visible for deutans. We classified subjects according to the HRR instructions for humans, based on the proximity of absorption peaks in human and cones of Cebus (Jacobs 1996). First we considered the group of plates in which the subject had the most correct responses. If these plates were from protan group, we classified the subject as protan; if they were from the deutan group, we classified the subject as deutan. Next we looked at the plate in which the subject made his last error (now considering both groups). If the last error was on plates 19 or 20, we rated the severity ranking as strong; if it was on plates 16–18, we rated the severity as medium; and if it was on plates 11–15, we rated it as light. We cut the plates from the HRR book and inserted them into transparent plastic cases for protection. We changed the cases once a month to avoid any visual and T.S. Altavini et al. olfactory cues. We used a version of the Wisconsin General Test Apparatus (WGTA), assembled on a portable table in front of the cage, to run the experiments. The apparatus, made of acrylic, had two windows that could be opened by the experimental subjects and in which we presented HRR shapes (Fig. 1). The apparatus stood at a distance that allowed the monkeys to touch the HRR plates with the tip of their fingers. This distance varied 15–20 cm according to the length of the subject’s arm. A wooden screen prevented the subjects from observing us setting up the HRR plates in the apparatus.

Procedure

We divided the procedure into three phases: modeling, training, and testing. None of the phases exceeded 60 min of testing per day. In the modeling phase, we allowed the monkey to manipulate freely the windows of the apparatus, which at this stage did not contain HRR plates. Once the subject had learned how to operate the apparatus, we moved to the training phase. In the training phase we placed the SD– plate in one of the windows and SD+plates 2 or 3 in the other. We presented the SD– and SD+on the left or right in a pseudorandom sequence, according to a modified version of the Gellerman table (Gellerman 1933). We recorded a correct response when the subject opened the SD+window and we rewarded the monkey with a piece of grape through that window. We offered no reinforcement if the subject opened the SD– window. The monkeys started the testing phase once they attained 43 correct responses in three consecutive training sessions of at least 53 trials each, or 81.1 % correct responses (P<0.0001 according to the binomial test). If the subject persisted in the choice of one side, we repeated the wrong attempts until the monkey selected the other side.

Fig. 1 Apparatus used for the HRR presentations. a Frontal view, b experimenter view, and c side view with a subject’s arm making a choice. Hard, Randy, and Rittler Test in Capuchins

The testing phase consisted of presenting all HRR plates paired with the SD–.We conducted 20 sessions for each subject and presented each plate twice per session (i.e., we presented each plate 40 times). We considered a subject to have discrimi- nated a plate successfully when it made a correct response ≥27 times (P<0.02, according to the binomial test). After all phases were completed, we submitted the monkeys to a control session that involved 53 trials of SD– vs. SD–. We expected that the subjects would perform randomly in this case, ensuring that they were not using any clues other than the presence of the geometric shapes.

Genetic Analysis

We collected blood samples from the subjects via femoral venipuncture during routine healthcare exams. We extracted DNA from blood samples using a purification kit (PURE- GENE® DNA, Gentra System). We used the polymerase chain reaction (PCR) to amplify the exons 3 and 5 of the X-linked opsin genes, as described by Mancuso et al. (2006). The sequences of the forward and reverse primers for exon 3 were 5′GGAT CACGGGTCTCTGGTC and 5′CTGCTCCAACCAAAGATGG, respectively. For exon 5 the forward primer was 5′GTGGCAAAGCAGCAGAAAG and the reverse primer was 5′CTGCCGGTTCATAAAGACATAG. We sequenced the PCR products directly using the DYEnamic™ ET Dye Terminator kit on a MegaBACE™ 1000 sequencer (GE Health- care). We used the amino acids at the position 180 (expressed by exon 3), 277 and 280 (both expressed by exon 5) to predict the maximum absorption peak of the visual pigment.

