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First published online October 5, 2007
Journal of Experimental Biology 210, 3559-3567 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005488
Circadian timed episodic-like memory – a bee knows what to do when, and also where
1 BEEgroup, Biozentrum, Universitaet Würzburg, Am Hubland, D-97074
Würzburg, Germany
2 ARC Centre of Excellence in Vision Science, Research School of Biological
Sciences, Australian National University, PO Box 475, Canberra ACT 2601,
Australia
Author for correspondence (e-mail:
shaowu.zhang{at}anu.edu.au)
Accepted 14 August 2007
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Summary |
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Key words: honeybee, memory, contextual learning, circadian rhythm, pattern vision
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;
Lehrer et al., 1995;
Chittka et al., 1993;
Vorobyev and Menzel, 1999;
Collett et al., 2003). The
species of flowers that are in bloom, say this week, are likely to be replaced
by a different species at a different location next week, and different flower
species have different peak times of nectar secretion during the day
(Kakutani et al., 1989). So
the bee needs not only spatial information, such as the features and location
of the flowers, but also temporal information, and has indeed evolved an
impressive ability to learn colours, odours, shapes and routes, within a time
frame, quickly and accurately.
Bees can learn the time of day when flowers start secreting nectar. In an
early study, when bees were trained to visit a feeder at a particular hour of
the day, almost all of the trained bees visited the feeder during the
hour-long reward period (Behling,
1929). This `Zeitgedächtnis' or time-sense persists for
6–8 days, and thus can outlast short periods of bad weather
(Wahl, 1932). It was also
shown that bees can recall 9 different times per day, with an accuracy of 20
min (Koltermann, 1971). In
Koltermann's experiments, the bees could associate scents with an artificial
feeder at a particular time.
Honeybee foragers possess a circadian rhythm, with an activity period
during the day and a sleep-like state at night
(Lindauer, 1975;
von Frisch, 1993;
Bloch and Robinson, 2001;
Bloch et al., 2001;
Moore, 2001). A special
feature of the honeybees' circadian rhythm is its flexibility. In typical
circadian rhythms, a particular behaviour is fixed to a special phase of the
cycle. The honeybee `Zeitgedächtnis' enables the bee to continuously
adjust its behaviour according to its memory and the time of day
(Chalifman, 1950;
Lindauer, 1954;
Wittekindt, 1955).
Honeybees have the ability to flexibly change their preference for a visual
pattern according to the context in which a discrimination task is carried
out. Context cues help to carve up the world into distinct regions, and so can
aid animals to cope with possible confusions
(Colborn et al., 1999;
Fauria et al., 2002;
Cheng, 2005;
Dale et al., 2005). Honeybees
can learn to treat the same stimulus in different ways, depending on the
context in which the stimulus is presented
(Gould, 1987;
Menzel et al., 1996;
Srinivasan et al., 1998;
Colborn et al., 1999;
Zhang et al., 2006). Menzel et
al. investigated whether and how contextual parameters, such as time of day
and features characterizing the location, can be utilized to determine choice
behaviour (Menzel et al.,
1996). They claimed that time of day cannot by itself elicit a
conditioned response, but can control different behaviours, such as
image-matching, navigation and timing of motivation to forage, and thus act as
an occasion setter for a sensory-motor routine
(Menzel et al., 2006).
There has, however, been little experimental work investigating bees'
abilities to modulate their behaviour in response to multiple contextual cues
in the spatial and/or temporal domain. In a previous study, we showed that
honeybees are able to reverse their pattern preference according to the task
at hand and the time of day (Zhang et al.,
2006). In these experiments, the bees learned to make opposite
decisions when foraging and when homing (task), and also in morning and
afternoon (time). These contextual cues help the bees to memorize the rules
for navigating an experimental maze, and to recall the correct memory in the
associated context. In the present study, we further investigated how the
colour, shape and location of patterns could be memorized within a time frame,
and examined the importance of different contextual cues.
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Materials and methods |
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Maze set-up
Three compound Y-mazes were used in the experiments. Each was made of four
cylinders of 25 cm height and 25 cm diameter, and covered by a PerspexTM
lid. The four cylinders were connected by holes, 4 cm in diameter, through
which the bees could fly from one cylinder to the next. The holes were
positioned in the middle of the cylinder wall, halfway up from the base (12.5
cm from both ends). The first cylinder carried two holes on opposite sides.
