honeybees
Dans ce papier, nous avons étudié les capacités de généralisation de couleurs des abeilles. Nous avons cherché à savoir comment deux gradients de généralisation de type excitateur, produits par des stimuli visuels récompensés, interagissent afin de déterminer une réponse de généralisation résultante.
Dans ce but, nous avons entraîné différents groupes d’abeilles avec une (groupes contrôle) ou deux couleurs récompensées (groupe expérimental). Nous avons ensuite observé les réponses des abeilles aux couleurs entraînées et à de nouvelles couleurs, interpolées ou extrapolées dans l’espace perceptif de couleurs de l’abeille, par rapport aux couleurs préalablement entraînées. Ceci nous a permit de comparer les performances de généralisation de groupes d’abeilles afin de voir comment interagissaient les deux gradients excitateurs.
Nos résultats montrent comment les abeilles généralisent leur choix à des couleurs nouvelles mais ne permettent pas de trancher de façon claire quant aux différents modèles d’interpolation et d’extrapolation proposés. Ils suggèrent, cependant, que les gradients de généralisation générés par chacun des deux stimuli récompensés s’additionnent linéairement pour donner un gradient de généralisation résultant. Nous avons également observé un résultat très intéressant qui montre des asymétries dans les gradients de généralisation, indépendantes de la distance perceptive entre stimuli. Ainsi, les abeilles ont regroupé comme similaires certains stimuli et non pas d’autres, à l’encontre même de leur distance perceptive. Ce résultat pourrait notamment suggérer l’existence de catégories de couleurs chez l’abeille.
Article I
The cognitive implications of asymmetric color
generalization in honeybees
Julie Benard & Martin Giurfa (*)
Centre de Recherches sur la Cognition Animale (UMR 5169), CNRS - Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 4, France.
(*)
Corresponding author: Mail: giurfa@cict.frTelephone: +33 561 55 67 33
Fax: +33 561 55 61 54
Keywords: Generalization; Vision; Color Vision; Honeybee; Color Categories
Published on Animal cognition (in press)
Abstract
Generalization occurs when a conditioned response formed to one stimulus is also elicited by other stimuli which have not been used in the course of conditioning. Here we studied color generalization in honeybees Apis mellifera trained to two rewarded colors, S1+ and S2+. After
training, bees were tested with non-rewarded novel stimuli, which lay between the trained stimuli in a honeybee color space (Int) or outside the range defined by the trained stimuli (E1
and E2). We analyzed whether bees interpolated their choice to Int and/or extrapolated it to E1
and E2. We compared the performances of the group trained with S1+ and S2+ to those of
control groups trained only with S1+ or S2+. Bees trained with S1+ and S2+ responded
similarly and highly to all test stimuli. These results do not allow discerning between generalization models based on the presence of interpolation and/ or extrapolation. Nevertheless, bee’s performance was consistent with a linear summation of the two generalization gradients generated by S1+ and S2+, respectively. These gradients were
asymmetric because control bees responded to the test stimuli as if these belonged to different similarity classes in spite of the fact that they had similar perceptual distances separating them. Stimuli treated as similar were located in the same half of the color spaces while stimuli treated as different were located in opposite halves. Our results suggest that color categories could exist in honeybees and may underlie the performance of the control groups. Under this assumption, color categories would be also present in simpler nervous systems, and would not require factors such as language to be expressed.
Article I
Introduction
Stimulus generalization is a behavioral phenomenon that is of paramount importance for understanding perceptual functions. First described and interpreted by Pavlov (1927) using classical conditioning, stimulus generalization has been studied extensively in humans and several animal species. In general, stimulus generalization is defined as "the behavioral fact that a conditioned response formed to one stimulus may also be elicited by other stimuli which have not been used in the course of conditioning" (Hilgard and Marquis, 1940). Generalization allows, therefore, treating different but similar stimuli as equivalent, thus promoting cognitive economy (Pavlov 1927; Shepard 1987; Ghirlanda and Enquist 2003). Such ability is fundamental as relevant natural signals seldom appear twice identically. Similarity along one or various perceptual dimensions determines the degree of generalization between stimuli (Shepard 1987). Determining such dimensions is fundamental for defining an animal’s perceptual space.
