How do bumblebees first find flowers? Unlearned approach responses and habituation

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1 ANIMAL BEHAVIOUR, 2004, 67, 379e386 doi: /j.anbehav How do bumblebees first find flowers? Unlearned approach responses and habituation VIRGINIA SIMONDS & C. M. S. PLOWRIGHT School of Psychology, University of Ottawa (Received 9 April 2002; initial acceptance 25 July 2002; final acceptance 10 March 2003; MS. number: A9328) To examine how bees distinguish between possible food sources and nonrewarding objects in the absence of previous experience with flowers, we presented flower-naïve bumblebees, Bombus impatiens, with unrewarding stimuli (colours or patterns) in a radial arm maze and compared their approach responses to each stimulus. Bees showed a significant preference for yellow and blue over other colours, and for radial patterns over concentric patterns or unpatterned discs. Habituation was demonstrated when the proportion of choices for the same pattern by the same bees decreased over two testing sessions. When an attractive novel pattern was presented in the third session, the trend was reversed. The results of this study confirm both that truly flower-naïve bees have unlearned colour and pattern preferences and that learning not to approach stimuli occurs in the absence of reward. Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Worker bees provide for themselves and their colony by foraging for nectar and pollen. Much of the literature on bee foraging behaviour has focused on foraging efficiency and how bees might achieve it. But before a worker bee can bring food back to the colony and learn how to find more, she faces the problem of identifying possible food sources: how do bees find flowers in the first place? In other words, how is it that they tend to approach flowers rather than cars, mailboxes, cows, rocks, and so forth. Although this question has been raised frequently, at least since Manning s (1956) study, it has received little attention in the literature until recently (see below). The behaviour of a worker on her very first foraging trip, before she has obtained any rewards from flowers, might be termed exploratory behaviour. We use the term here not to signify exploration in the teleological sense that bees have any conscious goal of information seeking, but rather in the sense that their behaviour serves to expose them to the conditions that are most likely to yield reinforcement and lead to subsequent learning of what is important for them (Shettleworth 1998). We examined two aspects of exploratory behaviours in bumblebees, Bombus impatiens (Hymenoptera: Apidae). Because bees perform these behaviours before obtaining a reward from flowers, they can be described as unlearned behaviours (a seldom used alternative expression is prefunctional ; Hogan 1988). The first aspect of exploratory behaviour that we examined is approach by the bees to particular colours and patterns. Some of the impetus for Correspondence: C. M. S. Plowright, School of Psychology, University of Ottawa, Ottawa, ON K1N 6N5, Canada ( cplowrit@uottawa.ca). 0003e3472/03/$30.00/0 this part of the research stemmed from our own anecdotal observations of bees landing on illustrated packets of flower seeds, blue and white patterned soccer balls, and clothing with floral prints. In our laboratory, a photograph of a flower elicited the strongest possible response from a naïve bee: approach, landing and proboscis extension. These unrewarding objects seem to have been mistaken for flowers, and so we began to investigate whether they had fooled the bees by virtue of some particular characteristics, such as their colour or pattern. The second aspect of exploratory behaviour that we examined was possible habituation to stimuli with these characteristics. Even if bees have unlearned preferences for some floral characteristics, these preferences would be expected to wane after a few unrewarded visits. Unless the preferences were modifiable, the bees might persist indefinitely in their unrewarding behaviours and never find food. While the overriding interest in bee behaviour has often been on learned behaviour, recently, the question of unlearned behaviour has received attention. This research has suggested, for example, that flower-naïve honeybees, Apis mellifera, have unlearned colour preferences for blue and green (Giurfa et al. 1995) and pattern preferences for radial flower-like patterns (Lehrer et al. 1995). These studies and others before them, however, have used methods that could have potentially interfered with an accurate assessment of unlearned responses, because they typically involved rewarding the bees in the experimental apparatus, such as a Y maze or a radial arm maze, before testing them. For example, bees might be rewarded for approaching an ostensibly neutral stimulus, such as a black and white checkerboard or a grey disc, and then 379 Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

