Odour Maps in the Brain of Butterflies with Divergent
Host-Plant Preferences
Mikael A. Carlsson1
*.
, Sonja Bisch-Knaden2.
, Alexander Scha¨pers1
, Raimondas Mozuraitis3,4
, Bill S.
Hansson2
, Niklas Janz1
1 Department of Zoology, Stockholm University, Stockholm, Sweden, 2 Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena,
Germany, 3 Department of Chemistry, Royal Institute of Technology, Stockholm, Sweden, 4 Laboratory of Chemical and Behavioural Ecology, Institute of Ecology, Nature
Research Centre, Vilnius, Lithuania
Abstract
Butterflies are believed to use mainly visual cues when searching for food and oviposition sites despite that their olfactory
system is morphologically similar to their nocturnal relatives, the moths. The olfactory ability in butterflies has, however, not
been thoroughly investigated. Therefore, we performed the first study of odour representation in the primary olfactory
centre, the antennal lobes, of butterflies. Host plant range is highly variable within the butterfly family Nymphalidae, with
extreme specialists and wide generalists found even among closely related species. Here we measured odour evoked Ca2+
activity in the antennal lobes of two nymphalid species with diverging host plant preferences, the specialist Aglais urticae
and the generalist Polygonia c-album. The butterflies responded with stimulus-specific combinations of activated glomeruli
to single plant-related compounds and to extracts of host and non-host plants. In general, responses were similar between
the species. However, the specialist A. urticae responded more specifically to its preferred host plant, stinging nettle, than P.
c-album. In addition, we found a species-specific difference both in correlation between responses to two common green
leaf volatiles and the sensitivity to these compounds. Our results indicate that these butterflies have the ability to detect
and to discriminate between different plant-related odorants.
Citation: Carlsson MA, Bisch-Knaden S, Scha¨pers A, Mozuraitis R, Hansson BS, et al. (2011) Odour Maps in the Brain of Butterflies with Divergent Host-Plant
Preferences. PLoS ONE 6(8): e24025. doi:10.1371/journal.pone.0024025
Editor: Frederic Marion-Poll, AgroParisTech, France
Received May 9, 2011; Accepted August 4, 2011; Published August 25, 2011
Copyright: ß 2011 Carlsson 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: The project was financed by The Swedish Research Council (NJ), the Stockholm University Faculty of Science (NJ), the Royal Swedish Academy of
Science (MAC), and the Max Planck Society (BSH). 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: mikael.carlsson@zoologi.su.se
. These authors contributed equally to this work.
Introduction
Larvae of phytophagous insects are dependent on the maternal
choice of oviposition site for survival and growth. Mated females
are guided to a potential host-plant primarily by visual or olfactory
cues. The final close-contact evaluation of an oviposition site may
rely on olfactory, gustatory or mechanical signals or a combination
of those [1]. Diurnal insects as butterflies are generally believed to
rely mainly on vision, while e.g. moths use olfaction to find a
suitable oviposition site [2]. However, whereas the olfactory
system is well studied in moths, their diurnal relatives have
attracted less attention. A handful of studies have reported odorant
evoked EAG responses from butterfly antennae [3,4,5]. However,
representation of odour information in the primary olfactory
centre, the antennal lobe (AL) has not previously been investigated
in butterflies.
Olfactory sensory neurons (OSNs) are housed in sensillae
located mainly on the antennae and labial palps [6]. All OSNs
expressing a specific type of olfactory receptor (OR) converge in a
spheroidal structure, glomerulus, in the AL [7,8,9,10,11]. The
number of glomeruli is therefore generally equivalent to the
number of different ORs. Functional imaging studies in the insect
AL have shown that different odorants evoke unique patterns of
activated glomeruli, which represent the activated OR types
[12,13,14,15,16]. The across-neuron principle and the convergence
of OSNs with identical OR type in the glomeruli create a
representation of the odour world in the brain, a ‘‘spatial code’’.
Results from studies in both vertebrates and insects suggest
common odour coding principles. Recently, it was shown that
there is indeed a high similarity of coding of aliphatic compounds
in ants, honeybees and rats [17]. This similarity does, of course,
not exclude that species-specific adaptations have evolved to
match requirements of the animal.
