The Time Course of Olfactory Working

Memory in Honey Bees (Apis mellifera)

Bachelor Thesis
Ludwig Sommer

University of Konstanz
Department of Biology Chair of Neurobiology
Professor G. C. Galizia
August 2008

Eidesstattliche Erklrung

Hiermit versichere ich, die vorliegende Bachelorarbeit eigenstndig

und unter ausschlielicher Verwendung und Kennzeichnung der
angegebenen Literatur verfasst zu haben.

Konstanz, August 2008

Ludwig Sommer

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ZUSAMMENFASSUNG .....................................................- 3 -

ABSTRACT ....................................................................- 4 -

INTRODUCTION ............................................................- 4 -

METHODS AND MATERIALS ............................................- 7 -

4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8

General Methods and Materials ..................................................- 7

Specifics of the Pilot Experiment ................................................- 9
Specifics of the First Period of the Main Experiment ......................- 9
Specifics of the Second Period of the Main Experiment ..................- 9
Data Acquisition and Processing............................................... - 10
Specific Data Processing Exclusive Data ................................. - 10
Specific Data Processing Two Periods of the Main Experiment .... - 10
Specific Data Processing Two Odors....................................... - 10

RESULTS AND DISCUSSION .........................................- 11 -

5.1
Pilot Experiment Trace Conditioning....................................... 5.2
Main Experiment Trace Conditioning and Backward Conditioning 5.2.1
Training......................................................................... 5.2.2
15 min Test.................................................................... 5.2.3
24 h Test ....................................................................... 5.3
Main Experiment Exclusive Data............................................ 5.4
Results of the First Period of the Main Experiment ...................... 5.5
Results of the Second Period of the Main Experiment .................. 5.6
Comparison of the Two Experimental Periods............................. 5.7
Comparison of the Two Odors.................................................. -

11
13
14
15
16
17
19
20
22
23

GENERAL DISCUSSION ................................................- 25 -

REFERENCES ..............................................................- 28 -

ACKNOWLEGMENTS.....................................................- 29 -

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1 ZUSAMMENFASSUNG

Das Arbeitsgedchtnis wird bentigt um eine mentale Reprsentation der Umwelt

herzustellen und damit komplexe Aufgaben in der gegenwrtigen Situation zu lsen. In
der mentalen Reprsentation der Umwelt werden zunchst auch biologisch
unbedeutende Reize reprsentiert. Nur dadurch knnen aus Reizsituationen der Umwelt
neue Kausalzusammenhnge abgeleitet werden, um schlielich Vorhersagen ber
zuknftige Ereignisse treffen zu knnen.
In

dieser

Arbeit

wurde

der

temporre

Speicher

des

olfaktorischen

Arbeitsgedchtnisses hinsichtlich dessen Dynamik und Dauer am Modeltier Honigbiene

(Apis mellifera) untersucht. Dazu wurde eine Pawlow-Konditionierung durchgefhrt, bei
welcher der bedingte Reiz (CS; Heptanon/Oktanol) mit dem unbedingten Reiz (US;
Zuckerwasser) zeitlich voneinander getrennt waren (kein berlappen der Stimuli).
Assoziatives Lernen sollte bei denjenigen Bienen eintreten, welche die Zeitspanne
zwischen den Stimuli (ISI; Inter-Stimuli Intervall) mit dem temporren Speicher
berbrcken knnen. Um die Dauer des temporren Speichers zu bestimmen, wurden
verschiedene Gruppen von Bienen mit verschiedenen ISI trainiert. Schlielich wurde
beobachtet, dass Bienen bis zu einem ISI von 6 s die Assoziation zwischen CS und US
herstellen konnten. Daraus wurde geschlossen, dass der temporre Speicher der
Honigbiene ber eine Dauer von 6 s die Duftinformation im Gedchtnis behalten kann.
Auerdem wurde herausgefunden, dass mit einem ISI von wenigen Sekunden der
Konditionierungserfolg am grten ist.

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2 ABSTRACT

Working memory is necessary to create a mental representation of the environment in

order to solve complex tasks in the present situation. This mental representation of the
environment contains also biologically irrelevant stimuli in the beginning. Only because
of this, new causal connections can be derived from a set of various stimuli. Out of
these causal connections, predictions of future events can be made.
In this project the temporary storage of olfactory working memory of the honey
bee (Apis mellifera) was studied in aspect to dynamics and duration. For that purpose we
used trace conditioning, in which the conditioned stimulus (CS; 2-Heptanone/
1-Octanol) and the unconditioned stimulus (US; sucrose solution) were separated in
time (no overlapping of the stimuli). Associative learning should occur in bees which
succeed in bridging the time gap between the two stimuli (ISI, inter-stimuli interval) by
keeping odor information in the temporary storage. In order to quantify the duration of
the temporary storage we varied the ISI in trace conditioning. We observed significant
numbers of bees responding to the CS even at an ISI of 6 s. Hence, we concluded that
odor information can be maintained in the temporary storage of working memory for at
least 6 s. Furthermore, we found that short ISI of a few seconds are best for
conditioning.

