J Comp Physiol A (2006) 192: 691700

DOI 10.1007/s00359-005-0091-4

O R I GI N A L P A P E R

Sayaka Hori Hideaki Takeuchi Kentaro Arikawa

Michiyo Kinoshita Naoko Ichikawa
Masami Sasaki Takeo Kubo

Associative visual learning, color discrimination, and chromatic

adaptation in the harnessed honeybee Apis mellifera L.

Received: 14 March 2005 / Revised: 22 December 2005 / Accepted: 22 December 2005 / Published online: 20 January 2006

Springer-Verlag 2006

Abstract We studied associative visual learning in har-

nessed honeybees trained with monochromatic lights
associated with a reward of sucrose solution delivered to
the antennae and proboscis, to elicit the proboscis
extension reex (PER). We demonstrated ve properties
of visual learning under these conditions. First, antennae
deprivation signicantly increased visual acquisition,
suggesting that sensory input from the antennae inter-
feres with visual learning. Second, covering the com-
pound eyes with silver paste signicantly decreased
visual acquisition, while covering the ocelli did not.
Third, there was no signicant dierence in the visual
acquisition between nurse bees, guard bees, and forag-
ers. Fourth, bees conditioned with a 540-nm light stim-
ulus exhibited light-induced PER with a 618-nm, but not
with a 439-nm light stimulus. Finally, bees conditioned
with a 540-nm light stimulus exhibited PER immediately
after the 439-nm light was turned o, suggesting that the
bees reacted to an afterimage induced by prior adapta-
tion to the 439-nm light that might be similar to the 540-
nm light.
Electronic Supplementary Material Supplementary material is
available for this article at http://dx.doi.org/10.1007/s00359-005-
0091-4 and is accessible for authorized users.
S. Hori H. Takeuchi (&) T. Kubo
Department of Biological Sciences, Graduate School of Science,
The University of Tokyo, 113-0033 Bunkyo-ku, Tokyo, Japan
E-mail: takeuchi@biol.s.u-tokyo.ac.jp
Tel.: +81-3-58414448
Fax: +81-3-58414448
K. Arikawa M. Kinoshita
Graduate School of Integrated Science,
Yokohama City University, 236-0027 Yokohama, Japan
N. Ichikawa M. Sasaki
Faculty of Agriculture, Tamagawa University,
194-8610 Machida, Tokyo, Japan
Introduction
The excellent visual capacity of the honeybee Apis
mellifera L. was rst discovered by von Frisch (1967)
who demonstrated that bees see and discriminate colors
in the appetitive context of food search. Honeybees are
able to discriminate various colors, shapes, and patterns
in a visual conditioning paradigm in which free-ying
bees are trained to y towards training/test targets and
are rewarded with sucrose solution if they select the
correct target (von Frisch 1967; Zhang et al. 2004;
Giurfa et al. 2001; Giurfa 2003; Horridge 2003; Stach
et al. 2004). Recently, Giurfa et al. (2001) used honey-
bees conditioned with a series of colors and patterns to
demonstrate that bees can form concepts of sameness
and dierence, and can build and generalize congural
pattern representations (Stach et al. 2004). Such high-
order cognitive visual performances of the honeybee are
supported by a remarkable small brain (approximately
1-mm3 volume) containing only approximately 9.6105
neurons (Witthoft 1967).
How does the honeybee brain perceive and process
visual information? Three types of photoreceptors have
been identied in the honeybee retina; Short (S),
Medium (M), and Long (L), with maximum sensitivity in
the ultraviolet (S or ultraviolet receptor; kmax=350 nm),
blue (M or blue receptor; kmax=440 nm), and green (L
or green receptor; kmax=540 nm) regions of the spec-
trum, respectively (Menzel and Blakers 1976). The
photoreceptor neurons project to the optic lobe, the vi-
sual center of the insect brain. Neurons that respond to a
specic color or motion have been identied in the optic
lobe (Menzel and Backhaus 1991). Some visual inter-
neurons project to the collar and/or a part of the basal
ring region of the mushroom body (MB), the insect
brain region is considered to be involved in high-order
sensory processing (Mobbs 1982; Ehmer and Gronen-
berg 2002). The molecular mechanisms and neural
network underlying visual processing in the honeybee,
however, are poorly understood.
692
In contrast, the mechanisms underlying olfactory
processing have been extensively characterized, be-
cause harnessed honeybees can be used in olfactory
conditioning of the proboscis extension reex (PER)
(Takeda 1961; Bitterman and Menzel 1983). The
advantage of this conditioning paradigm using har-
nessed bees is that, in contrast to visual learning, bees
are immobilized, thus allowing access to their nervous
system, which can be exposed by carefully cutting the
cuticle of the head capsule. In this way, olfactory
conditioning of PER can be coupled with experimental
techniques, such as electrophysiology (Mauelshagen
1993; Hammer 1993; Abel and Menzel 2001; Muller
et al. 2002), calcium imaging (Faber et al. 1999; Faber
and Menzel 2001; Sachse and Galizia 2003; Sandoz
et al. 2003; Galizia et al. 1999; Guerrieri et al. 2005),
pharmacology (Muller 1996, 2000; Grunbaum and