The butterfly family Nymphalidae contains about 6000 species
with divergent host plant range [18]. We have focused on two
species that differ in their degree of specialisation. The Comma
butterfly, Polygonia c-album, is a wide generalist feeding on herbs,
bushes and trees from families in four plant orders including the
stinging nettle, Urtica dioica [19], whereas the monophagous Small
Tortoiseshell, Aglais urticae, feeds mainly on U. dioica [20,21,22].
Specialisation on Urticales species seems to be an ancestral trait and
a widening of host plant range took place in a lineage in which P.
c-album belongs 20–30 million years ago [23]. This evolutionary
change may have been possible due to a pre-adaptation to a
polyphagous life-style or there may have been selection pressures
on modification of search mechanisms, e.g. olfaction, after a host
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shift. From earlier studies we know that P. c-album has a reduced
ability to discriminate between different qualities of U. dioica
compared to two specialist species, Inachis io and Vanessa indica [24].
In addition, an English population of P. c-album is more specialised
than the Swedish population and is actually better at discriminating
U. dioica from a non-host plant, Lamium album [25,26]. In these
behavioural studies it was not investigated if discrimination was
based on olfactory or other sensory cues.
The aim of the present study was to investigate the representation
of olfactory information in the ALs of the two butterfly species by
means of Ca2+
imaging. The major questions we wanted to address
were if the butterflies responded to plant related volatiles in an
odour-specific manner, i.e. with specific combinations of activated
glomeruli and if responses differed between the two species.
Results
Antennal lobe morphology is similar in the two species
First we performed an anatomical investigation of the ALs in the
two species. Figure 1 shows surface reconstructions of the ALs after
immunostaining with anti-synapsin. Both species have 60–65
glomeruli (n = 5 of each species). The glomerular diameter (widest)
was first measured in all visible glomeruli and then averaged in
each animal. The median diameter across animals was 46.56
2.2 mm (n = 5 animals) in P. c-album and 48.261.6 mm in A. urticae
(n = 5). Glomerular diameter did not differ between the species
(p = 1.0; Mann-Whitney-U test). Thus, the morphology of the ALs
in the two species is very similar and makes these species
appropriate for a physiological comparison.
Responses to plant extracts are more specific in A. urticae
Secondly, we used Ca2+
imaging to study AL responses to
antennal stimulation with plant extracts and single compounds.
Figure 2 shows typical examples from one individual of each
species of false-colour coded images of responses to the plant
extracts superimposed on wide-field images of the lobes. Each
response is scaled to its own intensity range and the false-colour
code ranges from 50% of activity to maximal activity. All extracts
activated multiple glomeruli. The solvent cyclohexane also evoked
responses but much weaker than the plant extracts.
We calculated correlation indices between all pairs of responses
to plant extracts. The similarity matrices in Figure 3A show the
median correlation values for P. c-album and for A. urticae. The
matrices were significantly correlated with each other (r = 0.64,
p = 0.0003, Mantel test). However, visual inspection of the
similarity matrices show that the responses were often less similar
in A. urticae than in P. c-album. Next we calculated the correlation
between responses to U. dioica and responses to all other plant
extracts. The correlation coefficients were significantly lower in A.
urticae than in P. c-album (Fig. 3B; p,0.001; Mann-Whitney-U test).
Responses to single compounds are generally similar
between the species
We used 18 different plant-related compounds (Fig. 4A). All
odorants except octanoic acid and nonanoic acid elicited reliable
and repeatable responses in all animals. Therefore, we excluded
these two compounds in the following analyses. The remaining 16
odorants evoked activity in different combinations of glomeruli.
Based on pairwise correlations of all odorants we constructed
similarity matrices (Fig. 4B). These matrices were significantly
correlated with each other (r = 0.75, p,0.0001, Mantel test). In
addition, we did a principal component analysis. Figure 4C show
graphic representations of the first two principal components,
which describe 94% and 81% of the total variance of the data,
respectively. The first component is significantly correlated between
the species (r = 0.80; p = 0.0002; Spearman rank correlation).
That is, in general odour-evoked activity is highly similar in
the two species.
