sequence learning by honeybees

J Comp Physiol A (1993) 172:693-706 Journal of

Neural, and

Physiology A " ' ~ "

�9 Springer-Verlag 1993

Sequence learning by honeybees T.S. Collett*, S.N. Fry, R. Wehner

Zoologisches Institut der Universit/it Zfirich, Winterthurerstrasse 180, CH-8057 Ziirich, Switzerland

Accepted: 10 February 1993

Abstract. Bees of several genera make foraging trips on which they visit a series of plants in a fixed order. To help understand how honeybees might acquire such routes, we examined whether (1) bees learn motor sequences, (2) they link motor instructions to visual stimuli, (3) their visual memories are triggered by contextual cues associated with the bees' position in a sequence.

1. Bees were trained to follow a complex route through a series of obstacles inside a large, 250 cm by 250 cm box. In tests, the obstacles were briefly removed and the bees continued to fly the same zig-zag trajectory that they had when the obstacles were present. The bees' complex trajectory could reflect either the performance of a sequence of motor instructions or their attempt to reach fixed points in their environment. When the point of entry to the box was shifted, the bees' trajectory with respect to the new point of entry was relatively un- changed, suggesting that bees have learnt a motor sequence.

2. Bees were trained along an obstacle course in which different flight directions were associated with the presence of different large patches of colour. In tests, the order of coloured patches was reversed, the trajectory followed by the bees was determined by the order of colours rather than by the learnt motor sequence suggesting that bees will readily link the performance of a particular trajectory to an arbitrary visual stimulus.

3. Bees flew through a series of 3 similar compartments to reach a food reward. Passage from one compartment to the next was only possible through the centre of one of a pair of patterns, e.g. white + ve vs. black - v e in the first box, blue + ve vs. yellow - v e in the second, vertical + ve vs. horizontal - ve in the last. In some tests, bees were presented with a white vs. a vertical stimulus in the front compartment, while, in other tests, the same pair of stimuli was presented in the rear compartment. Bees preferred the white stimulus when tested in the first

* Permanent address: Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton BN 1 9QG, UK

Correspondence to: T.S. Collett

compartment, but chose the vertical stimulus in the last compartment. Bees reaching a compartment are thus primed to recall the stimulus which they normally encounter there.

We argue that the elements which are linked together to form a route are "path-segments", each of which takes a bee for a given distance in a given direction.

Key words: Bees - Routes - Sequence learn ing- Memory retrieval

Introduction

Bees tend to forage in places where they were successful in finding pollen or nectar on previous occasions (Mfiller 1882). They often do not find enough food for a full load at a single site. They then visit several sites during one foraging trip and learn the location of each new site that they encounter (Manning 1956). Individual bees of several genera have been shown to follow fixed routes from one familiar site to another, visiting a sequence of plants in a set order (Manning 1956; Janzen 1971 ; Hein- rich 1976). A bee's route from site to site is not necessarily the shortest or most economical (Janzen 1971). This suboptimal order probably results from the limited flexi- bility of the bee's memory which means that new sites are inserted into a route with minimal disruption or rear- rangement of the preexisting sequence. The present paper asks: What is it that bees learn that enables them to follow stereotyped routes?

To follow a path means to obey a sequence of instructions. The instructions could be inherent in the external world, as when water flows down a river valley along a route imposed by the contours of the land. But the fact that bees can be trained along arbitrary routes (review: Collett 1993) and through mazes (review: Menzel 1990) suggests that to some extent bees do have an internal representation of sequences of instructions. We describe here three sets of experiments which investigate whether this is in fact the case.

694 T.S. Collett et al. : Sequence learning by honeybees

It is difficult to do controlled experiments over the many hundreds of metres that constitute a normal honeybee's foraging range. Our experiments were performed on a much smaller spatial scale, mostly in a box 250 cm by 250 cm, and it is of course hard to know the extent to which the mechanisms operating in the box are also at work in a large environment. However, the scale of our experimental apparatus is not completely unna- tural, bees flying through a thicket of branches to reach a flower may well learn routes over the kinds of distances we have investigated.

The first set of experiments asked whether bees learn a sequence of motor instructions of the kind: "fly East for x metres then fly South for y metres". There is much evidence that insects learn single vectors of this kind when, for instance, they fly between their hive and a single foraging site (e.g. von Frisch 1967; Wehner and Menzel 1990). So another way of framing our question is: "Do bees learn lists o f vectors ?" We approached this question by training large groups of bees to fly an obstacle course within the box to reach food at the other end. The obstacles forced the bees to keep to a defined and complex path, as in a maze (Weiss 1954). Once bees were skilled at this task, we occasionally removed all the obstacles to see whether the insects held to the same trajectory. As part of these tests, we varied the starting point of the bees' trajectory within the box to see whether the trajectory was determined by local visual cues or by an internal moto r sequence which was triggered at the bees' point of entry to the box.

