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First published online August 30, 2006
Journal of Experimental Biology 209, 3587-3598 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02423
Odour-evoked responses to queen pheromone components and to plant odours using optical imaging in the antennal lobe of the honey bee drone Apis mellifera L.
Research Centre for Animal Cognition, UMR 5169, Université Paul Sabatier, 118, Route de Narbonne, 31062 Toulouse cedex 4, France
e-mail: sandoz{at}cict.fr
Accepted 3 July 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Key words: olfaction, insect, calcium imaging, antennal lobe, sex pheromone, sexual communication
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). Queen pheromonal
components also play a crucial social role, since they maintain the colony's
cohesion, influencing both the physiology and the behaviour of worker bees
(Free, 1987;
Winston, 1987). Thus queen
pheromonal components act both as a primer pheromone, inhibiting the ovarian
development in workers, and as a releaser pheromone, attracting workers to
form a retinue around the queen, stimulating social pheromone production by
workers, controlling swarming, etc (Free,
1987; Winston,
1987). Two of the main components of the queen pheromone, produced
in the mandibular glands, are 9-keto-2 (E)-decenoic acid (9-ODA) and
9-hydroxy-2 (E)-decenoic acid (9-HDA). The 9-ODA, in particular, was shown to
be highly effective in male-attraction bioassays
(Gary, 1962;
Butler and Fairey, 1964).
However, the two compounds 9-ODA and 9-HDA, both alone or blended, are less
effective than a whole-queen extract in eliciting retinue behaviour in workers
(Slessor et al., 1988). Two
additional components, methyl p-hydroxybenzoate (HOB) and
4-hydroxy-3-methoxyphenylethanol (HVA), were discovered; they act
synergistically with 9-ODA and 9-HDA to elicit retinue attraction, but do not
work in isolation (Slessor et al.,
1988; Slessor et al.,
1990). The four-component blend also affects worker orientation
during swarming and inhibits queen rearing
(Winston et al., 1989).
The honey bee olfactory system displays a clear sexual dimorphism at both
the peripheral and the central level. Apart from clear differences in the
shape and size of worker and drone antennae (in the drone: shorter scapus but
thicker and longer flagellum, with eleven instead of ten segments, as in the
worker), the most impressive difference between the two sexes lies in the
number of pore-plate sensilla: around 18 000 on a drone antenna, and only 2800
on a worker antenna (Esslen and Kaissling,
1976). This difference translates into differences in the number
of olfactory sensory neurones (OSNs), which are around 330 000 in drones
vs 46 000 in workers (Esslen and
Kaissling, 1976). Electrophysiological studies have shown that
this increased number of OSNs in the drone is also related to an increased
probability of finding cells responding to 9-ODA than in workers
(Kaissling and Renner, 1968;
Vareschi, 1971). Moreover,
electroantennogram (EAG) recordings have shown a much higher response to 9-ODA
or to a whole-queen extract in drones than in workers
(Skirkeviciene and Skirkevicius,
1994; Vetter and Visher,
1997; Brockmann and
Brückner, 1998). Vareschi
(Vareschi, 1971)
electrophysiologically identified a type of receptor cell (type I), which
responded to 9-ODA and to a range of aliphatic acids. However, these cells
were extremely sensitive to 9-ODA, with a detection threshold at least three
orders of magnitude below that to other odours
(Vareschi, 1971). At the
central level, sexual dimorphism is present at the first olfactory centre, the
antennal lobe (AL). In worker bees, the AL consists of 
160 identified
functional units, the glomeruli, which are interconnected by approximately
4000 local, inhibitory interneurons (Fonta
et al., 1993). OSNs project onto such glomeruli via the
antennal nerve and processed olfactory information leaves the AL by
approximately 800 projection neurons, toward higher-order brain centres, such
as the mushroom bodies or the lateral protocerebrum
(Abel et al., 2001). In drones,
sensory tracts are thicker but project into a smaller number of glomeruli than
in workers (Arnold et al.,
1985). Most of them (103) correspond to glomeruli of a
similar size to those of workers (henceforth called `ordinary' glomeruli).
However, the most dramatic difference between the drone and the worker AL is
the presence of four hypertrophied glomeruli in the drone, the macroglomeruli
(Arnold et al., 1985). Based on
their impressive size and their similarity to the macroglomerular complexes of
males of several moth species, which are involved in the detection and
processing of female pheromone components, it was proposed that the
macroglomeruli of drone honeybees play a similar role and serve the detection
and processing of queen pheromonal components
(Arnold et al., 1985;
Masson and Mustaparta, 1990).
Besides pheromonal detection, the olfactory system of drones can also detect a
wide range of different odorants, including floral odours and social
pheromones, which it can learn to associate with a sugar reward in the
classical conditioning procedure of the proboscis extension response
(Vareschi, 1971;
Becker et al., 2000). Until
now, no study has addressed how pheromonal and plant odours are represented in
the drone antennal lobe, or evaluated the role of the macroglomeruli in queen
pheromone detection. The assumption that processing of queen pheromone
components is restricted to the drone macroglomeruli therefore remains
untested.
Optical imaging techniques provide a possible way to address this problem
experimentally. These techniques allow measuring the activity of numerous
neurons at the same time; in the worker honey bee, calcium imaging has been
successfully applied to record neural activity both from the ALs (e.g.