Muller 1998; Hammer and Menzel 1998; Lozono et al.
2001), and double-stranded RNA-directed RNA
interference (Farooqui et al. 2003, 2004). Electro-
physiologic techniques were used to identify some key
neurons involved in the CS and US pathways in
olfactory-PER associative learning (Hammer 1993;
Mauelshagen 1993; Muller et al 2002). Pharmacologic
methods were used to demonstrate that long-term
olfactory memory formation requires key signal
transduction molecules, such as nitric oxide synthase,
cyclic AMP-dependent protein kinase A, and protein
kinase C (Muller 1996, 2000; Grunbaum and Muller associative learning is expected. Moreover, the extent
1998; Lozono et al. 2001). Such a vast amount of
research on olfactory learning contrasts with the
scarcity of information concerning visual learning in
bees, especially with respect to the cellular and
molecular substrates. Clearly, progress in this area has
been impeded by the fact that visual learning in bees
requires free-ying bees, making it dicult to simul-
taneously access their brains. Nevertheless, the rst
study on PER conditioning was performed using vi-
sual stimuli instead of olfactory stimuli as the CS
(Kuwabara 1957). Kuwabara (1957) immobilized bees
by clipping their wings and elicited the PER by
touching the tarsae with sucrose solution contained in
a small spoon. Because the bees responded by PER to
the stimulation of water vapor from the spoon,
Kuwabara (1957) amputated the antennae to suppress
this confounding factor. Thus, Kuwabara used bees
that were deprived of their antennae and trained with
dierent spectral stimuli (from 410 to 630 nm). From
6 to 38 learning trials were necessary for the bees to
learn the light-reward association and such a large
range seemed to be wavelength-dependent, as learning
long-wavelength stimuli required fewer trials than
short-wavelength stimuli. The use of a small number
of bees per trained light condition (N=10) and the
lack of appropriate controls in which the CS and US
were presented unpaired in his study, however, makes
it dicult to draw denitive conclusions from the
results. Kuwabaras (1957) results suggested that
bees could broadly discriminate long wavelength (from
500 to 650 nm) from short wavelength (from 400 to
450 nm) regions of the spectrum. Interestingly, acqui-
sition in this visual conditioning paradigm seemed to
be robust as bees showed no sign of extinction even
after 30 consecutive CS presentations without any
reward at 5-min intervals (Kuwabara 1957).
Since Kuwabaras (1957) pioneering work, the idea
of conditioning PER in harnessed bees with light
stimuli was essentially abandoned. Fifteen years later,
Masuhr and Menzel (1972) also reported that bees
deprived of their antennae could learn to associate light
stimuli and sucrose reward, but no further research was
continued along this line. In 1998, Gerber and Smith
demonstrated that color illumination had an indirect
role in honeybee olfactory learning, but did not dem-
onstrate specic acquisition of light-reward associa-
tions. On the other hand, it is a common observation
that PER cannot be conditioned in harnessed bees
presented with colored cardboards. In such experi-
ments, however, antennal deprivation has generally
been neglected. It is therefore possible that such
deprivation constitutes a critical factor for acquisition
of the light-reward association, a fact that Kuwabaras
(1957) work did neither study nor consider. Addition-
ally, Kuwabaras (1957) results did not include the
appropriate controls for associative learning, such as
the unpaired presentation of CS and US, where no
to which bees learned to associate chromatic or ach-
romatic cues with sucrose reward also remains unclear.
In this context, separating the contributions of the
compound eyes and of the ocelli to the performances
observed could be a rst step in distinguishing between
these alternatives.
In the present study, we aimed at reproducing
Kuwabaras (1957) basic experiment in a controlled way
so that a robust protocol for visual associative learning
can be made available under laboratory conditions. We
modied some basic features of this original paradigm
and trained bees with dierent monochromatic lights
paired with sucrose solution delivered to the proboscis.
We then compared explicitly CSUS paired and
unpaired groups to determine whether changes in per-
formance over conditioning trials are truly associative.
We studied the eect of antenna deprivation on this
performance and determined the dierential contribu-
tions of ocelli and compound eyes, and whether or not
bees with dierent task specializations dier in their rate
of acquisition. Here, we demonstrated that bees can
learn to associate light stimuli with sucrose reward; this
learning is truly associative and that antennal depriva-
tion is critical for such learning to occur. We also
showed that visual input through the compound eyes is
necessary and that visual capabilities, such as color
discrimination and chromatic adaptation, can be ana-
lyzed in harnessed bees by exploiting the PER, as in
olfactory conditioning.