The bee would enter through the entrance hole, and fly through the next hole
into the second cylinder. The second cylinder had three holes, one serving as
entrance, and two others, 90° apart, as exits leading to the next two
cylinders. Each of the two holes carried a visual stimulus, between which the
bees had to choose (Fig. 1).
One of the two patterns indicated the position of the feeder reward. If the
bee made a positive decision by flying through the correct pattern (termed
positive), it would enter the third cylinder, and find a feeder with sugar
solution as a reward. If the bee chose the wrong (termed negative) pattern, it
found an empty cylinder, and was released to try again. A bee choosing between
visual patterns could not see whether the next cylinder contained the feeder
or not, because the feeder was placed on the floor of the maze, and a
cardboard baffle was placed behind the entrance holes of the reward cylinders.
This prevented the bees from seeing into the reward cylinder from the decision
cylinder. The entrance of the decision cylinder also had a baffle to slow the
bees down, which made observation easier, and gave the bees more time to look
at the visual stimuli. This maze set-up is well established in honeybee
behavioural research (Srinivasan and
Lehrer, 1988; Zhang et al.,
1992; Zhang and Srinivasan,
1994; Zhang et al.,
1995; Zhang et al.,
1996; Zhang et al.,
1999).
During training, the positions of the positive and negative patterns at the mazes were regularly swapped every 30 min, so that the bees could not use position as a cue to find the feeder. Similarly, the positions of the positive and negative patterns were interchanged every 10 min in the middle of the learning tests and every 5 min in the middle of the transfer tests.
Visual stimuli
The stimuli were presented as 18 cmx18 cm squares (grating patterns)
or 18 cm diameter circles (sector and ring patterns, details in Fig. S1 in
supplementary material) on the exits of the second cylinder. They were printed
on normal copy paper using a Fuji Xerox Document Centre C360 PS colour
printer. The training stimuli in maze A were always blue/white, and the
training stimuli in maze B were always yellow/white. The stimuli for the
transfer tests at the mazes A and B were black/white. The stimuli for the
transfer tests at maze C were blue/white, yellow/white or black/white.
Horizontal versus vertical gratings (Figs
1,
3,
4) and sector versus
ring patterns (Fig. S1a,b in supplementary material) were used as visual
stimuli with different groups of bees. The rewarded pattern, which provided
access to a feeder, was termed `positive', the unrewarded pattern was termed
`negative'.
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During training, the yellow vertical grating provided access to the feeder
at maze B in the morning, and the blue horizontal grating indicated the feeder
position at maze A in the afternoon (Fig.
1). When the sector and ring patterns were used, the yellow sector
pattern was positive at maze B in the morning, and the blue ring pattern was
positive at maze A in the afternoon (Fig. S1a in supplementary material).
Using the two colours blue and yellow at the two training mazes made learning
easier for the bees, probably because cues stay longer in memory when offered
in combination with other, simultaneously offered cues
(Lindauer, 1970;
Colborn et al., 1999;
Fauria et al., 2002;
Cheng, 2005).
Data collection
During the learning tests and the transfer tests at Maze A or Maze B, both
mazes were observed, and every positive and negative decision in the mazes was
recorded. Only the first choice of each bee during one foraging flight was
included in the data. The reward continued to be offered during all tests, to
prevent bees from losing their motivation to visit the apparatus
(Zhang et al., 1999). The
learning tests lasted for 20 min, and the transfer tests lasted for 10 min. In
the middle of each testing period (after 10 min in the learning tests, and
after 5 min in the transfer tests), the positions of the patterns were swapped
in order to cancel out any effect of a possible side bias. All transfer tests
were followed by at least 30 min of normal training at maze A or B. This
shorter testing period, and subsequent training under normal conditions,
ensured that the bees did not learn during the transfer test conditions. Each
bee was allowed to make a maximum of three rewarded visits in each of the
transfer tests, which is not enough to learn a pattern discrimination task.
Additionally, there was a break of at least 24 h before a transfer test
condition was repeated. When testing the bees at maze C, mazes A and B were
disassembled, in order to make the bees visit maze C.
Tests at mazes A and B
The performance of individual bees was recorded in the learning tests.
During training, the bees learned for each of the mazes A and B where and when
to go, and what to do there. The constant control of the learning level
ensured that the bees were well trained throughout the transfer tests. These
tests were repeated with a different group of bees, using the sector and ring
patterns (Fig. S1a in supplementary material).