Joerges et al., 1997;
Sachse and Galizia, 2002;
Sandoz et al., 2003) and the
mushroom bodies (e.g. Faber and Menzel,
2001; Szyszka et al.,
2005). In the ALs, odours elicit combinatorial activity patterns
across glomeruli (Joerges et al.,
1997) and odour quality is represented by a specific distributed
code, conserved between individuals
(Galizia et al., 1999;
Sachse et al., 1999). Activity
patterns in the AL clearly correspond to a perceptual representation of
odorants, as physiological similarity between activity patterns correlates
with perceptual similarity as deduced from generalisation performances of bees
conditioned to a wide spectrum of selected odours
(Guerrieri et al., 2005).
Using calcium imaging, I measured odour-evoked responses in the glomeruli of
the drone AL to understand how information about queen pheromone components
and other odours is represented in the drone brain. Odours belonging to three
main classes of stimuli were presented: (i) queen pheromonal components,
potentially used by drones for the recognition of queens during nuptial
flights, (ii) social pheromonal components produced by workers and used for
social cohesion in the colony, and (iii) floral odours, which are present in
the food stores of the hive and/or brought back by foragers.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Sandoz et al., 2003), although
some adaptations were required due to differences in the conformation of the
drone head (in particular the big compound eyes and the short antenna scapi).
The antennae were fixed to the front of the chamber using fine plastic threads
and a two-component silicon glue (Kwik-Sil, World Precision Instruments).
Small pieces of plastic foil (0.5 mm thick) were then waxed at an angle to the
front, and vertically to the sides of the chamber, to create a small pool
around the brain region. Thus the antennae remained in the air, whilst the
brain region could be kept in saline solution. A window was then cut in the
cuticle of the head, as a triangle between the eyes to the sides, and the
antennae to the front. Because the antennal lobes are less accessible in the
drone than in the worker bee, the cuticle constituting the articulations of
the antennae (antennal sclerite) was removed very carefully. Glands and
trachea were removed to reveal both antennal lobes, and the oesophagus was
removed. The brain was then washed thoroughly with saline solution (in mmol
l-1: Nacl, 130; KCl, 6; MgCl2, 4; CaCl2, 5;
sucrose, 160; glucose, 25; Hepes, 10; pH 6.7, 500 mOsmol; all chemicals from
Sigma-Aldrich, Lyon, France). For staining, the saline solution was gently
removed, and the brain was bathed with 50 µl of dye solution. The dye
consisted of Calcium-Green-2AM (50 µg) dissolved with 50 µl Pluronic
F-127 (20% in dimethylsufoxide, DMSO) in 800 µl Ringer (all from Molecular
Probes, Eugene, OR, USA). The piece of head cuticle was then replaced onto the
opening and the drone was left for 1 h on ice. After staining, the brain was
thoroughly washed with saline.
Optical recordings of odour-evoked activity
In vivo calcium imaging recordings were carried out using a
T.I.L.L. photonics imaging system (Martinsried, Germany). Stained bees were
placed under an epifluorescent microscope with a water-immersion objective
(20x, NA 0.5), and the head region was immersed in saline solution. The
preparation was slightly tipped to the front to offer a view of the antennal
lobe surface with as few focus differences as possible between the different
lobe regions. Images were taken using a 640x480 pixel 12-bit monochrome
CCD-camera (T.I.L.L. Imago) cooled to -12°C. Each measurement consisted of
100 frames at a rate of 5 frames s-1 (interval between frames 200
ms), and the integration time was 15 ms. Odour stimuli were given at the 15th
frame for 1 s. Pixel image size corresponded to 5x5 µm after
4x4 binning on chip. Monochromatic excitation light at 475 nm was
applied using a monochromator (T.I.L.L Polychrom IV). The filter set on the
microscope was composed of a 505 nm dichroic filter and an LP 515 nm emission
filter. Under the microscope, a constant air-stream, into which odour stimuli
could be injected, was directed to the drone's antennae (distance 2 cm).
During odour stimulation, a secondary airflow was diverted from the main
airflow and passed through an interchangeable glass pipette containing the
odour. Stimulations were controlled by the imaging system's computer. The
floral odours used were 1-hexanol, 1-nonanol, limonene and +/-linalool (from
Sigma-Aldrich) and clove and orange essential oils obtained from a drugstore
(Berlin-Dahlem, Germany). Some social pheromonal components such as citral,
geraniol (aggregation), isoamyl acetate and 2-heptanone (alarm) were only
tested in a few animals (N=3). The queen pheromone components tested
were: 9-keto-2 (E)-decenoic acid (9-ODA), 9-hydroxy-2 (E)-decenoic acid
(9-HDA), 4-hydroxy-3-methoxyphenylethanol (HVA) and methyl
p-hydroxybenzoate (HOB). Another component, 10-hydroxy-2 (E)-decenoic
acid (10-HDA), which is present in high amounts in both workers and virgin
queens (Plettner et al., 1997)
was also included. Odour sources were prepared by applying 5 µl of
substance (floral odours and social pheromones) or 5 µl of substance
diluted in isopropanol (queen pheromone components, concentration of 5 µg
µl-1) onto a 1 cm2 piece of filter paper inserted in
a Pasteur pipette. When preparing the pheromone sources, the solvent was
allowed to evaporate for 30 s before the pipette was closed. As control
stimuli, pipettes containing a clean piece of filter paper (air control) or a
filter paper with 5 µl of isopropanol (2prop; also evaporated) were used.
