Simultaneous mastering of two abstract concepts by the miniature brain of bees.

Abstract

Sorting objects and events into categories and concepts is a fundamental cognitive capacity that reduces the cost of learning every particular situation encountered in our daily lives. Relational concepts such as "same," "different," "better than," or "larger than"--among others--are essential in human cognition because they allow highly efficient classifying of events irrespective of physical similarity. Mastering a relational concept involves encoding a relationship by the brain independently of the physical objects linked by the relation and is, therefore, consistent with abstraction capacities. Processing several concepts at a time presupposes an even higher level of cognitive sophistication that is not expected in an invertebrate. We found that the miniature brains of honey bees rapidly learn to master two abstract concepts simultaneously, one based on spatial relationships (above/below and right/left) and another based on the perception of difference. Bees that learned to classify visual targets by using this dual concept transferred their choices to unknown stimuli that offered a best match in terms of dual-concept availability: their components presented the appropriate spatial relationship and differed from one another. This study reveals a surprising facility of brains to extract abstract concepts from a set of complex pictures and to combine them in a rule for subsequent choices. This finding thus provides excellent opportunities for understanding how cognitive processing is achieved by relatively simple neural architectures.

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Simultaneous mastering of two abstract concepts by
the miniature brain of bees
Aurore Avarguès-Webera,b,c,1, Adrian G. Dyerc,d, Maud Combea,b, and Martin Giurfaa,b,2
aUniversité de Toulouse, Centre de Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France;bCentre National de la Recherche Scientifique
(CNRS), Centre de Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France;cDepartment of Physiology, Monash University, Clayton VIC 3800,
Australia; anddSchool of Media and Communication, RMIT University, Melbourne VIC 3001, Australia
Edited* by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved March 16, 2012 (received for review February 13, 2012)
Sorting objects and events into categories and concepts is a funda-
mental cognitive capacity that reduces the cost of learning every
particular situation encountered in our daily lives. Relational
concepts such as “same,” “different,” “better than,” or “larger
than”—among others—are essential in human cognition because
theyallowhighlyefficientclassifyingofeventsirrespectiveofphys-
icalsimilarity.Masteringarelationalconceptinvolvesencodingare-
lationship by the brain independently of the physical objects linked
by the relation and is, therefore, consistent with abstraction capaci-
ties. Processing several concepts at a time presupposes an even
higher level of cognitive sophistication that is not expected in an
invertebrate. We found that the miniature brains of honey bees
rapidly learn to master two abstract concepts simultaneously, one
based on spatial relationships (above/below and right/left) and an-
other based on the perception of difference. Bees that learned to
classify visual targets by using this dual concept transferred their
choices to unknown stimuli that offered a best match in terms of
dual-conceptavailability:theircomponentspresentedthe appropri-
ate spatial relationship and differed from one another. This study
reveals a surprising facility of brains to extract abstract concepts
from a set of complex pictures and to combine them in a rule for
subsequent choices. This finding thus provides excellent opportuni-
ties for understanding how cognitive processing is achieved by rel-
atively simple neural architectures.
concept learning|vision|insect|Apis mellifera
C
daily lives (1–5). Instead, we group specific instances in broad
classes that allow responses to novel situations, which are deemed
equivalent to the classified items. Stimulus classification can be
governed by perceptual similarities: The belonging to a given class
is defined, in this case, by common physical features predictive of
the category (6). Alternate classifications that are independent of
perceptual similarity can be achieved by using relations as
groupingcriterion(6).Relationssuchas“same”or“different”and
“better than” or “worse than” can serve as the basis for a form of
classification that can be considered abstract because it involves
learning beyond perceptual generalization. Given that the terms
“category” and “concept” are often used indiscriminately (7), we
will use here the former to refer to perceptual classification and
the latter to relational classification. Manipulating concepts pre-
supposes a high level of cognitive sophistication because the re-
lational concept has to be coded as a specific entity by the brain
independently of the physical nature of objects linked by this re-
lation (8). Such ability requires time to develop during human
infancy and is not necessarily suspected to be possible with in an
insect brain (3, 5).
