Prior classical olfactory conditioning improves odour-cued flight orientation of honey bees in a wind tunnel
1 Laboratoire de Physiologie Cérébrale, CNRS UMR 8118, 6 Rue
des Saint-Pères, 75006 Paris, France
2 Laboratoire Fonctionnement et Evolution des Systèmes Ecologiques,
UMR 7625, Université Pierre et Marie Curie, Box 237, 7 Quai Saint
Bernard, 75252 Paris Cedex 05, France
3 Direction des Relations Internationales, CNRS, 3 Rue Michel-Ange, 75794
Paris, France
* Author for correspondence (e-mail: antoine.chaffiol{at}univ-paris5.fr)
Accepted 17 July 2005
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Summary |
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Key words: honey bee, orientation in flight, olfactory cue, wind tunnel, olfactory conditioning, information transfer
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Introduction |
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Honey bees experience a wide range of olfactory information throughout
their life. This information can be gained inside the hive, for example from
the stored pollen and nectar or during dances, as recruited bees make antennal
contact with the dancing bee that is impregnated with floral scents. It can
also be acquired outside the hive during the foraging bouts. The ability to
use olfactory information acquired in various contexts during subsequent
orientation tasks might contribute to the enhancement of orientation
performance. Reporting that flower volatiles, carried by returning foragers
from the sites they have recently visited, could be used by the recruits to
search for food, von Frisch
(1967) and Wenner et al.
(1969
) were the first to
demonstrate that olfactory information acquired in the hive can be used in an
orientation context. Since then, some studies have shown that bees are able to
transfer information from a foraging situation to an associative context
(Gerber et al., 1996
) and from
an associative context to a walking orientation response in an olfactometer
(Backchine et al., 1992
;
Sandoz et al., 2000
).
Recently, Reinhard et al.
(2004
) showed that a scent
blown into the hive can trigger trajectory memories of a site the bees had
previously visited. This impressive finding of an associative recall also
confirms that orientation of the bees can take into account various memorised
information.
In the present work, we investigated the role of odours in the orientation
behaviour during short-range flights, and we tested the effect of a prior
olfactory experience on subsequent flight orientation. One goal in
investigating such types of orientation behaviour is to measure not only the
final outcome but also how an individual actually succeeds in the task.
Indeed, quantifying the number of visits to a site gives little information on
the difficulty the bees had in finding it, since it does not document the
method they used to achieve the task, and it might underestimate some types of
exploratory strategy based on trial-and-error repetitions. Wind tunnel devices
enable the investigator to detail the individual approach flight and to
control for the various cues that could be used by the insects under natural
conditions. This type of device has been extensively used to study olfactory
orientation in flying insects (e.g. Baker
et al., 1984; Fadamiro et al.,
1998
; Jang et al.,
2000
). It was shown to be particularly suitable for analyzing the
orientation mechanisms involved in sexual attraction, in the search for plants
by phytophagous insects or in host research by parasitoids. A wind tunnel was
previously used to study the energetics of flight on suspended honey bees
(Hanauer-Thieser and Nachtigall,
1995
) but it has never been exploited for the study of flight
orientation in this species, although some types of flight room refer to a
similar approach (Poppy and Williams,
1999
; Laloi et al.,
2000
). Consequently, we adapted a wind tunnel originally designed
for the observation of moths and parasitoids for use with honey bees. We
established reliable flight descriptors prior to using this device to study
how worker bees can orientate in flight towards two different volatiles: an
odour of nestmates, which can be part of the olfactory cues driving the
returning flight of a bee to the hive, and linalool, a common floral odour. We
then investigated whether olfactory cues acquired through classical olfactory
conditioning of the proboscis extension reflex
(Bitterman et al., 1983
) could
influence a bee's orientation within a 2-m range.