An experimental run consisted of three fully randomised series of 10-13 odour
presentations with 1-2 min inter-trial intervals.
Mapping activity onto glomeruli
During optical imaging, the glomerular structure of the antennal lobes is
not visible and fluorescence is homogeneous over the whole AL surface. To
reveal the glomerular AL structure after performing the calcium imaging
recordings, standard techniques developed for worker bees were used
(Galizia et al., 1999).
Briefly, a mixture 125:1 (v/v) of a protease solution (from Bacillus
licheniformis in propylene glycol; Sigma Aldrich) for digesting the brain
sheath, and of the dye RH795 (dissolved in absolute ethanol) for staining cell
membranes (Molecular Probes), was applied to the brain for 1 h. Afterwards,
the brain was rinsed with saline solution and fluorescent photographs were
taken at 5-10 different focal planes under 530-540 nm excitation light, using
a filter set containing a 570 nm dichroic mirror and a LP 590 nm emission
filter. One particularly large activity spot situated on the dorsolateral side
of the antennal lobe was clearly identified as the second macroglomerulus MG2,
based on its location [(Arnold et al.,
1985); see Fig. 1A]
and on the direct comparison of imaging data and anatomical stainings carried
out after imaging. Its direct medial neighbour, MG1, was recognised only on
the basis of anatomical stainings, as no signals were recorded in this
macroglomerulus (see Results). Concerning ordinary glomeruli, anatomical
stainings after imaging showed limited success in revealing their layout and
numbers. In a few cases in which the glomerular layout could be resolved,
activity spots clearly corresponded to individual glomeruli (see
Fig. 1). Later, additional
stainings on drones not subjected to calcium imaging were performed, replacing
RH795 by a 4% solution of Neutral Red in distilled water. These stainings gave
much better results and allowed confirmation of the general layout of the
drone AL (see Fig. 1A).
|
F/F)
were calculated as (F-F0)/F0,
taking as reference background F0 the average of three
frames before any odour stimulation (here frames 5-7). Third, to correct for
bleaching and possible irregularities of lamp illumination in the temporal
dimension, a subtraction was made at each pixel of each frame, of the median
value of all the pixels of that frame. Such a correction stabilizes the
baseline of the recordings, without removing pertinent signals. Odour-evoked
signals were the typical stereotyped biphasic signals usually obtained with
bath application of Calcium Green, with first a fast fluorescence increase,
followed by a slow fluorescence decrease below baseline
(Fig. 1E);
(Galizia et al., 1997;
Stetter et al., 2001;
Sandoz et al., 2003). The
maximum signal was obtained 1.0-1.2 s after odour application and the minimum
8-10 s after odour application (Fig.
1E). For visual observation of the signals in the different AL regions (Figs 1, 2 and insets in Fig. 3), activity maps are shown with the best possible spatial definition of odour-induced signals. Therefore, the full signal amplitude was used. Each pixel represented the mean of three frames after 1 s (i.e. from 0.6 to 1.2 s) minus the mean of three frames after 9 s (i.e. from 8.6 to 9.2 s). Activity maps are presented in a false-colour code, from dark blue (no signal) to red (maximum signal).
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For quantitative analysis of signal amplitudes (Figs
3 and
4), the analysis focused, as in
previous work (Galizia et al.,
1999; Sachse et al.,
1999; Sandoz et al.,
2003), on the fast (positive) signal component evoked by odour
stimulation. There are several reasons for this choice. First, this calcium
increase on odour stimulation can be ascribed to an intracellular calcium
increase from the extracellular medium, directly related to neuronal activity
(see also Galizia and Kimmerle,
2004). It reflects most probably pre-synaptic neuronal activity
from OSNs (Galizia et al.,
1998; Sachse and Galizia,
2003). Second, studies recording neuronal responses downstream
along the olfactory pathway showed that these neurons [projection neurons and
clawed Kenyon cells (Sachse and Galizia,
2002; Szyszka et al.,
2005)] respond well within the first second after odour
application. For each activity spot studied, the time course of relative
fluorescence changes was calculated by averaging 25 pixels (5x5). The
amplitude of odour-induced responses was calculated as the mean of 3 frames
after odour onset (i.e. 0.6-1.2 s) minus the mean of 3 frames just before the
odour stimulus (i.e. -0.8 to -0.2 s). This value was then used in all
computations.
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Measuring odour similarity in drones
One aim of this study was to measure the similarity between different
odorants based on the signals obtained in the region of ordinary glomeruli.