Honey bees are a powerful model for studies of visual cognition
becausethey canbereliably trainedtoassociate visual stimuliwith
sucrose reward (9–11). In this experimental framework, bees can
learn, through experience, to navigate in complex mazes (12), to
categorize objects based on coincident visual features (11, 13–15),
and to master discriminations based on identity (16, 17) and
oncepts andcategories promote cognitive economyby sparing
usthelearningofevery particular instanceencountered in our
numerosity (18–20). Bees can also learn the concept of above/
below with respect to a constant reference (e.g., a horizontal line)
(21), but whether bees can reach a level of abstraction allowing
them to master multiple concepts simultaneously is an intriguing
question that currently remains unknown and that goes beyond all
previous attempts to characterize cognitive power of insect brains
(22). Here we ask whether bees can learn to choose variable
complex stimuli that fulfill simultaneously two conceptual
requirements: being always placed in a consistent spatial re-
lationship and, in addition, being always perceptually different.
Results
To answer this question, marked honey bees were individually
trained to fly into a Y-shaped maze where they had to discrim-
inate between two stimuli presented vertically on the back walls
of the maze. Stimuli were composed of two different elements,
which were arranged in one of two spatial configurations: above/
below (one above the other) or left/right (one on the right of the
other). Importantly, stimulus components were always different
from one another, be they achromatic patterns or chromatic
discs (Figs. 1A, 2A, and 3A, training). Bees were trained to enter
the Y maze and were rewarded with sucrose solution for
choosing either the above/below stimuli or the left/right stimuli.
A drop of quinine solution was delivered for any incorrect choice
because it significantly enhances visual attention while learning
perceptually difficult tasks (23). Thus, during 30 learning trials (i.
e., 30 foraging bouts between the hive and the Y maze), half of
the bees (n = 14) were rewarded for recognizing the above/below
relationship (and penalized for choosing the left/right relation-
ship), with the conditions reversed for the other half (n = 14)
(i.e., reward: right/left; punishment: above/below). Within each
group, bees were either trained with achromatic patterns (half of
the bees) or with color discs (Fig. S1). Stimulus features and
positions were systematically varied between trials (Fig. S1 and
Figs. 1A, 2A, and 3A), maintaining only the above/below or left/
right relationship as the only predictor of reward, while also
preserving the requirement that simultaneously presented stim-
uli contained different elements. To uncover the nature of the
solutions inculcated by the training, we performed nonrewarded
transfer tests in three different experiments in which bees were
presented with novel stimuli (Figs. 1A, 2A, and 3A, transfer
tests). To exclude the possibility that bees may potentially use the
global center of gravity of the stimuli as a cue to resolve the task
(24), all stimuli used in the nonrewarded tests were equally
Author contributions: A.A.-W., A.G.D., and M.G. designed research; A.A.-W. performed
research; A.A.-W. and M.C. analyzed data; and A.A.-W., A.G.D., and M.G. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1Present address: Biological and Experimental Psychology Group, School of Biological and
Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom.
2To whom correspondence should be addressed. E-mail: giurfa@cict.fr.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1202576109/-/DCSupplemental.
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centered, thus presenting their center of gravity at exactly the
same position.
During training, bees learned to choose the rewarded spatial
relationship between stimuli, irrespective of the specific relation-
ship (above/below or left/right) or the type of visual stimulus
(achromatic patterns or chromatic discs) (P < 0.01 in all cases; see
acquisition curves in Fig. S2). Learning was robust despite a sys-
tematic trial-to-trial variation of both stimulus features and the
absolute position of stimuli in the bees’ visual field.