2 380 ANIMAL BEHAVIOUR, 67, 3 tested for preferences on new colours or patterns where all the new stimuli were thought to be equally similar or dissimilar to the neutral ones. Using this approach, it is not possible to rule out the possibility that the rewarded experience on the neutral stimuli may have affected the behaviour on the new stimuli; indeed, in explicit tests of stimulus generalization, some colour choices by bumblebees are controlled by generalization from learned colours (Gumbert 2000). Even if there were no training on neutral stimuli, and bees were tested for their choices of equally rewarding stimuli, reward could still interfere with the assessment of unlearned preferences: behaviour after the first visit might be influenced by the first reward experienced. Even one rewarded visit on any one stimulus might in principle have a self-amplifying effect: a bee might return to that stimulus because it was previously rewarded there, and the other stimuli might remain unvisited even though they contain undiscovered reward. One way to obviate these difficulties is not to use reward at all in the testing apparatus. Unlearned ( innate ) preferences of bumblebees have been investigated in this way (Lunau 1990, 1991, 1992; Lunau & Maier 1995; Lunau et al. 1996) by testing the bees reactions to artificial flowers in a flight cage. Lunau (1990) found, for example, that optical flower signals, such as gradients of spectral purity, elicited approach flight from 1 m and antennal touching. Although this method rules out any associative learning from training stimuli, the dependent measure of approach flight can itself be intractable. At a distance of 1 m, it may be difficult to discern whether a bee is actively approaching the test stimuli or whether it is simply in free flight. In the present series of experiments, we used a combination of the methods described above to test for unlearned behaviour in bumblebees: bees were tested in a radial arm maze, which provided a clear-cut and easily measurable dependent variable of entrances into a corridor, but unlike previous maze studies, the bees experienced no reward in the maze. We addressed the following two questions. (1) Do naïve bumblebees have preferences for pattern and colour? (2) If so, are these preferences altered by repeated exposure? Subjects GENERAL METHODS Five colonies of bumblebees, B. impatiens Cresson, were used for the full series of experiments. The colonies were raised in the laboratory from captured queens according to the procedure developed by Plowright & Jay (1966). All bees were identified by gluing numbered labels of various colours on to the thorax. In the colony, the bees were provided with an ad libitum supply of pollen and honey solution (honey and water 2:1 by volume). Materials and Apparatus Each colony was housed in a wooden domicile (30! 15! 15 cm). The domicile was attached to a screened flight cage (1:83! 1:83! 1:83 m) by a wooden walkway (33 cm). Glass plates were placed on the top of the walkway with two vertical gates to allow easy identification and corralling of bees leaving and entering the colony. Three panels of fluorescent light fixtures illuminated the flight cage. We constructed a radial arm maze, modelled after Lehrer et al. (1995), with 12 corridors (14! 15! 15 cm, W! H! L) that opened on to a central area (22 cm wide, 15 cm high). The entrance to each corridor was 6 cm wide. The floor and ceiling of the maze were made with 1/8-inch (0.32 cm) clear Plexiglas and the external walls and corridors were made with 1/8-inch opaque grey Plexiglas. All pieces were fixed with a Plexiglas epoxy except for the ceiling, which was removable. Testing stimuli (discs: 12-cm diameter; rectangles: 14! 15 cm) were positioned vertically at the end of each corridor. Procedure Training to forage in the flight cage We deprived the colony of honey solution for 2 days before training. On the first day of training, we allowed bees leaving the colony for the first time to forage freely from eight pots filled with honey solution (which was translucent and almost colourless), and we noted the identifying numbers of these bees. The pots were small, round cylinders made from white plastic (1.5-cm diameter, 0.5 cm deep). They were placed on a wooden tray approximately 1 m from the floor in the far right corner (relative to the colony entrance) of the flight cage. After approximately 2 h, we returned the bees to the colony; we implemented this foraging session to select foragers for further training the next day. On the second day, we allowed the same bees that had foraged on the previous day to forage again for 1 h, then returned these bees to the colony. Only bees that were observed foraging on both training days were selected for testing. Testing for preferences We removed the tray of pots from the cage and placed the 12-arm radial maze in its place, on top of a 1-m-high stool in the centre of the flight cage. The stimuli were placed at the ends of the corridors in the maze. As each of the selected bees exited the walkway, we captured it in a small vial and immediately placed it in the centre of the maze. We tested bees at this point in the procedure to ensure that they were still in foraging mode. Although the bees experienced reward from the tray of pots, they never experienced reward in the maze, which is why we use the term flower-naïve throughout (Giurfa et al. 1995). Once the bee began to fly in the maze, we recorded the bee s first 20e30 choices. A choice was determined to be made when the bee flew across the entrance and into the corridor towards the stimulus (almost all bees flew well into the corridor and some landed on the stimulus). Between testing sessions, we rotated the maze by 90( to control for position of stimuli, shadows and other inconsistencies in lighting, and we changed the experimenter s position in relation to the maze. This procedure was repeated in its entirety with two more colonies.

