Olfactory learning by means of trophallaxis in Apis mellifera
1 Departamento de Fisiología, Biología Molecular y Celular,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, Pabellón II, C1428EHA, Buenos Aires,
Argentina
2 Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie,
Institut für Biologie-Neurobiologie, Berlin D-14195, Germany
* Author for correspondence (e-mail: rjdm02{at}yahoo.com.ar)
Accepted 27 October 2004
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Summary |
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Key words: honeybee, Apis mellifera, trophallaxis, olfactory conditioning, associative learning
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Introduction |
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In 1923, Karl von Frisch (von Frisch,
1923) demonstrated that floral scents clinging to the body of an
experimental bee (returning to the nest from a natural nectar source) can
stimulate other individuals of the same colony to leave the nest and visit the
prospective source. Yet, within the broad spectrum of the floral scents nectar
is also perfumed with specific fragrances. In view of that, von Frisch
(1946
) extended his analyses
by considering nectar odours as possibly also being olfactory cues that might
enhance recruitment. Specifically, he took into account that each forager
carries a scented crop of nectar in its honey stomach each time it enters the
nest after a successful bout. Thus, since nectar is distributed rapidly among
colony members by means of trophallaxis, i.e. the exchange of liquid food by
mouth (Doolittle, 1907
;
Rösch, 1925
;
Nixon and Ribbands, 1952
;
Free, 1956
), its distribution
might enhance recruitment on the basis of olfactory learning occurring during
trophallaxis. In other words, forager-mates receiving nectar samples inside
the nest might learn (and later recognize) the odour of the nectar being
collected and search for the prospective nectar source by using its particular
olfactory cues in their subsequent flights.
Thus, von Frisch (1946)
presented marked bees with sugar solutions scented with the particular
fragrance of a given flower species `A'. In doing this, however, he used an
artificial feeder designed in such a way that bees contacted the offered
solution only with their tongues (i.e. animals gathered the offered reward
without exposing their bodies to the scented solution). In addition, this
feeder was externally enwreathed with scented petals of a second flower
species `B'. As a result, the marked foragers returning to the nest carried
sugar solutions that contained the odour `A' (the nectar scent belonging to
the first flower species) and, at the same time, their bodies carried
externally odour `B' (the floral scent belonging to the second flower
species). Afterwards, he analysed the effects of both odours (A and B) on
recruitment by placing two different outdoor flower dishes (each presenting
flowers of the first or the second flower species) and then counting the
number of recruited bees arriving at each one of the dishes. He found that
recruited bees searched preferentially for the flower species whose odour (A)
was diluted in the offered solution, especially when the training feeder was
placed at a distance several hundred meters from the nest. Following a series
of complementary experiments, von Frisch concluded that, in addition to the
floral odours attached externally to the forager bodies, specific olfactory
information (about the flower species being exploited) is transferred to
nest-mates on the basis of nectar scents. Subsequent experiments also
demonstrated that foragers usually receive nectar samples before being
recruited (Dirscheld, 1960
) and
that learned scents blown artificially inside the nest can trigger visual
memories of specific locations that trained bees have previously visited
(Reinhard et al., 2004
).
Indeed, trophallaxis, the behaviour through which returning foragers
transfer their nectar crops to other members of the colony
(Doolittle, 1907;
Rösch, 1925
;
Nixon and Ribbands, 1952
;
Wilson, 1971
), might allow
nest-mates to learn the specific olfactory cues of the nectar they receive.