First Experiment: Bees Transferred the Learned Spatial Relation to
Novel Stimuli. We assessed the bees’ ability to transfer the learned
spatialrelationshiptonovelstimulithatdifferedperceptuallyfrom
those used during training (Fig. 1A, transfer test). We thus de-
termined whether stimulus similarity or a true relational concept
wasusedbythebees.Beestrainedwithachromaticpatterns(group
1; n = 5) were tested with colors, whereas those trained with col-
ored disks were tested with achromatic patterns (group 2; n = 5).
Inbothcases,beeschosetheappropriatespatialrelationshipinthe
respective transfer tests (Fig. 1B; P < 0.001 for both groups 1 and
2). No significant differences were found between transfer per-
formances of groups 1 and 2. This result shows that the bees
learned to use the relational spatial concept (above/below or left/
right) irrespective of the perceptual similarity between stimuli
defining this concept.
Second Experiment: Bees Did Not Use the Global Orientation of the
Stimuli as a Lower-Level Cue to Resolve the Task. We then tested
whether bees did indeed use a spatial concept or alternatively
responded to broad orientation cues (25, 26). Specifically, we
Fig. 1.
trained with color discs. Within each group, half of the bees were rewarded on the above/below relation, whereas the other half were rewarded on the right/
left relation. Novel stimuli were presented in the transfer test. (B) Performance in the transfer test (percentage of correct choices) of groups 1 and 2. Bees
preferred the novel stimuli arranged in the trained spatial relationship. Data shown are mean + SEM (n = 5 for each bar; ***P < 0.001).
Experiment 1. (A) Example of conditioning and testing procedure for two groups of bees: group 1 trained with achromatic patterns and group 2
Fig. 2.
Within each group, half of the bees were rewarded on the above/below relation, whereas the other half were rewarded on the right/left relation. Transfer
tests shown correspond to bees rewarded on the above/below relation. (B) Performance in the transfer tests (percentage of choices for the option displaying
two stimuli). Bees preferred the two stimuli to the bar if they were linked by the appropriate relationship (transfer test 1) but not if they displayed the
inappropriate relationship (transfer test 2). Data shown are mean + SEM (n = 5 for each bar; ***P < 0.001; NS, P > 0.05).
Experiment 2. (A) Example of conditioning and testing procedure for group 1 trained with achromatic patterns and group 2 trained with color discs.
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aimed to distinguish between learning that two stimuli were
connected by a specific relationship (e.g., one above the other or
one on the right of the other) and learning that a global cue (e.g.,
vertical or horizontal, respectively) was associated with sucrose
reward. Two groups of bees (n = 5 each) were trained either with
achromatic patterns (group 1) or colors (group 2) to choose
a specific spatial relationship (above/below or left/right). In two
transfer tests (Fig. 2A, transfer tests), bees were confronted with
novel stimuli that were not used during the training. In transfer
test 1, they had to choose between a large bar (Fig. S1C), vertical
for above/below-trained animals and horizontal for left/right-
trained animals, and two stimuli that were placed in the appro-
priate spatial relationship but which were shifted with respect to
each other (Fig. 2A, transfer test 1) to avoid the global vertical or
horizontal visual stimulation. In transfer test 2, the same large
bar was presented versus two stimuli exhibiting the nonrewarded
spatial relationship (left/right for above/below-trained animals
and vice versa) (Fig. 2A, transfer test 2).
In transfer test 1, bees of both groups significantly preferred the
two stimuli overthe largebar (Fig. 2B; P < 0.001 for both groups 1
and 2; transfer test 1), thus showing that they were guided by the
presence of two stimuli in a specific spatial relationship (even if
stimuliwere slightlyshiftedwithrespecttoeachother)ratherthan
by using a strong orientation cue. There was no significant dif-
ference on transfer test performance for groups 1 and 2 nor be-
tween bees trained for above/below and bees trained for left/right
[not significant (NS)in all cases]. In transfer test 2, bees from both
groupsexhibitedno significant preference foreither stimulus(Fig.