3 SIMONDS & PLOWRIGHT: UNLEARNED RESPONSES/HABITUATION 381 Statistical Analysis We carried out a log-linear analysis of the count data from each experiment using GLIM version 4.0 statistical software (Francis et al. 1993). An a level of 0.05 was used in tests of significance. When post hoc comparisons were made, we used the Bonferroni approach to adjust the a level (0.05 divided by the number of comparisons): at most, two comparisons were made, with an a level of for each comparison. EXPERIMENT 1A: TESTING FOR COLOUR PREFERENCES We tested naïve bumblebees responses to blue, yellow, white and red stimuli where no reward was offered in association with the stimuli. We name colours (blue, yellow, red) as they appeared to us but define colours in terms of physical characteristics (i.e. dominant wavelength, spectral purity and intensity), and therefore do not draw conclusions about the bees perceptual experience of colour (i.e. hue, saturation and brightness). That bees have a preference for blue and yellow has been confirmed in the literature (e.g. Lunau & Maier 1995). Testing for these preferences was necessary to validate our method and was also a prerequisite for subsequent testing of the effects of repeated exposure (experiment 1b). However, because our objective was not to investigate the details of colour perception per se, we could not determine whether observed preferences for blue and yellow, if they did emerge, were due to differences in hue, saturation or brightness. Methods The methods were as described under General Methods. We tested 33 naïve bumblebees from three colonies (colony 1: N Z 13 bees; colony 2: N Z 10 bees; colony 3: N Z 10 bees) using three replicates of four differently coloured discs (yellow, blue, white, red) made from construction paper. The colours were assessed by eye on three dimensions according to Munsell s (1967) specifications (hue Z wavelength, chroma Z spectral purity, value Z intensity); yellow: hue Z 5Y, chroma Z 8, value Z 8.5; blue: hue Z 5PB, chroma Z 8, value Z 5; red: hue Z 2.5R, chroma Z 10, value Z 5. We used a within-subjects design to record the first 20 colour choices made by each bee (33 bees! 20 choices Z 660 choices). Results We found a significant main effect for colour (c 2 3 Z 205:90, P! 0:001; Fig. 1): about 75% of the time, the bees chose yellow or blue over white or red. The effect of colour interacted with colony (c 2 6 Z 23:08, P! 0:001): in colony 2, the bees favoured yellow over blue more than in the other colonies, but both blue and yellow were still more attractive than either red or white. Figure 1. Mean number of choices for each colour out of 20 choices (N Z 660 observations) in experiment 1a. EXPERIMENT 1B: TESTING FOR HABITUATION TO COLOUR Habituation consists of a response decrement resulting from repeated stimulation (Harris 1943): behaviour changes in such a way that time and energy are not wasted in unnecessary, inappropriate, or ineffective behaviours (Shettleworth 1998, page 142). Habituation has been well documented in a wide variety of invertebrates and vertebrates (e.g. Harris 1943). It is a form of learning, and as such, it does not include response decrements due to fatigue, trauma, ageing, and so forth (Thompson & Spencer 1966). Such explanations can be ruled out by presenting the animal with a novel stimulus: if an increase in responding is observed, then factors such as fatigue cannot have been responsible for the waning in behaviour. In Harris (1943) review of the literature on habituation, he concluded that it had not yet been systematically studied in Hymenoptera. In the ensuing years, the study of habituation in this order has remained all but untouched. In experiment 1b, we tested whether habituation takes place when stimuli are repeatedly presented without reward. We repeatedly exposed bees to the same stimulus to determine whether their response would decrease in the absence of reward. After 60 presentations of the original stimulus, we presented a novel stimulus to assess whether the decrease in response was due to fatigue or other confounding variables. Evidence for habituation would be obtained if the bees responded to the novel stimulus more frequently than to the habituated stimulus. We set up three conditions to test these hypotheses: a control condition in which six of the 12 arms of the maze had yellow discs at the end of each corridor and six of the arms had no stimuli (grey corridors) over three test sessions, and two conditions in which the yellow stimuli were replaced with a different colour on the third test session. The initial assignment of colour to corridor was random.

