Lateralization in the Invertebrate Brain: Left-Right
Asymmetry of Olfaction in Bumble Bee, Bombus
terrestris
Gianfranco Anfora1*, Elisa Rigosi1,2, Elisa Frasnelli2, Vincenza Ruga2, Federica Trona1, Giorgio
Vallortigara2
1 Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy, 2Centre for Mind/Brain Sciences, University of Trento, Rovereto, Italy
Abstract
Brain and behavioural lateralization at the population level has been recently hypothesized to have evolved under social
selective pressures as a strategy to optimize coordination among asymmetrical individuals. Evidence for this hypothesis
have been collected in Hymenoptera: eusocial honey bees showed olfactory lateralization at the population level, whereas
solitary mason bees only showed individual-level olfactory lateralization. Here we investigated lateralization of odour
detection and learning in the bumble bee, Bombus terrestris L., an annual eusocial species of Hymenoptera. By training
bumble bees on the proboscis extension reflex paradigm with only one antenna in use, we provided the very first evidence
of asymmetrical performance favouring the right antenna in responding to learned odours in this species.
Electroantennographic responses did not reveal significant antennal asymmetries in odour detection, whereas
morphological counting of olfactory sensilla showed a predominance in the number of olfactory sensilla trichodea type
A in the right antenna. The occurrence of a population level asymmetry in olfactory learning of bumble bee provides new
information on the relationship between social behaviour and the evolution of population-level asymmetries in animals.
Citation: Anfora G, Rigosi E, Frasnelli E, Ruga V, Trona F, et al. (2011) Lateralization in the Invertebrate Brain: Left-Right Asymmetry of Olfaction in Bumble Bee,
Bombus terrestris. PLoS ONE 6(4): e18903. doi:10.1371/journal.pone.0018903
Editor: Thomas Burne, University of Queensland, Australia
Received January 3, 2011; Accepted March 11, 2011; Published April 27, 2011
Copyright:  2011 Anfora et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the Provincia Autonoma di Trento (http://www.provincia.tn.it) and the Fondazione Cassa di Risparmio di Trento e
Rovereto (http://www.fondazionecaritro.it). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gianfranco.anfora@iasma.it
Introduction
Research on anatomical and functional side-related specializa-
tions of the brain has mainly focused on vertebrates [1,2,3].
Recently, evidence for brain and behavioural lateralization among
invertebrates has been reported [4,5,6,7,8,9,10].
Olfactory asymmetries have been shown in honey bees by
Letzkus et al. [11]. When conditioned, using the proboscis
extension reflex paradigm (PER) [12], with only one antenna in
use, bees showed better learning with their right rather than their
left antenna. Evidence for lateralization of olfactory learning in the
honey bee has been subsequently confirmed and extended
exploiting the same paradigm, using different scents and without
any coating of the antennae, i.e. with lateral presentations of odour
stimuli [13,14,15,16].
In natural conditions, asymmetries may occur at the population-
level when more than 50% of the individuals are lateralized in the
same direction, whereas lateralization at the individual level occurs
when most of the individuals are lateralized with either a left- or
right- bias equally distributed in the population [1]. Recently, the
issue has been tackled of the advantages for an individual in a
population of being bound into directional behavioural asymme-
tries [2]. Ghirlanda and Vallortigara [17] showed, using
mathematical game theory, that in a prey-predator ecological
context, population-level lateralization might represent an evolu-
tionary stable strategy (ESS) driven by social pressures, i.e. when
asymmetrical organisms have to coordinate their asymmetries in
behaviour among each other. A well-fitting example might be the
turning behaviour to escape from a predator in shoaling fish
species. In a large number of teleost fishes the shoaling species
appeared to be lateralized at the population level, while the
majority of non-shoaling species were lateralized at the individual
level [18].
Strictly related species of bees (Superfam. Apoidea) with
different levels of intraspecific social interactions may provide
important evidence in order to evaluate the hypothesis that
population-level asymmetries are more likely to occur among
social species. Anfora et al. [14] recently reported that two
different species of bees, Apis mellifera, the most sophisticated
eusocial species, and Osmia cornuta, a solitary species, showed
different olfactory asymmetry behaviours. The eusocial species
appeared to be lateralized at the population level, whereas the
solitary species appeared to be lateralized only at the individual
level.
