Eli Scharlat

341394757

Associative Olfactory Learning in Western honeybees ( Apis mellifera) using

Proboscis Extension Reflex (PER) conditioning

Abstract
Due to their marked ability to learn and form associations based on memory, honeybees are
important organisms for reseach in the field of animal learning. In this study we investigated the
associative learning ability of the Western honeybee ( Apis mellifera) using Proboscis Extension
Reflex (PER) conditioning. The bees were exposed to neutral stimuli in the form of odorous
compounds, each paired with a positive or negative stimuli. Our results showed a trend of learned
association between the positive/negative stimuli and the neutral stimuli, and suggested that the
short-term associations that the bees formed were stronger than the longer term associations. We
conclude that the bees demonstrated the ability to associate odors with stimuli, however due to
inconsistently controlled expirmental condtions, our results deviate slighty from previous studies.

Introduction
The Western Honeybee (Apis mellifera) is a domesticated species which is critical for commercial
pollination1, along with its well known production of honey. One area of interest is the honeybee’s
ability to learn to associate stimuli with a reward, a form of classical conditioning. This ability is
crucial for the success of the bee and of the hive as a whole, as the honeybee’s ability to associate
floral odors with the nectar from flowers on which they land allows them to recognize these
flowers by odor in later foraging attempts.2
One method to measure the honeybees associative learning ability is by observing its Proboscis
Extention Reflex (PER) in response to stimuli. PER is a an extension of the proboscis of an insect
in response to stimulus of the the antennae, and has been used to study olfactory learning in bees
since the 1960’s.3 In this type of conditioning experiment, the bees proboscis extension in response
to a conditioned stimulus (CS) is measured. The CS is applied and then followed by an application
of an uncoditioned stimulus (US), in the form of a sugar solution, to the bees antennae. If
succeeding application of the CS elicits proboscis extension, this suggests that the bee has
learned to asscociate the two stimuli. This method has been used to measure the learning ability of
honeybees, as well as to compare the learning ability of Apis mellifera to other members of the
Apis genus.4
Our experiment seeks to measure this associative ability in honeybee workers using both positive
and negative stimuli each paired with a neutral stimulus. We hypothesize, based on the findings of
similar studies pairing postive and neutral stimuli 5 6, that the bees will succeed at forming
associations between the negative/postive stimuli and the paired neutral stimuli.
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Methods and Materials

Collection of the bees
The worker bees (Apis mellifera) for the experiment were captured from hives from the Center for
Bee Research at the Faculty of Agriculture of the Hebrew University of Jerusalem, in Rehovot,
Israel.

Experimental Conditions
The experiment was conducted in the Ariovitch lab building on November 23 rd from 13:00-15:00.
The lab conditions at the time were 52% humidity at a temperature of 23.3° Celsius. The bees
were kept in sealed glass vials and refrigerated for between one to two minutes to induce sedation
and starved to induce hunger. Since the bees where captured separately and as closely to the
experiment as allowed, a precise staving interval cannot be stated. Each vial contained between 1
to 3 bees.

Experimental Process
Each bee was placed into a groove that was cut out on the end of a plastic drinking straw and fixed
in place by wrapping tape around the bee’s thorax, allowing for the head and front legs to protrude
over the edge of the straw.
The bees were split into two groups, each group with four bees, and kept separately on wooden
stands with nails embedded in them that allowed for the fixation of the straws holding the bees.
To avoid confusion between the groups, the tape use for fixation was a different color for each
group. At this point bees that showed signs of extreme fatigue were fed sucrose solution with a
cotton swab for a duration of 3 seconds, to prevent death.
Each group of bees received a conditioned stimulus (CS) in the form of an odorous compound not
known to elicit PER in honeybees: either Benzyl Acetate (B) or Geranyl Acetate (A), which were
applied using a straw with cotton wool inside that was soaked in the respective compound, and
then aimed at the bee and blown by a student. Each conditioned stimulus was followed by an
application of an unconditioned stimulus (US): for Benzyl Acetate a 2M NaCl solution was
applied to the bee’s antennae, representing the negative stimulus (-) and for Geranyl Acetate a 1M
sugar solution was applied, representing the positive stimulus (+). In the case of the sugar solution
the bees were allowed to feed for 3 seconds. The application of the US was done using cotton
swabs that had been soaked in the respective solutions.
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For the application of the stimuli, the bee receiving the treatment was isolated on a separate
wooden stand. 10 seconds after being transferred, the bee was exposed to the CS and subsequently
the US, and 10 seconds after exposure was returned to the group block. This was repeated for each
bee in a group and the procedure was then repeated on the second group, with one group receiving
the sugar pairing (+) first (AB), and the other receiving the NaCl pairing (-) first (BA).
The Intertrial Interval was 6 minutes, with 5 minutes for application of the stimuli pairings and the
final minute for feeding the bees who did not receive the sugar pairing. 21 pairs of students
conducted the experiment. To control for the variable of differential waiting intervals between
reception of stimuli, 11 of the groups began with the AB treatment and 10 began with the BA
treatment.