2B: NS for both groups 1 and 2; transfer test 2), showing that the
barprovidingthesalientorientationcuewasnotperceivedbybees
asrepresentingtherewardedstimuli.Thisfindingthusallowsusto
excludethispotential low-level explanation for thebees’ behavior.
These results confirmed that bees learned to choose two stimuli
arranged in a specific spatial relationship, irrespective of the
particular stimuli used to build this relationship.
Third Experiment: Bees Extracted an Additional Relational Concept
from Training and Used It in Combination with the Spatial Concept.
Did bees also learn from training that the stimuli had to be dif-
ferent? To answer this question, we performed a third experiment
to determine whether bees could additionally learn the concept
that stimuli elements must always be different while also choosing
on the basis of specific spatial relationships. Bees were trained
with either achromatic patterns (group 1; n = 4) or color discs
(group 2; n = 4) and then presented with three transfer tests (Fig.
3A) in which the bees had to choose between two different stimuli
orthesamestimulusrepeated.Intransfertest1,thecorrectspatial
relationshiplinkedbothkindsofstimuli,whereasintransfertest2,
theincorrectspatialrelationshippertained.Finally,intransfertest
3, conflictive information was presented as the correct spatial re-
lationship linked two identical stimuli, whereas the incorrect
spatial relationship linked two different stimuli.
In transfer test1, bees from both groups preferred two different
stimuli arranged in the appropriate spatial relationship over two
identical stimuli arranged in the same relationship (Fig. 3B; P <
0.01 for both groups 1 and 2; transfer test 1). Significant differ-
ences were notobservedbetween transferperformancesofgroups
1 or 2 nor between bees trained for above/below and bees trained
for left/right (NS in all cases). In transfer test 2, bees presented
withstimuliintheincorrect spatialrelationshipalsodemonstrated
a significant preference for the stimuli with two different compo-
nentsoverstimuliwithtwoidenticalcomponents(Fig.3B;P<0.01
for both groups 1 and 2; transfer test 2). No significant differences
were found between the transfer performances of groups 1 and 2
nor between bees trained on the above/below or left/right con-
figurations(NSinallcases).Theseresultsconfirmthat,inaddition
to the specific spatial relationship, bees learned to attend to the
requirement for different stimulus components. Note that pref-
erence for difference over sameness was so strong that, in transfer
test 2, it even led the bees to choose a stimulus that was explicitly
associated with a penalty of quinine solution during the training.
This result admits two different interpretations: either difference
dominatesoverspatial relationshipor is at least treatedseparately
in the definition of the rewarded category or the negative stimulus
wasnotlearnedperseduringtraining.Thelatterpossibilitycanbe
excluded because studies on visual discrimination learning in the
same setup andwithidenticalreinforcements showthatbeeslearn
both the positive and the negative stimulus (23). Finally, in
transfer test 3, bees presented with the dilemma of choosing be-
tweenthecorrect relationshiplinkingtwoidentical stimuliandthe
Fig. 3.
Within each group, half of the bees were rewarded on the above/below relation, whereas the other half were rewarded on the right/left relation. Transfer
tests shown correspond to bees rewarded on the above/below relation. (B) Performance in the transfer tests (percentage of choices for the option displaying
two different stimuli). Bees preferred two different to two identical stimuli both in the appropriate (transfer test 1) and the inappropriate (transfer test 2)
relationships and chose randomly in a conflict situation (transfer test 3). Data shown are mean + SEM (n = 4 for each bar; **P < 0.01; ***P < 0.001; NS, P > 0.05).
Experiment 3. (A) Example of conditioning and testing procedure for group 1 trained with achromatic patterns and group 2 trained with color discs.