Here we studied an annual social species of Apoidea, Bombus
terrestris L. (Hymenoptera: Apidae). These bumble bees exhibit
primitive eusocial behaviour as they have an annual cycle with
single queens founding new annual nests. Therefore, bumble bees
can represent one of the last evolutionary steps in the taxonomic
group of Hymenoptera towards the complete development of
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eusociality [19,20]. We tested olfactory learning in bumble bees
with only one antenna in use. Given that in honey bees
behavioural lateralization in olfactory learning has been associated
with anatomical and electrophysiological asymmetries at the
peripheral level in the olfactory neural pathway [11,14,16], we
also measured the number of putative olfactory sensilla in the left
and the right antennae using scanning electron microscopy and
the electrophysiological responses of the two antennae when
stimulated by odours behaviourally relevant to bumble bees.
Materials and Methods
Insects
For all the experiments bumble bee foragers were collected from
the same colony of B. terrestris, supplied by Bioplanet s.c.a., Cesena,
Italy. The tested individuals were not age-marked but they could
be considered to have had similar olfactory experiences because of
prior exposition to the same odours inside the colony and because
they were not allowed to forage outside the nest.
We used female foragers of similar size (mean body size: 1.7 cm)
in order to minimize naturally occurring antennal sensitivity
variations [21].
Test compounds
The test synthetic chemicals were two odours behaviourally
relevant to bumble bees: isoamylacetate (Sigma-Aldrich, Milano,
Italy; .99.7% purity), both component of their pheromone blends
and a floral compound, and (-)-linalool (Sigma-Aldrich, .98.5%
purity), a common floral compound, [22,23].
Behavioural experiments
Behavioural methods made use of the experimental procedures
developed in honey bees [11,12,13] and bumble bees [23] for
olfactory learning. After 12 hours of food deprivation, bumble
bees were cooled in 750 ml containers until immobilized and
secured in metal holders. The insects were randomly assigned to
three different groups; with the left (N = 10) or the right (N = 10)
antenna coated with a two-components silicon compound
(Silagum-Mono, DMG, Germany), or with both the antennae
uncoated (N = 10) [11,14]. Training started one hour after the
antennae had been coated. Each animal in its holder was in turn
placed in front of an exhaust fan and trained using (-)-linalool, plus
1 M sucrose solution (reward) as a positive stimulus (10 ml of (-)-
linalool dissolved in 3 ml of the sugar solution). The negative
stimulus was unscented saturated NaCl solution. Three learning
trials were given every 6 min. On the first trial a drop of the
positive stimulus solution at the end of a 23 gauge needle was held
1 cm over the antennae and after 5 s the antennae were touched,
which led to PER. The bumble bee was then allowed to ingest the
drop of (-)-linalool sugar solution as reward. The procedure was
immediately repeated with the saline solution, which did not
trigger PER but avoidance by moving the antennae away from the
negative stimulus. On the two subsequent training trials the
procedure of the first trial was repeated (and usually PER occurred
without the need to touch the antennae). To check possible
behavioural differences among the three groups during the
training procedures, we performed analysis of variance (ANOVA)
with antenna in use as a between-subject factor, considering the
number of proboscis extensions over the total conditioning trials.
Recall of short-term odour memory was tested 1 hour after the
end of training. Both (-)-linalool, dissolved in distilled water at the
same concentration as used in training, and saturated salt solution
were presented holding a drop of these solutions over bumble bee’s
antennae for 5 s being careful not to touch them. Each animal was
tested in a total of 10 such paired trials, presented in random order
and separated by an inter-trial interval of 60 s. It has been
demonstrated, in fact, that habituation in PER responses does not
occur over 20 trials [13]. We recorded every time the bumble bee
extended the proboscis. The percentage of correct responses was
calculated as number of proboscis extensions to the (-)-linalool over
the total (-)-linalool presentations per animal (no proboscis
extensions to salt solution occurred).
Data were analyzed by analysis of variance (ANOVA) with
antenna in use as a between-subjects factor.
Electroantennography (EAG)
Absolute EAG responses (mV) were recorded from right and left
isolated antennae of B. terrestris foragers (N = 20) with a standard
EAG apparatus (Syntech, Hilversum, The Netherlands). Animals
were anaesthetized, antennae were cut at the level of the scape and
the uppermost part of the antennal tip was removed. The base of
the antenna was placed inside a glass micropipette filled with
Kaissling saline solution [24] and the tip put into the recording
glass micropipette electrode. The first antenna tested was chosen
randomly and the animal was kept alive until the second antenna
was used.