Other factors in addition to a learnt sequence of motor instructions could help determine a bee's direction when it leaves one foraging site and makes for the next. The next experiment was designed to discover whether there is a linkage between a bee's current visual surroundings and its subsequent heading. There are several reports that bees fly towards intermediate beacons such as prominent trees which lie close to the direct pa th to a goal (von Frisch 1967). Our question is rather different. Do bees store internal instructions of the kind: "Head due Nor th on seeing the enchanted wood". More formal- ly, do bees form an association between a familiar scene at one foraging site and the direction that they must take in order to reach the next site on their route? Sensori- motor links of this kind are a useful supplement to sequences of motor instructions. They would, for instance, enable bees to continue on their route after a motor sequence that they were following has been inter- rupted.

To see whether such links are forged, we trained bees along a fixed route inside the same large box. The direction to be taken at different points within the box was signalled by large patches o f colour, as in Weiss's (1953) experiments on walking honeybees. . In tests we altered the sequence of the coloured patches and asked whether the bees' trajectory was determined by the colour which they saw or by the motor sequence which they may have learnt.

The final series of experiments explored whether bees store sequences of visual memories. Once bees are close to a goal, their search is guided by their memory of local

visual landmarks (reviews: Wehner 1981 ; Collett 1992). In part this memory is triggered by seeing the landmarks themselves (Collett and Kelber 1988). However, it would be good if there were additional triggers to memory recall, and an obvious one is position in a temporal sequence. When bees visit a sequence of places, they must retrieve the correct memory at the correct location. It is then risky to rely solely on immediate sensory input for retrieval. The world is repetitive; one clump of trees may resemble another, particularly for an insect with low spatial resolution which confronts the clump from a new vantage point. To increase the chances of being guided by the right memory, it helps for that memory to be primed, so that when the insect arrives close to its goal, it already expects to see a particular landmark configura- tion.

Some evidence that this occurs was given earlier (Col- lett and Kelber 1988). The experiments described here approach the problem differently. Bees were trained to fly through a series of three identical compar tments to reach a sucrose feeder. At the back of each compar tment was a pair of visual stimuli, one of which marked the exit f rom the compartment . The positions of the positive and the negative stimuli were frequently exchanged. After bees had learnt to fly directly to the correct stimulus in each compar tment , we tested their response to the sequence of stimuli. To do this, the negative stimulus in one compar tment was replaced by a copy of the positive stimulus f rom another compartment . I f bees have learnt to expect a particular pattern in each compartment , they should prefer the stimulus appropriate to that compart - ment over a positive stimulus taken f rom another compartment.

Methods

Experiments on a local race of Apis mellifera were conducted inside a large laboratory. One wall of the laboratory was almost entirely glass and contained a pair of glass doors which opened on to a balcony. Throughout the summer, bees foraged at a feeder on the balcony which was kept filled with a low concentration of sucrose. Bees recruited from this feeder and from a nearby hive were readily persuaded to forage inside the laboratory by providing a more concentrated sucrose solution than that available outside.

Motor sequences. A group of between 30 to 50 bees was trained to enter a large box through a 15 cm diameter hole in the side nearest to the external door to the lab. The box was 250 cm by 250 cm with 50 cm high walls. It was roofed with 2 large sheets of transparent plexiglas through which the bees' flight path was recorded using a videocamera suspended from the ceiling. The walls and floor of the box were painted white with random dark markings to provide visual stabilizing cues. Vertical slots in the side walls spaced 30 cm apart allowed the front and back walls to be put in various positions. The slots also accepted partitions which were used to divide the box into separate compartments. In this experiment two sheets of transparent plexiglas which extended from side to side and from floor to ceiling divided the box into 3 compartments. The only way bees could pass through the partitions was by flying through a 15 cm hole drilled in each. The centres of all 3 holes were 25 cm above the floor and they were arranged so that the bee had to fly in a zig-zag trajectory from the start hole to a 2 cm exit hole located 10 cm below the roof in the back wall of the last compartment (e.g., Fig. 1). This hole gave bees access to a feeder. Bees did not retrace their entry

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T.S. Collett et al.: Sequence learning by honeybees

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Fig. 1. Sample flight trajectories in box. Top: training. Left: plan view of arrangement of partitions and holes. The whole of the box is not shown. En- try hole to the box is at the bottom of each diagram and is marked with an e. Coordinates of final hole are (160, 180). Right: plan view of 4 flight trajectories through holes. Middle: standard tests in the ab- sence of partitions. Left: position of entry hole, e. Right: 5 trajectories through box; circles mark expected positions of bends. Bottom: displaced tests. Axes show distances in cm. To avoid confusion, some trajectories are drawn with a dotted line. Here and elsewhere number on the axes denote cms

path on leaving the feeder. Instead they flew back through the 2 cm hole and up to the plexiglas ceiling from where they were released.