Materials and methods
Preparation of the bees
European honeybees (Apis mellifera L.) maintained at
the University of Tokyo were used for the experiments.
Colonies were purchased from the Kumagaya Honeybee
Farm (Saitama, Japan). For all the experiments except
the one in which workers were compared according to
the division of labor, ying workers were randomly
collected near the hive entrance on sunny days from
spring to autumn. In the winter, the honeybee colonies
were kept in a greenhouse maintained at 1325C and
the workers were collected similarly. To test for poten-
tial dierences in acquisition due to dierent task spe-
cialization, we collected foragers, guard bees, and nurse
bees. Worker bees exhibit age-polytheism such that the
labors of workers change according to their age after
eclosion: young workers (nurse bees) are engaged in
nursing their brood, middle-aged workers (guard bees)
in guarding the colony from natural enemies, and older
bees (forgers) in foraging for nectar and pollen (Winston
1987). Foragers were captured at the hive entrance and
were collected as workers with pollen loads on their hind
legs. Guard bees were collected as workers that attacked
tweezers presented as a decoy at the hive entrance. Nurse
bees were collected as workers that had inserted their
heads into brood combs at least twice successively or for
more than 20 s. To collect nurse bees, frames holding the
sheets of honeycomb were taken out and observed out-
side of the hive box.
Captured bees were anesthetized by cooling on ice.
Following Kuwabaras (1957) procedure, both antennae
were cut with ne scissors at the base of the scapus,
taking care not to pull them. Bees with leaking hemol-
ymph were omitted (<1%) from the experiments. The
bees were then harnessed in 500-ll plastic centrifuge
tubes with small amounts of cotton at the bottom to
support the bees weight. The harnessed bees were sta-
bilized using a collar placed between their heads and
thoraces. The bees were then kept on a 16h:8h light
photoperiod (photo-phase 05:0021:00 h) at 25C and
6070% humidity, and fed 10 ll of 1.4 M sucrose
solution every morning and evening. Two days after
being placed in the tubes, harnessed bees that performed
prompt PER when sucrose solution touched the pro-
boscis were selected for conditioning experiments. More
than 90% of the total bees passed this screening.
Associative visual PER conditioning
The selected bees were trained with 10 conditioning
trials per day for 2 days (total 20 trials). Each of the
harnessed bees was placed in the experimental arena in
a room illuminated with 40 W uorescent lamps. The
arena consisted of a tube holder (1072.5 cm3) and a
sheet of translucent light-projecting screen that was
693
attached as a roof providing a 5 cm high ceiling in the
center of the arena (Fig. 1). One side of the arena was
closed with a piece of white paper (76 cm2) to mini-
mize interference by uncontrolled visual information.
Light was provided by a 150 W high stable Xenon
lamp (Hamamatsu Photonics LC5 and L8253). A light
guide was used to illuminate the arena. The exit pupil
of the light guide was set 32 cm above the head of the
harnessed bee. Monochromatic light was provided by
attaching an interference lter (618, 540, or 439 nm;
half-bandwidth=ca. 10 nm) at the exit pupil. In most
experiments, bees were conditioned with a 618-nm light
(Figs. 3, 4, 5) whose intensity was measured at the
bees head (Sanso Radiometer 470-D) and adjusted to
2.411014 photons/cm2/s using the shutter. This wave-
length was chosen because it selectively activates L
receptors. In free-ying honeybees, L receptors are
important for visual detection and recognition of
chromatic stimuli (Giurfa et al. 1996, 1997; Hempel de
Ibarra and Giurfa 2003) and for orientation detection
(Giger and Srinivasan 1996; Stach et al. 2004). The
upper wavelength limit of L receptors is 650 nm,
whereas that of M receptors is 550 nm (Menzel and
Blakers 1976). Therefore, L receptors are selectively
stimulated by light in the 550 to 650-nm wavelength
range. For wavelength discrimination and the color
adaptation experiment (Figs. 6, 7), the intensity of each
monochromatic light was measured at the bees head
and adjusted to 2.411014 photons/cm2/s. The temper-
ature change in the arena due to the monochromatic
illumination was less than 0.2C, even after 10 min of
continuous illumination.
Approximately 30 s before each conditioning trial, a
honeybee was placed in the arena (i.e., under the light-
projecting screen). Two groups of bees were condi-
tioned. In the explicitly paired group, light (CS) was
temporally paired with sucrose solution (US). In the
unpaired groups, the CS was not paired with the US
(Fig. 2a). In the paired groups, bees were exposed to
either a 618- or 540-nm light (CS) for 7 s. Four seconds
after the onset of the CS, 1 ll of 1.4 M sucrose
solution (US) was delivered to the honeybees by means
of a micropipette directly touched to the proboscis to
evoke the PER, and the bees were fed for 3 s (Fig. 2b).
To avoid a response to the remaining sucrose solution,
the collar was covered with white plastic tape, which
was changed whenever sucrose solution was spilled on
it. In the paired group, 20 conditioning trials were
presented with a 20-min inter-trial interval (ITI). In the
unpaired groups, the CS and US were presented in
separate, alternating trials separated by 10 min. As for
the paired group, the CS lasted 7 s and the US 3 s. To
compare both groups in an equivalent condition with
respect to an ITI and the number of placements in the
conditioning arena, we added 20 additional placement
trials in the paired group interspersed with the condi-
tioning trials. In such placement trials, bees were placed
in the arena, but did not receive any stimulation.
We measured the percentage of PER over trials (see
694