In a generalization experiment, an animal is conditioned to a given stimulus, and then presented in a test with novel stimuli that may or may not have elements or dimensions in common with the conditioning stimulus. If the animal does not respond to the same degree to the test stimuli, it is concluded that there are perceptual differences between the stimuli trained and the novel test stimuli. Usually, the response function of an animal in a generalization experiment follows a gradient in which maximal responses are expected for the conditioned stimulus and a progressive decrement of responses is observed for test stimuli as these become less similar to the trained stimulus (Guttman and Kalish 1956; Shepard 1958). The resulting generalization function is therefore often bell-shaped.
In natural conditions, animals do not learn a single stimulus but multiple stimuli, thus raising the question of how generalization gradients may combine in order to produce an adaptive response. Some of these gradients may be excitatory (i.e. the animal will tend to respond to stimuli similar to a stimulus signaling availability of reinforcement, S+) while other may be inhibitory (i.e. the animal will tend to avoid stimuli similar to a stimulus signaling lack of reinforcement, S-). Their intradimensional combination may give origin to the phenomenon of ‘peak shift’ (Hanson 1959; Lynn et al. 2005; Baddeley et al. 2007), which leads subjects to respond more to a test stimulus that is different than the S+ in the direction away from the S-.
Other studies have recently focused on how intradimensional excitatory gradients may combine in order to produce a generalization response. To reduce the level of complexity of this question, such studies have considered the situation in which an animal has been trained with only two stimuli, S1+ and S2+, which both signal availability of reinforcement (Enquist
and Johnstone 1997, Jones et al. 2001). Different models have been proposed to account for an animal’s response under such training circumstances (see Jones et al. 2001 for review). Some models predict that the animal presented with a novel stimulus lying between S1+ and
S2+ in a perceptual space will interpolate, thus choosing equally or more this novel stimulus.
Other models posit that animals interpolate and extrapolate (i.e. generalize their response to stimuli lying beyond the range defined by S1+ and S2+). Jones et al summarized these
possibilities in four basic models: 1) linear summation of the generalization gradients generated by S1+ and S2+ (red and blue Gaussian distributions in Fig. 1, respectively); in this
model (Spence 1937; Enquist and Johnstone 1997), neither interpolation (i.e. a heightened response to intermediate test stimuli) nor extrapolation is expected beyond the gradients of S1+ and S2+ (see green distribution in Fig. 1a); interpolation may occur only if S1+ and S2+
are not too different (Enquist and Johnstone 1997); 2) estimation of a continuous Gaussian distribution to which S1+ and S2+ would belong (Fried and Holyoak 1984; see green
distribution in Fig. 1b); if S1+ and S2+ are separated in the stimulus continuum, one should
observe higher interpolation (the animals should prefer the interpolated stimulus to S1+ and
S2+) and high extrapolation (variability should be large, giving extrapolation beyond the
limits set by S1+ and S2+); 3) weighted average of all possible models consistent with S1+ and
S2+ (Tenenbaum and Griffiths 2001); this model (see green distribution in Fig. 1c), proposed
in a general Bayesian framework, generates uniform interpolation between S1+ and S2+ and
low or no extrapolation beyond them; 4) same as 3) but adding internal noise, which results in higher interpolation (the animals should prefer the interpolated stimulus to S1+ and S2+) and
low or no extrapolation (not shown).