The holes in the transparent dividers were almost invisible to the bees and during training we marked the holes either by black rings around them or by small 2 cm disks placed just above them. In order to prevent the bees from becoming too reliant upon these visual markers, the latter were periodically removed. After a day's training many of the bees slalomed fluently through this obstacle course. The bees were then ready for testing.

Tests were administered infrequently, about once every 60 to 90 min. First, all the bees in the lab were caught and released outside the lab. The doors to the lab were shut and the plexiglas roof and dividers removed. The plexiglas roof was then replaced, the videore- corder started and the lab doors opened. The trained bees entered the box through the start hole as usual and flew through the now empty box to the exit hole where they fed as normal. We controlled the flow of bees in various ways to try to prevent more than a few from flying through the box at the same time. After we thought that


most of the bees had entered, we restored the training arrangement. Training then resumed until the next test. In some tests, the position of the first wall was changed in order to shift the start hole from its training position.

The video camera caught most of the bee's flight path between start and exit holes. The bees' trajectories were recorded on videotape, later transcribed onto acetate sheets and information from these tracings stored on computer. Trajectories in which bees in- teracted with each other were ignored, as were those in which the bees flew very much more slowly than they did during training. A total of 514 trajectories was transcribed for the analysis presented in Figs. 3 to 5. Uncertainties of various kinds which included straight flights through the box forced us to discard 74 of the transcribed trajectories from the analysis.

Sensori-motor sequences. Two separate experiments were performed. Again a group of ca. 30 to 50 bees was trained to pass through a series of compartments in the box to reach a feeder at the other end. Bees entered the box through a 6 cm diameter centrally located hole, and flew 60 cm through the first compartment to a white painted divider with another centrally located 6 cm hole (inset in Fig. 8a, b). From this hole they could see the next divider which was coloured. In the first experiment this divider was blue and in the second it was coloured yellow. In both cases, two holes, 130 cm

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Fig. 2. Trajectories during standard and displaced tests showing that when the position of the first bend is misplaced in a certain direction so is that of the second. * point to the positions of bends to illustrate how bends were defined for the plots in Figs. 3 and 4

apart, were drilled in the divider. One of these was open to provide an exit to the next compartment. In the first experiment the holes were 6 cm in diameter and the right one was open. In the second experiment the holes were 2 cm in diameter and the left one was open. Exit from the third compartment was through a single centrally located, 6 cm hole in another white painted divider. From this hole, bees could see the back wall of the final compartment. This wall was coloured yellow in the first experiment and blue in the second experiment. It contained two holes which, like those in the first coloured divider, were positioned 130 cm apart and of which one gave access to the feeder (left hole in the first experiment and right hole in the second). Thus, in both experiments sight of a blue divider at the back of a compartment was a signal to fly to the right through that compartment while yellow was a signal to fly to the left (Fig. 8).

After several hours of training, bees were tested every 90 min to see whether they had learnt the association between colour and flight direction. To this end, we reversed the order of coloured dividers within the box. In the first experiment, the two coloured dividers were simply swapped and both holes in both dividers were open. In the second experiment, the yellow divider was moved to the position of the blue divider. The yellow divider was itself replaced by a blue divider in the middle of which had been cut a 3 cm high horizontal slit which extended from one side wall to the other. The slit was used to prevent the bees from aiming at a target, as might have happened when the divider had holes. The bees' trajectories through the box were recorded on videotape. At the end of each test the training arrangement was replaced and the bees continued to collect sucrose until the next test.

Sensory sequences. Bees were trained to fly through a series of boxes for a sucrose reward which was available after the bees had left the last box. The boxes were arranged end to end on a long narrow table one end of which was placed close to the glass door to the balcony. Each box was 40 cm high, 60 cm wide and 50 cm long with white painted walls and floor covered with random scribbles. The boxes were roofed with transparent plexiglas. Bees entered the first box through a 5 cm wide hole, shown to the left in Fig. 10. Two visual patterns (each 25 cm by 25 cm) were fixed to the back of each compartment (Fig. 10, bottom). Passage from each box to the next was through a 2 cm hole in the centre of one of the patterns which we term the positive stimulus. The hole in the other pattern, the negative stimulus, led to a blocked compartment. The positive stimulus was switched from side to side every 10 to 15 min. Movable sliders behind the patterns controlled which compartment behind the pattern was open and which was closed. Thus, the bee could only fly directly through the boxes if it learnt to aim at the positive stimulus in each box.

Preliminary experiments showed that discriminations learnt in each box were not independent of each other. For instance, we trained bees in one box using a black-white vertical grating as the positive stimulus and a similar horizontal grating as the negative stimulus. The bees became confused if in another box in the sequence they had to treat a horizontal grating as the positive stimulus and a vertical grating as the negative stimulus. This indicates that the contextual cues differentiating the boxes are not very powerful for under other circumstances bees will readily learn such state-dependent discriminations. Thus, Kelber (1989) trained bees in the open air to discriminate between horizontal and vertical gratings which were placed side by side on a vertical board. The bees were rewarded at the vertical grating when the pair of gratings faced south and at the horizontal grating when the pair faced north. Tests showed that they had no difficulty in learning to choose the appropriate grating in each condition.