Fig. 1 Schematic diagram of

the apparatus used for light-
PER associative learning. Bees
are harnessed in a small tube
and placed in the experimental
arena. The light stimulus is
indirectly irradiated from above
the light-projecting screen using
a Xenon lamp with a
monochromatic lter

Electronic Supplementary Material Table S1) and
analyzed whether performance changed signicantly
using repeated measures ANOVA. The data for bees
that died during the experiments were not included in
the analysis.
The contribution of ocelli and compound eyes to visual
PER conditioning
Corporation, AG-400-100, Japan) to suppress light
input. Paint was applied at least twice using a ne brush
and checked under a dissection microscope to conrm
that the eyes were entirely thickly covered. A non-treated
group of bees served as a control. We measured the
percentage of PER over trials and analyzed whether
performance changed signicantly using a one-way
repeated measures ANOVA.

To determine the visual input mediating visual PER Wavelength discrimination and chromatic adaptation
conditioning, we trained bees in an explicitly paired
manner (see previous), but painted either the compound A group of bees was conditioned with a 540-nm light
eyes and/or the ocelli with silver paste (The Nilaco stimulus explicitly paired with sucrose solution. A bee

Fig. 2 a Stimulus conditions.

The paired group rst received
a light stimulus (conditioned
stimulus, CS; 7 s) and was then
presented with sucrose
(unconditioned stimulus, US;
3 s). Note that the CS and US
co-terminated. The CSUS
pairings were presented with 20-
min inter-trial intervals (ITIs).
In the unpaired group, the CS
(7 s) and US (3 s) were
administered consecutively
every 10 min. b Response of a
conditioned honeybee under
the paired-stimuli condition. 1
Before the light stimulus (CS); 2
PER was evoked by the 618-nm
light stimulus as indicated by a
yellow arrow within the rst 4 s
of the stimulus; 3 the bee was
then fed with sucrose solution
for 3 s. A conditioned bee can
be viewed in Electronic
Supplementary Material Video
S1
 695

Results

Establishment of light-PER associative learning

We analyzed the performance of the explicitly paired

Fig. 3 Light-PER associative learning in the antennae deprived
honeybee using 618-nm monochromatic light. In the conditioning
procedure, 20 conditioning trials were presented with an approx-
imately 20-min ITI (N=28). In the unpaired group (10 trials/day;
N=28), the CS and US were presented consecutively at 10-min
intervals. Proboscis extension was recorded during the rst 4 s of
each CS period without US presentation. The data for bees that
died during the experiments were not included in the analysis. *
Signicant dierence, P<0.001
and the unpaired groups of bees deprived of their
antennae. Both groups were exposed to a 618-nm stim-
ulus. In the paired group, light stimulation was followed
by sucrose solution; in the unpaired group, light and
sucrose were not temporally associated. Figure 3 shows
the performance of both groups in terms of percentage
PER over the 20 trials. The reactions of individual bees
are shown in Electronic Supplementary material Table
S1. A conditioned bee can be viewed in Electronic
Supplementary Material Video S1. In the paired group,
the responsiveness to the CS increased signicantly over
trials (one-way repeated measures ANOVA: F=6.2124,
P<0.00001), and reached a level of approximately 40%