Fig. 2 Performance of each subject as absolute number of correct responses across the training sessions. The dotted line represents the upper limit for random responses (performance below the line is considered random) according to the binomial test. There is some variance in the upper limit because the number of trials per training session varied. Training sessions were 55 trials or 1 h in duration. Subjects needed to achieve ≥80 % correct responses in at least three consecutive sessions of 55 trials each to proceed to the test phase. T.S. Altavini et al.

All procedures (including behavioral testing and blood collection) were approved by the Ethics Committee for Animal Use of the University of Brasília.

Results

Behavioral Tests

All six subjects completed each phase of the behavioral evaluation procedure suc- cessfully. Male 1 needed 12 training sessions to reach the test phase; male 2, 15 sessions; male 3, 10 sessions; female 1, 6 sessions; female 2, 14 sessions; and female 3, 9 sessions (Fig. 2; all males and female 3 performed extra training sessions). Based on the test phase, we classified males 2 and 3 as strong protons; male 1, female 3, and female 2 as strong deutans; and female 1 as a trichromat (Table I). All individuals performed randomly during the control phase.

Table I Summary of the results of the test phase

Plate type Plate number and shape Female 1 Female 2 Female 3 Male 1 Male 2 Male 3

Protan 11º 1 0 1 0 1 0 13Δ 1 11110 14º 1 0 1 1 1 0 15X 1 0 0 0 1 0 16Δ 1 00011 17º 1 1 1 0 1 1 18Δ 1 00011 19X 1 0 0 0 0 0 20º 1 0 0 0 1 1 Total protan 9 2 4 2 8 4 Deutan 11Δ 1 11011 12X 1 1 1 1 1 1 14X 1 0 0 1 0 0 15º 1 1 1 1 1 0 16º 1 1 1 1 0 0 17Δ 1 11110 18X 1 1 1 1 0 0 19º 1 1 1 1 0 0 20Δ 1 11100 Total deutan 9 8 8 8 4 2 Classification – Trichromat Deutan Deutan Deutan Protan Protan severity ––strong strong strong strong strong

The number 1 indicates discrimination (≥27 correct responses in 40 trials) while 0 indicates a lack of discrimination (random performance in 40 trials). The color vision classification is based on the type of plate in which the subject achieved more discriminations. Severity is based on the last error in either type of plate Hard, Randy, and Rittler Test in Capuchins

Table II Description of opsin genotype, absorption peaks, and phenotypes for six Cebus libidinosus

ID Exon 3 Exon 5 Allele 1 Allele 2 λmax-1 λmax-2 Phenotype

180 277 280 Genetic Behavioral

Female 1 Ser Tyr/Phe Thr/Ala SYT SFA 560–563 534 Trichromat Trichromat Male 2 Ala Phe Thr AFT – 542–547 – Protanope Protan strong Male 3 Ala Phe Thr AFT – 542–547 – Protanope Protan strong Male 1 Ser Tyr Thr SYT – 560–563 – Deuteranope Deutan strong Female 2 Ser Tyr Thr SYT – 560–563 – Deuteranope Deutan strong Female 3 Ser Tyr Thr SYT – 560–563 – Deuteranope Deutan strong

Genetic Analysis

All three males and females 2 and 3 were homozygous, expressing only one allele for the M/L opsin gene, characterizing the individuals as dichromats (Table II). Males 2

Fig. 3 Spectral sensitivity curves predicted for M/L opsin from the amino acid sequences found in Cebus libidinosus. a Two males had the allele AFT with λmax around 542–547 nm and were classified as protanope. b Another male and two females had a deuteranope phenotype due to the expression of allele SYT with λmax around 560–563 nm. c One female was trichromat, expressing two alleles with λmax around 534 nm (SFA) and 560–563 nm (SYT). T.S. Altavini et al.

and 3 expressed an opsin with wavelength peak (λmax) around 542–547 nm (Fig. 3a). Male 1 and females 2 and 3 expressed a visual pigment with λmax around 560– 563 nm (Fig. 3b). Female 1 was heterozygous for the M/L opsin gene with a trichromat phenotype, expressing alleles with λmax at 534 nm and 560–563 nm (Fig. 3c). The total number of alleles was 3 and allele frequency varied among individuals: the most frequent allele had λmax in 560–563 nm (N04), followed by 542–547 nm (N02) and 534 nm (N01).