In experiment 1, we investigated whether honeybees can distinguish the patterns at two locations without the colour cue, using black patterns in mazes A and B (Fig. 1). If the bees were still able to choose the positive pattern, we could be certain that they had used the maze location cue (where), the shape cue (what), and the time cue (when), independently of the colour cue. These tests were repeated with a different group of bees, using sector and ring patterns (Fig. S1b in supplementary material).
Transfer tests at maze C
In experiment 2, we examined whether honeybees can choose the learned
training patterns at a novel location, namely in maze C
(Fig. 1a). The bees had never
visited maze C before, and thus the maze location cue was excluded. They had
to base their decision on the pattern colour and shape (what) and the time of
day (when), transferring their knowledge of what to do in a certain timeframe
to a new `where'.
In experiment 3, we examined whether honeybees can choose the trained colour independently of the location and shape cue. Yellow and blue vertical gratings were used in the morning, and yellow and blue horizontal gratings were presented in the afternoon (Fig. 1b). Thus, the bees could not use the pattern shape and the location cue for decision making. Since both patterns had the same (positive) shape, the bees had to decide between yellow and blue according to the time of day at the new location.
In experiment 4, we examined whether honeybees can choose the trained shape independently of the location and colour cue. We excluded the colour and location cues by presenting black gratings at maze C (Fig. 1c). The bees had to choose a pattern shape according to the time of day, without relying on pattern colour or maze location as cues.
Analysis of performance
We first performed analyses of variance (ANOVA) across all repeated tests
for individual bees and for each type of experimental condition, using the
statistical software SYSTAT 11 to check the homogeneity of the data. Once the
data were found to be homogeneous, the performance of each bee was evaluated
separately by pooling its correct choices and visits over all repeated tests,
and calculating the ratio of the number of correct choices to the number of
visits. Then, the average performance for a particular experimental condition
was obtained by averaging choice frequencies across bees. The sample size
(N) was the number of bees, rather than the number of individual
choices, ensuring that the samples were truly statistically independent. Mean
values of choice frequency, standard deviation (s.d.) and standard error of
the mean (s.e.m.) were calculated. In the text and in the figures, performance
is indicated by the mean choice frequency (± s.e.m.). In the analysis,
we included only bees that visited both of the mazes regularly. A visit at the
correct maze was counted when the bee entered the maze and made a decision. A
visit at the wrong maze was counted when the bee entered the maze, and also
when it hovered in front of the maze.
The performances in the morning and the afternoon tests were compared by GraphPad Prism statistical software, using two-way repeated measurement ANOVA (Time: morning versus afternoon; Repeated measurements) to determine whether performance changed significantly during the time and between the repeated measurements. Post-hoc comparisons were done by means of the Bonferroni t-tests, which compared each repeated measurement in the morning and in the afternoon (for example, percentage of choices for the vertical pattern in the afternoon, compared with the percentage of choices for the vertical pattern in the morning). Control experiments were carried out at the end of experiments to test for a possible side bias. We conducted a simple dual choice test at Maze A and Maze B, for which 2x2 McNemar tests were used for statistical analysis.
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The transfer test conditions (black patterns at mazes A and B) did not affect the bees' location preference. During the morning transfer tests, most visits (0.92±0.06) from 8 tests were recorded at maze B (n=8, total visits N=124); whereas only a small number of bees (total 10 visits in 8 tests) approached maze A. In the afternoon transfer tests, most visits (0.97±0.03) from 8 test times were recorded at maze A (n=8, total visits N=440; Fig. 2B), whereas a very small number of bees (total 13 visits in 8 tests) visited maze A. These bees visited the wrong maze, i.e. maze A in the morning and maze B in the afternoon and confined their visit to a quick fly-over, only occasionally entering the maze. The bees still preferred maze B in the morning, and reversed their preference to maze A in the afternoon (t=39.1, d.f.=14, P<0.001).
The bees learned to choose the yellow vertical grating at maze B in the morning, and the blue horizontal grating at maze A in the afternoon
The learning tests were conducted to ensure that the bees had a constant
learning level throughout the experimental period. Once again, the bees had to
fly to maze B in the morning, and choose the yellow vertical grating there. In
the afternoon, they had to forage at maze A, and choose the blue horizontal
grating. The results are shown in Fig.
3A.