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Materials and methods |
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Wind tunnel
The wind tunnel (Fig. 1)
constituted a transparent vault (Plexiglas), 62 cm central height, on a
200x72 cm floor. An input fan created an airflow by driving the air of
the experimental room into the flight chamber, and an extraction fan drove the
air outdoors. A fine mesh net was positioned at the entrance of the flow to
obtain a laminar airflow in the flight chamber (as checked using white
NH4Cl vapour). The following conditions were set up to ensure
satisfactory honey bee flight: the input and output flow speed was adjusted to
0.3 m s-1; the temperature was set to 23±1°C, which was
shown to induce good honey bee flight activity
(Hanauer-Thieser and Nachtigall,
1995); the light was adapted to the high flicker fusion of 150 Hz
(van Praagh, 1972
) by using
four fluorescent neon lights (36 W, 120 cm length each) set on high-frequency
connectors (Mazda ref 136 HFR). The lights were arranged in pairs, 38 cm above
the top of the flight chamber vault, the two pairs being separated by a
distance of 60 cm. To reduce phototropism, indirect light was obtained by
setting a piece of opaque cardboard under the neon lights so that most of the
light was reflected onto the walls of the experimental room. Under these
conditions, the luminosity was of 6.6 µmol photons m-2
s-1 (400 lux) within the whole flight chamber. In our experimental
conditions, the UV light component was highly reduced. This may affect the
flight structure although little is known about the role of UV in short-range
flight. Nevertheless, since our experiments were always based on comparisons
between a test group and a control group, both exposed to the same tunnel
conditions, a putative change in the flight structure would have been balanced
between groups and would not affect our conclusions. Our tunnel could be
improved by using UV-transmitting Plexiglas instead of normal Plexiglas and by
using lights with a UV component. Preliminary experiments, intended to improve
displacement in flight in the tunnel, showed that visual marks were useful to
facilitate the orientation of the bees, since bees need image motion to
estimate distances (Kirchner and
Srinivasan, 1989
). Thus, vertical bands (black paper, 2 cmwidth
x 40 cm length) were added to the sides of the Plexiglas vault. Trap
doors on the tunnel floor allowed manipulations inside the flight chamber.
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Volatiles were delivered to the flight chamber through a glass tube (diameter 0.5 cm) emerging 18 cm above the tunnel floor. To obtain a regular flow of volatiles, the odour was delivered through a constant outflow using a pump (1085 ml min-1). The output of the odour source was positioned in the middle of a metallic cardboard disk (5 cm diameter) that could be used as a visual cue and a possible landing area.
General procedure for flight activity recording
Each bee was placed in a small individual cage (3x4x5 cm) for
at least 1 min before introduction into the flight chamber. The cage was
positioned on a platform set 18 cm above the floor and 120 cm from the odour
source (Fig. 1). Once the trap
doors were locked, the cage could be opened from the outside. After the air
flow was switched on, we let the honey bee get familiarized with the tunnel
conditions for at least 15 s. The experiment started when the bee was released
from the cage. Behaviour was observed for 5 min. Bees that took more than 1
min to fly out of the cage were excluded from further analysis.
Two types of parameters were recorded during the experiments. First, we recorded the total flight duration of each bee and its flight duration around the source, i.e. in a volume of 20x25x72 cm centred at the odour source. This volume was chosen after preliminary experiments, since it corresponded to the distance where pre-landing behaviour was obvious. Appropriate marks on the Plexiglas vault allowed the visualization of this zone. Second, we recorded the proportion of bees performing orientated flights (upwind zigzag flight) and the proportion of bees circling (i.e. bees exhibiting circling flights or stationary flights around the source for more than 2 s).
Conditioning of the proboscis extension reflex
The experimental procedure for classical conditioning of the proboscis
extension was the standard procedure detailed elsewhere
(Bitterman et al., 1983;
Sandoz et al., 1995
). Each bee
was mounted in a glass holder and starved for four hours. Bees were then
individually subjected to three conditioning trials (C1, C2, C3) with 15-min
inter-trial intervals. Before each trial, bees were positioned for 15 s in the
airflow to familiarise them with the mechanical stimulation. Then the odour
stimulus (conditioned stimulus) was presented for 6 s and then, 3 s after the
onset of the odour, the antennae were touched with a 30% sucrose solution
(unconditioned stimulus). The subsequent proboscis extension was rewarded with
a drop of the same sucrose solution. Bees that showed spontaneous responses at
the first presentation of the odour were discarded from the following steps
since later responses of such individuals could not be interpreted as purely
associative.
Experiment 1: orientation toward nestmate odour and linalool
The first experiment was designed to study worker bees' orientation towards
two different volatiles, an odour of nestmates and a floral odour. The
nestmates' odour source consisted of a sealed box (11.2x17.5x13
cm) containing 50 bees originating from the same rearing cage as the
tested individual. To avoid visual and acoustic cues, this box was placed
outside the tunnel, and the stimulating air flow passed through the box before
being released in the flight chamber. Thus, only volatiles were delivered into
the tunnel. The floral odour source was linalool (Sigma, St Quentin Fallavier,
France; 9597% purity), a common floral compound
(Knudsen et al., 1993
). The
stimulating flow passed through a glass vial containing 200 µl of pure
linalool. Three groups of bees were thus subjected to the following
stimulations: bees exposed to nestmates' odour (N=25), bees exposed
to linalool (N=25) and bees exposed to no odour (control group;
N=25).