Relation between structural similarity and neural
response similarity
To assess the relation between molecular structure and neural
response we used an odour panel consisting of three series of
aliphatic compounds (alcohols, aldehydes and ketones) with
differing chain lengths (6, 8 or 9 carbon atoms). We calculated
pairwise correlations between compounds with the same functional
group but different chain length. With the exception of alcohols
in A. urticae, responses to two compounds differing by only one
carbon atom were more correlated than compounds differing by 3
carbon atoms, disregarding the functional group (Fig. 5A; Kruskal
Wallis test followed by Dunn’s Multiple Comparison Test). In A.
urticae, however, responses to alcohols differing by 2 carbon atoms
differed from responses to alcohols differing by 3 carbon atoms.
That is, compounds with similar chemical structure generally
evoked more similar activity patterns than compounds with less
similar structures.
In addition, we compared responses to compounds sharing
chain length but with different functional groups. The correlation
between responses to 6-alc and 6-ald was significantly lower in A.
urticae than in P. c-album (Fig. 5B; p,0.05; Mann-Whitney-U test).
No other pairs were significantly different.
Species differences in sensitivity to green leaf volatiles
In the test above we found that responses to two commonly
occurring green leaf volatiles, GLVs [1], 6-alc and 6-ald differed
between the species. Therefore we tested sensitivity over a range of
doses to these compounds and, as a control, to the floral compound
geraniol (Fig. 6). We identified the glomerulus with the strongest
activity at each dose and compared this response to the response to
the solvent (two-tailed Wilcoxon matched-pairs signed rank test). P.
c-album responded to 6-alc down to 4.861022
mg compared to
4.86100
mg for A. urticae. With 6-ald the sensitivity was reversed with
A. urticae being more sensitive than P. c-album (4.861022
mg and
4.86100
mg, respectively). Sensitivity to geraniol was similar in the
two species (threshold 5.26100
mg).
Figure 1. Antennal lobe reconstructions. Frontal views of surface
reconstructions based on optical sections of the ALs. Outlines show the
borders of the ALs. L, lateral; D, dorsal. Scale bar = 50 mm. AN, antennal
nerve.
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In summary, we found that responses to plant extracts and
single plant related compounds were generally similar in the two
species. However, we showed that A. urticae responded more
discriminatively than P. c-album to the common host plant U. dioica.
In both species we found a correlation between structural
similarity and the similarity of responses, whereas both correlation
between hexan-1-ol and hexan-1-al and sensitivity to these
compounds differed between the species.
Discussion
Searching for a suitable host plant may involve input from
different sensory modalities. As opposed to e.g. moths, diurnal
insects as butterflies are believed to mainly rely on vision when
trying to locate an oviposition site [2]. However, in the present
study we showed that two species of Nymphalid butterflies have
the neuronal capacity to respond to extracts of host- and non-host
plants and to a number of plant-derived compounds. This is the
first study showing odour-evoked activity in the butterfly AL. Not
surprisingly, we found that the tested compounds generally evoked
unique combinations of activated glomeruli. Furthermore, even
though responses to the tested odorants generally were similar
between the species we also found species-specific differences.
Despite that butterflies diverged from moths at least 100 Ma ago
[27,28] and that they have adapted to a diurnal lifestyle,
anatomical and physiological characteristics of the olfactory
system seem to be conserved. The ALs of the butterflies in our
study consist of 60–65 glomeruli, which is numerically comparable
to moths [29,30,31,32]. To our knowledge, the only previously
published anatomical study of the AL of butterflies showed that the
number of glomeruli in Pieris brassicae is 6263 [33]. If we can
extrapolate the rough one-to-one relationship of receptors to
glomeruli in Drosophila [8,11] we can assume that these butterflies
have approximately 60 receptor types. The odour-evoked
responses in the AL showed similarities to what has been observed
in other animals [13,14,15,16,17,34,35]. Most of the odorants
evoked unique, though overlapping, patterns of activated glomeruli.
Another feature that has been observed in several species is the
correspondence between structural similarity and the similarity of
neural responses [17,36,37]. One aspect of structural feature that
Figure 2. Responses to plant extracts. False-colour coded images of odour-evoked activity to plant extracts and control (solvent) superimposed
on wide-field images of the lobes. Each response is scaled to the upper 50% of its intensity range. D, dorsal; M, medial. AN, antennal nerve; AL,
antennal lobe. Scale bar = 100 mm.