Analysis of variance showed no significant differences in the performances of individual bees on all testing days either in the morning (ANOVA, d.f.1=5, d.f.2=84, F=1.14, P=0.362) or the afternoon sessions (ANOVA, d.f.1=13, d.f.2=75, F=0.787, P=0.672) at mazes A and B. When the bees visited maze B in the morning during training (Fig. 3A), they chose the yellow vertical grating in most of the visits (0.88±0.02, N=28). In the afternoon at maze A (Fig. 3A), their choice frequency for the blue horizontal grating was 0.80±0.02 (N=29). Results of ANOVA tests are as follows: d.f. (interaction)=7, F=0.573, P=0.778; d.f. (time)=1, F=853.6, P<0.001; d.f. (repeated tests)=1, F=1.737, P=0.102. All the Bonferroni post tests showed performance in the morning and in the afternoon are significantly different. Thus, they learned to reverse their pattern preference between morning and afternoon. The transfer tests were started with this performance as a baseline.
Experiment 1
The bees could distinguish black patterns at mazes A and B
Analysis of variance showed no significant differences between the
performances of individual bees on all testing days either in the morning
(ANOVA, d.f.1=6, d.f.2=25, F=1.584, P=0.193) or in the
afternoon (ANOVA, d.f.1=14, d.f.2=44, F=0.52, P=0.908). When
the bees were tested for transfer to black gratings at mazes A and B
(Fig. 3B), they chose the
vertical grating at maze B in the morning in most of the visits
(0.74±0.04, N=10). In the afternoon at maze A, the choice
frequency for the horizontal grating was 0.72±0.04 (N=20).
Results of ANOVA tests are as follows: d.f. (interaction)=2, F=0.05,
P=0.949; d.f. (time)=1, F=75.12, P<0.001; d.f.
(repeated tests)=2, F=2.257, P=0.113. All the Bonferroni
post tests showed that performances in the morning and in the afternoon are
significantly different. The bees significantly reversed their pattern
preference from vertical in the morning to horizontal in the afternoon, even
without the colour cue, choosing a grating orientation according to the time
of day (when) and the maze location (where).
Experiments 2–4 at maze C
Maze C had a neutral position between the training mazes A and B
(Fig. 1), and the bees had
never foraged in this maze before. To avoid any further learning at maze C
during the transfer tests, the testing time was kept short, so that each bee
did not visit more than three times in each testing session. Moreover, the
transfer tests were followed by at least 30 min of normal training at mazes A
and B.
Experiment 2
Honeybees can apply the learnt rules from mazes A and B to a new location within a temporal context
In the first transfer experiment at maze C, the bees were tested with the
usual training patterns, but with the location cue excluded. They encountered
yellow gratings in the morning and blue gratings in the afternoon. Analysis of
variance showed no significant differences between the performances of
individual bees on all testing days either in the morning (ANOVA, d.f.1=5,
d.f.2=15, F=1.334, P=0.303) or in the afternoon (ANOVA,
d.f.1=7, d.f.2=6, F=0.918, P=0.55). The results of this
experiment are shown in Fig.
4A. The choice frequency for the yellow vertical grating in the
morning was 0.94±0.02 (N=18), while the choice frequency for
the blue horizontal grating in the afternoon was 0.73±0.06
(N=9). Results of ANOVA tests are as follows: d.f. (interaction)=1,
F=2.736, P=0.108; d.f. (time)=1, F=220,
P<0.0001; d.f. (repeated tests)=1, F=0.283,
P=0.598. All the Bonferroni post tests showed that performances in
the morning and in the afternoon are significantly different. They reversed
their pattern preference in the same (neutral) location, choosing a grating
according to the time of day (when) and the pattern colour (what).
Experiment 3
Honeybees can use colour cues alone to make a correct decision at maze C within a temporal context
In the next experiment at maze C, the bees were tested for colour
preference by presenting blue and yellow vertical gratings in the morning, and
blue and yellow horizontal gratings in the afternoon. Thus, we excluded the
pattern orientation and the maze location cues. Analysis of variance showed no
significant differences between the performances of individual bees on all
testing days either in the morning (ANOVA, d.f.1=9, d.f.2=12,
F=0.625, P=0.756) or in the afternoon (ANOVA, d.f.1=7,
d.f.2=8, F=0.170, P=0.985). The results of this experiment
are shown in Fig. 4B. The
choice frequency for the yellow grating in the morning was 0.87±0.04
(N=19). In the afternoon, the bees preferred the blue horizontal
grating over the yellow horizontal grating, with a choice frequency of
0.95±0.02 (N=11). Results of ANOVA tests are as follows: d.f.