During a single trophallactic interaction, a food-donor opens its mandibles
broadly, keeping its antennae downward and close to the head, while a variable
number of recipient nest-mates start contacting its prementum with their
protruded proboscis to sip the nectar it proffers, also moving their antennae
towards the donor (Free, 1957
,
1959
). Thus, the recipients
receive both olfactory and gustatory stimulation. However, although early
reports indicated that trophallaxis may allow honeybees to assign nectar
odours with predictive values (Butler,
1951
; Ribbands,
1955
; von Frisch,
1965
), the prospective olfactory learning involved in trophallaxis
has been never addressed directly. As a consequence, the idea that foragers
transfer nectar-related olfactory information by delivering scented nectar
inside the nest relies basically on indirect evidence
(Ribbands, 1955
;
von Frisch, 1965
). That is,
the role of trophallaxis in learning nectar-related olfactory cues has not
been analysed by measuring the trophallactic behaviour of the foragers and its
possible correlation with well-quantifiable learning performances. Instead, it
has been inferred from the ensuing choice behaviour of the animals
(von Frisch, 1946
). Obviously,
the latter perspective relies on the fact that nectar is distributed by means
of trophallaxis within honeybee colonies. However, it is a complex set of
visual and chemical stimuli which, alongside innate strategies improving the
gathering of energy as well as specific memories, determines the choice
behaviour of free-flying honeybees (von
Frisch, 1965
). Hence, to address directly the possible olfactory
learning involved in trophallaxis and its function in the context of the
foraging task requires, initially, a detailed quantification of both
trophallaxis and learning under controlled experimental situations that
resemble natural conditions as closely as possible. Such an analysis does not
yet exist. Furthermore, the effects of both the odour and sugar concentration
present in the transferred nectar on the possible olfactory learning occurring
during trophallaxis are entirely unknown. In addition, if honeybees acquire
specific olfactory memories by means of trophallaxis, it would be extremely
important to identify whether these are short- or long-term memories. This
distinction might have important implications on the foraging strategies
arising both at the individual and the group-level.
The first aim of the present study was therefore to examine whether a
single trophallactic interaction might serve a forager to associate the odour
(as the conditioned stimulus or CS) and the sugar (as the unconditioned
stimulus or US) present in the sucrose solution it receives through
trophallaxis. During the experiments, pairs of animals were first isolated to
induce trophallaxis under controlled conditions. Afterwards, classical
olfactory conditioning of the honeybee's proboscis extension response (PER
conditioning), a well-developed method used extensively to analyse different
aspects of appetitive learning and memory formation
(Kuwabara, 1957;
Takeda, 1961
;
Bitterman et al., 1983
;
Menzel, 2001
), was used to
investigate the possible role of trophallaxis in learning olfactory cues. We
argued that if a honeybee perceives an odour stimulus diluted in the sucrose
reward it receives during trophallaxis (immediately before sucrose or even
simultaneously), it must form an association between the two stimuli such that
the odour may trigger the animal's proboscis extension in subsequent tests (as
the conditioned response or CR). In addition, we addressed three further
important questions: (i) the effect of the odour concentration (CS intensity),
(ii) the effect of the sugar concentration (US intensity) and (iii) the time
course of the olfactory learning by means of trophallaxis. Finally, the
relevance of learning through trophallaxis in the task of successful foraging
is discussed.
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Materials and methods |
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Experimental subjects
Foragers were labelled and trained to collect unscented sucrose solution at
an ad libitum feeder placed 10 m away from the hive. At the beginning
of each trial, two arriving foragers were captured at the feeder (without
allowing them to make contact with the offered solution) and carried to the
laboratory by using individual plastic tubes. Afterwards, one of the foragers
(the donor bee) was fed with sucrose solution (up to satiation) while the
other one (the recipient bee) remained unfed inside the plastic tube. Each
donor was fed through the end of the plastic tube, which was covered by a soft
mesh with slots whose diameters (1 mm) were larger than the cross section
of the honeybee proboscis. Thus, donors placed individually inside the tubes
were easily able to extend their proboscis through the mesh in order to get
the sugar solution offered (from outside) via a graded capillary
tube. In this way, they contacted the offered solutions only with their
proboscis. During single trials, the time elapsed between both animals being
captured and the donor being fed was approximately 5 min. The sugar solutions
offered to donors presented different odours and sucrose concentrations
according to the experimental series described below.
Recording trophallaxis during a single experimental session
Once the donor had been fed, both donor and recipient were placed inside a
transparent experimental arena to induce trophallaxis (for a detailed
description of the arena, see Farina and
Núñez, 1991). Once in the arena, animals were
initially separated by a sliding door placed in the centre of the arena. Each
session started when the door was removed (allowing the animals to get in
contact with each other) and finished when a single trophallaxis had occurred
or when no trophallaxis occurred after the first 10 min following the
beginning of the session. Following trophallaxis, the recipient only was
further used, testing its proboscis response as described below. If no
trophallaxis occurred the animals were removed and the procedure was repeated
with a different pair of animals. The duration of trophallaxis was recorded.
We obtained 10-20 animals per day that received sucrose solution during a
single trophallactic event.