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incorrect relationship linking two different stimuli showed no
preference for either situation (Fig. 3B; NS for both groups 1 and
2; transfer test 3). No differences were found between transfer
performances of groups 1 and 2 nor between bees trained for
above/below and bees trained for left/right (NS in all cases). Thus,
because the bees had learned that stimuli elements both had to be
differentand hadtohave thecorrect spatialarrangement,the rule
guiding the bees’ decisions consisted of two combined concepts.
Discussion
These results demonstrate that the miniature brain of honey bees
is capable of extracting at least two relational concepts from
experience with complex stimuli and that these concepts can be
combined to determine sophisticated performances, which are
independent of the physical nature of the stimuli. Irrespective of
whether stimuli were chromatic or achromatic, bees learned that
they had to choose stimuli arranged in a specific spatial re-
lationship and that all stimuli were composed of different visual
elements. Thus, a dual concept based on two distinct relational
rules rather than perceptual similarity guided the honey bees’
choices in these experiments.
Sophisticated multiple-concept processing is not necessarily
expectedinaninsectbraingivenitsreducedsizeandsmallnumber
of neurons relative to larger organisms (e.g., 960,000 neurons in
a honey bee brain vs. 100 billion in a human brain). It seems,
therefore, that views emphasizing relational thinking as a corner-
stone of human perception and cognition by underlying among
others analogy, language, or mathematical abilities (8) need to be
reconsidered and extended to relatively simpler brains (22).
Notably, honey bees extracted the concept of difference even
if they were not specifically trained to do so because both
rewarded and punished stimuli were composed of two different
elements. Moreover, bees required only 30 training trials to ex-
tract and master both relational concepts (i.e., ∼3 h of training),
whereas several thousand of trials are generally required for
similar concepts to develop in some primates (27). However,
comparisons between bees’ and primates’ performances are not
necessarily pertinent: Whereas bees could freely move in our
experiments and their constant returns to the experimental setup
revealed a high foraging motivation, conceptual learning in pri-
mates is usually tested in artificial laboratory environments in
which animals are individually immobilized and far from bi-
ological relevant situations. Alternative research strategies fa-
voring the study of primates’ cognitive abilities in seminatural
environments and preserving social groups (28) may introduce
drastic differences in terms of the easiness with which these
animals solve conceptual problems. However, the relative facility
of bees to master relational concepts should serve as the basis of
a reassessment of the considerate requirement of high brain
complexity involving multiple processing steps to resolve concept
learning tasks (22).
Low-level explanations should be considered as potential
accounts of some visual performances of insects (22). Focusing
on generalization along a perceptual dimension such as color or
achromatic pattern properties (11), differences in the center of
gravity of images (24), or broad orientation cues (25) was ex-
plicitly excluded by our experiments. Template matching has
been proposed as a simple mechanism by which insects com-
pared learned images with images that are currently perceived
(29–32). A strict pixel-by-pixel comparison (32) can be excluded
in our experiments because bees transferred appropriately their
choice to novel visual images that differed in spatial details and/
or color. However, template matching can adopt more sophis-
ticated forms compatible with generalization to novel stimuli.
Correlation analysis can underlie flexible forms of template
matching because it allows statistic quantification of similarity
between two or more different images (33). To determine
whether this type of visual strategy potentially accounted for the
bees’ performance in our experiments, we computed average
left/right and above/below stimuli, both for all stimuli displayed
to the bees during the training and for those whose position
during the training matched the position of the test stimuli (Fig.
S3). In both cases, coefficients of correlation between average
training stimuli and actual test stimuli were calculated by using
fast-normalized cross-correlation, which provides best template
coincidence (33). Fig. S3 shows that, although template matching
can potentially account for transfer performances in experiment
1, it can be definitely discarded for experiments 2 and 3, where
bees chose against the predictions of this simple visual strategy.
The simultaneous mastering of two visual concepts by bees
reflected, therefore, a higher level of complexity than just pe-
ripheral image coincidence.