The test synthetic compounds were isoamylacetate and (-)-
linalool. For each compound, 25 ml of five decadic steps hexane
solutions (ranging from 1022 to 102 mg/ml) were absorbed on
1 cm2 pieces of filter paper, inserted into individual Pasteur
pipettes and put into the constant air flow tube directed to the
antenna (50 cm3/s). Stimuli of 500 ms were presented in
ascending order of dosage with 30 s inter-stimuli intervals, using
a stimulus controller (CS-55, Syntech). Control pipettes (loaded
with 25 ml of hexane and an empty pipette) were used before and
after each series of stimuli. Data were log transformed to account
for the heterogeneity of variances and analyzed by analysis of
variance (ANOVA) with antenna, scent and dose as within-subject
factors.
Scanning Electron Microscopy (SEM)
Bumble bees (N = 14) were anaesthetized and their left and right
antennae were cut at the base of pedicel. The basal segments of
each pair of antennae were attached to a circular stub by double-
sided conductive tape (TAAB Laboratories Equipment Ltd.
Aldermaston, UK). All samples were gold coated for guaranteeing
electrical conductivity and scanned with a XL 30, Field Emission
Environmental Scanning Electron Microscope (FEI-Philips, Eind-
hoven, The Netherlands). Each antenna was imaged from four
different viewpoints: ventral (holder at 0u), right (sample tilted at
275u), left (sample tilted at +75u) and dorsal (after removing
antenna from stub and replacing upside). Because of the lack of
olfactory sensilla on the first two segments of the flagellum of B.
terrestris, only segments from 3rd to 10th were scanned. Each
segment from 3rd to 9th was scanned longitudinally at a
magnification of 600 times (Figure 1a). A magnification of 800
times was used for the 10th segment (apex). For each segment four
images were collected according to the different viewpoints.
Both putative olfactory sensilla, i.e. sensilla placodea (Figure 1b–
c), trichodea type A (Figure 1b), coeloconica (Figure 1c), and
basiconica (Figure 1d), and non-olfactory sensilla, i.e. sensilla
trichodea type B (Figure 1b), and ampullacea (Figure 1c), were
identified according to their specific morphological characteristics
as described in Frasnelli et al. [16] and in A˚gren and Halberg [25].
Each type of sensillum was then tagged and counted on all
acquired images by using image analysis software (UTHSCSA
ImageTool Version 3.0). Data were clustered according to the four
viewpoints, eight antennal segments, two antennae and six
Lateralization of Olfaction in Bumble Bee
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sensillum types. Data were analyzed by analysis of variance with
antenna, segment and type of sensilla as within-subjects factors.
Each sensillum type was analyzed by analysis of variance
(ANOVA) with antenna and segment as within-subjects factors.
Results
Behavioural experiments
No behavioural differences emerged among the three groups
during the training procedures (F2,27 = 1.02, p = 0.375).
The analysis of variance of correct responses 1 h after training
revealed a significant effect of the antenna in use (F2,27 = 80.86,
p,0.001) (Figure 2). Post hoc comparison using Tukey HSD test
revealed a significant difference between bees using their right and
their left antenna (p,0.001), and between bees using their left
antenna and those using both antennae (p,0.001) and between
bees using their right antenna and bees using both antennae
(p,0.01).
Electroantennography (EAG)
The results of electroantennography are shown in Figure 3. The
EAG responses elicited by the tested odours, isoamylacetate and (-
)-linalool, were not significantly different between the right and the
left antenna (F1,19 = 2.72, p = 0.12). Though not lateralized at the
population level, 12 out of 20 individual bumble bees showed
significantly stronger responses (estimated by one-tailed binomial
test, p,0.05) either with the right (9 animals) or the left (3 animals)
antenna (one-tailed binomial test, p = 0.054). The ANOVA also
revealed a significant increase in EAG responses with increasing
doses of both tested odours (F4,16 = 42.52, p,0.001), a significant
effect of the type of odours (F1,76 = 107.61, p,0.001) and a
significant interaction between type of odours and dose
(F4,76 = 20.49, p,0.001).