Early experiments also showed that learning in these boxes only occurred if the edges in black-white patterns had the same orientation over the whole pattern. Thus, we failed to teach bees the difference between two stimuli, one of which was a series of black and white concentric rings and the other a circle divided into black and white sectors. We eventually decided upon three easily discrimi- nated pairs of stimuli: (1) black and white vertical stripes vs black

T.S. Collett et al. : Sequence learning by honeybees 697

and white horizontal stripes (each stripe 2 cm wide); (2) blue paper vs yellow paper; (3) black paper vs white paper.

After a day's training 4 min tests were administered ca. every 30 min. The patterns in one box were then replaced by two positive stimuli. One was identical to the positive stimulus from that box and the other was identical to the positive stimulus from another box. (We tried testing with two negative stimuli, but the bees were then reluctant to pass through either hole.) Different sets of stimuli were used for training and testing. When tests were given in the front or middle boxes, bees were rewarded normally after leaving the rear box. For tests in the rear box, the sucrose was removed and bees made several passes through the sequence. We used two hand-held counters to record the number of bees flying through each hole during the test. The positive stimulus belonging to the tested box was on the left in half of the tests and on the right in the other half. At the end of the test, the training situation was restored immediately.

R e s u l t s

Motor sequences

Bees flying within the large box were trained to pass through holes in a series of dividers in order to reach a sucrose reward. We ask Whether the bees' trajectory retains the same form in tests when the dividers are removed.

Some individual trajectories of bees flying through the box during training and testing are shown in Fig. 1 (right). The left hand column of this figure indicates the position of the entry hole in plan view, marked with an 'e'. During training (top), when the dividers are in place, bees fly directly from hole to hole. In standard tests, the two dividers are missing. Circles superimposed on the trajectories mark the positions of holes during training. Bees continue to turn in roughly the right place even though no divider is present (see also Fig. 3 and Table 1).

Bees in these tests could be turning in the correct location either because they are replaying a motor pro- gramme or because they are flying to particular locations in the box. To distinguish between these possibilities, bees were tested with the front wall and the entry hole displaced. Expressed in the coordinate system of the figure, the hole was shifted 30 cm down the y-axis and 40 cm to the left along the x-axis. The bees thus entered the box and began their flight from an unusual position. The circles show where the turns should be if the insects were flying a motor sequence that began at the new start position. By eye, it seems that the bends in the trajectories are shifted downwards and leftwards, as would be the case if the bees were recalling a list of motor commands.

The next few figures present a more quantitative analysis of this point. To do this, we needed a simple

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Fig. 3. Positions of first (o) and second (A) bends in standard and displaced tests for bees trained as in Fig. 1. Filled circles mark positions of holes during training defined with respect to the entry hole in the test. Right column shows the position of second bends when these are normalised by shifting all first bends to 160, 60 (top) and 120, 30 (bottom). Statistical details are given in Table 1


Table 1. Positions of holes during training and positions of bends during standard and displaced tests

Entry point 2 "d hole/1 st bend 3 rd hole/2 "d bend

x y x y x y

mean s.d. mean s.d. N mean s.d. mean s.d. N

Holes in training 130 30 160 60 90 120 Standard bends 130 30 156.7 15.5 63.9 12.5 77 102.8 24.1 108.0 19.6 72 Normalised on 1 st bend 160 60 106.0 25 .2 103.6 16.2 72 Displaced bends 90 0 123.4 21.6 39.4 16.9 120 88.4 17.8 74.1 20.4 98 Normalised on 1 st bend 120 30 84.5 16.8 64.6 14.4 98

Holes in training 90 0 160 60 90 120 Standard bends 90 0 136.1 27 .0 49.6 16.0 129 84.1 18.2 95.0 29.3 89 Normalised on 1 st bend 160 60 106.4 18.9 105.9 23.2 89 Displaced bends 130 30 162.3 19.4 75.4 15.9 114 107.9 22 .4 123.9 20.5 67 Normalised on 1 st bend 200 90 144,3 22 .3 140.2 16.4 67

method of compressing the spatial information in the trajectories. The technique we adopted was to neglect everything except the position of the turns. The asterisks in Fig. 2 give examples of where we judged the turns to be. We placed turns at the point of greatest excursion to the left or to the right. When the vertical coordinate of this point was ill-defined, because, for instance, the bee flew straight up the page, we took the point of max imum inflexion. With these criteria we could usually determine the bend's position without debate. The total number of discarded trajectories is given in the Methods. Very occasionally, the first turn was made to the right instead of to the left and was positioned on the left of the entry hole. When this happened we recorded the position of the first bend, but ignored subsequent bends.