Discussion

A comparison between human color vision phenotypes diagnosed by color vision tests and primate color vision genotypes (Osorio et al. 2004) suggests that squirrel monkeys (Saimiri) with a M/L opsin absorption peak of 535 nm or 562 nm behave similarly to a human protanope (missing L-cone) or deuteranope (missing M-cone), respectively, and that individuals with absorption peaks of 535 and 562 nm, 535 and 550 nm, or 550 and 562 nm can be compared to normal trichromat, protanomalous, or deuteranomalous humans, respectively. Thus, we can infer that male 2 and male 3 have M/L opsins with absorption peaks at 536 nm (although the result for male 3 is not as clear as that for male 2); male 1, female 3, and female 2 have opsins with absorption peak at 563 nm; and female 1 is probably a trichromat (the HRR test does not predict the trichromat genotype). Genetic analysis agreed with the color vision phenotypes derived from the HRR results. Female 1 is truly a trichromat; male 1, female 3, and female 2, which have opsins with peak absorption 560–563 nm, exhibited a strong deutan phenotype; and male 2 and male 3, whose opsin absorption peaks around 542–547 nm, are strong protans (alleles for which Osorio et al. [2004] made no predictions of behavior). Although we did not test any dichromat subjects expressing alleles with absorption peaks around 552 nm and 535 nm, we suspect that they would behave as protans. A plate-by-plate statistical analysis of performance might help to differentiate between individuals with the same HRR test classification. Cebus that are trichromats expressing opsins with distant absorption peaks, such as female 1, should discriminate all HRR test plates correctly, as do trichromatic humans. We cannot be sure, however, that heterozygous females expressing opsins with closer absorption peaks, i.e., 545/552 nm or 552/560 nm, would be classified as normal trichromats by the HRR test. It is possible that they would be classified as a weak deutan or a weak protan, a classification suggested to be similar to anomalous human trichromats (Neitz et al. 1996). It might be possible, therefore, to differentiate trichromat phenotypes this way, although there is no evidence that different trichro- mat phenotypes show different discrimination behavior in nature. The mean number of training sessions required by all tested subjects to perform >80 % correct responses was 11±3.05 sessions. However, the only trichromat needed just six sessions to achieve this performance. If this result is confirmed in other subjects it means that trichromats can be classified simply by the number of training sessions required to achieve the 80 % threshold. The major difference between the results provided here for the HRR test and those provided by the Ishihara test (Saito et al. 2003, 2005a, b) is the possibility of using Hard, Randy, and Rittler Test in Capuchins behavioral evidence for the qualitative distinction of dichromats (either deutan or protan). Thus, the HRR test appears to be a reliable and inexpensive method for the classification of primate color vision.

Acknowledgments We thank Cintia Carla da Silveira for helping to conduct the experiments; the Primate Center’s veterinary physicians Danilo Teixeira and Raimundo Silva de Oliveira for providing subject health care; and Geinaldo Vieira da Silva and Adão Pedro Nunes da Silva from the Primate Center for maintenance care of the monkeys. We received some important suggestions from Dr. Maria Clotilde Henriques Tavarez. We thank the reviewers for suggestions that improved the text and made it more accurate. T. S. Altavini was the recipient of a CAPES Master’s Scholarship; D. M. O. Bonci, a FAPESP Postdoctoral Fellowship (Processo 2011/17423-6); and D. F. Ventura, a CNPq Productivity Fellowship and Grant. The project received financial support from FINATEC and from FAPESP (Projeto Temático 2008/ 58731-2 and FAPESP 2009/06026-6).

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