4 382 ANIMAL BEHAVIOUR, 67, 3 Methods Subjects, materials and procedure The methods were as described in General Methods, with the following exceptions. We tested 27 naïve bumblebees from three colonies under three conditions (3 bees per colony! 3 colonies! 3 conditions): one control condition in which the colour array remained the same during all three test sessions and two conditions in which the colour array was changed on the third test session. For each condition, bees made a binary choice between six coloured rectangles (14! 15 cm) and six corridors with no stimuli (grey end of the corridor). The number of choices allowed was increased from 20 to 30 to increase the opportunity for habituation (27 bees! 30 choices! 3 test sessions Z 2430 choices). If the bee stopped choosing before 30 choices were recorded, no data points were used (N Z 4 bees). Each day, three bees were tested and returned to the colony after testing. When test session 1 was completed, the tested bees were allowed to forage from the pots of honey solution for at least 15 min. Once the bees returned to the colony after foraging and re-entered the walkway, they were tested a second time using the same procedure as above. Again, after test session 2, when the tested bees re-emerged from the colony, they were allowed to forage for at least 15 min. Once they returned to the colony and re-emerged again, test session 3 commenced using the same procedure as above. Testing began at approximately the same time each day and terminated on the same day when each of the three bees had completed three testing sessions. Test conditions Yelloweyellow array (control). Bees chose between six yellow rectangles and six corridors that contained no stimuli (grey corridor) during all the three test sessions. The assignment of colour to corridor was random. Yelloweblue array. Bees chose between six yellow rectangles and six corridors that contained no stimuli (grey corridor) during test sessions 1 and 2. In test session 3, we replaced the yellow rectangles with six blue rectangles. Yellowered array. Bees chose between six yellow rectangles and six corridors that contained no stimuli (grey corridor) during test sessions 1 and 2. In test session 3, we replaced the yellow rectangles with six red rectangles. Design We used a 2 (coloured stimuli versus grey)! 3 (test sessions) within-subjects design to record the first 30 colour choices made by each bee in each condition, where condition was a between-subjects factor. Results and Discussion Yelloweyellow array Bees colour choices depended on test session (interaction: c 2 2 Z 36:10, P! 0:001; Fig. 2a). This interaction Figure 2. Mean G SE number of choices out of 30 over three test sessions, separated by 15 min, for three conditions in experiment 1b. (a) Control: yellow stimuli were used for all three test sessions. (b) On the third test session, the yellow stimuli were replaced with blue. (c) On the third test session, yellow stimuli were replaced with red. B: blue; G: grey; R: red; Y: yellow. was not dependent on colony (c 2 10 Z 8:94, NS). Bees showed a preference for yellow stimuli over no stimuli (grey corridor) in test session 1, but not in test sessions 2 and 3. The decline in the proportion of choices for yellow from test session 1 to 2 was significant (c 2 1 Z 30:39, P! 0:001). Yelloweblue array Again, bees choices for yellow over grey varied according to testing session (interaction: c 2 2 Z 25:95, P! 0:001; Fig. 2b). This interaction was not dependent on colony (c 2 10 Z 6:87, NS). The bees chose yellow over grey in test session 1, but not in test session 2 (c 2 1 Z 19:71, P! 0:001). When the blue stimuli were introduced in test session 3, the number of choices for grey did not decrease compared to test session 2 (i.e. there was no increase in interest when the new stimulus was introduced). Yellowered array As in the first two conditions, the preference for yellow over grey depended on test session (interaction:

5 SIMONDS & PLOWRIGHT: UNLEARNED RESPONSES/HABITUATION 383 c 2 2 Z 23:66, P! 0:001; Fig. 2c). This interaction was not dependent on colony (c 2 10 Z 2:873, NS). Bees chose yellow over grey in test session 1, but this preference waned in test session 2 (c 2 1 Z 6:01, P! 0:025). When the red stimuli were introduced in test session 3, the number of choices for grey did not decrease; again, the new stimulus did not elicit renewed approach behaviour). In test session 1, the most frequent choice for all of the bees was yellow except for one bee, which showed no preference for yellow. In summary, bees chose the yellow stimulus over no stimulus (grey) in test session 1 in all three conditions. Although the response decrement between test sessions 1 and 2 suggests that habituation may have occurred, neither the introduction of a previously attractive blue stimulus, nor the introduction of a previously unattractive red stimulus (experiment 1a) produced any renewed responding in test session 3. Therefore, fatigue may account for the response decrement, but this seems unlikely given how frequently and actively bees forage in a natural environment. Alternatively, the novel colour stimulus may not have been sufficiently different to provoke responses; that is, one colour may generalize to another. Perhaps in order to trigger an increase in responding, the novel stimulus must differ along other dimensions besides colour, or perhaps along more salient dimensions (e.g. pattern, shape, size). EXPERIMENT 2A: TESTING FOR PATTERN PREFERENCES In this experiment we tested bumblebees preferences for radial versus circular patterns. The patterns used were based on the stimuli used by Lehrer et al. (1995) in a series of tests for pattern preferences in honeybees. We chose one pattern that produced marked preference (a radial pattern) and one pattern that produced marked avoidance (a concentric pattern). We used blue and yellow patterned stimuli since these colours were shown to be attractive to bees in experiment 1a. Methods We tested 33 naïve bumblebees from three colonies (colony 1: N Z 9; colony 2: N Z 19; colony 3: N Z 5) using four replicates of three stimuli (a radial pattern with six alternating wedges of blue and yellow; a concentric circle with four alternating bands of blue and yellow; a plain blue disc) made from construction paper. We used a within-subjects design to record the first 20 choices made by each bee (33 bees! 20 choices Z 660 choices). Results We combined the number of choices made for the plain blue disc and the concentric circle pattern and compared these with the number of choices made for the radial pattern. Bees chose the radial pattern significantly more often (c 2 1 Z 29:07, P! 0:001) than the concentric and plain blue discs combined (Fig. 3). The effect of pattern did not interact with colony (c 2 2 Z 2:06, NS). Figure 3. Mean number of choices for each stimulus type out of 20 choices (N Z 660 observations) in experiment 2a. Bees chose the radial pattern significantly more often than the other two stimulus types combined (P! 0:001). EXPERIMENT 2B: TESTING FOR HABITUATION TO PATTERNS Although the bees in experiment 1b showed a response decrement over time, because there was no increase in responding to a novel stimulus, we cannot conclude that there was habituation. Thus, we applied some changes in experiment 2b: we lowered the number of choices per bee per test session from 30 to 20 in test sessions 1 and 2 to minimize possible effects of fatigue and we used test stimuli that were not only coloured but also patterned. Methods Subjects, materials and procedure The methods were as described in General Methods, with the following exceptions. We tested 45 naïve bumblebees from three colonies: under five conditions (3 bees per colony! 3 colonies! 5 conditions): two control conditions in which patterned stimuli remained the same during all three test sessions and three conditions in which patterned stimuli were changed on the third test session. For each condition, bees made a binary choice between six patterned stimuli and six blue discs (shown to be attractive in experiment 1a). The patterned stimuli (radial, floral, concentric) varied for each condition (Fig. 4). Test conditions Radialeradial array (control). Bees chose between six radial discs of alternating blue and yellow wedges and six plain blue discs in all three test sessions. Assignment of discs to corridors was random. Two bees stopped choosing before 20 choices were recorded and so none of their data points were used.