Here we have studied stimulus generalization after training to two S+ using the honeybee Apis mellifera as a model, and color vision as the perceptual framework for our work. Honeybees constitute a good model for addressing the question of color generalization because they are flower constant pollinators that exploit the same floral species as long as it provides nectar and/or pollen reward (Grant 1951; Chittka et al. 1999). Floral constancy is based on the bees’ ability to learn and memorize distinctive features of flowers, among which colors play an essential role (Grant 1951; Hill et al. 1997; Chittka et al. 1999). This capacity is
Article I
amenable to the laboratory as bees can be easily trained to choose a given color target, associated with a drop of sucrose solution (Benard et al. 2006). Since the discovery that bees see colors (von Frisch 1914) much research has been performed to characterize the bees’ color vision both at the behavioral and physiological level (Menzel and Backhaus 1991). 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 (Wakakuwa et al. 2005), 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 (Peitsch et al. 1992). These photoreceptors feed into different types of color opponent neurons present in the visual neuropiles of the honeybee brain (Kien and Menzel 1977; Yang et al. 2004).
Previous studies have focused on color discrimination capacities in honeybees, either to determine a Δλ function, i.e. stimulus discriminability as a function of wavelength (von Helversen 1972) or to relate these capacities to the conditioning procedure employed (Giurfa 2004). Although generalization to colors after training to a neutral stimulus (an evenly reflecting aluminum disk) has been studied in bees (Giurfa 1991), no study has, to our knowledge, trained bees with two rewarded colors (S1+ and S2+) in order to study
systematically color generalization in terms of interpolation or extrapolation. Here we trained honeybees with two color stimuli and tested them with novel stimuli in color generalization tests. These novel stimuli lay between the trained stimuli in a honeybee color space, so that generalization, if any, would reflect an interpolation performance. Alternatively, the novel stimuli lay outside the range defined by the trained stimuli, so that generalization, if any, would reflect an extrapolation performance (Jones et al. 2001).
Methods
Free-flying honeybees, Apis mellifera L., were trained to collect 30% (w/w) sucrose solution on a feeder 30 m distant from the hive. Foragers were individually marked with a colored spot on the thorax or the abdomen and trained to fly into the experimental setup to collect 50% sucrose solution (weight/weight) therein. Only one bee was trained and tested at a time.
Experimental Setup
The setup was a wooden Y-maze with a UV-transparent Plexiglas ceiling to ensure natural daylight conditions within the maze (Srinivasan and Lehrer 1988; Giurfa et al. 1996). A sliding door guaranteed that only one bee at time could enter the maze. Once in the maze, the bee had to pass through an entrance hole (5 cm in diameter) in the middle of a frontal panel to enter into the decision chamber. Only when the insect was in this small chamber, could it see both back walls of the maze simultaneously. The back walls measured 20 x 20 cm and had a central orifice, 0.6 cm in diameter, through which sucrose solution could be delivered.
In one of the arms, a rewarded colored stimulus was presented vertically on a grey background. A bee landing on it was rewarded with sucrose solution. In the other arm, the grey background alone was visible and non-rewarded. The two arms were interchanged after one or two visits in a pseudorandom way to ensure that the bees did not associate the reward with any particular side.
Stimuli
In our two experiments, the reflectance spectra of each colored stimuli was measured by means of a S2000 Miniature Fiber Optic Spectrometer from Ocean Optics, sensitive in the range from 200 to 1100 nm. Because there is a dispute concerning the appropriateness of the two main color spaces used to represent honeybee color vision, the color opponent coding space (COC space; Backhaus 1991) and the color hexagon (Chittka 1992), we have represented colors and evaluated color similarity in both spaces in order to avoid sterile discussions. The results of our work were unaffected by the kind of space used.
Article I
Each colored stimulus was presented on a grey background (20 x 20 cm) cut from HKS-92N paper (K + E Stuttgart, Stuttgart-Feuerbach, Germany). This grey background was common both to rewarded and non-rewarded stimuli so that it had no predictive value in terms of its outcome. It could therefore not introduce inhibitory biases resulting in peak shift (see Introduction). In Experiment I, five different colored stimuli were used, referred as E1
(extrapolated 1), S1+ (trained 1), Int (interpolated), S2+ (trained 2) and E2 (extrapolated 2).