What do bees gain from extracting and combining concepts of
different nature? Identifying spatial relationships between objects
is useful for learning about generic configurations that underlie
specific stimulus categories in naturalistic environments. For ex-
ample, bilateral flowers share a common layout with upper and
lower petals and a left/right symmetry so that spatial relationships
may facilitate the construction of a general configuration corre-
sponding to what a bilateral flower should be (34–36). Further-
more, honey bees are central-place foragers, navigating between
a fixed place (the hive) and surrounding food sources (flowers).
Navigational strategies implemented by these insects are ex-
tremely sophisticated and may even rely on map-like representa-
tions of space (37). In this navigational context, dealing with
abstract spatial relationships such as left/right or above/below may
be extremely helpful for identification of expected landmarks en
route to the goal in a complex environment where there may be
a large amount of distracting information that must be ignored
(38). If, in addition, the concept of “difference” is available, an
abstract but rich representation of space can be achieved even by
a miniature brain. For instance,bees may havelearned that a food
source is located between two landmarks that are adjacent either
in the horizontal (i.e., left/right) or in the vertical (i.e., above/be-
low) dimension and that, besides this spatial requirement, are also
sufficiently different to be discriminated from other potential
distracter stimuli present in the environment. Learning to com-
bine the spatial and the difference concept then allows a bee to
quickly apply the acquired rules to new scenarios that might occur
as foraging conditions change. This finding is thus likely to be of
very high value for understanding how brains can learn complex
cognitive-type tasks and how modeling of miniature brain visual
processing may provide useful solutions for the development of
artificial visual systems (39, 40).
Materials and Methods
Free-flying honey bees were marked with a color spot on the thorax and
individually trained to collect 1 M sucrose solution within a Y maze (41). The
maze was covered with an UV-transparent Plexiglas ceiling and illuminated
by natural daylight. During both training and tests, only one bee was
present in the apparatus. The entrance of the maze led to a decision
chamber defined by the intersection of the maze arms. Once in the decision
chamber, a bee could choose between the stimuli presented on the back
walls of both arms. The back walls (20 × 20 cm) were placed at a distance of
15 cm from the decision chamber.
Toimprovevisuallearning,adifferential-conditioningprocedurewasused
(41) so that one stimulus was rewarded with 1 M sucrose solution whereas
another stimulus was penalized with 60 mM quinine hydrochloride dihy-
drate solution (23). Solutions were offered in a micropipette inserted in the
center of the back walls. Each bee was trained for 30 trials (i.e., 30 foraging
bouts between the hive and the laboratory) during which the sides of
rewarded and nonrewarded stimuli (left or right) were interchanged in
a pseudorandom sequence. In each trial, the first choice of a bee was
quantified after it entered the maze. Tests lasted 45 s, during which the
number of contacts with the surface of the stimuli was recorded. Each test
was completed twice, interchanging the sides of the stimuli. Neither sucrose
nor quinine was offered during tests. Three to six refreshing training trials
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were intermingled between the tests. The sequence of tests was randomized
from bee to bee.
Stimuli. Stimuli were presented on a gray background that covered the back
walls of the maze. The background was cut from a neutral gray HKS-92N
paper (K+E Stuttgart). Pairs of vertically or horizontally aligned achromatic
black patterns or color discs were used to establish above/below or left/right
relationships (Fig. S1).
