Ce travail a été motivé directement par notre étude précédente sur la généralisation de couleurs. Dans ce papier, nous avons voulu savoir si les abeilles étaient capables de diviser le continuum du spectre de longueurs d’ondes, générant ainsi des catégories de couleurs. Nous avons voulu voir si les abeilles pouvaient établir deux catégories assez larges, « jaunâtre » et « bleuâtre » à l’œil humain, correspondant respectivement à des catégories « moyenne longueur d’ondes » et « longue longueur d’ondes » pour l’abeille.
Dans une première série d’expériences, nous avons vérifié que les abeilles étaient parfaitement capables de discriminer entre les différentes couleurs utilisées appartenant à une même catégorie. Pour cela nous avons entraînées les abeilles de façon différentielle, avec les différentes couleurs utilisées. Dans une seconde série d’expériences, nous avons entraîné les abeilles, toujours de façon différentielle, à catégoriser les couleurs « jaunes » vs. « bleu ». Nous avons ensuite testé leurs performances de transfert vers de nouveaux stimuli préservant la différence perceptive « jaunes » vs. « bleu ». Les abeilles ayant réussi le transfert, nous avons réalisé une troisième expérience afin de démontrer de façon claire l’existence de catégorisation de couleurs : nous avons comparé les performances des abeilles dans une tâche de discrimination impliquant 2 couleurs d’une même catégorie (intra-catégorielle, jaune vs. jaune) et dans une tâche de discrimination impliquant 2 couleurs de catégories différentes (inter-catégorielles, jaune vs. bleu). Les couleurs choisies sont dans tous les cas discriminables et présentaient la même distance perceptive entre elles.
Nos résultats montrent que la discrimination était facilitée lorsque les 2 couleurs appartenaient à des catégories différentes (bleu vs. jaune), malgré le fait la différence perceptive entre ces couleurs était la même séparant les deux couleurs de la même catégorie (jaune vs. jaune). Ainsi, nous montrons que les abeilles sont effectivement capables de catégoriser les couleurs utilisées en deux catégories « jaunâtre » vs. « bleuâtre ».
Article II
Color Categories in an Insect: Studies on Honeybees
Julie Benard and Martin Giurfa (*)
Centre de Recherches sur la Cognition Animale (UMR 5169), CNRS - Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 9, France.
Running head: Color Categorization in Honeybees
(*)
Corresponding author: Mail: giurfa@cict.frTelephone: +33 561 55 67 33 Fax: +33 561 55 61 54
To be submitted to Proceedings of the National Academy of Sciences
Summary Background
Humans categorize colors as they divide the physical continuum of the color spectrum into discrete classes. Color categorization has been the subject of intense debates opposing ‘nature’ vs. ‘nurture’ arguments. These debates have focused, for instance, on the determination of color categories by physiological constraints in the visual system as opposed to the notion that color categories may be related to the presence of language as structuring facror. Investigating whether or not an insect endowed with trichromatic color vision and color learning capabilities can categorize colors beyond simple color generalization may represent an important contribution to this debate.
Methodology / Principal Findings
Here we studied whether honeybees can be trained to categorize bluish vs. yellowish stimuli that cluster in different regions of the honeybee color space. We show that bees trained with 16 successively-changing bluish vs. yellowish color pairs transferred appropriately their choice to novel bluish or yellowish stimuli that were distinguishable from the training stimuli. Thus, bees assigned the novel colors to the appropriate color classes in a performance consistent with color categorization. As expected for a categorization task, a sharp boundary was found between the bluish and the yellowish classes. Moreover, a critical test was performed to test whether color discrimination was better between colors belonging to different categories than between colors belonging to the same category even when the perceptual distance between these colors was the same. We found that, in the absence of such differences, color discrimination was better in between-category stimuli than in within- category stimuli.
Conclusions
Our results show that an insect can master a color categorization task despite having a relatively simple nervous system. Bees treated bluish and yellowish stimuli as belonging to different classes. Such a performance could not be accounted for by color distance as categorization occurred even under conditions of equal distance between test stimuli. We conclude that the presence of color categories is a basic feature, also present in simpler nervous systems, and which does not require structuring factors such as language to be expressed.
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Introduction
Categorization, the ability to group distinguishable objects or events in classes on the basis of a common attribute or set of attributes [1, 2, 3], is a fundamental cognitive ability as it allows overlooking differences for the sake of generality. Categorization differs from generalization, the transfer of a response from one stimulus to a similar one [4, 5] because in generalization a gradual decrease of responding along a perceptual similarity scale can be observed, while sharper boundaries (not gradual transitions) are found between categories [6, 7]. Within a category, stimuli are treated as similar even though they are in principle, distinguishable [8].