(interaction)=1, F=2.827, P=0.102; d.f. (time)=1,
F=344.5, P<0.0001; d.f. (repeated tests)=1,
F=3.646, P=0.065. All the Bonferroni post tests showed
performance in the morning and in the afternoon are significantly different.
The bees were able to reverse their colour preference from yellow in the
morning to blue in the afternoon in a neutral location, basing their decision
in the maze on the temporal context.
Experiment 4
Honeybees can use orientation cues without colour cues, to make a correct decision at maze C within a temporal context
In the last experiment of this series, the location and colour cues were
excluded by presenting black horizontal and vertical gratings at maze C
(Fig. 4C). The bees had to
choose a grating orientation according to the time of day. Analysis of
variance showed no significant differences between the performances of
individual bees on all testing days either in the morning (ANOVA, d.f.1=10,
d.f.2=8, F=0.539, P=0.822) or in the afternoon (ANOVA,
d.f.1=12, d.f.2=10, F=1.638, P=0.221). In the morning, the
bees preferred the vertical grating (0.84±0.04, N=12). In the
afternoon, they significantly reversed their pattern preference to the
horizontal grating (0.69±0.06, N=11). Results of ANOVA tests
are as follows: d.f. (interaction)=1, F=1.631, P=0.209; d.f.
(time)=1, F=52.4, P<0.0001; d.f. (repeated tests)=1,
F=0.243, P=0.625. All the Bonferroni post tests showed that
performances in the morning and in the afternoon are significantly different.
The bees could reverse their pattern preference solely within a temporal
context; no other cue was present to influence the bees' choices for the
horizontal or vertical patterns. The same was true for the experiments with
sector and ring patterns (see Fig. S2 in supplementary material).
To negate the possibility that the bees had an unexpected preference for the vertical pattern, all experiments at the mazes A and B and the black pattern transfer test at maze C were repeated with a different group of bees and a set of central symmetric sector and ring patterns. The results are shown in Figs S1 and S2 in the supplementary material.
Control tests
Control tests were conducted to ensure that the honeybees did not develop a
preference to a particular side of the maze. In these tests, the decision
cylinders carried the same visual patterns on both sides, and no food reward.
The bees' decisions for the left or right side were monitored.
At maze A, the choice frequency for the right side was 51.7%, while that for the left side was 48.3%. Thus, the bees did not have a preference for a particular side of maze A (2x2 McNemar test, d.f.=1, P=0.961, see Fig. S3a in supplementary material). At maze B, the bees chose the right side in 52.8% of the visits. The left side was chosen in 47.1% of the visits. There was no significant difference in the bees' choices for the left or the right side of maze B (2x2 McNemar test, d.f.=1, P=0.936, see Fig. S3b in supplementary material).
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). In our
experiments, during training, the bees learned to choose the yellow vertical
grating at maze B in the morning, and the blue horizontal grating at maze A in
the afternoon (Figs 1,
2). The very low error rate in
this training indicates that visiting two or more feeding places at fixed
times is an easy task for foragers, and might be a common strategy in
honeybees. The aim of experiment 1 was to investigate whether honeybees can still find the correct pattern shape, without the colour cue. The bees extracted the orientation information of the coloured patterns, and applied it to the novel black patterns (Fig. 3A,B). The same was true for the sector and ring patterns (see Fig. S3b in supplementary material). They showed a clear preference for the correct pattern at the respective maze and time of day.
Foraging at a novel feeding site
In all the transfer tests at the neutral maze C (experiments 2–4),
the location cue was excluded. This set-up further allowed us to artificially
remove all other cues, except the temporal cue, as required. Under these
conditions, the bees had to rely on their time-sense when deciding between
visual patterns that possessed only partial features, such as colour or
shape.
The aim of experiment 2 was to investigate whether honeybees can transfer learnt rules to a novel location. The data show that the bees recalled the rewarded patterns according to the pattern colour and the time of day, independent of the location cue (Fig. 3A). In this case, the colour and the time could prime the visual memory of shape, i.e. choosing the vertical grating if they saw yellow, and the horizontal grating if they saw blue, or choosing the vertical shape in the morning and the horizontal shape in the afternoon. The next experiment was carried out to check the importance of pattern colour.