was used for wavelength discrimination and chromatic

adaptation experiments, if the CS induced PER three
successive times prior to the presentation of the US
during trials (see Electronic Supplementary Material
Table S2). The bees were tested with a 439-nm mono-
chromatic light, a 540-nm monochromatic light (the
CS), and a 618-nm monochromatic light. Stimulus pre-
sentations lasted 7 s each and were separated by a
20-min interval. These three tests were performed under
extinction conditions (i.e., no reward delivered after
light stimulation). We recorded the number of bees that
exhibited light-induced PER and the number of bees
that extended their proboscis within 7 s after the light
was turned o. We analyzed whether percentage of PER
in the six conditions (439-nm ON, 540-nm ON, 618-nm
ON, 439-nm OFF, 540-nm OFF, and 618-nm OFF)
changed signicantly using a one-way repeated measures
ANOVA.

Statistics

Repeated measures ANOVA was used for between-

group (two-way ANOVA) as well as within-group (one-
way ANOVA) comparisons. Although parametric
analysis of variance is usually not allowed for dichoto-
mous data, such as those of the PER, the Monte Carlo
studies indicate that it is permissible to use ANOVAs for
dichotomous dependent variables under certain condi-
tions, which are met by our data: equal cell frequencies
(Lunney 1970, Giurfa and Malun 2004). ANOVA,
however, can only indicate whether there is a signicant
dierence among the levels of the groups. If a signicant
main eect or interaction was found, the nding was
followed with post hoc tests, the Newman-Keuls tests
for repeated measures. The a-level was set to 0.05 (two-
tailed) for all analyses.
Fig. 4 a Eect of antennae deprivation on light-PER associative
learning. Proboscis extension rate for the antenna + (N=20) and
antenna (N=20) bees were plotted. *Signicant dierence,
P<0.01. b Eect of ocelli and/or compound eye occlusion on
light-PER associative learning. Proboscis extension rates for the
four groups were plotted. Number of bees tested were: N=29 for
bees with covered compound eyes (compound eye ocellus +);
N=29 for bees with covered ocelli (compound eye + ocellus );
N=29 for bees with both eyes covered (compound eye ocellus );
N=29 for intact bees (compound eye + ocellus +). * Signicant
dierence, P<0.05, **P<0.005
696
Fig. 5 Comparison of learning ability of nurse bees, guard bees,
and foragers. Proboscis extension rates for the three groups were
plotted. Number of bees tested were: N=20 for nurse bees; N=20
for guard bees; N=20 for foragers. There were no signicant
dierences (P>0.1)
on the 16, 17, and 19th trials, even though decay in
performance occurred on the 18 and 20th trials. Trials
14 and 16 through 20 were signicantly dierent from
trial 1 (Newman Keuls, post hoc tests, P<0.01). In
contrast, there was no signicant increase in the
unpaired group (one-way repeated measures ANOVA:
F=0.8201, P=0.683). Furthermore, two-way repeated
measures ANOVA indicated a signicant dierence be-
tween the paired and the unpaired groups (F=17.1941,
P<0.001). The dierence indicated that the increase in
responsiveness in the paired group was due to associa-
tive learning of the lightsucrose reward contingency.
Thus, PER can be conditioned using monochromatic
lights paired with a sucrose reward.
Eect of antennae deprivation
To analyze whether antenna deprivation is a critical
factor for successful visual PER conditioning, we tested
two groups of bees using a 618-nm light as the CS: one
group was completely deprived of their antennae and the
other was not (Fig. 4a). Although the responsiveness to
the CS over trials in the intact group increased slightly
(one-way repeated measure ANOVA: F=1.7504,
P<0.05) and reached a level of 520%, none of the
trials diered signicantly from trial 1 (Newman Keuls,
post hoc tests). Two-way repeated measures ANOVA
indicated signicant dierences in the PER rates
between these two groups (F=7.9502, P<0.01). To
exclude the possibility that physical damage due to
antenna deprivation by scissors might enhance the
acquisition of a visual learning, we also tested another
control group of bees that were deprived of the distal
regions (one segment) of both antennae. The bees were
assumed to have experienced physical damage similar to
that in the test group, but to still receive sensory input
from the antennae. As a result, the increase in CRs over
trials was not observed in the control group (Electronic
Supplementary Material Fig.S1. Two-way repeated
measures ANOVA indicated signicant dierences in
the PER rates between the control (N=10) and test
group (N=10) (F=5.690, P<0.05). These results
strongly suggested that sensory input from the antennae
(olfactory and/or mechanical stimulation) interferes
with the light-PER associative learning in this condi-
tioning paradigm. This might explain why attempts to
achieve visual PER conditioning without antennae
deprivation have been generally unsuccessful.
Contribution of ocelli and compound eyes
To examine the possible dierential contribution of
ocelli and compound eyes to visual PER conditioning,
we compared the performance of four groups of bees:
one in which the ocelli were painted with silver paste,
another in which the compound eyes were painted, a
third group with both the ocelli and compound eyes
painted, and a fourth group of intact bees (Fig. 4b).
Responsiveness to the CS over trials in two of the groups
(the intact group and the ocelli painted group) increased
signicantly (one-way repeated measures ANOVA:
F=7.976 and 4.0526, respectively, P<0.00001), while
that in the other two groups (the compound eyes painted
group, and the compound eyes and ocelli-painted group)
did not (one-way repeated measures ANOVA:
F=1.3952 and 1.4493, respectively). Two-way repeated
measures ANOVA indicated that there were signicant
dierences between the compound eye-painted group
compared with intact bees (F=11.7170, P<0.005),
whereas there were no signicant dierences between the
ocelli-painted group and intact bees (F=0.2225,
P>0.5). These results indicate that the visual informa-
tion received by the compound eyes was crucial in this
conditioning paradigm.
Comparison of the visual learning capacity according
to the division of labor of the workers
In the above experiments, we used ying workers col-
lected randomly near the hive entrance. As the PER rate
was not greater than 4050% in our conditioning par-
adigm, we hypothesized that visual learning ability
might dier depending on the division of labor of the
workers. To examine whether the learning ability of the
workers diered depending on their labor, we collected
nurse bees, guard bees, and foragers separately based on
their behavior, and subjected them to the conditioning
paradigm under the paired condition using a 618-nm
light stimulus (Fig. 5). Responsiveness to the CS over
trials in all three groups increased signicantly (one-way
repeated measures ANOVA, F=4.1271 (nurse bees),
F=4.0427 (guard bees), and F=2.2561 (foragers),
P<0.00001). Two-way factor ANOVA demonstrated
that there were no signicant dierences among the