Because anatomical stainings did not allow all glomeruli present at the AL
surface to be distinguished during imaging, the study was focussed on the
activity spots (between N=10 and N=18) observed in each
drone. As a measure of similarity between odours, the Euclidian distance
between odour representations in a putative n-dimensional space in
which the activity of each spot represents one dimension was calculated. The
higher the calculated distance between odour representations, the less similar
were the odour representations. For each odour pair, distances calculated in
each individual were averaged and subjected to a correlation analysis with
previous data on honeybee workers (Galizia
et al., 1999; Sachse et al.,
1999; Sandoz et al.,
2003). For calculating distances between odour representations
based on imaging data (Galizia et al.,
1999; Sachse et al.,
1999), a method was used that gave good results in a previous
report (Guerrieri et al.,
2005): AL activation maps (as presented on
http://neuro.unikonstanz.de/)
were thus transcribed into activation levels for each glomerulus from 0 to 3,
according to the following signal scale: activity below 40%, 0; 40-60%
activity, 1; 60-80% activity, 2; >80% activity, 3. As a second comparison
between odour similarity in drones and workers, data from a previous study,
using naïve bees only (Sandoz et al.,
2003), were used. As this study carrying out bilateral optical
imaging of both ALs showed that odour-induced activity of naïve bees is
symmetrical, the signals from right and left antennal lobes were averaged
within each individual. The signal amplitudes recorded in the 24 AL glomeruli
recognized in Sandoz et al. (Sandoz et
al., 2003) were used to calculate Euclidian distances between
odour representations as was done with drone data. The significance of all
linear correlations was assessed by calculating Pearson's r, and
using Student's t-test.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Odours evoke calcium signals in the glomeruli of the drone antennal lobe
Thirty four drones were prepared for optical imaging experiments, 12 of
which allowed recording of calcium signals in response to odours.
Fig. 1B,C present a typical
example of odour-evoked calcium signals in the drone antennal lobe. Calcium
signals were always observed in topical foci, which were different for
different odours (Fig. 1B). The
location of the activated foci corresponded to two distinct anatomical
regions: the ventral region of ordinary glomeruli, and the dorsolateral region
of the lobe corresponding to MG2. No odour-induced responses were observed in
the dorso-medial region corresponding to MG1. Although anatomical
after-stainings usually gave limited success in revealing the layout of
ordinary glomeruli, in some instances it could be clearly seen that the
activity foci in the dorsal region originated in individual ordinary glomeruli
(Fig. 1B). Control odours (air
or 2-propanol) usually induced no or very low signals. In all imaged drones,
the calcium signals showed typical biphasic time courses like the ones
observed with the same staining method in workers [bath-applied Calcium Green
2-AM (Galizia et al., 1997)].
This time course was similar in all glomerular types (ordinary glomeruli or
macroglomerulus), and was the same for pheromonal and floral odours. After
odour application (Fig. 1C,
example with 9-ODA), fluorescence increased topically, reaching a maximum
after 1.0-1.2 s (fast positive component), before decreasing to a minimum,
about 8-10 s after odour application (slow negative component). Apart from a
small bleaching effect in some recordings, the signal increased again to
near-baseline within 20 s. Because other studies have shown that neuronal
responses downstream along the olfactory pathway respond well within the first
second after odour application [projection neurones and clawed Kenyon cells
(Sachse and Galizia, 2002;
Szyszka et al., 2005)],
further analysis concentrated on the fast positive component.
Odour-induced calcium signals were reproducible within an individual as
well as between individuals (Fig.
2A) and were also symmetrical between brain hemispheres.
Fig. 2A presents the responses
observed in five different individuals (three left and two right antennal
lobes) to 1-hexanol and to the pheromone component 9-ODA. Clearly, 1-hexanol
induced activity in one large ordinary glomerulus (H in the
Fig. 2A) on the ventro-lateral
side of the antennal lobe, and in two to three smaller glomeruli (A, B and C
in Fig. 2A) on the
ventro-medial side. This arrangement was found symmetrically in left and right
antennal lobes (Fig. 2A).
Similarly, response to 9-ODA was in all drones found in the same region,
corresponding to MG2. When optical recordings were carried out simultaneously
on the two antennal lobes (Sandoz
et al., 2003), a symmetrical arrangement of active glomeruli was
clearly found (Fig. 2B).
Queen pheromonal components activate both ordinary glomeruli and one macroglomerulus
The drones were presented with five queen pheromonal components: 9-ODA,
9-HDA, 10-HDA, HVA and HOB. Responses to these components were of three
types.
First, as shown above (Fig.
2A), the major component of the queen pheromone 9-ODA consistently
induced calcium signals in the dorso-lateral region corresponding to MG2. This
response was very clear and appeared in all individuals that showed calcium
signals (N=12). Moreover, it was very specific: 9-ODA did not induce
signals in other AL regions, and no other odour tested activated MG2
efficiently. In some cases, other odours could be seen to induce very low
responses in MG2, but these could not be distinguished from air or 2-propanol
controls. To document the specificity of MG2 responses to 9-ODA,
Fig. 3A shows the responses
recorded to the five queen pheromonal components and the two control stimuli
(air control, and 2-propanol solvent) (N=11 drones, overall 32
stimulations with each odour). In MG2, the only response above baseline was to
9-ODA (0.52±0.08%) as confirmed by a two-way
stimulusxmacroglomerulus ANOVA (both repeated factors): both stimulus
and macroglomerulus factors were significant (F6,60=14.4,
P<0.001; F1,10=11.5, P<0.007
respectively), and the only significant heterogeneity detected by
post-hoc Scheffé contrasts was between 9-ODA and each of the
other stimuli (P<0.001 in all cases). In some recordings, signals
in MG2 seemed spatially heterogeneous, with some areas showing higher activity
than others (see, for instance, the response to 9-ODA in
Fig. 1B). Based on this
observation, and because anatomical findings suggested that the drone
macroglomeruli may contain different subunits, and therefore correspond
functionally to a macroglomerular complex
(Arnold et al., 1985), it was
important to check whether different zones of the macroglomerulus may respond
differentially to the presented odours. Three different foci were placed in
the five best drone recordings, which allowed seeing the borders of MG2. The
first focus (F1) was the point of activity to 9-ODA, which was at the most
frontal-lateral part of MG2. The other two foci (F2, F3) were regularly placed
along the length of MG2 in a medio-lateral axis.