The categorization of colors has deserved wide attention because of the relative ease with which Humans divide the physical continuum of the visual spectrum in discrete classes (e.g. blue, green, red, etc). Color categorization has been the subject of much debate opposing “nature vs. nurture” arguments [9, 10]. This debate has focused, for instance, on the determination of color categories by physiological constraints such as color opponent processes in the visual system [11] as opposed to the notion that color categories may develop through experience. In the light of this discussion, the existence of well-controlled color conditioning protocols in animals that are commonly used as models for the study of color vision allows studying the role of experience in color categorization, providing that this capacity can be demonstrated.
Honeybees Apis mellifera provide a good model for addressing the question of color categorization because they are flower constant pollinators [12, 13] that exploit the same floral species as long as it provides nectar and/or pollen reward. Floral constancy is based on the bees’ ability to learn and memorize distinctive features of flowers [14], among which visual cues play an essential role [15, 16]. Honeybees can categorize black and white visual patterns as they learn to choose to similar, distinguishable patterns and group such stimuli into defined categories [see 17 for review]. Although the ability to categorize achromatic patterns has been shown in honeybees, no study has tackled so far the issue of color categorization [18] despite the fact that bees see colors and that their color vision has been extensively studied at the behavioral and physiological level [19]. Honeybees are trichromats and see colors in the range from 300 nm to 650 nm. Three kinds of photoreceptors have been identified in the bee retina [20], which are maximally sensitive in the ultraviolet (S receptor: 344 nm), blue (M: receptor: 436 nm) and green (L receptor: 544 nm) regions of the spectrum, respectively [19] (Fig. 1A). Color opponent neurons processing color information at a central
level have also been found in honeybees [21, 22] although the kind of opponent processing underlying color vision in bees remains unclear.
Here we ask whether honeybees learn to categorize colors so that they discriminate between color classes but generalize within classes of otherwise distinguishable stimuli. We show that bees extensively trained with several bluish vs. yellowish pairs learned to assign correctly novel, distinguishable stimuli to the appropriate color category. The boundary between these two color categories was sharp and separated bluish from yellowish stimuli. Accordingly; it was easier for the bees to distinguish between bluish and yellowish than between two yellowish targets even if the distance separating these alternatives was the same in the bee color space. Our results suggest that even simple brains can group color stimuli in functional classes.
Results
Experiment 1: Bees discriminate within color classes
A prerequisite for categorization experiments is to show that animals are capable of discriminating stimuli within a given category. Otherwise, generalization between members of the category may simply reflect a failure in discrimination and not real categorization. In our experiments, we established two potential color classes using ten color stimuli, five of which were bluish and the other five yellowish to the human eye (Fig. 1B; Table 1). In two different perceptual color spaces postulated for the honeybee, the color opponent coding space [23] and the color hexagon [24], both groups of stimuli occupied distinct areas (see Fig. 1C for the color hexagon and Fig. S1 in supporting information). In both spaces, bluish stimuli cluster in the upper quadrants while yellowish stimuli cluster in the lower quadrants. Figure 1a,b and Table 1 show that bluish stimuli excited predominantly the M (‘blue’) photoreceptor type (mean receptor excitation: 0.44 ± 0.03; mean ± S.E.) while yellowish stimuli excited predominantly the L (‘green’) photoreceptor type (0.57 ± 0.07; mean ± S.E.) .
We studied whether or not honeybees discriminated colors within the bluish and the yellowish classes. Bees were trained to collect sugar solution on a dual-choice setup [25, 26] allowing the simultaneous presentation of two colors, both bluish or both yellowish, one rewarded with sucrose solution (indicated as ‘+’) and another non-rewarded (indicated as ‘-’). Within each color class, ten color pairs were trained and tested using an independent group of bees for each pair. Experiments were balanced as, on average, 3 bees experienced one
Article II
reinforcement contingency (e.g. Bluish 1+ vs. Bluish 2-) and 3 other bees experienced the reversed contingency (e.g. Bluish 1- vs. Bluish 2+). Thus, on average, 6 bees were trained with each color pair (118 bees were studied in total).
Bees discriminated all stimuli within the bluish and the yellowish classes. They significantly chose the stimulus previously rewarded, independently of the color trained and of the non-reinforced color presented as alternative. Figure 2 shows that the bees’ color choice was therefore significantly different from a random choice proportion of 50% (Student’s t test: p < 0.05 for all 20 discriminations). Representing color choices as a function of color distance in the hexagon (Fig. 2) shows that more distinct colors (colors that are more separated in a color space) were better discriminated than colors that were more similar [27] (Table 2). This result is independent of the color space considered as an identical trend is found using the color opponent coding space (Fig. S2 in supporting information). Thus, honeybees could distinguish between all bluish and between all yellowish stimuli employed in our experiments.