Experiment 2: effect of odour conditioning on the orientation task
A second experiment was designed to analyse the influence of prior
olfactory learning on the olfactory orientation of the bees in the wind
tunnel. Bees (N=25) were conditioned to linalool using the proboscis
extension paradigm. In parallel, control bees (N=25) were subjected
to the whole procedure (identical conditions of harnessing, starvation and
stimulation) but without odour delivery. At the end of the conditioning
session, bees were gently removed from the glass holders, with special care
given to their wings, and then caged individually with food for one hour. They
were then tested in the flight tunnel in the presence of linalool, following
the conditions described above.
Data recording and statistical analysis
During the observations, the different flight parameters were recorded
using The Observer software version 3.0 (Noldus Inc., Wageningen, The
Netherlands). After control for normality and homoscedasticity, total flight
durations were compared amongst groups using a one-way analysis of variance
(ANOVA). With respect to flight duration around the source, data did not meet
the assumptions of normality and homoscedasticity. Thus, this variable was
analysed using non-parametric tests: a MannWhitney test was used to
compare two groups (experiment 2: conditioned bees and control bees) and a
KruskalWallis test was used to compare three groups (experiment 1: bees
stimulated with linalool, bees stimulated with nestmate odour, and control
bees). In the cases in which the KruskalWallis test indicated a
significant difference among groups, non-parametric pairwise comparisons
(Conover, 1980) were applied
in order to identify the groups that differed at the experimentwise alpha
level of 0.05. With respect to the number of bees that performed orientated
flights and the number of bees that performed circlings, groups were compared
using a
2 test. When the proportion of individuals was found
to differ amongst more than two groups (experiment 1), pairwise comparisons
were conducted using
2 tests with 1 d.f., in order to identify
the groups that differed. To ensure that the experimentwise alpha level was
0.05, the alpha level of each pairwise test was adjusted downward according to
the DunnSidak correction (Sokal and
Rohlf, 1995
).
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Results |
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Experiment 2: effect of odour conditioning on the orientation task
Bees previously conditioned to linalool in the proboscis extension
procedure were compared with control bees. The total flight duration
(Fig. 3A) did not differ
significantly between the two groups (ANOVA, F=0.36,
P>0.05, N=25), flight durations being similar to those
observed in the first experiment (136.3±9.6 s for the conditioned bees,
127.3±11.7 s for the control bees). By contrast, the two groups
differed with respect to their flight duration around the source
(Fig. 3A; MannWhitney
test, z=4.75, P<0.001, N=25), conditioned bees
spending more time around the odour source (7.00±1.86 s) than control
bees (0.36±0.14 s). Conditioned bees exhibited significantly more
orientated flights than control bees (Fig.
3B; 2=5.56, P<0.05, N=25).
The proportion of circlings also differed between the two groups
(Fig. 3B;
2 =
9.44, P<0.01), conditioned bees exhibiting more circlings (40%)
than control bees (4%). Thus, there was a strong effect of conditioning on the
flight behaviour in the wind tunnel, conditioned bees showing an increase in
the orientation response towards the odour.
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Discussion |
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We first analysed the behaviour of the bees in response to two types of
olfactory stimuli: (1) linalool, a common floral compound
(Knudsen et al., 1993) that is
not known to have any pheromonal value to the honey bee, and (2) a kin odour
from nestmates. In all experiments, bees placed in the tunnel without odour
(control groups) and bees subjected to odorant stimulations did not differ
with regard to the total flight duration, all of them flying for approximately
half the observation period (5 min). This suggests that the mere presence of
an odour stimulus did not enhance flight willingness. In the first experiment,
the two odours induced orientated flights but they differed slightly with
regard to the extent of this effect. Nestmate odour induced orientated flights
in 68% of the workers, and circling behaviour around the source in 60%
(compared with only 28% and 0%, respectively, for the control group), thereby
demonstrating a strong influence of this social odour. Under the same
conditions, linalool induced orientated flights in 48% of the bees and
circlings in 28%. One can question whether the vapour pressure and the
diffusion range of the odours might affect the performance of the bees. With
regard to the nature of the social odour, the volatiles produced by caged
worker bees contained at least Nasanov pheromone, the main active components
of which are geraniol, citral and nerol. All these components, as well as
linalool, have approximately the same volatility (boiling point at 760 mmHg;
linalool 198200°C; geraniol 229230°C; citral
228229°C; nerol 225228°C). We can thus assume that the
diffusion of the tested odours within the tunnel did not differ significantly
and that this factor did not affect our results.
The social odour produced by caged nestmates was supposed to mimic the
social cues that can drive the approach flight of a bee returning to the hive.