Scanning Electron Microscopy (SEM)
The results of SEM analysis are shown in Figure 4. The overall
number of sensilla analyzed appeared to be higher on the right than
on the left antenna (F1,13 = 22.56, p,0.001). The analysis of
variance also revealed significant effect of segment (F7,91 = 43.20,
p,0.001), sensillum type (F5,65 = 396.40, p,0.001) and antenna per
sensillum type interaction (F5,65 = 17.89, p,0.001). Separate
analyses for each sensillum type revealed a significant right antenna
dominance in the number of olfactory sensilla trichodea type A
(F1,13 = 21.26, p,0.001); no significant antenna effects were found
in the number of sensilla basiconica (F1,13 = 1.47, p = 0.247), sensilla
coeloconica (F1,13 = 3.61, p = 0.08) and sensilla placodea
(F1,13 = 0.97, p = 0.342). Analyses of non-olfactory sensilla did not
reveal any significant difference between right and left antennae in
the number of sensilla trichodea type B (F1,13 = 3.45, p = 0.086) and
sensilla ampullacea (F1,13 = 0.10, p = 0.755).
Figure 1. Scanning electron micrographs of Bombus terrestris foragers. (a) ventral view of a medial segment of the flagellum; (b) details of
sensillum trichodeum type A, type B and sensillum placodeum; (c) details of sensillum coeloconicum, ampullaceum, trichodeum type B and setae; (d)
detail of sensillum basiconicum. Am, sensillum ampullaceum; Ba, sensillum basiconicum; Co, sensillum coeloconicum; Pl, sensillum placodeum; Se,
seta; TA, sensillum trichodeum type A; TB, sensillum trichodeum type B.
doi:10.1371/journal.pone.0018903.g001
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Discussion
The results extended previous findings on olfactory asymmetries
in hymenopteran insects [11,14], by showing a right side
dominance in short-term recall of olfactory memory in another
Apoidea species, B. terrestris. Bumble bees conditioned to extend
their proboscis (PER) revealed better learning performance when
trained with their right rather than their left antenna, with a
magnitude comparable to that previously found in A. mellifera
[11,14].
In honey bees lateralization of olfactory learning is associated
with morphological and electrophysiological asymmetries: the
number of olfactory sensilla and the electroantennographic
responses have been shown to be higher in the right than in the
left antenna [11,14,16]. In the present study no significant overall
differences in EAG responses between the right and the left
antenna of bumble bees were observed. Nevertheless, there was a
considerable trend when looking on the number of individuals
showing significant lateralization (12 vs. 20), and among this subset
of individuals the majority showed stronger responses with the
right antenna (9 vs. 3). Since electroantennography records the
sum of responses of all olfactory receptor neurons housed in the
sensilla of a single antenna, the results obtained using SEM might
explain the difference with the data obtained in honey bees. Only
one class of bumble bee olfactory sensilla, trichodea type A,
exhibited an anatomical asymmetry, being more abundant on the
surface of the right antenna than on the left one, and a slight
tendency emerged for a second class, i.e. sensilla coeloconica. On
the other hand, sensilla placodea, the most common olfactory
organs in Apoidea species, did not show any considerable
asymmetrical distribution in B. terrestris. This can explain why no
overall asymmetry was observed in EAG responses in bumble
bees. Other factors may have also contributed to the species
difference, i.e. the number of receptor neurons in each sensillum
category and the number of receptor sites in each olfactory
neuron, that could be independently associated with the gain or
loss of asymmetry in the mechanisms of peripheral perception.
The nematode Caenorhabditis elegans provides a striking example of
the multiple factors contributing to lateralized odour detection in
invertebrates. In this species it has been observed that a
symmetrical distribution of olfactory sensory neurons hides an
asymmetrical pattern on their surface of the G-protein-coupled
olfactory receptors responsible for functional odour lateralization
[4].
Kells and Goulson [26] noticed that three species of bumble
bees, Bombus lapidarius, Bombus lucorum and Bombus pascuorum,
showed preferences in the directions of circling when they visited
florets arranged in circles around a vertical inflorescence.
Interestingly, they did not observe any lateralization in B. terrestris.