3 INTRODUCTION

The honey bee is an exceptional phenomenon of evolution. However, evolution does

not yield prefect organisms. Instead organisms are restricted in their abilities by being
adapted to an ecological niche. But nevertheless these specializations are as many and as
various as there are ecological niches. Especially the behavioral strategies that enable
animals to emancipate from environmental selective pressure are one of the most
beautiful and impressing creations of the evolutionary game of chance. A bee colony is
probably the most wonderful way of nature, to organize matter and energy in space and time (Tautz,
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2007). The organization of the bee colony has to be highly efficient. The main feature of
the colony to solve this task is the division of labor. For neuroscientists the most
interesting group of bees are the foragers, because they have to deal with complex tasks,
which highly claims the cognitive abilities like spatial orientation and memory. In this
respect, time is a parameter that is not to be underestimated. In order to manage tasks in
the present an animal has to generate a perception of the present by holding
information in a temporary storage. This temporary storage is a compartment of
working memory, which in the end enables animals to cope with present tasks. The
term working memory is commonly defined as follows: A memory system that holds
information in temporary storage during the planning and execution of a task (Dudai, 2004).
We were interested in the dynamics of olfactory working memory in honey bees. To
study the time course of working memory we performed olfactory trace conditioning.
Conditioning techniques date from Ivan Petrovitch Pavlov, a Russian psychologist,
physiologist and physicist. In classical conditioning animals learn to associate an
originally neutral stimulus (CS; conditioned stimulus) with a biologically relevant
stimulus (US; unconditioned stimulus) (Pavlov, 1927). In trace conditioning the CS and
the US are presented separated in time by a trace interval. The temporary storage that is
used in a trace conditioning task is called trace memory. Trace conditioning is well
established in the vertebrate behavioral research (Shors, 2004). The association in a trace
conditioning task is more difficult to learn, as the stimuli are not overlapping in time.
Furthermore, in vertebrate research trace memory is often considered a declarative
memory (Clark & Squire, 1998), because of its dependency on the hippocampal
formation (Baylin et al., 2001) and association with awareness. Working memory and
trace memory of honey bees have been studied by various researchers. So did Menzel
and Bitterman in 1983. Similar as in our study, they investigated the temporary storage
of odor information using a trace conditioning on harnessed bees. They found, that
short inter-stimuli intervals (ISI) are best for conditioning and that the trace memory
spans at least 5 seconds (Menzel & Bitterman, 1983). Similar results arose in a project
about the honey bees working memory by Zhang and colleagues in 2005. They used
free flying bees, which had to cope with a visual delayed matching to sample (DMTS)
task. In such a task the bee does not have to learn an element like an odor or a certain
color (elementary learning), rather it has to learn a rule (non-elementary learning). In this
process a sample stimulus is presented to the bee, which is to be kept in a temporary
storage until the bee is confronted with two further stimuli. Now the bee has to match
the correct new stimulus to the stimulus presented before. The correct behavior is
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usually reinforced by a sucrose reward. Zhang and colleagues sized the span of the
temporary storage by enlarging the delay between sample and comparison stimuli.
Performance of the bees is significantly better than random-choice level even at delays
as long as 5 s (Zhang et al., 2005).
The aim of our project was mainly to find out how long bees are able to retain odor
information in the temporary storage of working memory. It is well known that bees
can associate an odor with a reward, like nectar, just by a few pairings of the two stimuli
and are able to maintain the knowledge about the predictive value in a stable long-term
memory that is resistant to different forms of interference (Giurfa, 2007). A neutral
stimulus gets a predictive value, if it precedes or appears at the same time of the reward.
Physiological research on associative learning in honey bees showed, that one cell plays
an important role in this. It is the so called Vummx1-neuron, which mediates the reward
function in appetitive olfactory learning (Hammer, 1993). But what happens when the
unknown stimulus precedes the reward? And how long can the two stimuli be separated
in time to be still associated with each other? For the research on this question precise
ISI are necessary. Therefore a short conditioned stimulus, an odor pulse of 0.5 seconds,
was used in our experiment. As the ISI is commonly defined as the interval from the
onset of the CS to the onset of the US, the discrepancy between the ISI and the time in
which no stimulus is presented is reduced by shortening the CS. Information about the
working memorys temporary storage is certainly crucial for further research on the nonelementary properties of working memory, like solving a DMTS task. But it is also basic
for other kinds of conditioning, when CS and US do not overlap.

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4 METHODS AND MATERIALS

4.1 General Methods and Materials

The subjects were free flying honey bees (Apis mellifera) from hives on the roof of the
department of biology at the University of Constance. These bees were immobilized by
cooling and instantly harnessed into plastic tubes, which surrounded the abdomen and
hold the head visible to the observer at the end of the tube, almost immovable but
allowing free movement of the antennae and
mouthparts (including proboscis). The harnessed bees
were fed with 1 M sucrose solution, which was
renewed at least every week. Over night the bees were
kept in a box humidified by moistened Kleenex. The
following morning (from 8 to 12 oclock) the bees were
placed on the training site (a 48 cm hard plastic bar
with 16 cavities for the tubes separated by plastic
baffles) (see Fig. 1). Then the appetitive motivational
state and/or intact reflexes of the bees was tested by
touching only the antennae with sucrose solution. Bees

Figure 1

not executing an proboscis extension reflex (PER) were

Olfactometer ejects odor pulses

excluded from the experiment as negative responses during

into a permanent air stream

acquisition and/or retrieval can be due to sensory-motor deficits

directed

instead of learning and/or memory deficits (Giurfa, 2007).

Only extending the proboscis beyond a virtual line

Training site.

to

the

bee.

Accumulation of scented air is

prevented by an exhaust.

between the opened mandibles was defined as PER. The training site was displaceable
along a steel base plate orientated rectangular to the air stream, between an olfactometer
and an exhaust. Bees were on a defined distance of 2 cm from the orifice of the
olfactometer.
The olfactometer was constructed by Dr. Paul Szyszka and Benjamin Birnbach
(2008). It contained syringes (3 ml; Norm-Ject, Henke Sass Wolf, Tuttlingen, Germany)
that could be filled with an odor by placing a 0.7 x 3.5 cm piece of cellulose strips (Sugi,
Kettenbach, Eschenburg, Germany) soaked with 200 l of the odor solution.
Furthermore it was important that the olfactometer ejects well-defined odor pulses.
Consequently, special attention was paid on this. The odor pulse was therefore
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controlled by magnetic valves positioned downstream of the syringes and was injected
into a continuous air stream. The olfactometer valves were triggered by a customwritten computer program (Alexander Galkin, Berlin). The carrier air used was
humidified (40%). Flowmeter were used to regulate the carrier air to 2600 ml/min, and
the odor loaded air to 300 ml/min. The orifice of the olfactometer had a diameter of
1 cm. A second separate air stream directed to the bee next to the training position
allowed additional habituation (see Fig. 1).
In order to determine how long bees are able to keep odor information in trace
memory, we varied the inter-stimuli interval (ISI) in a trace conditioning. For US with
an overall duration of 3 s the bees were rewarded by manually touching both antennae
with the tip of a metal stick, which had been dipped in a 1 M sucrose solution before,
and feeding the bee. For CS, an odor pulse of 0.5 s was injected into the permanent air
stream of the olfactometer. Two odorants were taken alternatively in order to control
odor specific effects. These odorants were 2-Heptanon and 1-1-Octanol (both: Fluka,
Sigma-Aldrich) diluted in mineral oil to concentrations of each 10-2.
Measurements were carried out in the morning (from 9 to 12 oclock) or additional
in the afternoon (from 12 to 15 oclock). For each setup 16 bees were arranged in
groups with different ISI. Therefore, ISIs ranging from -6 s to +15 s were used. The
pilot experiment and the two periods of the main experiment differed in the selection of
ISIs (see specifics below). Since the number of 16 bees could often not be divided
through the number of ISI groups, the partition of the groups was
regularly changed in order to end up with an equal number of data
points for each ISI.
6 training trials were carried out with inter-trial intervals (ITI) of
each 10 min. The length of each trial depended on the length of the
ISI. At the beginning of a trial each bee was moved to the training
position and stayed there for 25 s in order to habituate to the air
stream. The CS was presented for 0.5 s at the 25th second. Following
the time slot of the ISI the US was presented for 3 s (see Fig. 2). The
trial was finished by moving the bee away from the training position