Fig. 3B presents the amplitude
of responses of these three foci to the range of odours tested in these drones
(eight odours and two control stimuli). In all three parts of the
macroglomerulus, significant responses were only recorded for 9-ODA, and no
qualitative difference in odour responses appeared among the three foci. A
two-way ANOVA stimulusxfocus (repeated measures) indicated that both
factors were significant (F9,36=6.63, P<0.001,
and F2,8=6.6, P<0.05), but most importantly
that no interaction was found between the factors
(F18,72=0.71, NS). Thus the significant difference between
foci is attributable to intensity differences (the third focus showing less
response to 9-ODA than the other two) and not to different odour response
spectra. Thus, all parts of MG2 accessible to optical imaging responded
specifically to 9-ODA and appeared to correspond to a single functional
unit.
In contrast to 9-ODA, the queen pheromone components HOB and HVA induced activity mostly in two ordinary glomeruli in the centre of the frontal region (N=7/12 drones): the HOB glomerulus was usually a more ventral neighbour of the HVA glomerulus. In contrast to 9-ODA in MG2, the HOB and HVA glomeruli commonly showed responses to floral odorants. Fig. 3C presents response profiles of these two glomeruli in five individuals in which the two glomeruli were identified. While glomerulus 1 responded to HOB, glomerulus 2 responded to HVA. However, glomerulus 1 responded clearly to 1-hexanol and to limonene, and glomerulus 2 mostly responded to 1-hexanol and to the clove oil and orange oil blends. Thus, these glomeruli did not respond specifically to HOB and HVA. Lastly, 9-HDA and 10-HDA induced only very low and diffuse signals in some ordinary glomeruli and/or in MG2. However, these signals were inconsistent and could not be differentiated from control stimuli. Therefore they were not quantified.
A combinatorial response table in the drone antennal lobe
As indicated above, floral odours and mixtures presented to the drones
induced focal calcium responses on the ventromedial side of the antennal lobe
(N=12 drones), a region rich in ordinary glomeruli. Different odours
activated specific sets of ordinary glomeruli (from one to six) in a
combinatorial manner. Different odours could also activate the same
glomerulus, as shown in Fig.
1B, where most odours activated the upper-right glomerulus, while
only 1-hexanol and orange oil activated the glomerulus on the right, above
MG1. Social pheromonal components were only tested in a few individuals.
Geraniol and citral induced signals only in ordinary glomeruli while isoamyl
acetate and 2-heptanone did not give any measurable signals (N=2-3
individuals). The combinatorial responses obtained to floral odours in
ordinary glomeruli are represented as the responses of activity foci (size
>25 µm i.e. 5 pixels) that responded to any of the odour stimulations.
An example of such a combinatorial activity table is given in
Fig. 4A (mean of three
stimulations with each odour in one drone). Most odour-induced activity is
visible in ordinary glomeruli (numbered 1-10; see
Fig. 4B for localisation),
which responded in a combinatorial manner to the six floral odours. The only
pheromone components that induced responses in ordinary glomeruli were HOB and
HVA. MG2 responded to 9-ODA and not to other odours. Responses to the diluted
queen pheromone probes usually had lower amplitudes than responses to the pure
floral odours, which explains how pheromone responses only reach the green
category (40-60% of maximum response) in
Fig. 4A.
The search for similar glomeruli in different individuals was difficult due to the fact that no glomerular atlas of the drone antennal lobe is yet available. However, five ordinary glomeruli were tentatively described in the five best drones, according to (1) their relative position in the antennal lobe; (2) their size; (3) their odour response profiles. These glomeruli are presented in Fig. 4B on the same drone as in Fig. 4A: the most prominent ordinary glomerulus was localised on the ventro-lateral side, was egg-shaped and responded strongly to 1-hexanol and to orange oil (corresponds to glomerulus H in Fig. 2A). Two other glomeruli were the HOB and HVA glomeruli described above, placed ventrally and in the centre of the AL. Lastly, two glomeruli on the medial side responded strongly to limonene (and in some drones to linalool) in the case of the more ventral one, and to 1-hexanol in the case of the more dorsal one.