Harnessing
Once a single trophallaxis occurred between donor and recipient, the
recipient was induced to enter a small plastic harness (a cone-shaped tube 4
cm long with an open end 4 mm in diameter) from which its head remained
protruded, by exploiting the natural positive phototaxis of honeybees. A light
bulb was placed 30 cm above the arena and the plastic roof of the arena was
covered with a sheet of paper while the harness remained illuminated.
Following trophallaxis (when both donor and recipient remained separated by
the sliding door), the light was turned on and the recipient rapidly moved
from the inner arena into the illuminated harness. Afterwards, it was fixed in
the harness by two pieces of thin tape, one placed on the top between the head
and the thorax and the other horizontally behind the thorax. Thus, the animal
could freely move its antennae, mandibles and proboscis. Once fixed in the
harnesses, recipients were placed in racks within a dark humidified chamber.
In the evening following trophallaxis, they were fed up to satiation
(unscented 1.8 mol l-1 sucrose solution) and kept inside the
chamber until tested.
Testing the proboscis extension response of the recipient bees
By testing the subsequent proboscis extension of the recipients we could
determine whether an association had been established between the odour (CS)
and sucrose (US) present in the sugar solution received through trophallaxis.
Each recipient was tested 21, 27 and 46 h following trophallaxis (first,
second and third test, respectively). During each of the three different
tests, animals were presented with two different odours. Hence we always
tested their responses to both linalool, the odour added (or not) to the
solution they had received during trophallaxis (see the experimental series
described below), and eugenol, a second odour to which the animals were never
exposed.
Half of the bees were presented with the sequence linalool-eugenol and the remaining half with the sequence eugenol-linalool. Odours were presented via an air stream delivered through a 20 ml plastic syringe that contained a piece of filter paper soaked with 4 µl of pure odorant (the odour source). A fan placed behind the animal extracted the odours released in the test room. Each of these trials lasted approximately 40 s. Removing bees from the racks to the test site was followed by 20 s accommodation period, after which the respective 5 s stimulation started. After stimulation bees remained at the test site for other 15 s and were then placed back in the racks.
Prior to the beginning of each of the three tests (30 min), animals were stimulated by applying sucrose solution (1.8 mol l-1) to their antennae to determine whether or not they responded to the US. Recipients that failed to respond were excluded from the analysis. These trials were performed outside the test room to avoid possible associations among unspecific features of the test room and the US. Spontaneous responses to the air stream were also tested prior to odour stimulation. Animals that responded positively to the air stream were excluded from the analysis as well. In between successive tests, bees were kept in the dark humidified chamber and only fed up to satiation (as described above) in the evening following the first two tests.
Measuring the learning performances
Throughout the experiments, each animal was considered to show a
conditioned response (CR) when it only responded to linalool. Animals that
responded to eugenol, i.e. a second control odour (see above) and not to
linalool, as well as those that responded to both odours (0 and 4.6%,
respectively, throughout all the experiments described here) were excluded
from the analysis.
Next, for each of the three different tests, we calculated the percentage of positive proboscis extensions (%PE1, %PE2 and %PE3, corresponding to the first, second and third tests, respectively) as the proportion of animals that showed a CR, as calculated from the total number of tested animals after excluding (1) animals that responded to the control odour, (2) animals that failed to respond to the US prior to the test and (3) animals that responded to the air stream prior to the test.
In addition, we calculated a general percentage of positive proboscis extensions (%PEG) as the proportion of animals that showed a CR in any of the three different tests, calculated from the total number of tested animals, after excluding (1) animals that responded to the control odour in any of the single tests, (2) animals that failed to respond to the US in all the single tests and (3) animals that responded to the air stream in all the single tests.
Experimental series
Three different experimental series were performed to vary both the odour
concentration and sucrose concentration of the sugar solution that recipients
received during trophallaxis, and the sucrose concentration they had
experienced previously at the feeder (where animals were captured at the
beginning of the experiments). After donors had been fed (see above), the
experimental procedure was identical in all series.
Series 1: Olfactory conditioning by means of trophallaxis
In this experimental series we addressed whether olfactory conditioning
occurs during a single trophallactic interaction. First, foragers were allowed
to collect unscented 1.8 mol l-1 sucrose solution at the feeder.
Next, donors were fed with either scented (50 µl of linalool per litre of
sucrose solution) or unscented 1.8 mol l-1 sucrose solution.
Finally, following trophallaxis, the conditioned responses of the recipients
were tested as described above.