Figure 2
Rewarding of a
bee. US is a 1 M
sucrose

5 s after the onset of the US and thereby bringing the next bee to the

solution

training position. The PER-performance was noted throughout the

presented to

training for all intervals except the ISI of -6 s and 0 s, because at these

antennae and

ISI CS-evoked PER could not be distinguished from US-evoked PER.

Two tests followed the last training trial, one after 15 min and another
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proboscis for a
total of 3 s.

after 24 h, by a single presentation of the 0.5 s odor pulse without rewarding the bee.
Afterwards, the motivational state of the bees was tested by touching the antennae with
sucrose solution again. Those which did not execute a PER were excluded of the
evaluation of the tests results. The timing of the events during the experiment was
electronically controlled by auditory signals.
The ISI of -6 s and 0 s were used as they are useful for comparison. It is known,
that conditioning with negative ISI, so called backward pairing of stimuli, does not cause
appetitive learning, but contrary even produces aversive learning. We as well found that
bees respond more often to the CS after a forward pairing than after training with
backward pairing. ISI groups in which significantly more bees responded to the CS
compared to the -6 ISI group, should indicate associative learning and consequently,
that the bees have retained the odor information all along the time of the ISI. The ISI of
0 s was used because in this case the two stimuli are simultaneously presented, and a
high percentage of bees responding could be expected. This could be used to estimate
the success of associative learning in all the other ISI groups by comparison.

4.2 Specifics of the Pilot Experiment

The olfactometer used in the pilot experiment did not have a second air stream for
habituation. Moreover, the valves were positioned upstream of the odor-syringes. At
this, diffusion of odor molecules into the permanent air stream had to be considered.
Only four ISI groups of bees were trained in the pilot experiment. Therefore the
ISIs of 0, 3, 6, 15 s were used. The bees were only tested once after 15 min. No test
after 24 h was executed in the pilot experiment.

4.3 Specifics of the First Period of the Main Experiment

A newly constructed olfactometer was used (see general methods and materials). The
ISI of -6, 0, 1, 2, 3, 6, 10 and 15 s were used for trace conditioning.

4.4 Specifics of the Second Period of the Main Experiment

Two further ISI groups (4 s and 5 s) were introduced. Eventually, the ISIs of -6, 0, 1, 2,
3, 4, 5, 6, 10 and 15 s were used for trace conditioning. Group strengths were adjusted,
in order to end up with an equal number of data points for each ISI group. Still, all ISI
groups were trained and tested together in each experimental run.
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4.5 Data Acquisition and Processing

The PER was measured by so called one-zero sampling (Martin & Bateson, 2007),
where 1 was noted representing a response, but only if a PER was executed at the same
time as the CS presentation, and 0 (no response) in all other cases.
During training, data from bees was taken down except for the -6 and the 0 s ISI. In
the tests, data from all bees was noted. However, bees that did not respond to the US
with a PER more than two times were defined as dead and were completely excluded
from evaluation, because they obviously received a quite anomalous training compared
to the other bees. In contrast, data of bees that only once did not respond to the US,
was still evaluated, in the tests, as this aberration is not seen as relevant. But it was not
evaluated in the training.
For data analysis we used custom written routines in R (www.r-project.org; routines
written by Ana Florencia Silbering, Konstanz) and the statistical software SigmaStat 3.0
(Statcon). The results of the 15 min test and 24 h test were investigated towards
significances compared to the ISI of -6 s, by One-Way-ANOVA. Furthermore, in the
15 min test and the 24 h test, results of each ISI group were tested against its
spontaneous response level in the first trial of training using a t-test.

4.6 Specific Data Processing Exclusive Data

Only data from bees, which survived al along the whole experiment, was taken for data
processing.

4.7 Specific Data Processing Two Periods of the Main Experiment

Data was separately processed depending on the period in which the data was collected.

4.8 Specific Data Processing Two Odors

Data was separately processed depending on the odor used in the experimental runs.

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5 RESULTS AND DISCUSSION

In order to study the time course of the temporary storage (trace memory) of working
memory in honey bees we performed trace conditioning with different ISIs and
measured responses during training and tests using the proboscis extension reflex (PER).
Increase of response to the CS during training and tests indicate associative learning.
During trace conditioning CS-US association can only be achieved if the bee has access
to odor trace memory at the time of the sucrose reward (US). Therefore, associative
odor learning can be used to study trace memory. Differences were expected between
the ISI-groups during training in terms of acquisition of memory and in the tests in
terms of consolidation. Moreover, the longest ISI, which still can be bridged by the bee
using its temporary storage to build an association, should be found. Therefore, the
results are shown in detail for the pilot experiment and the main experiment.