Data Collection and Processing

The observations of the bee’s proboscis responses were recorded on pre-prepared worksheets
distributed to each pair of students conducting the experiment, and then transferred to an excel
document by each pair at the end of the experiment (Table 1). The results where then collated by
the course staff and distributed to the students in an excel document. For this paper, the provided
results where further processed to express the results in percentages.

Table 1: Example of format of data collection sheet

used for the experiment; 0 was marked for lack of
PER, 1 for PER

Treatmen Bee/Trial 1 2 3 4 5 6 7
t BA CS U Fed CS U Fed CS US Fe CS US CS US CS US CS US
S S d
B - + B - + B - + A + A + A + A +
BA 1 1 0 1 0 0 1 0 1 1 1 1 0 1 0 1 0 1
BA 2 0 0 1 0 1 1 0 0 1 0 1 0 0 0 1 0 1
BA 3 0 0 1 0 1 1 0 1 1 0 1 0 1 0 1 0 1
BA 4 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1
Treatmen Bee/Trial 1 2 3 4 5 6 7
t AB CS US CS US CS US CS U Fed CS U Fed CS US Fe CS US
S S d
A + A + A + B - + B - + B - + A +
AB 5 1 1 1 1 1 1 0 1 1 0 1 1 0 1 1 0 1
AB 6 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1
AB 7 1 1 0 1 1 1 0 1 1 0 1 1 0 1 1 0 1
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AB 8 0 1 0 1 0 1 0 0 1 0 1 1 0 1 1 0 1

Results
For the unconditioned stimuli, the overwhelming majority of the worker bees (91.82%) displayed
PER when exposed to the sucrose solution, and 23.55% fewer (68.27%) displayed PER when
exposed to the NaCl solution (Figure 1).

100.00% 91.82%
90.00%
80.00%
68.27%
70.00%
60.00%
PER%

50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
1M Sucrose STIMULUS 2M NaCl

Figure 1: Percentage of bees displaying proboscis

extension reflex (PER) in response to unconditioned
stimuli

For the conditioned stimuli groups that first received the negative pairing (BA), a negative
association between the NaCl and the Benzyl Acetate was observed, as displayed by the reduced
percentage of Bees displaying PER when exposed to the Benzyl Acetate over the first three
rounds. As well, the BA treatment groups showed increasing positive association between
Sucrose and Geranyl Acetate between rounds 4-6, as measured by an increased percentage of bees
displaying PER each round, although this increase was not completely consistent and dropped
significantly in round 7 for the BA→AB group (Figure 2).
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33%
18%
3%
1B 2B 3B 4A 5A 6A 7A

PER%
BA(B 0.405405 0.270270 0.162162 0.25 0.305555 0.388888 0.25
A→A 40540540 27027027 16216216 55555555 88888888
B) 5 2 6 9
BA(A 0.357142 0.285714 0.119047 0.073170 0.219512 0.170731 0.219512
B→B 85714285 28571428 61904761 73170731 19512195 70731707 19512195
A) 7 6 9 71 1 3 1

Repetition

BA(BA→AB) BA(AB→BA)
Figure 2: Percentage of bees displaying PER per-repetition for
negative-pairing initiated experiment (BA)

For the conditioned stimuli groups that first received the positive pairing (AB), a positive
association was observed between the Sucrose and the Geranyl Acetate across the first three
repetitions, as measured by an increase in the percentage of bee’s displaying PER over the rounds,
and for one of the groups in the final repetition as well. Accordingly, a negative association was
observed between NaCl and Benzyl acetate across repetitions 4-6, although for the BA→AB
group the association was more pronounced, as the drop in the percentage of bees displaying PER
per-round was more dramatic (Figure 3).