Series 2: Effect of CS intensity
In this experimental series we analysed the effects of CS intensity on the
subsequent CR. As in the previous series, foragers collected unscented 1.8 mol
l-1 sucrose solution at the feeder. Next, six different
experimental groups were defined based on the odour concentration of the sugar
solution (1.8 mol l-1 sucrose solution) used to feed the donors,
i.e. 0 (unscented), 0.1, 1, 5, 50 and 100 µl linalool l-1
solution. Donors were offered the different solutions quasi-randomly over
successive experimental days. As before, the conditioned responses were tested
following trophallaxis.
Series 3: Effect of US intensity
We also analysed the effects of US intensity on the subsequent CRs. In this
experimental series, foragers collected unscented 0.5 mol l-1
sucrose solution at the feeder. Next, donors were fed with scented solutions
that presented sucrose concentrations of either 0.5 mol l-1 or 1.8
mol l-1, employing an odour concentration of 5 µl linalool
l-1 solution, which had elicited intermediate response levels in
the second series (see Results). In addition, a third group of donors (i.e.
control group) was fed with unscented 0.5 mol l-1 sucrose solution.
The different solutions were used quasi-randomly. As before, the conditioned
responses were tested following trophallaxis.
Statistical analysis
The percentages of positive proboscis extensions were compared using
G-tests (comparisons among groups) and McNemar tests (comparisons
among different tests). Data on the duration of trophallaxis were analysed
using t-tests, analysis of variance (ANOVA) and Tukey-Kramer
comparisons (Zar, 1984).
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Results |
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Series 2: Effect of CS intensity
Next we extended the analysis in order to evaluate the effects of CS
intensity on the subsequent CRs. We thus compared responses from six different
groups of animals defined on the basis of the odour concentration present in
the sugar solution they received during trophallaxis (ranging from 0, i.e.
unscented, up to 100 µl linalool l-1 solution, see Materials and
methods). As shown in Fig. 2A
(see the insert, hatched bars), the higher the odour concentration the higher
the general response level (% PEG: G(5)=91.8,
P<0.0001, N=211; data from 0 µl l-1,
unscented, and 50 µl l-1 correspond to the first experimental
series). Hence, an odour concentration of 0.1 µl l-1 gave
response levels that did not differ statistically from the spontaneous
responses elicited by the animals that received unscented solutions. Odour
concentrations of 1 and 5 µl l-1 gave increasing intermediate
values and, finally, the highest odour concentrations assayed (50 and 100
µl l-1) gave maximum response levels.
|
Fig. 2B shows the effects of
CS intensity on the more specific response levels obtained for each of the
three different tests (%PE1: G(5)=43.7,
P<0.0001, N=193; %PE2:
G(5)=71.6, P<0.0001, N=141;
%PE3: G(5)=41.6, P<0.0001,
N=136; G-test). Interestingly, for the highest odour
concentrations (50 and 100 µl l-1), responses increased
significantly between the first and the second tests (performed 21 and 27 h
following trophallaxis, respectively). Since animals were tested in a
cumulative fashion (see Materials and methods), we expected that the first
extinction test would lead to a similar or even a reduced response in the
second test, but instead we found an increase (see
Fig. 2B, white and grey bars,
50 µl l-1 group: 2=6.13, P=0.01, 100
µl l-1 group:
2=5.14, P=0.02; McNemar
test). In the third test, the responses did not differ significantly from the
first or the second test for both 50 and 100 µl l-1
concentrations (see Fig. 2B,
black bars, 50 µl l-1: %PE1 vs
%PE3,
2=1.5, P=0.2, %PE2
vs %PE3,
2=0.12, P=0.9; 100 µl
l-1: %PE1 vs %PE3,
2=3.13, P=0.8, %PE2 vs
%PE3,
2=0.25, P=0.6; McNemar test).
Series 3: Effect of US intensity
To analyse the effects of US intensity we compared responses from two
different groups of animals that received different concentrations of scented
sucrose solution during trophallaxis (either 0.5 mol l-1 or 1.8 mol
l-1). A control group of animals (which received unscented 0.5 mol
l-1 sucrose solution) was included in the analysis (see Materials
and methods). Results showed that the general response level increases
together with the sucrose concentration
(Fig. 3A, %PEG:
G(1)=8.7, P=0.003, N=64;
G-test). No responses were found for the control group throughout the
different tests (Fig. 3A,B).