5.1 Pilot Experiment Trace Conditioning

The pilot experiment was used for testing the materials and methods that were designed
for the study on the time course of olfactory working memory. As an indicator for
associative learning the proboscis extension reflex (PER) of the bee is used. Having
trained the bees, they in fact showed responses to the conditioned stimulus (CS), an
odor pulse of only 0.5 s (Fig. 3). This means that the animals were using a certain
storage, the trace memory, for the maintenance of odor information during the ISI until
the association could be built.
During training the responses of the 0 s ISI group could not be measured (see
methods & material). In the 1st trial of the training none of the bees responded to the
CS. This could be interpreted in the way that the CS was unknown or did not have any
predictive value to the bees. But already in the 2nd trial, responses could be seen with the
6 s ISI curve to the top while around 10% of bees responded to the CS. Overall, the
training curve of the 3 s ISI was most of the time at the top. No saturation of the curve
could be recognized, which indicates a non scooped potential. The curve of the 6 s ISI
ran close to the 3 s ISI curve and showed saturation at a percentage of around 30% in
trial 5 and 6. The 15 s ISI curve stayed on a subordinate level throughout the training.
However, there were little maxima (around 10% of bees responding) at the 3rd and the
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6th trial. These little maxima could

be explained by sensitization.
During

the

conditioning

procedure the bee gets sensitized

by the sucrose reward and may
extend the proboscis to the CS
without
association.

having
But

made

the

sensitization

should be independent from the

ISI and hence, the method of
conditioning bees with different
ISI is still appropriate to study the
temporary storage of working
memory. Consequently long ISI
that do apparently not create
acquisition of memory could be
seen as reference for sensitization
effects by the US.
15 minutes after the last trial
of training a test was executed, in

Figure 3 Pilot experiment. Trace conditioning to odor

with different ISI in harnessed bees. CS last for 0.5 s and
is presented before the US. US is a sucrose solution
presented to antennae and proboscis for a total of 3 s.
Percentage of bees responding is given by the ordinate.
Left plot: Training curves. Bees were conditioned to odor
by six trials. Right pot: Test 15 min after the last trial of
the training. CS is singly presented. To simplify matters
brackets assign only few exemplary leaps within two ISI
groups (statistical test: One Way ANOVA; more details
are given in the text).

which only the CS was presented to the bee. The test results showed the percentage of
bees responding in the 0 s ISI group, which was not measured during training. The 0 s
ISI was the group with the highest percentage of bees responding in the test with
around 84%. The CS was responded to by 40% of the bees in the 3 s ISI group, which
was a little less than in the 6th trial of the training and which was significantly less than
the 0 s ISI percentage of bees responding (0 s ISI vs. 3 s ISI: t=4.968, p<0.001). The 6 s
ISI group reached around 20%, which was less than its result in the 6th trial of training.
It was significantly less than the 3 s ISI test result (3 s ISI vs. 6 s ISI: t=2.074, p=0.039).
In the 15 s ISI group none of the bees responded to the CS at all, which was less than
the responses it achieved in the 6th trial of the training and also significantly less than the
6 s ISI percentage in the test (6 s ISI vs. 15 s ISI: t=2.031, p=0.044). The test results
showed that acquisition of memory was induced after training with ISI of 0, 3 and 6 s,
but no acquisition of memory took place in the 15 s ISI group. Hence, one could
assume can be retained in trace memory for 6 s.

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In the test a decreasing tendency of the percentages of bees responding was already
visible from the shortest to the longest ISI. The training curves confirmed this finding,
with the shortest ISI (3 s) creating the steepest curve, the 6 s ISI in the middle and the
longest ISI (15 s) staying at a subordinate level.
The results of the pilot experiment showed that the method is suitable to study
trace memory in honey bees on a behavioral level, in order to find differences in the
effects of ISIs on the retention of odor information in trace memory.
But also improvements of methods and materials were derived from the results. So
the olfactometer was optimized by adding a second air stream for habituation of the bee
on the position next to the training position. This was necessary as in the first trial of
training no responses were noted, which was probably due to the fact that extending the
proboscis before the onset of the CS was defined as not responding to the CS. Because
of that, the number of responses in the whole experiment was less than it should be. In
contrast to that, a stronger habituation offers reduced biases. Especially in the first trial
it is important to detect spontaneous responses to the CS, which then can be used as
reference to the training and test results. Furthermore, doubts in the precision of the
0.5 s odor pulse arose, as the valves of the olfactometer were positioned upstream of the
syringes. So the assumption, that trace memory last for 6 s could be disputed at this
point. The positions of the valves were changed in the new olfactometer (see methods
and materials) in order to reduce diffusion of odor molecules into the permanent air
stream and in order to receive more precise odor pulses for trace conditioning.

5.2 Main Experiment Trace Conditioning and Backward

Conditioning
For the main experiment new ISI were introduced in order to get information about the
characteristics of trace memory in terms of time. From there on ISI with durations of -6,
0, 1, 2, 3, 4, 5, 6, 10 and 15 s were used. Moreover, a new olfactometer should extend
habituation of the bees and reduce biases. Another test, the 24 h test, the day after the
training was established to study the effects of different ISI on the consolidation of
long-term memory, based on associative learning that depends on trace memory.
And indeed bees responded to the CS the day after the training (see Fig. 4). Also in
the training and in the test after 15 min the new ISI evoked responses, which differed in
occurrence frequencies dependent to the ISI used for training. In general percentage of
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bees responding rose from the first to the sixth trial of training. This effect is stronger at
short ISI and less evident or even not significant at long ISI. Comparing both tests the
results were similar. Percentage of bees responding decreased from the shortest ISI to
the longest. Contrary bees of the backward pairing ISI of -6 s showed no response at all.

Figure 4 All data of the main experiment. Trace conditioning to odor with different ISI in harnessed bees.
CS last for 0.5 s and is presented before the US. US is a sucrose solution presented to antennae and
proboscis for a total of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training
curves. Bees were conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the
training. CS is singly presented. Right plot: Test 24 h after the last trial of the training. CS is singly
presented. Little stars assign ISI groups that are significantly different towards the -6 s ISI group
(statistical test: One Way ANOVA). To simplify matters brackets assign only few exemplary leaps within
two ISI groups (statistical test: One Way ANOVA; more details are given in the text). Filled bars assign
significantly different test results compared to the 1st trial of the training (statistical test: t-test).