Six different achromatic patterns printed with a high-resolution laser
printer on UV-reflecting white paper of constant quality (a checkerboard,
a radial four-sectored pattern, a horizontal grating, a vertical grating, and
two concentric patterns; Fig. S1A) and six different color discs (cut from HKS
papers 1N, 3N, 29N, 32N, 68N, and 71N; Fig. S1B) were used. Patterns were
7 × 7 cm and subtended a visual angle of 26° to the eyes of a bee located in
the decision chamber of the maze. Grating stripes, checkerboard squares,
and concentric squares and rings were all 1 cm width, which corresponded to
a visual angle of 4° from the decision chamber. Sectors in the radial pattern
covered 2.5 cm in their largest extension, thus subtending a visual angle of
10° to the bees’ eye when deciding between stimuli. Experiments using
horizontal and vertical black-and-white gratings of varying spatial fre-
quencies have shown that single stripes can be resolved if they subtend
a threshold angle of 2.3° (42). Thus, all achromatic patterns could be well
resolved for honey bees. Color disks were 7 cm diameter and subtended
a visual angle of 26° to the eyes of a bee in the decision chamber. An angular
threshold of 15° has been reported for color-disk detectability so that our
color disks could be well detected and discriminated in terms of their color
information (43). Colors could be well discriminated from each other
according to both the color opponent coding space (44) and the color
hexagon (45), two perceptual spaces proposed for honey bee color vision.
Large achromatic and chromatic bars 7 × 18 cm were used in transfer tests of
the second experiment. They could be presented with their main axis vertical
or horizontal. Achromatic bars were extensions of the training patterns (Fig.
S1C). Chromatic bars (Fig. S1C) were cut from the same HKS-N papers used
for the training color disks.
During training, two groups of bees were trained in parallel in each ex-
periment, one with achromatic patterns and the other with color discs. From
the six stimuli available within each category, four were randomly selected as
training stimuli, thus leaving two other stimuli for the transfer tests. The four
training stimuli were arranged in pairs to define above/below and left/right
relationships. All six possible combinations between the four training stimuli
(sixstimulus pairs) were used during the training, i.e.,each pair was presented
five times (30 learning trials in total). Additionally, vertically (above/below)
and horizontally (left/right) aligned stimuli were presented in three different
positions,eitheron thecentral axis or on both distal axes ofthe backwall(Fig.
S1D). Stimulus pairs and their positions were randomly varied from trial to
trial. In this way, bees were precluded from using absolute spatial locations
or the center of gravity of patterns (24) to solve the discrimination task. This
training procedure has been shown to promote concept formation in bees
and offers the advantage of training and testing multiple stimulus combi-
nations (21). Only bees that completed the entire training and test sequences
were included in the results; no other selection criterion was applied.
Statistics. Acquisition was measured in terms of the percentage of correct
choicesduringthree blocksoftrainingtrials. Performance ofbalancedgroups
during training was compared by using ANOVA for repeated measurements
with groups and trial blocks as factors of analysis. During the tests, both the
first choice and the cumulative contacts with the surface of the targets were
counted for 45 s. The choice proportion for each of the two test stimuli was
then calculated. Performance during the tests was analyzed in terms of the
proportion of correct choices per test, producing a single value per bee to
exclude pseudoreplication. A one-sample t test was used to test the null
hypothesis that the proportion of correct choices was not different from
a random value of 50%. A binomial test was used to analyze whether the
first choices were randomly distributed between test stimuli. Test analyses
refer to the cumulative contacts recorded during 45 s. Similar results were
obtained when the first choice was considered.
Data of all bees (above/below- and left/right-trained bees as well as color-
and achromatic pattern-trained bees) were pooled upon analysis of test
performances and no significant differences were found between these
different conditions. Between-group and between-test comparisons were
made by using t tests for independent and paired samples, respectively. The
α level for all analyses was 0.05.
ACKNOWLEDGMENTS. We thank Tom Collett, Jochen Zeil, Lars Chittka,
Lalina Muir, David Reser, and Shao-Wu Zhang for helpful criticisms and
suggestions on previous versions of the manuscript. A.A.-W. and M.G. thank
the French Research Council (Centre National de la Recherche Scientifique),
the University Paul Sabatier (Project APIGENE), and the National Research
Agency (Project Apicolor) for generous support. A.A.-W. was supported by
a Travelling Fellowship of The Journal of Experimental Biology and by the
fellowship “Actions Thématiques” of the University Paul Sabatier (ATUPS).
A.G.D. acknowledges the Australian Research Council for Grants DP0878968
and DP0987989.
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