Nest olfactory recognition mainly relies on the orientation pheromone produced
by the Nasanov gland (Pickett et al.,
1980; Ferguson and Free,
1981
), which could be the basis of the attraction observed in our
experiments. Studies on the production of geraniol, a main active component of
the Nasanov pheromone, have shown high variation with age
(Boch and Shearer, 1963
): very
young bees do not produce geraniol, but appreciable amounts are produced after
the bees are more than 12 days old. Thus, our caged bees actually released
Nasanov pheromone. Consequently, they might have experienced this odour (or
other compounds belonging to their nestmates) before testing, in an
imprinting-like process. Indeed, the recognition of the nest odour is known to
involve early learning of various odours such as cuticular hydrocarbons and
odours of stored food (Breed and Stiller,
1992
; Breed et al.,
1995
,
1998
). Thus, the observed
attraction might rely on an innate response to the compounds and/or on a
previous experience during the development of the bee. With the exception of
the nestmate odours, we can assume that our laboratory rearing conditions led
to a much reduced olfactory exposure, ensuring no exposure to linalool at
least from the time of emergence. By contrast, we cannot reject the
possibility that the bees had experienced linalool during their development.
Indeed, the effects of passive exposure to environmental cues during
development are well documented in insects. This exposure most often occurs at
the early adult stage, but some work has also indicated preimaginal
experiences (Isingrini et al.,
1985
; Dobson,
1987
; Carlin and Schwartz,
1989
). In the honeybee, Sandoz et al.
(2000
) have found an effect of
early adult exposure but no effect of preimaginal exposure. An exposure to
linalool, before we collected and caged the bees, could thus explain why this
floral compound induced orientated flights. More generally, similarities
between the responses to nestmate odour and linalool also suggest that
olfactory orientation at short range might be first based on a non-specific
response to the presence of an odour, which could be modulated according to
the biological value of the odorant stimulation (e.g. floral or pheromonal
volatiles) or to the previous experience of the bee.
The second experiment was designed to consider whether a previous
experience (classical olfactory conditioning of the proboscis extension) could
actually modify odour-cued orientation at short range. Some bees exhibited
spontaneous proboscis extension to linalool at the first presentation of this
odour. For our purpose, these bees were discarded from the experiment since
their later responses could not be attributed to associative learning alone.
The bees that responded spontaneously represented 20% of all the tested bees,
which is similar to the values reported in the literature
(Bhagavan et al., 1994;
Sandoz et al., 1995
;
Laloi et al., 2001
). Our
results showed that, with linalool diffused upwind, only 20% of the
non-conditioned bees made orientated flight, and only 4% of them exhibited
circling behaviour. The orientation performance towards this floral odour was
strongly enhanced (up to 52% of orientated flights and 40% of circlings) by
prior classical olfactory associative learning. Moreover, the patterns of
response observed in the first experiment are similar to those obtained after
olfactory conditioning. This suggests that the results of the first experiment
could also reflect some kind of associative experience.
This result demonstrates, under controlled conditions, that honey bees can
use olfactory information gained in a previous classical conditioning
procedure in a subsequent flight orientation context. This complements
previous studies in the honey bee on information transfer from one context to
another. First, Jakobsen et al.
(1995) showed that free-flying
bees could be attracted to artificial feeders by an odour added in the hive.
Thereafter, Gerber et al.
(1996
) showed a possible
information transfer of learning in a free-flying foraging situation
(instrumental context) to the reflex response of proboscis extension
(Pavlovian context). In situations more closely related to our experiment, two
studies have shown that olfactory information acquired in a classical
conditioning of the proboscis extension reflex can influence the walking
orientation performance of a bee in an olfactometer device. Bakchine et al.
(1992
) found that conditioning
to geraniol, a pheromonal/floral compound, increased the orientation response
of bees towards this odour in a four-armed olfactometer. Sandoz et al.
(2000
) obtained similar
results with two floral compounds, linalool and phenylacetaldehyde. More
generally, these results suggest that a forager could gain information about
the odours it can use to navigate and search for food not only during foraging
(von Frisch, 1967
;
Menzel et al., 1993
) but also
within the hive before its first foraging bout. Indeed, worker bees returning
to the hive bring odours and food that constitute, at least for a certain time
period, a good indication of the available resources. Future foragers should
thus have already acquired some kind of olfactory preferences that could
improve their subsequent foraging performance.
Our results show the necessity of considering the influence of learning when addressing the question of olfactory orientation even at short distance. For this, the wind tunnel could be a powerful tool for examining precisely the variation of flight structure and orientation performance in response to various factors. These factors could be the nature and the complexity of odorous stimuli, the distance at which compounds are detected, as well as various types of learning procedure such as differential conditioning in which bees are conditioned to discriminate a rewarded odour from a non-rewarded one. The wind tunnel could also be particularly suitable for studying the role of odours in honey bee decision making during foraging.
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Acknowledgments |
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