5.2.1 Training
Responses of the -6 s ISI and 0 s ISI groups could not be recorded in the training (see
methods & material). In the 1st trial of the training all percentages of bees responding
were below the 10% level (see Fig. 4). In the 2nd trial percentages ranged from 5% to
23% with the 15 s ISI at the top. In the 3rd trial the range was from 12% to 40%. The
1 s ISI rose steeply to around 40%. The 2 and 3 s ISIs went up to about 25%. All the
- 14 -

other ISIs concentrated on a percentage level of 15%. In the 4th trial percentages were
within 10% to 50%. The 4th trial decentralized the concentration of curves, which could
be seen in the 3rd trial at the level of 15%. But the ISIs from 2 to 15 s were still grouped
in a range from 10% to 30%, in which the shorter ISI (2, 3, 5 and 4 s) were positioned
in the upper part and the longer ISI (15, 6 and 10 s) at the lower end. In the 5th trial the
range was from 5% to 58% with the 1 s ISI still at the top rising linear like to 58%. In
the 6th trial the range was enlarged spanning from 12% to 65%. In the end of the
training there were four groups of ISI percentage levels. The 1 s ISI as a single to the
top, the 3 and 4 s ISIs following, the 2, 5, and 6 s ISIs in the middle and the two longest
ISIs (10 and 15 s) with the lowest percentages of bees responding. The training curve of
the 1 s ISI stacked out of the others by having highest percentages in most of the trials
and showing no saturation. This meant that the 1 s ISI was the best ISI for associative
learning. The upholding of information about the CS in trace memory was no problem
over a time of 1 s. No striking differences within the ISI from 2 to 6 s could be seen.
The 10 and 15 s ISI remained at the same percentage level of bees responding and
therefore there was no indication of associative learning.

5.2.2 15 min Test

In the test, which was executed 15 minutes after the last trial of the training, none of the
bees belonging to the -6 s ISI group responded to the CS. The percentage of bees
responding in the 0 s ISI group was around 69%, which was the highest percentage in
the test. The 1 s ISI was around 67%, which was quite the same as it achieved in the 6th
trial of the training. However, it was little less than the percentage of bees responding in
the 0 s ISI group in the test (but no significant difference). In the 2 s ISI group around
45% of bees responded, which was more than in the last trial of training. But also it was
significantly less than the percentage of bees responding in the 1 s ISI group in the test
(1 s ISI vs. 2 s ISI: t=2.021, p=0.044). In the 3 s ISI group about 35% of bees
responded, which was lower than in the 6th trial of the training and also less than the
percentage of bees responding in the 2 s ISI group in the test (but no significant
difference). The 4 s ISI reached 28% of bees responding, which was less compared to
the 6th trial of the training and also less than the 3 s ISI result (but no significant
difference). Surprisingly the 5 s ISI showed a percentage of 36%, which was higher than
in trial 6 of the training and higher compared to the 4 s ISI (but no significant
difference). It was thus on the same level as the 3 s ISI. But there were no significant
- 15 -

differences detected between the ISI of 4 and 5 s. In the 6 s ISI 18% of bees responded,
which was less than in the 6th trial of the training and even significantly less than the 5 s
ISI result (5 s ISI vs. 6 s ISI: t=2.035, p=0.043). The 10 and 15 s ISI were around 9% in
the test, which were both less than in trial 6 and also less than the 6 s ISI result (but no
significant difference).
An obvious effect was that shorter ISIs led to higher percentages of bees
responding. Because of that, ISI within a few seconds are best for associative learning
and acquisition of memory. The 1 s ISI was even at the same level compared to the 0 s
ISI. A significant leap could be seen from 1 s ISI to 2 s ISI (1 s ISI vs. 2 s ISI: t=2.021,
p=0.044). After that, percentages of bees responding declined regularly. However no
significant differences between each neighboring ISI groups could be found, except the
leaps from 1 to 2 s ISI and from 5 to 6 s ISI. A striking result was the percentage of the
5 s ISI, which could be seen as a peak within the ISI-function, but even at this, no
significant differences between the neighboring ISI could be detected. Significantly
different percentages of bees responding, compared to the -6 s ISI, were seen in the ISI
groups of 0, 1, 2, 3, 4 and 5 s. (-6 s ISI vs. 0 s ISI: t=6.978, p<0.001; -6 s ISI vs. 1 s ISI:
t=6.687, p=<0.001; -6 s ISI vs. 2 s ISI: t=4.652, p=<0.001; -6 s ISI vs. 3 s ISI: t=3.701,
p=<0.001; -6 s ISI vs. 4 s ISI: t=2.847, p=0.005; -6 s ISI vs. 5 s ISI: t=3.826,
p=<0.001). Thus, associative learning took place and trace memory could be used in an
ISI of at least 5 s. No associative learning took place at the training with 6, 10 and 15 s
ISIs.

5.2.3 24 h Test
In the 24 h test it was of interest, whether the association built up on the basis of the
odor information in the temporary storage, is transferred into long-term memory as well
as the association caused by a synchronous pairing of the stimuli in the 0 s ISI group.
To check up bees were tested one day after the training and responses to the single odor
were recorded in schedules.
Bees responded to the CS even one day after the training (see Fig. 4). Short ISIs
evoked higher percentages than long ISIs.
The -6 s ISI bees did not respond to the CS at all. The 0 s ISI was again at the
highest level of percentages (around 60%). But, it was less strong than in the test the day
before. The 1 s ISI was around 42%, which was less than in the test the day before and
less than the 0 s ISI (but no significant difference). The 2 s ISI was around 36%, which
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was less than in the test the day before and less than the 1 s ISI (but no significant
difference). Surprisingly the 3 s ISI was around 50%, which was more than in the test
the day before and also more than in the 2 s ISI group (but no significant difference).
The 4 s ISI was around 36%, which was more than in the test the day before, but less
than the 3 s ISI and same percentage than the 2 s ISI (no significant differences). The
5 s ISI was around 28%, which was less than in the test the day before and a little less
than the 4 s ISI (but no significant difference). The 6 s ISI was around 25%, which was
more than in the test the day before, but almost the same as the 5 s ISI result (but no
significant difference). The 10 s ISI was around 18%, which was more than in the test
the day before and less than the 6 s ISI result (but no significant difference). The 15 s
ISI was around 9%, which was the same as in the test the day before and less than the
10 s ISI (but no significant difference). Significantly different percentages of bees
responding, compared to the -6 s ISI group, were reached by the ISI of 0, 1, 2, 3, 4, 5,
and 6 s (-6 s ISI vs. 0 s ISI: t=5.334, p=<0.001; -6 s ISI vs. 1 s ISI: t=3.745, p<0.001; -6
s ISI vs. 2 s ISI: t=2.967, p=0.003; -6 s ISI vs. 3 s ISI: t=4.445, p<0.001; -6 s ISI vs. 4 s
ISI: t=3.040, p=0.003; -6 s ISI vs. 5 s ISI: t=2.481, p=0.014; -6 s ISI vs. 6 s ISI: t=2.205,
p=0.028). Thus associative learning took place and trace memory could be used in an
ISI of at least 6 s, which is a different result compared to the 15 min test result. No
associative learning took place at the training with 10 and 15 s ISIs.