The performances of animals that received scented 0.5 mol l-1
sucrose solution did not differ statistically from the performances of the
control group (%PEG: G(1)=1.2, P=0.3,
N=64; G-test) but those that received scented 1.8 mol
l-1 sucrose solution gave higher responses in comparison to the
control group (% PEG: G(1)=12.4,
P=0.0004, N=58; G-test).
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With respect to the responses obtained in the different tests (Fig. 3B), animals did not respond to CS presentation in the first test. In the second test (%PE2), response levels were 4.8% and 29.4% for animals that received 0.5 mol l-1 and 1.8 mol l-1 scented sucrose solutions, respectively (G(1)=4.5, P=0.03, N=38; G-test). In addition, in the third test (%PE3) results gave values of 0% and 14.8% for animals that received 0.5 mol l-1 and 1.8 mol l-1 scented solutions, respectively (G(1)=6.6, P=0.01, N=59; G-test). The performances of the animals that received either unscented (i.e. the control group) or scented 0.5 mol l-1 sucrose solution did not differ when the second (%PE2) and the third (%PE3) tests are compared (%PE2: G(1)=0.9, P=0.3, N=33; G-test, %PE3: animals did not respond in either one condition or the other). By contrast, the performances of the animals that received scented 1.8 mol l-1 sucrose solution were significantly higher than those of the control group in the case of the same two tests (%PE2: G(1)=6.1, P=0.01, N=29; %PE3: G(1)=5.7, P=0.02, N=53; G-test). In this series, data did not allow within-test comparisons, due to the high proportion of animals that did not show conditioned responses.
Effects of the recent previous foraging experience
Next, we evaluated possible effects of previous recent foraging
experiences, i.e. the sucrose concentration offered at the training feeder, on
the subsequent learning performances of the recipients (data correspond to the
second and the third experimental series). We thus compared responses from two
different groups of animals that collected either 0.5 mol l-1 or
1.8 mol l-1 unscented sucrose solutions (see Materials and methods)
prior to their trophallactic interactions. Once in the arena, all the animals
received scented (5 µl linalool l-1 solution) 1.8 mol
l-1 sucrose solution by means of trophallaxis. As
Fig. 4 shows, animals that
collected 0.5 mol l-1 sucrose solution prior to trophallaxis did
not respond to CS presentation during the first test, whereas the
%PE1 was significantly higher (30.4%) for animals that collected
1.8 mol l-1 sucrose solution
(Fig. 4, white bars,
%PE1, G(1)=10.9, P<0.001,
N=46; G-test). The responses of both groups did not differ
statistically in the second and the third tests, although a tendency was
found, suggesting higher response levels for animals that had collected 1.8
mol l-1 sucrose solution at the feeder
(Fig. 4, grey bars,
%PE2=29.4% (0.5 mol l-1) vs 31.2% (1.8 mol
l-1), G(1)=0.15, P=0.7, N=33;
black bars, %PE3=14.8% (0.5 mol l-1) vs 30%
(1.8 mol l-1), G(1)=1.57, P=0.2,
N=47). Likewise, the general response level (%PEG, not
illustrated) did not differ statically between both groups
(G(1)=1.59, P=0.2, N=54;
G-test), although a tendency also suggested higher response levels
for the animals that collected 1.8 mol l-1 sucrose solution prior
to trophallaxis (27.6% and 44.0% for 0.5 and 1.8 mol l-1,
respectively).
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The duration of trophallaxis
The duration of trophallaxis ranged from 1.2 s up to 23.6 s throughout the
totality of the experiments (11.7±0.6 s, mean ±
M.E.M.). In the second experimental series,
analysing the effects of CS intensity, no differences were found among groups
(F(5,223)=0.88, P=0.5, one-way ANOVA). By
contrast, in the third experimental series, addressing the effects of US
intensity, differences appeared among groups
(F(5,241)=10.1, P<0.001, one-way ANOVA after
log transformation). The higher the sucrose concentration the lower the
duration of trophallaxis when animals exchange scented sugar solutions
(P<0.001, Tukey-Kramer comparison). No differences appeared
between these groups and the control group (unscented 0.5 mol l-1
vs scented 0.5 mol l-1: P=0.1, unscented 0.5 mol
l-1 vs scented 1.8 mol l-1: P=0.06,
Tukey-Kramer comparisons).