Similarity among odours measured in drone and worker antennal lobes
Even though individual glomeruli can be tentatively recognised in different
drones, building a physiological response atlas, as was successfully carried
out for the worker bee (Sachse et al.,
1999) is not possible. Nevertheless, the similarity in calcium
activity patterns elicited by different odorants can be evaluated within each
individual. Such a measure is independent of the identity of each particular
glomerulus, as long as the same glomerular ensemble is considered in the
different individuals, which is the case in this study. Based on activity in
ordinary glomeruli, similarity relationships among odours were compared in
drones and in workers [datasets (Galizia
et al., 1999; Sachse et al.,
1999)]. Cluster analyses based on glomerular responses in both
castes showed clearly different arrangements
(Fig. 4C). In workers, three
main clusters appeared, which grouped (1) two alcohols (1-hexanol and
1-nonanol), (2) two terpenes (linalool and limonene) and the orange oil
mixture, and (3) the clove oil mixture, respectively. In contrast, in drones,
two clusters appeared, which grouped (1) 1-hexanol, limonene and orange, and
(2) nonanol, linalool and clove oil, respectively. Correlation analyses
representing Euclidian distances between odour representations in drones in
function of the same measure in workers did not show any hint of correlation
(data not shown, r=0.08, NS, 15 odour pairs). A similar analysis
performed with the odours common in this and a previous study [all odours
except linalool (Sandoz et al.,
2003)] also did not show any correlation between drone and worker
odour-similarity relationships (data not shown, r=0.08 also, NS, 10
odour pairs).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Among the identified queen pheromone components
(Slessor et al., 1988), three
gave clear signals in the drone antennal lobe: 9-ODA, HOB and HVA. By
contrast, 9-HDA and the virginqueen and worker component 10-HDA showed no
consistent signals in the glomeruli that could be imaged. The specificity of
MG2 responses to 9-ODA confirms the hypothesis that the very conspicuous drone
macroglomeruli would be involved specifically in pheromone detection and
processing (Arnold et al.,
1985; Masson and Mustaparta,
1990). The fact that MG2 is the most voluminous macroglomerulus of
the drone AL (Arnold et al.,
1985; Brockmann,
1999; Brockmann and
Brückner, 2001) fits well with previous electrophysiological
studies, which showed that an important part of the drone peripheral olfactory
system is dedicated to the detection of 9-ODA
(Kaissling and Renner, 1968;
Vareschi 1971;
Skirkeviciene and Skirkevicius,
1994; Brockmann and
Brückner, 1998; Vetter
and Visher, 1997). However, a previous anatomical study suggested
that MG2 is not originally a single hypertrophied glomerulus, but rather the
result of the fusion of several glomeruli
(Arnold et al., 1985). In some
recordings, signals in MG2 were not spatially homogenous, but showed what
appeared to be small activity regions along the macroglomerulus' long axis.
However, no indication was found of any heterogeneity in the responses of MG2
along this axis. Rather, the different regions responded to 9-ODA but not to
other stimuli. Therefore, similar to moths, in which a clear odotopic
projection of pheromone-specific OSNs to the macroglomerular complex is found,
the results of the present study suggest that most (if not all) of the OSNs
tuned to 9-ODA project to MG2.
One intriguing result of the present study is the absence of odour-induced
signals in MG1, the second most voluminous macroglomerulus of the drone AL. It
is unlikely that this observation is due to specific loading problems of the
AM-dye in MG1, since during calcium imaging, fluorescence was found to be
homogeneous over the whole AL, including MG1. Since the odours were tested at
relatively high concentrations, concentration effects should not account for
this result. A more likely explanation is that the odorant(s) detected by
neurons projecting to MG1 were not among the panel of tested odours. An
interesting hypothesis is that MG1 would process information about still
unknown odorants involved in mating. Since the initial description of a queen
mandibular extract reproducing the retinue behaviour of worker bees, which led
to the use of 9-ODA, 9-HDA, HVA and HOB
(Slessor et al., 1988), new
components have been isolated that also have an effect on worker retinue:
methyl oleate, coniferyl alcohol, hexadecane-1ol and linolenic acid. These
components, which do not work isolated on retinue, but synergistically with
the synthetic queen mandibular pheromone
(Keeling et al., 2003), should
be tested in imaging conditions. However, it must be emphasised that the
search for queen pheromonal components has mainly focussed on creating blends
able to accurately reproduce workers' - but not drones' - behaviour
(Slessor et al., 1988;
Keeling et al., 2003). It is
generally acknowledged that drones are drawn to 9-ODA from long distances
(Gary, 1962) and that 9-ODA
alone does not always reproduce the effect of a complete queen extract in
attraction bioassays (Pain and Ruttner,
1963). Thus, while 9-ODA is clearly the main attractant, the
question of co-attractants is still unresolved. Interestingly, apart from
their important role in queen fighting
(Pflugfelder and Königer,
2003), it seems that once a drone is in the queen's vicinity the
initiation of mating is triggered, or at least enhanced, by olfactory
substances from the queen's tergite glands
(Renner and Baumann, 1964;
Renner and Vierling, 1977). A
hypothesized function for MG1 could be the detection of such a releaser
pheromone. All in all, future work should therefore test queen gland extracts
under imaging conditions in order to find possible new candidate odours that
may be detected and processed by MG1.
Responses to HVA and HOB occurred mainly in two ordinary glomeruli that
responded to several floral odorants (Fig.
3C). This observation suggests that the responses obtained to HVA
and HOB correspond to their detection by the general olfactory system and not
to a pheromonal subsystem. The fact that responses to queen pheromone
components were not circumscribed to the macroglomeruli and could be found in
ordinary glomeruli would underline functional differences rather than
similarities between the macroglomerular complex of male moths and that of
drone honeybees. However these differences may be only apparent. In fact,
virgin queens produce no HVA and very little HOB in comparison to mated queens
(Plettner et al., 1997) such
that these compounds could be only important for the induction of workers'
retinue behaviour by mated queens, and not for drone attraction by virgin
queens. In a similar way, 9-HDA and 10-HDA failed to induce consistent signals
in the present recordings. Overall, so far, one study found attraction to
9-HDA (Butler and Fairey, 1964)
but two subsequent studies failed to reproduce this finding
(Blum et al., 1971;
Boch et al., 1975). It could
thus be that, as for HVA and HOB, these compounds play a crucial role in queen
retinue but are not important in the mating process. Caution should, however,
be exercised, because two additional macroglomeruli, which were not accessible
in this imaging study, are present in the drone AL
(Arnold et al., 1985).