5.3 Main Experiment Exclusive Data

As the two tests of the main experiment evoked different results, data only of the bees
that survived the whole experiment were used for data processing. After this kind of
data processing, one could see that the percentage of bees responding in the 6 s ISI
group were quite at the same level (around 25%) in both tests (see Fig. 5). And in fact
both were significantly different compared to the -6 s ISI results (15 min test: -6 s ISI vs.
6 s ISI: t=2.263, p=0.024; 24 h test: -6 s ISI vs. 6 s ISI: t=2.187, p=0.030) and
compared to their respective 1st trial of training (1st trial vs. 15 min test: t=-2.043,
p=0.045); 1st trial vs. 24 h test: t=-2.043, p=0.045). Therefore bees were able to maintain
odor information at least 6 s in trace memory for associative learning. After the first
data procedure, which evaluated the data also from bees that died after the first test, no
significant percentage of bees responding could be detected. This demonstrated that
bees, which died afterwards, were less capable to associate the CS with the US at an ISI
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of 6 s. On the one hand this could mean that bees with higher cognitive capacities live
longer or at least are in good health. On the other hand, as the case may be that the bees,
which died, were just old bees the assumption would be, that age influences the capacity
of trace memory. In fact, research in human psychology revealed a negative correlation
between age and working memory performance (Oehm & Schneider, 1998).

Figure 5 Exclusive data. Trace conditioning to odor with different ISI in harnessed bees. CS last for 0.5 s
and is presented before the US. US is a sucrose solution presented to antennae and proboscis for a total
of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training curves. Bees were
conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the training. CS is singly
presented. Right plot: Test 24 h after the last trial of the training. CS is singly presented. Little stars assign
ISI groups that are significantly different towards the -6 s ISI group (statistical test: One Way ANOVA).
To simplify matters brackets assign only few exemplary leaps within two ISI groups (statistical test: One
Way ANOVA; more details are given in the text). Filled bars assign significantly different test results
compared to the 1st trial of the training (statistical test: t-test).

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5.4 Results of the First Period of the Main Experiment

As the data was collected in two periods separated in time and each contained different
numbers of ISI, it was needful to look at the results of the single periods. In the first
period the ISIs -6, 0, 1, 2, 3, 6, 10 and 15s were used.
In the training the curves of the ISI groups 1, 2, and 3 s could be seen in the upper
half (see Fig. 6). The ISI groups 6, 10 and 15 s ran within the under part.

Figure 6 Data of the first period. Trace conditioning to odor with different ISI in harnessed bees. CS last
for 0.5 s and is presented before the US. US is a sucrose solution presented to antennae and proboscis for
a total of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training curves. Bees were
conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the training. CS is singly
presented. Right plot: Test 24 h after the last trial of the training. CS is singly presented. Little stars assign
ISI groups that are significantly different towards the -6 s ISI group (statistical test: One Way ANOVA).
To simplify matters brackets assign only few exemplary leaps within two ISI groups (statistical test: One
Way ANOVA; more details are given in the text). Filled bars assign significantly different test results
compared to the 1st trial of the training (statistical test: t-test).

In the test 15 minutes after the training, the percentages of bees responding in the 0 and
the 1 s ISI groups were around 63%, slightly above the others (no significant difference).
2 and 3 s ISI reached percentages around 45%. And the 6, 10 and 15 s ISIs had low
percentages. There were only two significant leaps, one leap from -6 s ISI to 0 s ISI (-6 s
ISI vs. 0 s ISI: t=4.913, p<0.001) and another leap from 3 to 6 s ISI (3 s ISI vs. 6 s ISI:
- 19 -

t=3.519, p<0.001). Significantly different percentages compared to the -6 s ISI were

reached by the ISI of 0, 1, 2 and 3 s (-6 s ISI vs. 0 s ISI: t=4.913, p<0.001; -6 s ISI vs.
1 s ISI: t=4.673, p<0.001; -6 s ISI vs. 2 s ISI: t=2.941, p=0.004; -6 s ISI vs. 3 s ISI:
t=3.431, p<0.001).
In the test one day later, the 0 s ISI showed the highest percentage (around 50%) of
all groups. The 1, 2 and 3 s ISI groups were at a middle level at about 35%. The ISI 6,
10 and 15 s only had little percentages. Significances against the -6 s ISI were detected in
the ISI of 0, 2 and 3 s (-6 s ISI vs. 0 s ISI: t=3.390, p<0.001; -6 s ISI vs. 2 s ISI: t=2.247,
p=0.027; -6 s ISI vs. 3 s ISI: t=2.482, p=0.015). The percentages of bees responding in
the groups from 0 s ISI to 3 s ISI were not significantly different from each other. The
1 s ISI was not significantly different from the backward pairing ISI (-6 s ISI), but was
significantly different to the percentage of responses in the 1st trial of training (1st trial vs.
24 h test: t=-2.407, p=0.022). In the 3 s ISI group no significantly different percentage,
compared to the 1st trial of training, was detected.
All statistics in this part of the evaluation have to be seen critically, because the
number of bees was only the half of the number that should be taken for statistics,
while using a One Way ANOVA. The statistical test of an One Way ANOVA is usually
used for continuous distributions, but in our case discrete data were collected. An One
Way ANOVA can only be used for discrete data, if the number of data points is big
enough (at least n=40). In this part of evaluation, this was not the case.