45%
25%
5%
1A 2A 3A 4B 5B 6B 7A
PER%

AB( 0.36363 0.31818 0.45454 0.22727 0.13636 0.02272 0.25

BA 6363636 1818181 5454545 2727272 3636363 7272727
→A 364 818 455 727 636 2727
B)
AB( 0.38461 0.48648 0.51351 0.27027 0.16216 0.16216 0.51351
AB 5384615 6486486 3513513 0270270 2162162 2162162 3513513
→B 385 487 514 27 162 162 514
A)
REPeTITION

AB(BA→AB) AB(AB→BA)

Figure 3: Percentage of bees displaying PER per-repetition for

positive-pairing initiated experiment (AB)
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There was a trend of increased association of the CS with the US, with there being a drop in
appropriate response (PER for Geranyl Acetate and lack of PER for Benzyl Acetate) during the
repetition where the stimuli switched. After the repetition where the switch occurred the
association was again observed, with the exception of the final round, where the appropriate
response dropped below that of the baseline of 49.38% set in the first repetition, to 30.19% (Figure
4).

100.00%
a 90.00%
80.00%
66.67%
70.00%
55.63% 54.72% 56.60%
60.00%
Response %

49.38% 50.94%
50.00%
40.00%
30.19%
` 30.00%
20.00%
10.00%
0.00%
1 2 3 4 5 6 7
Repetition

Figure 4: Appropriate response per-repetition. Appropriate

response is defined as PER in the case of the positively paired CS
(Geranyl Acetate) and a lack of PER in the case of the negatively
paired CS (Benzyl acetate).

Discussion and Conclusions

In the experiment we set out to determine the worker bee’s ability to learn to associate a
neutral stimulus with a positive or negative stimulus. For the unconditioned positive stimulus,
our result of over 90% of bees displaying PER was expected, and the remaining percentage that
did not respond can be attributed to various factors, such as imperfect feeding, exhaustion of
the bees after having been refrigerated and deprived of food, or the manner in which the bees
where fixed to the straw impeding their function. The relatively high PER of the bees to the
negative unconditioned response, at almost 70%, was unexpected, however when taking into
account that the bees did learn to negatively associate the CS that was paired with the negative
stimulus, it is possible that the contact made with the bee’s antennae when administering the
NaCl was enough to elicit PER, and that those that displayed PER in this case where not
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responding to the NaCl but to the physical touch. Over the course of the repetitions, the bees
displayed increasing or decreasing percentages of PER in accordance with the positive and
negatively paired CS’s, respectively, displaying a trend of associative learning. There was some
variation in the degree of change between the groups that began with negative pairing and the
groups that began with positive pairing, however given the multitude of poorly controlled
variables in this experiment, we cannot conclude that this was a result of the order of the
protocol.
The exception to the observations we made of associative learning was the final repetition,
where the percentage of bees that correctly associated the CS with the US was even lower that
than observed in the initial repetition, where the bees hadn’t had a chance yet to learn any
association. This finding does not conform to results from other PER conditioning experiments
conducted with Apis mellifera, where learning increased sharply in the initial repetitions and
5 6
then remained stable in later repetitions. It is important to note however that these
experiments only dealt with a positive US or lack thereof, not a negative stimulus.
Possible explanations for this include aforementioned factors of imperfect feeding and
compromised integrity of the bees. Another possible confounding variable is the effect of the
blowing air onto the bees, independent of the smell, which was not measured nor controlled
for. Additionally, it is possible that the cotton wool soaked in the CS dried out at the end of the
experiment, and that this had an effect of the result of the final repetition.
Another notable finding was the drop in association that was observed during the repetition
where the stimulus was switched, showing that even after several rounds of conditioning the
bees had trouble retaining longer term associations. After this drop however, the bees again
displayed increased associated learning for the remaining rounds, suggesting that their shorter-
term associations where stronger.
This experiment suggests that worker bees are capable of associative learning through
conditioning and that their short-term associative learning may be more reliable than their
long-term associative learning, however given the many uncontrolled variables in the study,
these conclusions are not entirely robust. For future experiments, aside from increasing the
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sample size, variables such as the effect of physical contact to the antennae in triggering PER
and the effect of blowing air in triggering PER must be accounted for.

Works Cited
1
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2
Menzel, Randolf, et al. “Functional Organization of Appetitive Learning and Memory in a
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https://doi.org/10.1007/978-1-4615-2814-2_4.
3
JC;, Giurfa M;Sandoz. “Invertebrate Learning and Memory: Fifty Years of Olfactory
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4
Kaspi, Roy, and Sharoni Shafir. “Associative Olfactory Learning of the Red Dwarf Honey Bee
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0160-y.
5
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6
Jung, Jewon, et al. “Comparative Study of Olfactory Learning and Memory in Apis Cerana and
Apis Mellifera Foragers.” Journal of Apiculture, vol. 32, no. 4, 2017, pp. 275–280.,
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