The left-hand column of Fig. 3 plots the positions of the first (�9 and second (A) bends of the trajectories f rom bees which had been trained with the dividers arranged as shown in Fig. 1 and tested with the entry hole in the standard position (top) or displaced position (bottom). To determine whether the bends in the displaced tests were located differently f rom those in the standard tests, we computed the centres of gravity of the clusters of points belonging to each bend. The centres of gravity are shown on the bent arrows in Fig. 4 (top). The elbow in the solid arrow shows the average position of the first turn in the standard tests and the tip of that arrow gives the average position of the second turn. The dashed arrow shows the same for the displaced tests. Means and standard deviations of the x and y coordinates of the bends are given in Table 1 (top), as are the number of data points. T-tests calculated independently for the x and y coordinates show that the positions of the 1 st and 2nd turns differ significantly between the standard and displaced trajectories (P<0.001 in each case). It therefore seems likely that bees flying through the empty box are following a learnt sequence of motor instructions.

This conclusion gains further support f rom the "er- rors" made in individual flight paths. When the first bend occurs in the wrong position, the second bend tends to be displaced in the same erroneous direction. Examples in Fig. 2. show that, if the first bend of a trajectory is too

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Fig. 4. Top: means of clusters of bends shown in Fig. 3. Standard tests are indicated by solid arrows andfilled circles, displaced tests by broken arrows and open circles. Elbow in arrow shows mean position of first bend. Arrow-head shows position of second bend. Circles show positions of holes during training relative to entry hole. Arrow-head issuing from second hole shows mean position of second bend when data are normalised by superimposing positions of first bend. Bottom: the same for bends plotted in Fig. 5

T.S. Collett et al. : Sequence learning by honeybees

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Fig. 5. As Fig. 3, for bees trained through holes as shown in bottom diagram. Statistical details are given in Table 1

700 T.S. Collett et al.: Sequence learning by honeybees

far to the left (or to the right), the second turn is also too far to the left (or to the right). It is as though once the bees have completed the first component of their flight, they tend to perform a constant-sized second component without correcting for the inaccuracies of the first. To examine this point in more detail, trajectories were normalised by superimposing all the first bends on the expected position of that bend. This procedure caused the clustering of the second bends to become tighter (Fig. 3, right). Statistically, however, F-tests show that this effect is slight. It only reaches significance in one condition.

The whole experiment was repeated using a different training arrangement in which the distance between the entry hole and the hole in the first divider was lengthened (Fig. 5). The results of this experiment are shown in Figs. 4 and 5 and in the bottom half of Table 1, using the same format as before. In displaced tests, the entry hole was shifted upwards and to the right. Bend positions again differ significantly between the displaced and standard tests (P<0.001, Table 1).

If the bees have indeed learnt different routes in the two experiments, then the length of the vectors connecting the start hole to the first bend should be shorter in the first experiment than it is in the second experiment. A comparison of these vectors in the standard tests shows that the lengths of the vectors do differ significantly in the expected direction (P < 0.001). Predicted vector length in the first experiment is 42.4 cm and the measured mean length is 44.8 cm (s.d.= 15.7, n=77). Predicted vector length in the second experiment is 92.2 cm and the measured mean length is 70.6 cm (s.d. = 20.8 cm, n= 129). The value of Student's t between the two means is 9.4 (d.f. = 204). Thus, the bees' behaviour is not simply a result of the idiosyncracies of a particular training route.

In both experiments, as we have seen, the positions of bends in the displaced tests differ significantly from those in the standard tests suggesting that bees were following a set of motor instructions. However, this is probably not a complete description of their behaviour. In the first test, the vector connecting the entry hole to the first bend is significantly longer in the displaced tests than it is in the standard tests (standard test: mean = 44.8 cm, s.d. = 15.7 cm, n=77 ; displaced test: mean=53.6 cm, s.d.=23.2 cm, n=120; Student's t=2.95, d.f .=195, P<0.01). In the second experiment, the relative lengths of these vectors is reversed (standard test: mean=70.6 cm, s.d.=20.8 cm, n = 129; displaced test: mean=56.5 cm, s.d.=16.8 cm, n = l 1 4 ; Student's t=5.75, d.f .=241, P<0.001). In both cases, the length of the displaced vector shifts from that of the standard vector to bring the bee closer to the remembered position of the hole in the first divider. The bee may thus be driven by two sets of stored instructions, one deriving from a motor pro- gramme and the other from visual signals which guides it to the position of the training holes. During displaced tests the two instructions will conflict and the bee's trajectory will be a compromise.

In the two previous experiments, the routes which were imposed upon the bees forced them to make a turn in one direction followed by a turn in the other. In case alternating turns are a special case (e.g., Dingle 1965), a

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Fig. 6. Trajectory with two left turns. Top: arrangement of partitions during training. Dashed line shows imposed route. Sucrose is in box close to entry hole. Bottom: 4 trajectories with partitions removed. Filled circles mark positions of holes in training and square shows position of sucrose. Arrow gives direction of flight

group of ca. 50 bees was trained to a route in which they were required to make two turns in the same direction (Fig. 6). When bees were tested with the dividers removed, the general form of their trajectories resembled that of the imposed route, indicating that the ability to learn a trajectory is not limited to a particular sequence of turns. Trajectories were mostly examined by eye. A few were transcribed for illustrative purposes and some of these are shown in Fig. 6.