Calcium signals to a range of floral odorants in ordinary glomeruli of the
drone AL were similar to those usually observed in worker bees
(Joerges et al., 1997; Galizia
et al., 1997;
1999;
Sachse et al., 1999):
different odours induced signals in a specific subset of glomeruli. Although
comparison between individual drones was made difficult by the fact that
anatomical stainings did not give clear glomerular boundaries within ordinary
glomeruli of most imaged drones, obvious similarities between individuals
appeared. This suggests that the signals recorded in the anterior AL region
represent part of a conserved neural across-fibre pattern through which plant
odours are encoded in the drone brain. However, odour similarity relationships
were clearly different from those described in workers in previous studies.
This observation should be taken with caution. Because the macroglomeruli
represent about half of the anterior surface of the drone AL, the ordinary
glomeruli accessible to imaging are relatively few, about 20 out of 103
(Arnold et al., 1985). This
proportion is similar to the 30 glomeruli out of 160 routinely imaged in
the worker (Galizia et al.,
1999). Therefore this finding is probably due to the fact that the
glomeruli imaged in the two castes are not homologous, i.e. that they do not
correspond to the projection of OSNs expressing the same subsets of olfactory
receptors. In the present study, neither anatomical observation of glomerulus
position, size or shape, nor the respective arrangements of glomeruli
activated by the different odours, showed any similarity with the workers.
Future work will have to establish to what extent the functional arrangement
of glomeruli is conserved between drones and workers.
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Abel, R., Rybak, J. and Menzel, R. (2001). Structure and response patterns of olfactory interneurons in the honeybee, Apis mellifera. J. Comp. Neurol. 437,363 -383.[CrossRef][Medline]
Arnold, G., Masson, C. and Budharugsa, S. (1985). Comparative study of the antennal lobes and their afferent pathway in the worker bee and the drone (Apis mellifera). Cell Tissue Res. 242,593 -605.[CrossRef]
Becker, M. M., Brückner, D. and Crewe, R. (2000). Behavioural response of drone honey bees, Apis mellifera carnica and Apis mellifera scutellata, to worker-produced pheromone components. J. Apic. Res. 39,149 -152.
Blum, M. S., Boch, R., Doolittle, E., Tribble, M. T. and Traynham, J. G. (1971). Honey bee sex attractant: conformational analysis, structural specificity, and lack of masking activity of congeners. J. Insect Physiol. 17,349 -364.[CrossRef]
Boch, R., Shearer, D. A. and Young, J. C. (1975). Honeybee pheromones: field tests of natural and artificial queen substance. J. Chem. Ecol. 1, 133-148.[CrossRef]
Brockmann, A. (1999). Studies on the olfactory system of the honeybee drone, Apis mellifera. PhD thesis, University of Bremen, Germany.
Brockmann, A. and Brückner, D. (1998). The EAG response spectra of workers and drones to queen honeybee mandibular gland components: the evolution of a social signal. Naturwissenschaften 85,283 -285.[CrossRef]
Brockmann, A. and Brückner, D. (2001). Structural differences in the drone olfactory system of two phylogenetically distant Apis species, A. florea and A. mellifera.Naturwissenschaften 88,78 -81.[CrossRef][Medline]
Butler, C. G. and Fairey, E. M. (1964). Pheromones of the honeybee: biological studies of the mandibular gland secretion of the queen. J. Apic. Res. 3, 65-76.
Esslen, J. and Kaissling, K. E. (1976). Zahl und verteilung antennaler sensillen bei der honigbiene (Apis mellifera L.). Zoomorphologie 83,227 -251.[CrossRef]
Faber, T. and Menzel, R. (2001). Visualizing a mushroom body response to a conditioned odor in honeybees. Naturwissenschaften 88,472 -476.[CrossRef][Medline]
Fonta, C., Sun, X. J. and Masson, C. (1993).
Morphology and spatial distribution of bee antennal lobe interneurones
responsive to odours. Chem. Senses
18,101
-119.
Free, J. B. (1987). Pheromones of Social Bees. London: Chapman & Hall.
Galizia, C. G. and Kimmerle, B. (2004). Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology, calcium imaging and confocal microscopy. J. Comp. Physiol. A 190,21 -38.[Medline]
Galizia, C. G., Joerges, J., Kütnner, A., Faber, T. and Menzel, R. (1997). A semi-in-vivo preparation for optical recording of the insect brain. J. Neurosci. Methods. 76, 61-69.[CrossRef][Medline]
Galizia, C. G., Nägler, K., Hölldobler, B. and Menzel, R. (1998). Odour coding is bilaterally symmetrical in the antennal lobes of the honeybees (Apis mellifera). Eur. J. Neurosci. 10,2964 -2974.[CrossRef][Medline]
Galizia, C. G., Sachse, S., Rappert, A. and Menzel, R. (1999). The glomerular code for odor representation is species specific in the honeybee Apis mellifera. Nat. Neurosci. 2,473 -478.[CrossRef][Medline]
Gary, N. E. (1962). Chemical mating attractants in the queen honeybee. Science 136,1479 -1480.