5.5 Results of the Second Period of the Main Experiment

In the second period of the experiment two additional ISI (4 and 5 s) were also used for
conditioning.
In the training, the curve of the 1 s ISI group stacked out (see Fig. 7). At the end of
the training the curves of the ISI from 2 to 10 s were grouped in the middle ranging
from around 20% to 50%. The 15 s ISI showed only low percentages all along the
training trials.
In the test 15 minutes after the last trial of training highest percentages were
reached by the ISI of 0 and 1 s (around 70%). In the 2 s ISI around 50% of bees
responded. The ISI of 3, 4, 5 and 6 s were at a percentage level around 35%. Only
subordinate levels of percentage were reached by the 10 and the 15 s ISI, which were
less than 10%. Percentages were significantly different from the -6 s ISI in the ISI
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groups of 0, 1, 2, 3, 4, 5 and 6 s (-6 s ISI vs. 0 s ISI: t=5.114, p<0.001; -6 s ISI vs. 1 s ISI:
t=4.912, p<0.001; -6 s ISI vs. 2 s ISI: t=3.635, p<0.001; -6 s ISI vs. 3 s ISI: t=2.102,
p=0.037; -6 s ISI vs. 4 s ISI: t=2.282, p=0.023; -6 s ISI vs. 5 s ISI: t=3.054, p=0.003;
-6 s ISI vs. 6 s ISI: t=2.449, p=0.015). Neighboring response bars were not significantly
different from each other except the -6 s ISI against the 0 s ISI (-6 s ISI vs. 0 s ISI:
t=5.114, p<0.001).

Figure 7 Data of the second period. Trace conditioning to odor with different ISI in harnessed bees. CS last
for 0.5 s and is presented before the US. US is a sucrose solution presented to antennae and proboscis for
a total of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training curves. Bees were
conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the training. CS is singly
presented. Right plot: Test 24 h after the last trial of the training. CS is singly presented. Little stars assign
ISI groups that are significantly different towards the -6 s ISI group (statistical test: One Way ANOVA).
Filled bars assign significantly different test results compared to the 1st trial of the training (statistical test:
t-test).

In the test one day after the training, highest percentages of bees responding could bee
seen at the ISIs of 0, 1 and 3 s at a level around 60%. Middle percentages were reached
by the ISI groups of 2, 4, 5 and 6 s, which were around 35%. The 10 s ISI was around
18% and bees of the 15 s ISI showed no responses at all. Significantly different from the
-6 s ISI were the percentages of the ISI of 0, 1, 2, 3, 4, 5 and 6 s (-6 s ISI vs. 0 s ISI:
- 21 -

t=4.373, p<0.001; -6 s ISI vs. 1 s ISI: t=3.692, p<0.001; -6 s ISI vs. 2 s ISI: t=2.078,
p=0.039; -6 s ISI vs. 3 s ISI: t=3.784, p=<0.001; -6 s ISI vs. 4 s ISI: t=2.481, p=0.014;
-6 s ISI vs. 5 s ISI: t=2.006, p=0.047; -6 s ISI vs. 6 s ISI: t=2.572, p=0.011).
Neighboring percentage bars were not significantly different from each other except the
pair -6 s ISI and 0 s ISI (-6 s ISI vs. 0 s ISI: t=4.373, p<0.001).

5.6

Comparison of the Two Experimental Periods

Comparing the training curves only few striking difference could be seen. In the second
period of the experiment, the 1 s ISI curve stacked out of the other curves, showed the
steepest run and finally reached a percentage of bees responding around 75%. All this
could not be seen in the first period of the experiment. Another striking difference was
the run of the 6 s ISI curve. The 6 s ISI curve stayed at a subordinate level all along the
training trials in the first period of the experiment, but a clear increase could be seen in
the second period. The 6 s ISI curve showed a similar run compared to ISI curves like 2,
3, 4 and 5 s in the second period.
Comparing the test results from the two periods of the experiment the ISI of 0, 1
and 2 s increased their percentages of bees responding in the second period compared
to the first period. The ISI of 3 s decreased in the second period compared to the first.
The most striking different however could be seen in the 6 s ISI. None of the 6 s ISI
bees responded in the first period, but a significant percentage was reached in the
second period of the experiment (compare Fig. 6 and Fig. 7)
In the test one day after the training the ISI of 0 s, 1 s and 3 s increased in the
second period compared to the first period. The 2 s ISI showed similar percentages.
Bees of the 6 s ISI group did not respond at all in the first period, but showed a
significant percentage in the second period of the experiment.
Over all the most striking difference could be seen in the 6 s ISI group. The fact,
that none of the bees of the 6 s ISI responded in the first period of the experiment, but
a significant percentage of bees responded in the second period, could be explained by a
systematic improvement of the experimenter in handling and conditioning bees. But
also climate, temperature and seasonal effects, which were not controlled in the
experiment, could play a role.

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5.7 Comparison of the Two Odors

The pooled data were processed dependently on the odor used in the experiment.
Comparing the training curves only one striking difference could be seen. The curve of
the 1 s ISI in the 1-Octanol training showed clearly the steepest run of all curves and in
the 6th trial of the training around 75% of bees responded (see Fig. 9), which was more
than the 1 s ISI reached in the 2-Heptanone training (55%; see Fig.8).

Figure 8 2-Heptanone conditioning. Trace conditioning to odor with different ISI in harnessed bees. CS
last for 0.5 s and is presented before the US. US is a sucrose solution presented to antennae and proboscis
for a total of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training curves. Bees
were conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the training. CS is singly
presented. Right plot: Test 24 h after the last trial of the training. CS is singly presented. Little stars assign
ISI groups that are significantly different towards the -6 s ISI group (statistical test: One Way ANOVA).
Filled bars assign significantly different test results compared to the 1st trial of the training (statistical test:
t-test).

The steep run of the 1 s ISI could be interpreted as an 1-Octanol specific effect. In the
1-Octanol training the curves of the 2, 3, 4 and 5 s ISI could be seen grouped at a level
in the 6th trial, where about 40% of bees responded. In contrast to that, the curves of
the 6, 10 and 15 s ISIs were grouped in a range of 10% to 25% in the 6th trial. Such a
grouping effect, where the short ISI (2, 3, 4 and 5 s) were separated from the long ISI (6,
- 23 -

10 and 15) could not be seen in the 2-Heptanone training. Thus the grouping effect
could be another 1-Octanol specific effect.
In the test 15 minutes after the training the ISI of 2, 3, 4, and 5 s evoked a higher
percentage in the 1-Octanol experiments (see Fig. 9), rather than the same ISI in the
2-Heptanone experiments. Same percentages were reached in both odor tests at the ISI
of 0, 6, 10 and 15 s. Therefore one could say that conditioning with 1-Octanol is more
successful. Similar results could be seen in the test the day after the training.