Experiment 2: Bees learn to categorize bluish vs. yellowish colors
We next studied whether bees can group colors according to their spectral nature. Bees were trained in a dual-choice set-up with a random succession of color pairs, bluish vs. yellowish. For the training, four bluish and four yellowish stimuli were randomly arranged in all possible combinations so that each bee experienced 16 color pairs along 32 trials (each pair was presented twice). After completing the training, each bee was presented in a transfer test with the two remaining bluish and yellowish stimuli. These test stimuli were never experienced before and were non-rewarded. The novel color pair was different for each bee. Experiments were balanced as one group of bees was rewarded with sucrose solution on the bluish but not on the yellowish stimuli (n = 10), while another group received the reversed contingency (n =10).
Learning was similar for both groups of bees (2-factorial ANOVA for repeated measurements; F1,18 = 0.008, NS) so that acquisition curves were pooled and presented as a
single curve in Fig. 3A (black dots). This curve shows that bees increased significantly their percentage of correct choices during the four blocks of eight trials (1-factorial ANOVA for repeated measurements; F3,57 = 20.4, p < 0.0001), thus showing that they learned to
differentiate the bluish vs. the yellowish pairs (Fig. 3A). Performances of the two groups during the transfer tests were not different (difference between two proportions, p = 0),
therefore we pooled the data and presented them in Fig. 3B (black bar). In the transfer tests, bees preferred significantly the novel color belonging to the class previously rewarded (Fig. 3B; black bar; Student’s t test: t19= 8.72, p < 0.05), i.e. bees rewarded on bluish colors chose
the novel bluish stimulus while bees rewarded on yellowish colors chose the novel yellowish stimulus. Appropriate transfer to these novel colors was not due to a lack of discrimination as shown by the previous experiments. Therefore, bees grouped correctly new, distinguishable stimuli according to their bluish or yellowish nature consistently with a categorization performance.
In order to demonstrate that bees categorized colors and were not following simple generalization rules, we investigated whether or not a sharp boundary existed between color categories. In order to visualize the eventual presence of a boundary, we represented the performance of the bees measured in the transfer tests as a function of the position of the colors tested along the vertical opponent axis. This axis was chosen given that stimuli were mainly distributed along it (which was not the case for the horizontal axis), irrespective of the color space considered, thus yielding a linear scale for the representation of the performance of the bees. Figure 4A shows the detailed performance of bees in the transfer test using the Y axis of the color hexagon (see Fig. S3, upper panel, in supporting information for a similar representation using the B/UV Green opponency axis of the color opponent coding space). The blue curve corresponds to bees rewarded on bluish stimuli during the training; the green curve to bees rewarded on yellowish stimuli during the training. Each of the first five points of the blue curve shows the proportion of choices for a novel yellowish stimulus in the transfer test, while the last five points correspond to the choice of a novel bluish stimulus. For instance, the last point of the blue curve corresponds to the choice of two bees that in the transfer test were presented with the same correct (i.e. belonging to the category trained) bluish stimulus (Bluish 5 in Fig. 1C and Table 1) against two different yellowish stimuli (Yellowish 5 for one bee and Yellowish 3 for the other bee). On the same blue curve, the first point represents the choice of two bees that in the transfer test were confronted with the same incorrect (i.e. not belonging to the category trained) yellowish stimulus (Yellowish 1) against two different bluish stimuli (Bluish 2 for one bee and Bluish 3 for the other bee).
Bees rewarded on bluish stimuli responded significantly more to novel bluish stimuli while the opposite was found for bees rewarded on yellowish stimuli (Fisher Exact test: p < 0.00001). The transfer choices of the bees revealed the presence of a sharp boundary close to the 0 value (Fig. 4 and Fig. S3 in supporting information), as expected for a categorization performance [7].
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Experiment 3: within vs. between category discrimination
A critical experiment for deciding whether or not animals possess color categories implies comparing discrimination performances within vs. between categories. If the color pairs to be compared are separated by the same distance in a color space, one expects better discrimination between categories than within a color category. The conclusion is even more flagrant if the distance separating the well-distinguished colors of different categories is smaller than that between colors of the same category.