6 384 ANIMAL BEHAVIOUR, 67, 3 Figure 4. Mean G SE number of choices out of 20 over the three test sessions, separated by 15 min, for five conditions in experiment 2b. Two control conditions in which the stimulus array remained the same over all three test sessions: (a) radialeradial: bees chose between a radial pattern and plain blue discs in all three test sessions; (b) floralefloral: bees chose between a floral pattern and plain blue discs in all three test sessions. Three conditions in which the stimulus array was changed in the third test session: (c) radialefloral: the radial pattern was replaced with a floral pattern; (d) floraleradial: the floral pattern was replaced with a radial pattern; (e) radialeconcentric: the radial pattern was replaced with a concentric circle pattern. B: blue; C: concentric; F: floral; R: radial. Floralefloral array (control). Bees chose between six floral stimuli and six plain blue discs in all three test sessions. The floral stimuli were copies of a photograph of a wild flax (Linum perrene) that we had previously seen a bee probing. Radialefloral, floraleradial and radialeconcentric arrays. Procedures for these conditions were identical to those described above except that the stimuli used in the third test session differed from those in the first two sessions. In the radialefloral array, six radial discs were replaced with six floral stimuli in test session 3. In the floraleradial array, six floral stimuli were replaced by six radial discs in test session 3. In the radialeconcentric array, six radial discs were replaced by six yellow and blue concentric discs in test session 3. Design We used a 2 (patterned stimuli versus plain blue discs)! 3 (test sessions) within-subjects design to record the first 20 choices made by each bee in each condition, where condition was a between-subjects factor (9 bees! 60 choices! 5 conditions Z 2700 choices). Results and Discussion Controls Radialeradial array. The bees preference for a radial pattern over a plain blue disc depended on the testing session (interaction of pattern! test session: c 2 2 Z 37:37,

7 SIMONDS & PLOWRIGHT: UNLEARNED RESPONSES/HABITUATION 385 P! 0:001). The interaction with colony was not significant (c 2 10 Z 2:81). Bees showed a stronger preference for the radial pattern over the plain blue disc in test session 1 than in test session 2 (c 2 1 Z 29:15, P! 0:001), but showed no preference for the radial pattern in test sessions 2 and 3 (Fig. 4a). In other words, in test sessions 2 and 3, the bees chose the radial pattern and the plain blue disc in approximately equal proportions. Floralefloral array. Again, the bees preference for a floral pattern over a plain blue disc depended on test session (interaction: c 2 2 Z 46:21, P! 0:001). The interaction with colony was not significant (c 2 10 Z 4:54). Bees preferred the floral pattern over the plain blue disc in test session 1, but this preference waned by test session 2 (c 2 1 Z 26:28, P! 0:001). As in test session 2, bees showed no preference for the floral pattern over the plain blue disc in test session 3 (Fig. 4b). Array changes Radialefloral array. The bees preference for a pattern versus a plain blue disc varied according to test session (interaction: c 2 2 Z 23:55, P! 0:001). The interaction with colony was not significant (c 2 10 Z 5:95). Bees chose the radial pattern over the plain blue disc in test session 1 but not in test session 2 (c 2 1 Z 21:31, P! 0:001). When the floral pattern was introduced in test session 3, the proportion of choices for the plain blue disc decreased significantly compared with that in test session 2 (c 2 1 Z 12:17, P! 0:005; Fig. 4c). Floraleradial array. As in the radialefloral array condition, the bees preference for a pattern versus a plain blue disc depended on test session (interaction: c 2 2 Z 29:20, P! 0:001). The interaction with colony was not significant (c 2 10 Z 9:56). As in the floralefloral array condition, bees preference for the floral pattern over the plain blue disc decreased significantly from test session 1 to 2 (c 2 1 Z 17:23, P! 0:001). When the radial pattern was introduced in test session 3, the proportion of choices for the plain blue disc decreased significantly compared with that in test session 2 (c 2 1 Z 25:44, P! 0:001): the new pattern triggered an increase in responding (Fig. 4d). Radialeconcentric array. As in the other conditions, the bees preference for a pattern versus a plain blue disc depended on the test session (interaction: c 2 2 Z 20:65, P! 0:001). The interaction with colony was not significant (c 2 10 Z 6:32). Bees chose the radial pattern over the plain blue disc in test session 1, but this preference waned in test session 2 (c 2 1 Z 5:47, P! 0:025). When the concentric pattern was introduced in test session 3, the number of choices for the blue disc did not decrease (Fig. 4e). In all five conditions, bees showed a preference for patterns (either radial or floral) over plain blue discs in test session 1, but this preference diminished by test session 2. When a previously attractive stimulus was introduced in test session 3 (floraleradial, radialefloral), the number of choices for the plain blue disc decreased. However, not just any new stimulus elicited renewed responding: when an unattractive stimulus (concentric pattern) was introduced in test session 3, there was no increase in approach behaviour. GENERAL DISCUSSION How do bumblebees first find flowers? Our results elucidate two components of exploratory behaviour. The first component (experiments 1a and 2a) is an unlearned preference for