The loci of the color stimuli in the color spaces are depicted in Fig. 2 (COC space: Fig. 2A; color hexagon: Fig. 2B).
Color stimuli were printed using a high quality laser printer Xerox Phaser 860. Irrespective of the color space used, S1+ and S2+ defined a spectral range in the middle of
which was Int. E1 and E2 were in opposite directions beyond the limits set by S1+ and S2+.
Colors that were adjacent in the color space had approximately the same perceptual distance between them (Table 1).
In Experiment II, we used only three different colors referred as S1+ (trained 1), Int
(interpolated) and S2+ (trained 2), whose position in the color space is depicted in Figure 3
(COC space: Figure 3A and Figure 3B: color hexagon). The colored stimuli and the background were both cut out of HKS-N pigment papers. The color stimuli used were cut from HKS-50N (S1+), HKS-1N (Int) and HKS-3N (S2+) paper while the grey background was
cut from HKS-92N paper as in Experiment I. Int lay again between S1+ and S2+ in the color
space but the perceptual distance between adjacent stimuli was larger than in the first experiment (see Table 1).
In both experiments, control groups were trained with just one color stimulus (either S1+ or S2+). The experimental group was trained with a compound stimulus S1+/S2+ made of
S1+ and S2+. In this way, bees of the experimental group always experienced both S1+ and
S2+ simultaneously rewarded. When presented alone, S1+ and S2+ were colored squares each
having an area of 64 cm² (8x8 cm). The compound S1+/S2+ was a square with an area of 128
cm² as it was made of two adjacent rectangles, S1+ and S2+ each presenting an area of 64 cm²
(11.3x5.66 cm; see Fig. 4).
Training
We performed only individual training and testing, i.e. a single bee was present in the apparatus at a time. In this way, the experience of each individual bee on the training stimuli was precisely controlled (Giurfa 2007). In each experiment, bees were trained during 20 learning trials. Only the first choice of a trial was considered for computation of acquisition curves.
When the bee chose the rewarded stimulus (control groups: S1+ or S2+; experimental
group: compound S1+/ S2+), it could drink the sucrose solution ad libitum (60 µL) and
resumed afterward its foraging trip, thus returning to the hive. When the bee performed a wrong choice and flew into the arm presenting only the grey background, it was gently tossed away from the maze such that it had to enter again to get the sucrose solution. The procedure was repeated until the bee chose correctly the rewarded stimulus in that foraging bout (in this case, only the first choice was recorded as wrong).
In Experiment I, one group (group 1, n = 6) of bees was trained with color S1+
rewarded in one arm of the maze versus the non-rewarded grey background in the other arm of the maze. Another group (group 2, n = 6) was trained with color S2+ versus the grey
background. A third group (experimental group, n = 12) was trained with the compound stimulus S1+/S2+ versus the grey background (Fig. 4). The spatial orientation of the
compound stimulus S1+/S2+ varied randomly during training (i.e. changing the orientation of
its color components) in order to avoid that bees learned a specific spatial layout of colors. In Experiment II (Fig. 4), one group (group 1, n = 6) was trained with color S1+ versus
the non-rewarded grey background. Another group (group 2, n = 6) was trained with color S2+ versus the grey background and a third group (experimental group, n = 12) with the
compound stimulus S1+/ S2+ versus the grey background.
Tests
Tests were performed after completing the 20 conditioning trials and were identical for all groups of bees. Bees were tested in extinction conditions (no reward offered) in a dual choice situation. We recorded the first choice in a test and the cumulative number of touches for each stimulus during one minute (flights toward a target that ended with a contact of the target with the antennae of the bee; Giurfa et al. 1999).
Article I
In Experiment I, bees of groups 1 and 2 and of the experimental group experienced five tests in a random sequence which varied from bee to bee. Bees were tested with S1+,
S2+, Int, E1 and E2. All stimuli were presented versus the grey background in the alternative
arm of the maze (Fig. 4). In Experiment II, bees of all three groups (1, 2 and experimental) were tested with S1+, S2+ and Int. All stimuli were presented versus the grey background in
the alternative arm of the maze (Fig. 4).