Experiment 3 was conducted to check whether honeybees can transfer previously formed time–colour associations to a novel location. The high performance of the bees in this experiment (Fig. 4B) shows the importance of the colour cue in decision-making. As the pattern orientation always suggested a positive pattern, the bees had to decide on a colour according to the time-frame. The bees recalled the correct colour according to the time of day from their memory.
In experiment 4, we tried to determine if honeybees can transfer previously formed time–shape associations to a novel location. The lower performance in this experiment (Fig. 4C), compared to the colour experiment (Fig. 4B), shows that this task was more difficult for the bees. However, they still significantly reversed their pattern preference between morning and afternoon. The location and the colour cue were not necessary for the bees to make a correct decision in this test. While the shape cues (orientation of gratings) certainly made the learning process easier, the bees successfully extracted and memorized the rule `choose the vertical grating in the morning and the horizontal grating in the afternoon', and recalled it to solve the task, independent of the pattern colour, and without a location context. The same is true for the additional experiments with sector and ring patterns (see Fig. S3c in supplementary material).
The results confirm that bees would be able to forage from different kinds of flowers, at different times of the day, at the same feeding location. They could also select a particular kind of flower when visiting a new feeding location, recalling the memory of the most profitable flower species for a particular time of day.
These results cannot be an artefact caused by learning during a test,
because it takes 20–30 visits for a honeybee to learn a geometric
pattern (Zhang and Srinivasan,
1994; Zhang et al.,
2004). The transfer test periods were kept short, so that each bee
could make a maximum of three foraging trips per testing session. In addition,
the 10 min testing period was followed by at least 30 min of normal training,
which would also prevent a possible learning effect
(Fischer, 1957).
Context learning
Contextual cues offer the possibility of treating the same stimulus in two
or more different ways, thereby enabling the animal to interact more flexibly
with its environment. In the case of the honeybee, contextual cues are
essential for efficient foraging. When visiting a patch of flowers, the bees
can decide on a profitable flower within the temporal context, i.e. choose the
yellow flower in the morning and the blue flower in the afternoon, even when
exploring new foraging territories.
Pattern memory can be primed by two locations, in which a bee that is
trained to recognize one pattern at one site and another pattern at a second
site will choose A+ over B– at site A, but B+ over A– at site B
(Menzel et al., 1996). Bees'
memories can also be primed by the surrounding panorama, which includes
spatial and colour contextual cues (Cheng
et al., 1986; Collett and
Kelber, 1988; Collett et al.,
1997; Dale et al.,
2005). A familiar nectar scent, encountered at the hive entrance
before departing, can trigger specific route memories that expedite navigation
to one of two different food sources
(Reinhard et al., 2004). The
limit of memorisable scent–feeder associations seems to be two; to
distinguish three different feeder–scent combinations the bees need
additional cues (e.g. colours) (Reinhard
et al., 2006).
Finally, recent experiments have shown that honeybees are able to change
their visual pattern preference in the presence of two cues: the time of day
and the task at hand (Zhang et al.,
2006).
In our present experiment, location cues were excluded by testing the bees at a neutral position (in maze C), the task at hand was always foraging, and the only reliable rule for the bees was the time–colour or the time–shape association. The bees chose the correct patterns at maze C in two different experiments, at a level significantly different from random choice. This indicates that bees can still find the correct visual pattern according to the time of day, when all other available cues are excluded.
The honeybees' internal clock
Animals ranging from bees to rats routinely record the time of day at which
they have a noteworthy experience and make use of this record to time their
subsequent behaviour (Gallistel
,1990). Bees need a precise time-sense to compensate for the
movement of the sun in their dance language, which is performed in the total
darkness of the hive (Lindauer,
1960). Some `marathon dancers' perform the recruitment waggle
dance for hours, without leaving the darkness of the hive, accurately
indicating the direction of a food source with respect to the sun's azimuth at
any time of the day or night (Chalifman,
1950; Lindauer,
1954; Wittekindt,
1955). It was suggested that bees are innately informed of certain
general spatial and temporal features of solar movement
(Dyer and Dickinson, 1994).
Bees can synchronize their behavior with daily floral rhythms, foraging only
when nectar and pollen are at their highest levels. At other times, they
remain in the hive, conserving energy that otherwise would be exhausted on
non-productive foraging flights (Moore,
2001).