Fig. 6 Wavelength discrimination and color adaptation. Bees
conditioned to a 540-nm light stimulus were irradiated with 540-
nm (panels 13), 439-nm (panels 46), or 618-nm light stimuli
(panels 79). Panels 1, 4, and 7 bees before irradiation; panels 2, 5,
and 8 bees irradiated with light (CS); panels 3, 6, and 9 bees just
after the light was turned o. Note that PER was evoked during
three groups (Fig. 5), indicating that these workers have
almost equivalent ability for light-PER associative
learning, irrespective of their roles in the colony
(F=0.7101, P>0.1).
Wavelength discrimination and chromatic adaptation
Free-ying bees have visual capabilities, such as color
discrimination (Giger and Srinivasan 1996; Hempel de
Ibarra and Giurfa 2003) and chromatic adaptation
(Neumeyer 1981). We examined whether these visual
capabilities can be demonstrated using harnessed bees in
our paradigm. First, we irradiated bees that had been
conditioned to a 540-nm light stimulus with 618- and
697
irradiation at 540 nm (panel 2) and 618 nm (panel 8), whereas it
was evoked just after the 439-nm light was turned o (panel 6), as
indicated by the yellow arrows. Wavelength discrimination and
chromatic adaptation in a harnessed bee can be viewed in
Electronic Supplementary Material Video S2
439-nm monochromatic light adjusted to the same
photon numbers (2.411014 photons/cm2/s). We used
bees that had three successive CS-induced PERs prior to
presentation of the US during trials. Approximately
50% of the bees in the paired group with a 540-nm CS
(N=37) had three successive PERs during 20 trials,
indicating that honeybees can also be conditioned to
light stimuli with a 540-nm stimulus (see Electronic
Supplementary Material Table S2). There was no sig-
nicant dierence between the ratio of bees that exhi-
bited PER upon irradiation with a 618-nm (60%) or
a 540-nm light stimulus (78%) (N=18) (Figs. 6, 7,
Newman Keuls, post hoc tests, P>0.05). In contrast,
none of the bees conditioned to the 540-nm light stim-
ulus exhibited PER upon irradiation with a 439-nm light
698
Fig. 7 Number of bees that exhibited color discrimination and
chromatic adaptation. Light ON shows the percentage of bees
that exhibited PER during light irradiation, while Light OFF
shows the percentage of bees that exhibited PER just after the light
was turned o. *Signicant dierence, P<0.05
stimulus (Figs. 6, 7). These results indicate that the 540-
nm light-conditioned bees generalized between 618- and
540-nm light stimuli, whereas they did not generalize
between 540 and 439 nm (Newman Keuls, post hoc tests,
P<0.01). To exclude the possibility that bees used total
brightness or contrast dierences instead of wavelength,
we performed a similar experiment, changing the light
intensity. We irradiated bees that had been conditioned
to the 540-nm light stimulus (2.411014 photons/cm2/s)
with a 439-nm light stimulus with a higher intensity (1.4
times; 3.421014 photons/cm2/s) or decreased intensity
of 540-nm monochromatic light (0.41 times;
9.761013 photons/cm2/s, 0.15 times; 3.691013 pho-
tons/cm2/s). Only 4.5% of the bees exhibited PER upon
irradiation with an increased 439-nm light stimulus
(N=22). In contrast, 90.0 and 63.6% of bees condi-
tioned to the 540-nm light stimulus exhibited PER upon
irradiation with a decreased 540-nm light stimulus
(0.41 times, and 0.15 times, respectively; see Electronic
Supplementary Material Fig. S2. N=22; Newman
Keuls, post hoc tests, *P<0.05, **P<0.01). Further-
more, we also exposed bees to these test lights in a
dierent order and the result was similar to previous
results (data not shown). The results clearly indicated