Guerrieri, F., Schubert, M., Sandoz, J. C. and Giurfa, M. (2005). Perceptual and neural olfactory similarity in honeybees. PLoS Biol. 3,e60 .[CrossRef][Medline]
Joerges, J., Küttner, A., Galizia, C. G. and Menzel, R. (1997). Representations of odours and odour mixtures visualized in the honeybee brain. Nature 387,285 -288.
Kaissling, K. E. and Renner, M. (1968). Antennale rezeptoren für queen substance und sterzelduft bei der honigbiene. Z. Vergl. Physiol. 59,357 -361.[CrossRef]
Keeling, C. I., Slessor, K. N., Higo, H. A. and Winston, M.
L. (2003). New components of the honey bee (Apis
mellifera L.) queen retinue pheromone. Proc. Natl. Acad. Sci.
USA 100,4486
-4491.
Masson, C. and Mustaparta, H. (1990). Chemical
information processing in the olfactory system of insects. Physiol.
Rev. 70,199
-245.
Pain, J. and Ruttner, F. (1963). Les extraits de glandes mandibulaires des reines d'abeilles attirent les mâles lors du vol nuptial. C. R. Acad. Sci. Paris 256, 512.
Pflugfelder, J. and Königer, N. (2003). Fight between virgin queens (Apis mellifera) is initiated by contact to the dorsal abdominal surface. Apidologie 34,249 -256.[CrossRef]
Plettner, E., Otis, G. W., Wimalaratne, P. D. C., Winston, M. L., Slessor, K. N., Pankiw, T. and Punchihewa, P. W. K. (1997). Species- and caste-determined mandibular gland signals in honeybees (Apis). J. Chem. Ecol. 23,363 -377.[CrossRef]
Renner, M. and Baumann, M. (1964). Über komplexe von subepidermalen drüsenzellen (Duftdrüsen) der bienenkönigin. Naturwissenschaften 51, 68-69.
Renner, M. and Vierling, G. (1977). Die rolle des taschendrüsenpheromons beim hochzeitsflug der bienenkönigin.Behav. Ecol. Sociobiol. 2,329 -338.[CrossRef]
Sachse, S. and Galizia, C. G. (2002). The role
of inhibition for temporal and spatial odor representation in olfactory output
neurons: a calcium imaging study. J. Neurophysiol.
87,1106
-1117.
Sachse, S. and Galizia, C. G. (2003). The coding of odour-intensity in the honeybee antennal lobe: local computation optimizes odour representation. Eur. J. Neurosci. 18,2119 -2132.[CrossRef][Medline]
Sachse, S., Rappert, A. and Galizia, C. G. (1999). The spatial representation of chemical structures in the AL of honeybees: steps towards the olfactory code. Eur. J. Neurosci. 11,3970 -3982.[CrossRef][Medline]
Sandoz, J. C., Galizia, C. G. and Menzel, R. (2003). Side-specific olfactory conditioning leads to more specific odor representation between sides but not within sides in the honeybee antennal lobes. Neuroscience 120,1137 -1148.[CrossRef][Medline]
Skirkeviciene, Z. and Skirkevicius, A. (1994). Worker bee and drone (Apis mellifera L.) behavior and functional reorganization of their olfactory receptors. Pheromones 4,83 -92.
Slessor, K. N., Kaminski, L. A., King, G. G., Borden, J. H. and Winston, M. L. (1988). Semiochemical basis of the retinue response to queen honey bees. Nature 332,354 -356.[CrossRef]
Slessor, K. N., Kaminski, L. A., King, G. G. and Winston, M. L. (1990). Semiochemicals of the honeybee queen mandibular glands. J. Chem. Ecol. 16,851 -860.[CrossRef]
Stetter, M., Greve, H., Galizia, C. G. and Obermayer, K. (2001). Analysis of calcium imaging signals from the honeybee brain by nonlinear models. Neuroimage 13,119 -128.[Medline]
Szyszka, P., Ditzen, M., Galkin, A., Galizia, C. G. and Menzel, R. (2005). Sparsening and temporal sharpening of olfactory representations in the honeybee mushroom bodies. J. Neurophysiol. 94,3304 -3313.
Vareschi, E. (1971). Duftunterscheidung bei der honigbiene-einzelzellableitungen und verhaltensreaktionen. Z. Vergl. Physiol. 75,143 -173.
Vetter, R. S. and Visscher, P. K. (1997). Influence of age on antennal response of male honey bees, Apis mellifera, to queen mandibular pheromone and alarm pheromone component. J. Chem. Ecol. 23,1867 -1880.[CrossRef]
Winston, M. L. (1987). The Biology of the Honey Bee. Cambridge, MA: Harvard University Press.
Winston, M. L., Slessor, K. N., Willis, L. G., Naumann, K., Higo, H. A., Wyborn, M. H. and Kaminski, L. A. (1989). The influence of queen mandibular pheromone on worker attraction to swarm clusters and inhibition of queen rearing in the honeybee (Apis mellifera L.). Insectes Soc. 36,15 -27.[CrossRef]
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