We also tested for a possible correlation between the duration of trophallaxis and the subsequent learning performances of the recipients, using a post-hoc analysis. To this end, we considered the trophallactic interactions of the animals that showed subsequent conditioned responses. Thus, for all the series assayed, two different groups of animals were defined according to the responses they showed in the various tests: (1) bees that showed conditioned responses and (2) bees that did not. Afterwards, the durations of their respective trophallactic interactions were compared. In the second experimental series, no differences were found among groups (interaction term: F(5,216)=0.56, P=0.7; PE response factor: F(1,217)=0.53, P=0.5, odour concentration factor: F(5,217)=0.79, P=0.6, two-way ANOVA). After pooling data from the totality of the odour concentrations assayed in this series, durations (mean ± M.E.M.) were 11.8±0.7 s and 12.2±0.5 s for animals that responded and animals that did not respond during the various tests, respectively. In the third series, only the situation in which animals received scented 1.8 mol l-1 sucrose solution was analysed (sample sizes did not allow comparisons for the remaining treatments). As before, no differences were found in the mean duration of trophallaxis (t(1,27)=1.43, P=0.2, t-test). Values (mean ± M.E.M.) were 13.4±1.2 and 10.4±1.3 for animals that responded and those that did not respond, respectively.
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Discussion |
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Associative learning is usually characterized as either classical
(Pavlovian) conditioning (stimulus-stimulus and stimulus-response
associations) or operant (instrumental) conditioning (response-contingent
reinforcing; Pavlov, 1927;
Colwill and Rescorla, 1986
). In
classical conditioning, conditioned stimuli (CS) become predictive for
unconditioned stimuli (US). After conditioning (forward-pairing of CS and US)
CS elicit conditioned responses (CR). The CR can be considered as anticipatory
responses, as a training of behavioural habits, or as conditioned motivations
and emotions that are appropriate to the unconditioned reward stimulus
(Pavlov, 1927
;
Colwill and Rescorla, 1986
). In
operant conditioning, the animal's spontaneous responses are strengthened by
response-contingent reinforcement
(Skinner, 1938
;
Hebb, 1956
;
Rescorla, 1994
). During
trophallaxis, honeybees display active behaviour. Hence, one might ask whether
or not olfactory learning through trophallaxis constitutes operant
conditioning; however, trophallaxis represents an instinctive behaviour that
neither occurs by chance nor needs be learned. Hence, the idea of olfactory
learning through trophallaxis representing a classical associative
conditioning is undoubtedly more likely.
Under our experimental conditions, both stimuli (CS and US) are perceived
during trophallaxis. In addition, the odour (CS) is diluted in the sucrose
solution (US) that animals receive. Thus, although the temporal relationship
between CS and US is rather fixed it may vary with the performance of both
animals during trophallaxis. Yet the CS (odour) will always either precede the
US or occur at the same time as the US. In classical PER conditioning the
highest learning rates are observed when the CS is presented a few seconds
before the US (forward conditioning), but simultaneous paring of both stimuli
is also effective (Menzel,
1969,
1990
). In addition,
conditioned responses frequently develop with repetition of conditioning
trials, although single trial learning is known from a few examples. In the
classical PER conditioning, a single pairing of CS and US raises the animals'
responses from a very low spontaneous level (<10%) up to mostly >50%
(Bitterman et al., 1983
). In
trophallaxis, a single learning trial raised the responses from 3%
(spontaneous responses) up to 70%, indicating a fast and robust form of
learning. All our experiments were carried out with forager bees. By using
classical PER conditioning, however, Ray and Ferneyhough
(1999
) reported differences in
learning performances when foragers and younger bees are compared. Since
trophallaxis also occurs between young bees (nurse and guard bees), it will be
interesting to study learning through trophallaxis in younger workers.
Effect of CS intensity
We assume that the amount of odour diluted in the sucrose solution that
animals receive during trophallaxis is directly related to CS intensity. We
found that olfactory learning through trophallaxis improved with higher CS
intensities (Fig. 2). In the
classical PER conditioning, odour concentration also affects conditioned
responses (Pelz et al., 1997),
although odour application differed in our situation from that applied
normally in PER conditioning experiments. Pelz et al.