Figure 9 1-Octanol conditioning. Trace conditioning to odor with different ISI in harnessed bees. CS last
for 0.5 s and is presented before the US. US is a sucrose solution presented to antennae and proboscis for
a total of 3 s. Percentage of bees responding is given by the ordinate. Left plot: Training curves. Bees were
conditioned to odor by six trials. Middle pot: Test 15 min after the last trial of the training. CS is singly
presented. Right plot: Test 24 h after the last trial of the training. CS is singly presented. Little stars assign
ISI groups that are significantly different towards the -6 s ISI group (statistical test: One Way ANOVA).
Filled bars assign significantly different test results compared to the 1st trial of the training (statistical test:
t-test).

After the training with 2-Heptanone as well as 1-Octanol, significances could be seen in
the same ISI groups (0, 1, 2 and 3 s). Moreover the 3 s ISI group showed responses in
the 1st trial of the 2-Heptanone training as well as in the 1-Octanol training. In both
cases, the test results were not significantly different from the 1st trial of the training. In
the test one day after the training, the 2 s ISI did not evoke significant responses in the
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2-Heptanone experiments as well as in the 1-Octanol experiments. Taking this into

account one could conclude, that there is variability in the strength of the number of
bees responding dependent on the odor used in the experiment, but the dynamics of
trace memory stays the same.

6 GENERAL DISCUSSION

In order to determine the dynamics of trace conditioning, we varied the ISI. By

elongating the ISI a point should be reached where no association can be made anymore.
One has to take account of the possibility that the odor information could still be
represented in trace memory but at the same time no associative learning takes place for
several conceivable reasons. For example, the motivational state of the bee could be to
low, or its subjective perception of the odor might get less attractive the longer the
representation of the odor is kept in trace memory. Pavlov described conditioning
techniques as a tool to get insight in an animals subjective perception of stimuli
(Schwartz, 1988). That is why in our study two odors were used alternatively, in order to
see whether there are differences in the success of trace conditioning, which would
indicate a different subjective perception. And in fact, the results show differences in the
hedonistic behaviors of the bees, as 1-Octanol was over all more often responded than
2-Heptanone. But despite this hedonistic effect, the dynamics of trace memory stayed
equal. Therefore, it is reasonable that an association is made as long as any odor
information is maintained in trace memory independently from hedonistic effects.
Finally we can say that odor information is kept in trace memory ranging from 1 to 6 s,
but is gone after 10 s. The ISI of 7, 8 and 9 s should be studied in further research. But
given the results of previous research there is no indication that the odor information
can be kept longer than 6 s (compare: Menzel, 1983). Even quite different methods lead
to equal results. Zhang used a visual DMTS task with free flying bees to study the
temporary storage of odor information, whereas Menzel, like us, used trace conditioning
with harnessed bees. For comparison only, Menzel used an odor pulse of 2 s, whereby
the discrepancy between the ISI and the time, where no stimulus is present, is larger
especially at short intervals. But nevertheless, all results showed a trace memory up to
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6 s and no acquisition of memory at longer ISIs. Now one should raise the question
whether there is a difference in the temporary storage of working memory and trace
memory. Analogous results often reveal a basic phenomenon, sometimes even indicate
homologies. Taking this into account and following the principle of parsimony, where
results are explained with the simplest theory, one can say that trace memory is the
temporary storage of working memory. Moreover, evolution in general does not invent
new units, but uses old units to combine them to another arrangement, or to convert
their function by placing them into a new context. Hence, we can assume that working
memory exerts just the same trace memory which is also used for more simple tasks like
elementary learning. Also in human psychology there is no indication that different
systems are used for the temporary storage of working memory and other short-term
memories (Osaka et al., 2007)
Moreover, it is of interest, why the percentage of bees responding decreases when
the ISI is elongated and finally vanishes when ISIs longer than 6 s are used for training.
We consider three main possibilities to explain the result, that the longer the ISI the
smaller the number of bees responding to the CS.
First, the probability that bees do not associate the CS with the US could be higher
the longer the ISI was, due to neural processing, which would actively inhibit the neural
representation of the CS in order to estimate its importance for the present situation. In
our case, it would be the predictive value of the CS (predicting the sucrose solution),
dependent on which an association is formed. This would be favorable, because it is
plausible that two events closely following in time are implicated into a causal
connection. Besides, the inhibition could also stop the out-reading of information from
the temporary storage after a certain time. To find out, if there is any inhibition,
physiological research would be necessary. Therefore an inhibitory access to the
memory trace of the temporary storage should be found, which suppresses the
temporary storage activity or the out-reading of information at a certain time of a few
seconds.
Second, the decrease could be due to disturbance effects. The probability of
interference, due to disturbing stimuli within the time of the ISI, is higher the longer the
ISI is. But also stimuli that precede the CS presentation could disturb the storage of
odor information in the temporary storage during the ISI by changing the bees attention.
In order to test these considerations, further behavioral research would be appropriate.
One access would be to train three groups of bees by trace conditioning with a certain
ISI for all. One group trained with a distracting stimulus preceding the CS. Another
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group trained with a distracting stimulus within the ISI. And a last group trained without
the distracting stimulus. If significant differences could be seen an influence on
maintaining odor information in temporary storage by disturbing stimuli should be at
least one important factor.
Third, there are indications aroused by imaging research (Birnbach, 2008) that the
odor representation in the temporary storage of the bee changes in the course of time.
If so, the bees trained with long ISI would have been conditioned in fact to another
odor perception compared to bees trained with short ISI, which should be conditioned
to an odor perception being more alike the instantaneous CS perception. Thus, bees
trained with long ISI would recognize the CS in the test less often, which was shown in
this experiment already. An experimental approach to check the odor perception during
the ISI would be a generalization experiment. Here two groups of bees would be trained.
One group trained with classical conditioning, in which the stimuli are overlapping, and
another group trained with trace conditioning (no overlapping). After that a test would
be executed, where the response to a whole series of odors would be noted and a
generalization pattern could be drawn. A significant difference between the
generalization patterns of the two groups would indicate a different perception of the
CS in the classical conditioning group, compared to the trace conditioning group.
Finally the research on trace memory is of particular interest with respect to
learning issues, perception, sense of time, awareness and higher cognitive task solving.

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Prof.G.C.Galizia
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8 ACKNOWLEGMENTS

I am grateful to Prof. Dr. Giovanni Galizia and Dr. Paul Szyszka for their scientific
advice. Special thanks go to my fellow bachelors Jenny Plath and Benjamin Birnbach.

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