The greenhouse used in our experiments was covered by an opaque PVC sheet,
which was similar to the materials used to cover an extension of our All
Weather Bee Flight Facility (AWBFF). We have discussed the effect of UV and
polarized light on circadian rhythms in the honeybee in a previous publication
(Zhang et al., 2006). However,
we still measured the illumination spectrum in the greenhouse where the
present experiments were carried out, and found that UV light was tremendously
reduced to undetectable levels at the maze areas. In addition, the bees'
performance was not affected by large changes in the weather. The weather
record for the region shows that our experiments were sometimes carried out
under complete cloud cover. Even then, the bees could still perform as usual.
The consistent results obtained under different weather conditions indicate
that the honeybees did not need to use the sun's position, UV or polarized
sunlight as cues to change their preference according to time.
It is not clear, however, if the honeybee has a time-sense governed by a
circadian rhythm, connecting a specific memory to a certain phase in the 24
h-cycle, or if it is also capable of measuring the elapsed time between two
events. Interval-timing has been shown in vertebrates
(Richelle and Lejeune, 1980;
Gallistel, 1990;
Babb and Crystal, 2005), and
recently in an invertebrate from the same family as the honeybee, the
bumblebee Bombus impatiens
(Boisvert and Sherry, 2006).
Further experiments are planned to investigate the question of interval timing
in the honeybee. If honeybees show this ability, their memory of `what',
`where' and `when' could fulfil the behavioural criteria for episodic-like
memory in nonhuman animals, as shown in the food-caching scrub-jay
Aphelocoma coerulescens (Clayton
and Dickinson, 1998). Our experiments have shown that the honeybee
links together the elements of circadian time (when during a day), place
(where), and colour and pattern characteristics (what) in an integrated
fashion. This is akin to episodic-like memory, except that the temporal
element is circadian time, instead of interval timing. We have named this kind
of memory circadian timed episodic-like memory.
Cue ranking
In the learning tests (Fig.
3A and supplementary material Fig. S1a), the bees reached an
average performance (morning and afternoon sessions) of 83% correct choices.
Setting this as a baseline, and comparing it with the other tests where one or
several contextual cues were taken out, we can compare the difficulty of the
transfer tests, and thus determine the relative importance, to the bees, of
the different cues.
In the transfer tests at maze C, the bees reached their best average
performance of 91% in experiment 3, the colour discrimination task
(Fig. 4B). The performance in
the morning showed no significant difference between the learning test and the
transfer test (t=0.258, d.f.=45, P>0.8); however, in the
afternoon, the performance in this transfer test was even better than that of
the learning test (t=3.6, d.f.=38, P>0.001), regardless
of the missing location and pattern orientation cues. Thus, colour seems to be
the most important visual cue for honeybee choice behaviour. These findings
are consistent with previous reports that honeybees learn a new colour after
about five visits, whereas they normally require 20–30 visits to learn a
pattern (Zhang and Srinivasan,
1994).
Using the training patterns at maze C in experiment 2, the bees performed about the same as in the training tests at mazes A and B (83%, Fig. 4A). There was no significant difference in performance between the learning test and the transfer test in the morning (t=2.01, d.f.=44, P>0.05) and in the afternoon (t=1.25, d.f.=36, P>0.20). Here, the only missing cue was the maze location. This cue seems to have had almost no effect on the bees' choice performance in small scale navigation, when other contextual cues were available. The results of this transfer test demonstrate that pattern colour and the time of day were enough to allow a baseline level of performance at a new location.
When the colour cue and the location cue were both taken out in experiment 4, the bees' average performance was reduced to 72% (Fig. 4C and supplementary material Fig. S2). The performance in the morning was slightly worse in the transfer test than in the learning test, but not significantly so (t=1.10, d.f.=38, P>0.2). However, in the afternoon, the performance in the transfer test was slightly worse than that of the learning test (t=2.1, d.f.=40, P<0.05). The results indicate that the shape cue is more difficult for the bees to use than the colour cue. Pattern orientation or, in nature, the shape of different flowers, is thus more important than location for the bees' choice behaviour once they have reached their feeding site. The bees clearly used the former to distinguish between the patterns in all experiments except the colour discrimination task (where pattern orientation was unavailable).
Applying these findings to the natural situation, we can say that the colour and shape of flowers are the most important visual cues used by bees to choose between different flower species. When visiting different feeding sites, or different patches of flowers, they can recall their memory of the most rewarding species in conjunction with the time of day, and thus find the most profitable food source even at a new location.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work 
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References |
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