(1997
), for instance, used 10
µl of an odour per litre of solvent as the lowest initial dilution. In our
experiments, the minimal odour concentration that allowed learning
unambiguously was 5 µl of odour per litre of sugar solution
(Fig. 2). In the classical PER
experiments, however, the odours presented during conditioning were always
dissolved in air, but in our experiments they were mixed with water (i.e.
dissolved in the solution transmitted during trophallaxis). It is unknown how
these different procedures affect the final concentrations that reach the
chemoreceptors at the antenna, and thus a quantitative comparison is not
possible.
The effects of CS intensity on learning through trophallaxis might be
explained from two different perspectives. On the one hand, a low
concentration of diluted odour might not be detectable. As a result, the
corresponding pairing of CS-US cannot be achieved. On the other hand, although
perceived, a highly diluted odour might lead to an insufficient olfactory
stimulation, i.e. it might lie below a certain threshold value that must be
exceeded to assign the odour with a predictive value according to associative
learning processing. Based on several earlier results, the latter possibility
is more likely. First, it has been shown that honeybees can perceive scents
even when greatly diluted and, accordingly, their ensuing threshold of odour
perception is comparable to that of a man (von Frisch,
1919,
1965
;
Ribbands, 1954
). In this
study, the lowest odour concentration (0.1 µl l-1) was weakly
perceived by the researcher. Thus, it is reasonable to assume that the animals
were able to perceive all the odour concentrations used during the second
experimental series. Secondly, recent theories on associative learning have
introduced the concept of `salience', i.e. an experience-independent feature
of a conditioned stimulus that determines the rate at which it can enter into
associations with a given reward (Rescorla
and Wagner, 1972
; Sutton and
Barto, 1990
). Thus, learning depends on stimulus salience (for
alternative views, see Durlach,
1989
; Spear et al.,
1990
). Higher concentrations of an odour may lead to higher
salience and thus better learning. Intensity and salience effects on learning
can only be separated if the perceptual intensity is controlled as, for
example, in the study by Pelz et al.
(1997
), and if odours of equal
subjective strength are compared.
Memory over time
Animals were tested at three different times following trophallaxis: 21, 27
and 46 h. According to the well-studied temporal dynamics of memory formation
after PER conditioning (Menzel,
1999), animals were tested during early and later long-term
memory. Under our conditions, a single trophallactic interaction leads to high
levels of memory in both memory phases. Thus, a single trial of trophallaxis
induces long-term olfactory memories. This is different from PER conditioning.
In PER conditioning the memory after trial has usually already begun to decay
several hours after acquisition. Multiple conditioning trials, however, induce
a stable, long-lasting memory (Menzel,
1999
). It thus appears that the transition to long-term memory may
not always require multiple learning trials, as believed so far.
Interestingly, our data also show that responses increased over time for the
highest CS intensities but not for lower CS intensities. Improvement of
retention over time is usually interpreted as indicating a consolidation
process. Accordingly, it might be concluded that memory consolidation may be
stronger for high than for low CS intensities. In addition, although not
statistically significant, a tendency was found indicating lower response
levels in the latter test (only for the highest CS intensities). Further
experiments employing single tests distributed over time are required to
evaluate whether extinction underlies this tendency.
Effect of US intensity
Stronger US intensities usually lead to better learning
(Rescorla and Wagner, 1972).
The experiments of series 3 constitute the first attempt to investigate the
role of sucrose concentration (US intensity) in a task involving learning
through trophallaxis. We found that higher US intensity enhances conditioned
responses after a single conditioning trial. In addition, in evaluating the
effects of US intensity, we always employed an odour concentration (5 µl
l-1) that elicits intermediate response levels, and it will be
interesting to consider different combinations of both US and CS intensities
for further research.
In the honeybee, previous work also analysed the effects of US intensity
(sucrose solution) on appetitive learning. By means of classical PER
conditioning, for instance, Bitterman et al.
(1983) found similar learning
performances when sucrose solutions of 7, 20 and 40% w/w (i.e. mass of
sucrose/mass of solution) were used as US intensities, although the lowest US
intensity reduced the rate of acquisition. In addition, no differences in
retention were found when 0.5 mol l-1 and 2.5 mol l-1
sucrose solutions were used as US in classical PER conditioning
(Menzel et al., 2001
). The
effects of US intensity on associative learning were also tested in the case
of tactile learning and, interestingly, foragers differed with respect to
their responsiveness to different concentrations of sucrose. Hence, in
honeybees, the value of a sucrose concentration as the US has a relative
quality (Scheiner et al.,
1999
; Scheiner,
2004
).