that the bees responded to dierences in wavelength, but
not brightness or contrast.
During this experiment, the bees conditioned to a
540-nm light stimulus exhibited PER immediately after
the 439-nm light was turned o, although they did not
exhibit PER during the 439-nm light irradiation. The
percentage of bees that exhibited PER immediately after
the cessation of the 439-nm light was approximately
65% (N=18; Figs. 6, 7). After cessation of the 540- and
618-nm light, approximately 15 and 10% of bees
exhibited PER, respectively (Figs. 6, 7). These results
can be accounted for by the eect of chromatic adap-
tation demonstrated in free-ying bees (Hempel de
Ibarra and Giurfa 2003): the bees might exhibit PER
responding to an afterimage induced by a prior adap-
tation to 439-nm light, which is similar to 540-nm light.
Discussion
In the present study, we established light-PER associa-
tive learning using harnessed bees. The bees were con-
ditioned to light stimuli of two dierent wavelengths,
540 and 618 nm. The 540-nm wavelength is the sensi-
tivity maxima of L receptors and 618 nm is exclusively
received by L receptors. Our results are consistent with
previous reports of L receptor involvement in visual
learning and recognition in free-ying bees (Giger and
Srinivasan 1996; Giurfa et al. 1996; Hempel de Ibarra
and Giurfa 2003; Stach et al. 2004). We used this con-
ditioning paradigm to demonstrate some properties of
visual associative learning of the harnessed bees. First,
antennae deprivation signicantly enhanced visual
acquisition in this paradigm. The importance of antenna
deprivation in this conditioning paradigm has been
neglected for almost half a century since Kuwabaras
report (1957). Although removing the antennae with
scissors risks serious injury, the motivation of the bees
might not be severely aected, as shown by the fact that
they learned and showed normal responsiveness to the
US and survival rate. A general decrease in motivation
should result in no learning and/or a signicant decrease
of the response to the US. In our experiment, all bees
deprived of their antennae responded to the US during
training, demonstrating that it was not the case.
Next, we demonstrated that blocking input to the
compound eyes signicantly impaired light-PER asso-
ciative learning, suggesting that the visual input received
by the compound eyes is important for learning in this
paradigm. In the honeybee, ocelli perceive the general
luminosity of the environment, whereas compound
eyes allow color, pattern, and shape perception among
others. In our conditioning paradigm, from the various
cues, chromatic and achromatic, provided by the
monochromatic lights used as conditioned stimuli, the
bees probably learned to associate chromatic cues with
sucrose reward. Our results argue against the possibility
that the honeybees are conditioned with sensory stimuli
other than light, such as temperature change and/or
mechanical stimuli.
Next, we demonstrated that the workers have essen-
tially equivalent learning capacities, irrespective of their
role in the colony. In contrast, phototaxis of the workers
is more developed in forager bees than in nurse bees
(Ben-Shahar et al. 2003). In addition, nurse bees are
inferior to foragers in olfactory learning (Ray and
Ferneyhough 1997, 1999). Our results suggest that the
neural and/or molecular basis underlying visual learning
is not identical to that of phototaxis and/or olfactory
learning. There are two possible reasons why we cannot
detect any dierence between foragers and nurse bees in
this experiment. One is that the nurse bees might develop