certain floral characteristics. Certain colours and patterns seem inherently attractive to bumblebees. In our radial arm maze, in which the bees never obtained reward, blue and yellow were chosen over red or white (Fig. 1), and radial patterns were approached more frequently than concentric patterns and unpatterned stimuli (Fig. 3). Moreover, no colony differences in these preferences were obtained. These unlearned preferences are important for three reasons: (1) they show that bumblebees, like honeybees, seem to have an innate preference for flower-like patterns (Lehrer et al. 1995), which in itself is not surprising; (2) more importantly, they rule out the possibility that these preferences are attributable to reward obtained in the maze; and (3) they are a prerequisite for studying the effect of repeated exposure discussed below. The method described here could be used in future research to address other issues such as the roles of spectral purity (Lunau 1992) versus dominant wavelength (Gumbert 2000) in colour preferences. One methodological improvement for the future might be to measure time to choose in addition to choice. Colour and pattern preferences are advantageous to bees insofar as they facilitate landing on flowers rather than other objects, and obtaining food. If the preferences persisted for long in the absence of reward, however, they would soon become a liability instead of an asset. This second aspect of exploratory behaviour has received little if any attention in the literature. Any observations under naturalistic conditions that bees cease to visit unrewarding flower species are of little use in this regard: such observations are equally compatible with other interpretations having nothing to do with habituation. For instance, bees may learn to visit other flower species that do offer reward (i.e. they find food elsewhere), or perhaps they undergo extinction of learned preferences when rewarding flowers become empty. The study of habituation of pattern and colour preferences requires the repeated presentation of a stimulus without reward in a controlled environment, which is the contribution of experiments 1b and 2b. Although the bees approach behaviour to yellow waned over time in experiment 1b, they showed no renewed responding when a new stimulus (either blue or red) was presented. Habituation occurred in experiment 2b: not only was a decrease in responding to floral patterns (relative to plain blue discs) over three test sessions obtained (Fig. 4b), but also the introduction of a radial pattern in test session 3 triggered approach behaviour (Fig. 4d), ruling out fatigue or sensory adaptation. Similarly, a decrease over time in responding to radial patterns was obtained (Fig. 4a), and the introduction of

8 386 ANIMAL BEHAVIOUR, 67, 3 a floral pattern sparked exploration once more (Fig. 4c). Most interesting from an ecological point of view was that there seemed to be a limit on what would trigger the renewal in approach behaviour. Even after habituation to a radial stimulus occurred, the bees did not approach a concentric stimulus (Fig. 4e). Apparently, after a few unrewarding experiences with potential flowers, bees will move on, but only to other potential flowers. The results described above fill a void in the psychological literature on habituation, which has been neglected in Hymenoptera. A few examples in the literature have studied the behaviour of naïve bees. One such example is that of Bombus consobrinus, which specializes on Aconitum (monkshood) and shows unlearned behavioural adaptations to handling these flowers (Laverty & Plowright 1988). Compared with naïve workers of generalist species (B. fervidus, B. pennsylvanicus), which usually initiate probing in the wrong places and often fail to locate the nectary, naïve workers of B. consobrinus are more likely to probe in the vicinity of the nectary and to obtain reward quickly. Another example traces the developmental sequence of how unlearned behaviours in bees are fine-tuned by experience: blue is not only a colour that is preferred by naïve honeybees, but it is also learned faster than other colours (Menzel 1985). The interplay between rewarded experience and unlearned behaviour is relevant to the current interest in the cognitive ecology of pollination (Chittka & Thomson 2001). Our research underscores the further point that absence of reward is also effective in shaping unlearned behaviours into efficient foraging behaviours: bees can quickly learn not to approach that to which they were first drawn. Acknowledgments This paper was prepared as part of the first author s doctoral thesis and was presented at the Joint Annual Meeting of the Canadian Society for Brain, Behaviour and Cognitive Science and the European Psychological Society, Cambridge, U.K., July This research was supported by a research grant to C.M.S.P. and a graduate scholarship to V.S. from the Natural Sciences and Engineering Research Council of Canada. We thank Beth Elston for technical support. Pierre Mercier, Jane Ledingham, Chris Plowright, Robert Stelmack and George Fouriezos gave constructive advice on the design of the experiments and the manuscript. References Chittka, L. & Thomson, J. D Cognitive Ecology of Pollination. New York: Cambridge University Press. Francis, B., Green, M. & Payne, C GLIM: The Statistical System for Generalized Linear Interactive Modelling. Version 4.0. New York: Oxford University Press. Giurfa, M., Núñez, J., Chittka, L. & Menzel, R Colour preferences of flower-naive honeybees. 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