Effect of the recent previous foraging experience
Interestingly, under our experimental conditions, learning was also
affected by the recent previous foraging experiences of the animals, i.e. the
sucrose concentration they experienced at the training feeder prior to
trophallaxis (see Materials and methods). The higher the sucrose concentration
they experienced previously the higher the percentage of subsequent
conditioned responses, especially during the first test
(Fig. 4). It is well known that
sucrose modulates ongoing activities in honeybees. That is, it affects the
motivational level of the animals and may enhance the probability or even the
strength of several responses to other stimuli, leading to a status of
`arousal', i.e. a short-lived behavioural state that may accelerate the
gathering of information required for the formation of specific associative
memories (Hammer and Menzel,
1995). Thus, a stronger sucrose stimulation at the feeder may
arouse the animal and lead to better learning because of higher sensitivity to
the stimuli that will be perceived shortly during trophallaxis.
The duration of trophallaxis
The duration of the trophallactic interactions that led to learning ranged
from 1.2 s to 23.6 s. Thus, olfactory learning occurs even when trophallaxis
is very short. Experiments with free-flying honeybees raised the question
whether the strength of association increases with the duration of sucrose
stimulation (Buchanan and Bitterman,
1988; Couvillon et al.,
1991
; Menzel and Erber,
1972
). Only small effects of US duration were found. In PER
conditioning, the duration of sucrose stimulation (even beyond 1 s) appeared
not to affect the strength of the association, but more rigorous experiments
are necessary to be certain (Hoban et al.,
1996
). Our results similarly provide no indication that the
duration of trophallaxis might influence the strength of the response. These
findings are in agreement with the notion that CS/US pairing is the major
determinant of associative learning, independent of the duration of either
stimulus. Additional support comes from the finding that different stimuli
perceived throughout feeding are not associated with reward, but only those
experiences at the onset of reward (Opfinger,
1931
,
1949
;
Menzel, 1968
).
Olfactory learning through trophallaxis and foraging behaviour
We showed that associative learning of an odour occurs during a behavioural
performance, trophallaxis, which is very common in social insects. This kind
of learning leads to long-term olfactory memories. According to these
findings, employed foragers may train nest-mates by means of trophallaxis and
will therefore influence the subsequent search behaviour of bees flying out to
forage. Moreover, we found that olfactory learning through trophallaxis occurs
after a single conditioning trial, even when trophallaxis is brief. Previous
results indicate that foragers increase the number of their offering contacts
(i.e. the brief interactions in which they act as food donors during
trophallaxis) after experiencing an increase in reward
(De Marco and Farina, 2001).
Together with the present results, this means that highly rewarding nectar
sources may exhibit a high probability that their chemosensory cues will be
learned through trophallaxis by potential newly recruited foragers.
Furthermore, within the colony, nectar foragers perform offering contacts as
well as brief begging contacts (acting as food-receivers during trophallaxis;
von Frisch, 1965
). Recently,
De Marco and Farina (2003
)
showed that an increased resource uncertainty enhances the foragers' begging
behaviour. If an increased resource uncertainty enhances proboscis extensions
(as potential learning trials) and long-term olfactory memories can be formed
(or even retrieved) by means of trophallaxis, it will be then interesting to
study how the nectar-related chemosensory information transmitted during
trophallaxis (at any time within the colony) might affect the initial choice
behaviour of newly recruited foragers and the ongoing foraging process of
employed foragers. In addition, since trophallaxis also occurs between a
dancing bee and its followers, it is likely (but has not yet been proven) that
recruited bees seek the odour learned by imbibing samples from the dancer
(Lindauer, 1961
;
von Frisch, 1965
).
According to the present results, the strength of the associative learning
involved in trophallaxis increases together with both CS and US intensity as
well as the sucrose stimulation experienced previously by the animals.
Nectar-bearing flowers offer a variety of nectar odours as well as sugar
concentrations under natural environmental conditions. Since the combination
of the olfactory and gustatory stimuli provided by a given nectar source
constitute a primary source of guiding cues, olfactory learning through
trophallaxis may be crucial as long as nectar foragers use odours and sucrose
concentrations to optimise their foraging choices
(von Frisch, 1965;
Gould, 1993
). Our results
predict that high levels of nectar-scent concentrations as well as sugar
rewards will both enhance the number of aroused forager-mates and guide them
to the productive sources.
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Acknowledgments |
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References |
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