It could be that lateralization in circling is mainly due to antennal
Figure 2. Behavioural asymmetry during recall of short-term
odour memory in Bombus terrestris foragers, after trained on
the proboscis extension reflex. Mean percent correct responses 6
SE 1 h after (-)-linalool conditioning with both antennae in use (white
bars), right antenna in use only (grey bars), or left antenna in use only
(black bars). A significant effect of the antenna in use was found
(ANOVA: F2,27 = 80.86, p,0.001). Post hoc comparison using Tukey HSD
test revealed a significant difference between bees using their right and
their left antenna (p,0.001), and between bees using their left antenna
and those using both antennae (p,0.001) and between bees using
their right antenna and bees using both antennae (p,0.01).
doi:10.1371/journal.pone.0018903.g002
Figure 3. Mean EAG ± SE absolute responses (mV) of right (unbroken lines with black squares) and left (dotted lines with empty
squares) antenna of Bombus terrestris foragers (N=20) to isoamyl acetate (left) and (-)-linalool (right) at five different doses (Log10
mg/ml). No significant differences were found between the antennae (ANOVA: F1,19 = 2.72, p = 0.12). Significant effects of both dose (ANOVA:
F4,16 = 42.52, p,0.001) and scent (ANOVA: F1,76 = 107.61, p,0.001) were revealed.
doi:10.1371/journal.pone.0018903.g003
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Lateralization of Olfaction in Bumble Bee
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asymmetries (and not to higher level mechanisms associated with
learning and memory recall). Even in honey bees the evidence
suggests that peripheral asymmetries in receptors density and EAG
antennal responses could not entirely account for asymmetries in
memory recall as evinced from PER responses. Rogers and
Vallortigara [13] showed that at 1–2 hour after training using
both antennae, recall was possible only when the honey bees used
their right antenna but by 6 hours after training the memory could
be recalled only when the left antenna was in use. Clearly,
asymmetries in receptor density could not account for this time-
dependent shift in lateralization associated with memory consol-
idation [16].
It has been argued, however, that lateralization of function
initially evolved in bilateral animals to increase brain efficiency
[17]. Thus, morpho-physiological biases of the peripheral nervous
system in insects could be a consequence of the embedded brain
asymmetry [14]. Furthermore, the rates of olfactory lateralization
in bumble bees evidenced for the first time in the present work
corroborates the hypothesis of a link between levels of social
interactions and the alignment of the direction of asymmetries in a
population. Mathematical models of the evolution of population-
level asymmetries based on game theory [17] pointed out that
shared directionality in a population might arise as an evolutionary
strategy driven by living in a social group, where individually
asymmetrical organisms have to coordinate their behaviours with
the behaviour of other conspecific individuals. The key concept
would be that an individual within a social group benefits from
acting according to the behaviour of the majority of the group
individuals. According to the model the minority group (e.g. in this
case bumble bees with better performance with the left antenna)
would be maintained by frequency-dependent selection. Ghir-
landa et al. [27] extended the mathematical model examining
intraspecific interactions, with antagonistic-synergistic behaviours.
They showed that the consistency of direction of asymmetries in a
population should result from the most relevant of the two
interactions, in term of fitness contribution. Populations with
higher rates of synergistic interactions were shown to be more
strongly lateralized in the same direction. Thus, the involvement of
inter-individual interactions could have been a crucial factor for
the evolution of lateralization in the olfactory associative learning
also in B. terrestris. With respect to honey bees, the bumble bees
annual society represents a less developed system in individuals
exchanging information [19,20]. They show a complete lack of
both trophallaxis and transmission of definite geographical cues
but the communication between colony members appears to play
a key role in the nest as well. As a matter of fact, bumble bee
foragers release from tergal gland a foraging recruitment
pheromone that induces the inactive workers to leave the nest in
search of food sources [28]. Moreover, a learning process of the
currently rewarding floral odours occurs inside the nest driven by
the olfactory information flow carried on the successful incoming
bees in the honey pots; it has been suggested that the inter-
individual contacts significantly improve odour learning and
foragers recruitment [29,30].
In conclusion, the data described here add to increasing
evidence that lateralization of the nervous system is common in
invertebrate species. Likewise, our findings on strictly related
Apoidea species with different forms of social organization may
confirm altogether the hypothesis of the relationship between
social behaviour and the evolution of population-level asymmetries
also in arthropods. Future studies on other species of bumble bees
(Bombus spp.), or other Apoidea species characterized by different
social or pre-social behaviours, such as gregarism, may provide
additional insights to understand how strategic inter-individual
interactions in a population have been powerful forces in the
evolution of asymmetries.
Acknowledgments
We are grateful to Bioplanet s.c.a. for providing bumble bees. Many thanks
are due to Anna Eriksson for the critical reading of the manuscript.
Author Contributions
Conceived and designed the experiments: GA ER EF FT GV. Performed
the experiments: GA ER EF VR FT. Analyzed the data: GA ER GV.
Contributed reagents/materials/analysis tools: GA GV. Wrote the paper:
GA ER GV.
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PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e18903