Each test was repeated twice, alternating the sides of the test stimulus from one test to the next. Between each test, four refreshment trials were given to keep high the bees’ motivation to visit the maze after they were exposed to the non-rewarded tests. Fresh stimuli were used in all tests to ensure that test performances were not influenced by scent marks.
Statistical analysis
Proportion data used for representing acquisition curves were transformed using the arcsin √p transformation in order to normalize them. In this way, repeated measurement ANOVA could be used to test whether variation of acquisition curves was significant. We performed a one-factor ANOVA (factor: trial block, with four levels) to determine whether performance changed significantly during the training. Post hoc comparisons were done by means of the Tukey HSD test.
We analyzed the percent of correct touches recorded during the tests. These data were previously transformed using the arcsin √p transformation, and a Student’s t-test for single sample was applied to test whether the bees’ performance differed from a theoretical value of 50%. We analyzed the test performances of both the control groups trained with one color (S1+ or S2+), which showed the generalization gradient corresponding to their conditioning
stimulus, and those of the experimental group trained with both S1+ and S2+, which showed a
generalization gradient resulting from their experience with both rewarding colors. To this end, we used an ANOVA for repeated measurements for each group’s performance. In all analyses, the level of significance wasset at 0.05.
We used non-linear regression and dynamic fitting to adjust best Gaussian models to the test performances of bees trained with S1+ ( group 1) and with S2+ (group 2), both in
Experiments I and II. All data point (i.e. all choice proportions from all bees tested) were used to this end. An ANOVA corrected for the mean of observations was used as a measure of
goodness of fit between the models curves and our data points.
Results
Experiment I
The performances of the three groups of bees (two control groups, 1 and 2, n = 6 for each group, and one experimental group, n = 12) during the training are shown in Fig. 5 (left panel) in terms of the percent of correct choices along 4 blocks of 5 consecutive trials. There was a significant increase in the percentage of correct choices during the blocks of training for each group (repeated measurement ANOVA; group 1: F3,15 = 14.40, p < 0.001; group 2 : F3,15
= 6.72, p = 0.004; experimental group: F3,33 = 13.73, p < 0.001). The acquisition
performances of the three groups of bees did not differ significantly (F2,21 = 2.93, p = 0.08). In
each case, the mean performance in the last training block was significantly different from that in the first block, thus indicating that with ongoing training bees learned to choose the appropriate stimulus, S1+ (group 1), S2+ (group 2) or S1+/S2+ (experimental group).
Figure 5 (right panel) shows the performances of the three groups during the tests in which they were presented with their respective conditioning stimulus and with the novel stimuli. Bees of group 1 (black dots), which were trained with S1+, responded maximally to
S1+ but also generalized their choice to E1. These bees did not generalize their choice to Int,
S2+ and E2. An ANOVA for repeated measurements applied to the test performances showed
that bees of group 1 treated S1+ and E1 as equivalent and Int, S2+ and E2 as equivalent but
different from S1+ and E1 (F4,20 = 9.57, p < 0.001; post-hoc comparisons Tukey HSD) .
Bees of group 2 (white dots), which were trained with S2+, responded maximally to
S2+ but also generalized their choice to E2 and Int. These bees responded differently to the
stimuli tested (F4,20 = 28.78, p < 0.001; post-hoc comparisons Tukey HSD). Indeed, they
responded equally and differently from a random level to S2+, E2 and Int (S2+: t5 = 9.12, p <
0.001; E2 : t5 = 3.72, p = 0.014; Int: t5 = 3.84, p = 0.012) while their response to S1+ and E1
was also equivalent but not different from a random level (E1 : t5 = -2.51, p = 0.06; S1+: t5 = -
1.20, p = 0.28).Thus, the extent of the generalization gradients generated by S1+ and S2+ was
dissimilar.
Article I
Finally, the bees of the experimental group, trained with the compound S1+/S2+