Sensori-motor sequences

Bees, while flying through the box, clearly take note of their visual surroundings. This becomes very obvious if the visual appearance of one hole is altered. A bee will often turn around and fly back to where it entered the compartment before approaching the disturbed hole for a second attempt. Sometimes it flies back and forth several times before eventually passing through the hole. Possibly, it is relearning the appearance of the hole from a particular viewpoint. Figure 7a shows a particularly striking example of such behaviour. A 2 cm blue disc was substituted for the 2 cm black disc which had previously been fixed above the hole in the second divider. When the bee next arrived, it approached and retreated from the altered hole over a period of about 20 s before flying through. Figure 7b shows another much briefer episode of the same kind.


a /

% Fig. 7a, b. Bees sometimes retrace their path within a compartment. a 2 cm black disk above the top hole has just been replaced by a 2 cm blue disk. This mismatch between the expected and seen colour induces the bee to approach the hole and to turn way from it several times. It also spends time flying close to the new stimulus, b A less conspicuous example of the same behaviour. Distance between partitions is 60 cm. Entry hole is at bottom of each diagram

The question asked in this section is whether the scene, which the bee views through a hole on its arrival there, can come to determine the direction of its subsequent trajectory. To find out whether this is so, we trained bees to fly through a series of holes. A blue divider seen at the back of the compartment through the hole signalled that a trajectory was to be made to the right and a yellow divider that the flight path should be to the left. Bees were then tested with the order of coloured dividers reversed from the training arrangement. They flew in the direction indicated by the colour which they saw rather than that dictated by the motor sequence which they had stored.

Figure 8a shows superimposed tracings of trajectories recorded during tests in which both holes in the coloured dividers were open. The filled circles in the coloured dividers show the hole to which the bee should have

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Fig. 8a, b. Superimposed trajectories through compartments with coloured dividers at the back. These are labelled blue or yellow. The front compartment of the box is not drawn. Distance between dividers is 60 cm, whole width of box is not drawn. Insets show training and testing arrangement of dividers in plan view. Training route is shown by dotted line connecting holes, When the order of dividers is switched for tests, trajectories continue to be to the left when divider is yellow and to the right when divider is blue. Trajec- tory is controlled by colour, not by motor sequence, a Experiment in which first divider in test has two holes, b Experiment in which first divider in tests has long horizontal slit - shown in inset by dashed horizontal line

flown had it followed a motor sequence. The open circle shows the bee's predicted target on the assumption that its trajectory was governed by the colour that it saw. Bees during tests were predominantly guided by colour. Of a total of 54 trajectories through the compartment with the yellow divider at the back, 46 were to the left, while 47 out of 60 trajectories through the compartment with the blue divider were to the right.


Blue _ _ ~

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Fig. 9. Bees' flight-path through the 3rd and 4th compartment during tests in which the order of coloured dividers has been switched. A tortuous path in the 3rd compartment does not inter- fere with the performance of a direct trajectory in the final compartment. Breaks in the trajectory occur when the bee flew out of the field of view of the camera

The bees flying the trajectories illustrated in Fig. 8a could have been using one of the holes as a target and aiming directly at it. Are trajectories correctly directed when there is no explicit target? To answer this question, we repeated the experiment and tested bees with the blue training divider replaced by a yellow divider in which had been cut a narrow, horizontal slit extending the length of the divider (dashed line in plan view of box shown in inset to Fig. 8b). To help the eye, the positions of the training holes are drawn together with the trajectories, as in Fig. 8a. The initial directions of the trajec-

tories are only a little less accurate than those in Fig. 8a. But, when the bees approach the divider, they quite often turn as though they were confused by seeing the slit close-to.

Although we did not repeat the experiment and train bees with holes in different positions, it seems very un- likely that the bees had merely learnt to turn left to yellow and right to blue. The accurately directed trajectories in this and the previous experiments suggest that bees learn to perform trajectories in particular directions. We do not know what "compass" information might control the direction of travel. However, the most prominent direc- tional signal comes from the uneven light distribution in the room.

We conclude therefore that bees have learnt to link the production of a specific trajectory to the sight of a particular colour. Figure 9 emphasises how useful this association is to a bee which has been prevented from keeping to its normal route. In tests, the bees' motor pattern is disrupted so that they often enter the white compartment that lies between the two coloured ones by the hole on the "wrong" side. This does not necessarily confuse them and they sometimes fly straight to the central exit hole. But sometimes they are disturbed and follow a con- voluted route before reaching the exit hole. The interest- ing feature of the examples shown in Fig. 9 is that the final segment of the trajectory is quite unaffected by the preceding disturbance. Thus, once bees enter the back compartment and see the colour of the back wall they are reoriented and fly immediately in the direction specified by the colour.