In this experiment, we trained two groups of 10 bees in parallel. One of them had to perform a “within-category” discrimination (Yellowish 1 vs. Yellowish 5) while the other had to perform a “between-category” discrimination (Yellowish 5 vs. Bluish 1). The latter colors were chosen because they lay at the borders of the hypothetical category boundary (see Fig. 4). Yellowish 1 and 5 were then chosen because they were separated by practically the same distance (COC space: 5.42; color hexagon: 0.26) in the color spaces as Yellowish 5 and Bluish 1 (COC space: 5.09; color hexagon: 0.2). As can be seen from the exact distance values, Bluish 1 and Yellowish 5 were even closer to each other than the two Yellowish stimuli. Thus, if a color discrimination were to be more difficult for the bees, it should be that involving Yellowish 5 vs. Bluish 1.
Bees were trained to discriminate these color pairs in a dual choice set-up along 30 trials. Group I was trained to solve the “within-category” discrimination (Yellowish 1 vs. Yellowish 5). Half of the bees were trained with Yellowish 1 rewarded vs. Yellowish 5 non- rewarded (Yellowish 1+ vs. Yellowish 5–) while the other half experienced the reversed contingency (Yellowish 1– vs. Yellowish 5+). Group II was trained to solve the “between- category” discrimination. Half of the bees were trained with Bluish+ vs. Yellowish 5– while the other half experienced Bluish– vs. Yellowish 5+. Once the training was complete, bees were presented with fresh non-rewarded stimuli in a retention test. We compared the acquisition and the test performances of the two groups in order to reveal if, at equivalent perceptual distance, two colors belonging to different categories are better discriminated than two colors belonging to the same category.
The two groups of bees learned their corresponding discrimination task. Fig. 5A shows that in both cases the percentage of correct choices increased significantly during the three blocks of trials (1-factorial ANOVA for repeated measurements, Group I: F2,18 = 7.83,
p=0.004; Group II: F2,18 = 19.97, p=0.00003). However, the two groups of bees did not reach
the same level of correct choices at the end of training (F1,18 = 7.38, p = 0.01; post-hoc Tukey
HSD difference between the last block of training of the two groups: p = 0.038): bees trained with the within-category discrimination reached a significantly lower level of correct choices than the bees trained with the between-category discrimination. A similar result was found in the test performed after the last acquisition trial (Fig. 5B): although the performance of both groups in the test was different from a random choice, bees of group II responded significantly better than bees of group I (t18 = -3.85, p =0.001). These results show, therefore,
that the discrimination between Yellowish 5 and Bluish 1 was easier than that between Yellowish 1 vs. Yellowish 5 in spite of the fact that the latter were more distant in the bee color spaces. This result supports the previous conclusion that a sharp boundary exists between Yellowish 5 and Bluish 1. This boundary could be represented by the horizontal axis of the color space, which allows segregating our stimuli in two categories, bluish and yellowish or intermediate and long wavelength.
Discussion
Our results show that honeybees learned to group and discriminate color stimuli on the basis of intermediate (bluish) vs. long (yellowish) wavelength information when specifically trained to do so. As a consequence of this learning, bees transferred their choice to novel stimuli that they never saw before but that preserved the distinction bluish vs. yellowish (Experiment 2). This transfer occurred even if the novel stimuli that were correctly assigned either to the bluish or the yellowish category were in principle distinguishable from those that were used in the training (Experiment 1). In other words, bees treated all bluish or all yellowish stimuli as being equivalent, even if they could discriminate them. This result is consistent with the presence of two color categories, bluish and yellowish. Indeed, we found a sharp boundary between these two stimuli classes, as expected in categorization tasks (Experiment 2). Indeed, the existence of such boundary was confirmed by the finding that discriminating between the bluish and the yellowish colors at the borders of it was easier than discriminating two yellowish colors that were nevertheless more separated in the color space, and therefore, in principle, more distinguishable (Experiment III).
Our control experiments (Experiment 1) showed that bees discriminated between all stimuli belonging to the same class, either bluish or yellowish. This result is important as it shows that grouping within a class was not the consequence of a lack of discrimination. Such a high discrimination level was expected because bees trained under differential conditioning protocols achieve very fine color discriminations [28].
Article II
In our experiments bees were trained to categorize colors such that it is difficult to discuss whether categories evinced in our work are natural or experience-dependent. Even in Experiment III in which no category training was performed and yet differences in learning and retention were evinced between the within-category and the between-category color pairs, bees were not naïve foragers such that there was no control of their previous experience with flowers and colors in the field. Thus, the boundary found between yellowish and bluish colors could reflect physiological constraints or be experience-dependent. In favor of the first