their visual capacity during the 2-day incubation period
after xation, because they were kept under laboratory
conditions in a 16h:8h light photoperiod. Another is that
the appetitive motivation of pollen foragers is quite low
as their appetite is satised prior to returning to the hive.
It is possible that departing foragers have higher appe-
titive motivation and better acquisition performance.
Moreover, we were successful in demonstrating color
discrimination and chromatic adaptation for the rst
time in harnessed bees. The harnessed bees could dis-
tinguish between 540- and 439-nm light stimuli, but not
between 540- and 618-nm light stimuli in our condi-
tioning paradigm. In the honeybee, both 540- and 618-
nm monochromatic light stimulates L receptors almost
exclusively, whereas 439-nm light mainly stimulates M
receptors. Therefore, it is likely that the bees generalized
between the 540- and 618-nm light, based on their per-
ceptual similarity. Even more surprisingly, bees condi-
tioned to the 540-nm light stimulus exhibited PER as
soon as the 439-nm light was turned o. This suggests
that adaptation to the 439-nm light induced an after-
image that was indistinguishable from the 540-nm light.
This is probably equivalent to the successive color
contrast demonstrated in free-ying bees, which has
been explained by adaptation of the spectral receptors
(Neumeyer 1981). Our results further support that the
bees are not conditioned to an increase in light intensity
(i.e., number of photons) and/or an increase in temper-
ature in this conditioning paradigm.
What are the candidate neurons and brain regions
involved in visual PER associative learning? In olfactory
PER associative learning, the octopaminergic VUMmx1
neuron (ventral unpaired median cells of maxillary
neuromere 1) is the key neuron that mediates perception
of the US (Hammer and Menzel 1998). The VUMmx1
neuron might also mediate the US in light-PER asso-
ciative learning, as sucrose was also used as the US in
our conditioning paradigm. The VUMmx1 neuron
innervates the glomeruli of the antennal lobes, the lateral
procerebrum, and lips and basal rings of the MB calyces.
MB calyces can be subdivided into three zones: lips,
collars, and basal rings. The MB basal ring zone receives
input from both the antennal and optic lobes, whereas
the lip and collar zone receive main inputs from the
antennal and optic lobes, respectively (Mobbs 1982,
1984; Ehmer and Gronenberg 2002). As visual memory
formation was modulated by antenna deprivation in the
present conditioning paradigm, the MB basal ring is a
candidate brain region involved in light-PER associative
learning.
Using DNA microarray and dierential display
methods, we identied several genes selectively expressed
in honeybee MBs that are candidate genes involved in
honeybee behavior (Kamikouchi et al. 1998, 2000;
Takeuchi et al. 2001, 2002, 2004). Recently, analysis of
honeybee molecular biology has greatly progressed and
the Human Genome Sequencing Center at Baylor
College of Medicine is currently sequencing the honey-
bee genome (see the Human Genome Sequencing Center
699
web site for details: http://www.hgsc.bcm.tmc.edu/
projects/honeybee/). In this visual learning paradigm
using harnessed bees, in vivo analysis techniques, which
are used conventionally in olfactory learning (i.e.,
intracellular recording, calcium imaging, and pharma-
cologic methods), can be applied directly. In addition,
gene manipulation techniques, such as RNA interference
(Farooqui et al. 2003, 2004) and in vivo electroporation
(Kunieda and Kubo 2004), can also be applied to the
harnessed bees. A combination of behavioral and neu-
robiologic methods will contribute to clarify the neural
circuit and molecular mechanisms underlying visual
processing in honeybees.
Acknowledgments We would like to express our sincere gratitude to
Dr. Martin Giurfa (Universite Paul Sabatier) for his valuable
comments in writing this manuscript and instruction for the
statistical analysis. This work was supported by Grants-in Aid
from Bio-oriented Technology Research Advancement Institution
(BRAIN).
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