Many years ago, Weiss (1953) performed a similar experiment on walking honeybees in a coloured maze. He found that colour determined whether the bees went to the left or to the right corner of each compartment in the maze. The conditions of his experiment were different in that bees could have solved the problem by learning to aim at the appropriate corner of each compartment where a dark side wall on one side lies close to a coloured divider on the other.

Sensory sequences

The experiments described in this section ask whether bees which have been trained to learn a series of visual patterns come to know where in the series they will encounter a particular pattern. We begin with a sample experiment. Bees were trained on the series of visual discriminations shown in the top panel of Fig. 10. They flew through the boxes from left to right. The first pair of stimuli was black + vs. whi te - , the second pair was blue + vs. yel low-, and the last pair was vertical + vs. horizontal - . After bees had learnt these discriminations, they were tested either with white vs. vertical in the front box or with white vs. vertical in the back box. The bees' choices in these separate tests are shown as a bar graph in Fig. 11 and are also given numerically in group 4 line 1 of Fig. 12. A 2 by 2 Chi a test shows that the choices in the two boxes are significantly different (Fig. 12). The bees preferred to fly through the white stimulus in the

T.S. Collett et al.: Sequence learning by honeybees 703

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Fig. 10. Training apparatus for testing the priming of visual memories. Top: plan view of 3 boxes showing the order of stimuli. Middle: arrangement of boxes used for training groups 1 to 3 in Fig. 12. Bottom: Vertical and horizontal stimuli. Width of stripes is 2 cm. Calibration: 50 cm refers to plan view; 25 cm to front view

first box and through the vertical stimulus in the last box, as if they recognised which was the positive stimulus that they normally encountered in each box. Similar results were obtained in a further four experiments (Fig. 12: groups 1-3 and group 5 line 1) in which positive stimuli from the front and back boxes were presented in preference tests.

Although during training bees chose the correct stimulus in the middle box almost 100% of the time, the situation was different when bees were tested in the middle box with the positive stimulus from that box pitted against that belonging to one of the other boxes. For instance, Fig. 11 and group 4 line 3 of Fig. 12 display the bees' preferences when they were confronted with either vertical vs. blue in the middle box or with vertical vs. blue in the back box. When tested in the back box, bees strongly preferred to fly through the vertical stimulus, whereas in the middle box they flew equally often through the two positive stimuli. In all of the 6 experiments in which bees were tested in the middle box (Fig. 12: group 4 lines 2,3, group 5 lines 2,3 and group

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W V W V V B V B Fig. 11. Bees' choices when trained as in Fig. I0. (top) and tested with white vs. vertical stimuli in the front and rear boxes, and with vertical vs. blue stimuli in the middle and rear boxes. N is total number of bees passing through the respective holes

6), the bees either preferred the positive stimulus from the front or back boxes over that from the middle box or they showed no preference. The positive stimulus from the middle box was never favoured over the other one. This effect is reminiscent of the problems that humans have with the middle items of remembered lists. Middle items tend to be recalled less well than those at either end (review: Woodworth and Schlosberg 1955).

Nonetheless, the bees still treated test stimuli in the middle box differently from the way in which they treated the same stimuli presented in either of the flanking boxes. In 3 of these experiments the preference in favour of the positive stimulus from the front or back box was significantly weaker (P<0.01) when the two stimuli were presented in the middle box than it was when the same stimuli were shown in the front or in the back box (Fig. 12: group 4 line 3, group 5 line 2, group 6 line 2). Thus, it seems that bees do recognise when they are in the middle box and that this does affect their recall.

Taken together, these experiments tell us that bees learn to associate a given positive stimulus with a given box in the sequence and that they come to expect to see a particular positive stimulus in each box. What contextual cues tell them where they are in the sequence? We examined two possibilities. The first is that the bees had in some way discovered or manufactured differences between the boxes themselves, even though the boxes had been built and painted so that they were as similar to each other as possible. Bees might, for instance, have labelled the last box chemically with their own secretions because it was closest to the reward. To see whether this had happened, we swapped the middle and last boxes and tested bees in what was now the final box in the series. Bees were given a choice between blue (the positive stimulus in the middle box) and vertical (the positive stimulus in the last box). They chose the vertical stimulus 13 times and the blue stimulus once. In previous tests with the same stimuli placed in the middle box, when the latter was in its usual position, the blue stimulus was chosen 34 times and the vertical stimulus 31 times. This experiment suggests that the bees' sensitivity to the order of stimuli

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Fig. 12. S u m m a r y of exper iments conduc ted in 3 boxes. Each different t ra in ing a r r a n g e m e n t for a g roup of bees (left column) is separa ted by a solid line. Tests are shown in subsequen t co lumns . Numerals next to letters indicate n u m b e r o f bees pass ing t h r o u g h the indicated s t imulus in each box. Chi 2 value in the r ight c o l u m n

is tha t com pu ted for the tests in tha t row on the null hypothes i s tha t the preference between st imuli will be the same in each box. )~2 values above 6.7 m e a n tha t this null hypothes i s can be rejected with P < 0 . 0 1


does not rely upon their ability to recognise individual boxes.

We next asked whether bees were primed to expect the stimulus in one box by seeing the positive stimulus in the immediately preceding box in the sequence. In other words, do bees form a chain of associations between adjacent positive stimuli? In one of several experiments performed to investigate this question, bees were trained as shown in third line of Fig. 12 (see also Fig. 10, middle). The entry to the first box was marked by a yellow stimulus constantly positioned on the bee's left. Access to the middle box was through the white stimulus which alter- nated from side to side. Exit from the middle box was always on the right hand side and was marked with a blue stimulus. The positive stimulus in the last box was vertical.

Tests were administered in which bees chose between white and vertical in the front box, in the middle box and in the back box. As expected, bees tested in the front box strongly preferred white and when tested in the rear box they preferred vertical. When tests were to be administered in the middle box, we attempted to make the front box resemble the middle box. The usual white and black stimuli were removed and were replaced by a blue stimulus on the right. However, this change to the front box did not cause the bees which entered the middle box to behave as though they had reached the end and to choose the vertical stimulus. They preferred the white stimulus over the vertical one, but their preference for white was weaker than it was during tests in the front box. Thus, while we have been able to show that bees arrive in the front and rear boxes with the approporiate memory primed, we do not know what contextual cues tell the bees where they are.

Discussion

By forcing bees to fly along prescribed routes, we have been able to show that these insects possess several mechanisms which should help them in the efficient learning and performance of such routes. These are (i) the storage of motor sequences; (ii) the linkage of a motor trajectory to a visual stimulus; (iii) the expectation that particular visual stimuli will appear at specific points along a route. Although bees were rewarded only at the end of their passage through the box, it is not implausible that the mechanisms operating in these experiments will also be used when bees learn a sequence of feeding sites. In both cases, the route consists of a number of goals which are attained in a fixed order. The bees were funnelled through a series of holes and they could only progress to the end of the sequence if they could find each successive hole, just as they locate each successive feeding site along a "trap line".

Such routes with ordered goals are interestingly different from the stereotyped routes displayed by desert ants as they travel through a field of landmarks between a familiar feeding site and their nest. Although an individual ant sticks closely to a particular path through a collection of landmarks, it is not constrained to do so, and different individuals with the same starting point and

the same goal follow different, stable paths around the same landmarks (Collett et al. 1992). The underlying mechanisms which generate the two classes of stereotyped routes also seem to differ. There is no sign that ants in these conditions store a motor sequence. They are guided by a single vector which specifies the distance and direction of the nest from the feeding site (Miiller and Wehner 1988).

Our major conclusion is that bees remember sensory and motor information which allows them to reproduce a complex route. They do not simply rely on the external world to dictate their path; to some degree they have learnt a sequence of detailed instructions. The obvious next question is: What is stored and how is the sequence organised? The present experiments supply no definite answers. Nonetheless, they do hint that in part a sequence may consist of a linked series of vectors, each one of which is a command requiring the insect to fly for a certain distance in a certain direction.

There are several fragments of evidence favouring this hypothesis. First, when motor trajectories are normalised with respect to the first bend (Figs. 3 and 5), the spread of positions of the second bend is somewhat reduced. This suggests that the path-segment going from the entry hole to the first bend may be a unit which is encoded independently of that going from the first bend to the second bend. A similar conclusion can be drawn from the behaviour of bees which had the opportunity to link the trajectory within a compartment to a particular colour. The order in which path-segments are performed can then be switched by changing the order of the coloured dividers. The bees' trajectories (Fig. 8) show that individual path-segments are not greatly disturbed by this reordering, indicating again that individual path- segments are independent units. Furthermore, bees in this experiment entered the white compartment between the two coloured ones from an unexpected direction and their tortuous trajectories in this compartment indicated that they were then confused. However, once the bees reached the second coloured compartment, they flew without hesitation in the direction which they had learnt to associate with the colour (Fig. 9).

A route may thus be composed of individual path- segments which are separate items that become linked together. The linkage can be primarily internal as illustrated by motor sequences in which vectors follow each other despite changes to the external environment. The chaining between segments can also make use of external signals which appear at the appropriate time as the bee flies through a familiar environment.

Acknowledgements. We are very grateful to Brigitte Huber and to Hubert Kr/ittli for their help with the experiments, to Miriam Lehrer for valuable discussion and to Mike Land for helpful com- ments on the typescript. Financial support came from the Swiss National Science Foundation and the S.E.R.C. of the United King- dom


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