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can be varied systematically is chain length. We showed that the
distance between compounds with differing chain length generally
corresponded with the similarity of glomerular responses.
Even though the similarity matrices in Figure 2 did not differ
significantly between the two butterfly species we found that
responses to stinging nettle, U. dioica, was less similar to responses
to other plant extracts in A. urticae than in P. c-album. Whether this
neural response similarity also corresponds to perceptual similarity
[38,39,40] remains to be investigated. Even though A. urticae is
more discriminative when choosing a host plant than P. c-album
[20,21,22] we do not know if the choice is based on olfaction or
other sensory input. In a recent study it was, however, demonstrated
that another butterfly species, P. rapae, could behaviourally
distinguish a host plant from a non-host plant exclusively by
olfactory (but not visual) cues [41].
Both species responded to all single compounds except octanoic
and nonanoic acid. The reason for the lack of measurable
responses to these compounds may be either that the butterflies do
not possess receptors that can be activated, at least not at the
concentrations used in this study. An alternative explanation is
that glomeruli innervated by OSNs responding to these acids are
located on the caudal side of the AL, which is not accessible for
imaging. Responses to single compounds were generally similar
between the species. We found, however, that the correlation
between responses to two common GLVs differed between the
species in that glomerular activity patterns evoked by these
compounds were less similar in A. urticae than in P. c-album. We
further tested if sensitivity to these GLVs differed between the
species and found that P. c-album was more sensitive to a sixcarbon
alcohol than A. urticae. For a six-carbon aldehyde the
Figure 3. Responses to plant extracts. A Similarity matrices of responses to plant extracts in P. c-album (left, n = 12) and A. urticae (right, n = 9).
The colour coding depicts the median correlation index according to the scale. B The graph shows the correlation between responses to U. dioica
and responses to all other plant extracts in the two species (** p,0.001; Mann-Whitney-U test). A box plot shows the median correlation index of a
pair of odorants (horizontal line), the 25th and 75th percentile (lower and upper margin of the box) together with the minimum and maximum values
(whiskers). Colour coding as in A.
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situation was reversed, with A. urticae being more sensitive of the
species. When we tested the sensitivity to a floral compound,
which probably has no relevance for host plant search, there was
no difference between the species. Green leaf volatiles are emitted
in small amounts by intact green leaves [1] and it has been
demonstrated that these compounds are important cues for many
insects when trying to locate and identify a host plant. For
example, in the Colorado beetle, Leptinotarsa decemlineata, a correct
ratio between GLVs emitted from potato leaves is essential for
eliciting positive anemotaxis [42]. The plants used in our study
emit GLVs in different quantities and ratios. The two birch
species, e.g. emit 6-ald and 6-alc at markedly different ratios [43].
Preliminary data show that U. dioica emits more 6-ald than 6-alc
compared to R. nigrum and the Betula species ([43], R Mozuraitis,
personal communication). It should be noted that it is very difficult
to compare release of volatiles from different plants. Sampling
methods, all kinds of stress, seasonal variations and even variations
within different parts of a plant are factors that may influence the
analysis of released volatiles. However, in the present study we
always used the same extracts in both species and the fact remains
that A. urticae and P. c-album responded differently.
The diurnal lifestyle of butterflies and the evolutionary
divergence from moths more than 100 Ma ago [27,28] may have
generated some differences in the olfactory system. However, the
morphology of the AL is similar and most of odorants used in this
study has also been shown to elicit responses in different moth
species [12,13]. As moths are well-known for their extreme
sensitivity to odorants [44] it would be interesting to compare it
to butterflies. In a study investigating concentration dependency of
glomerular activity patterns in a moth [45] using the same
Figure 4. Responses to single compounds. A Molecular structures of the tested plant related compounds. B Similarity matrices of responses to
these compounds. Median correlation values are colour-coded according to scale. Correlation between the species r = 0.75 (p,0.0001, Mantel test). C
A principal component analysis showing the first two components for each species. Correlation of the first component (PC1) r = 0.80 (p = 0.0002,
Spearman rank correlation).
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methodology as in our study it was shown that the lowest dose of the
floral compound geraniol the moth responded to was 0.1 mg. In our
study we observed a significant response at 4.8 mg but not at
0.48 mg. This indicates that moths may be more sensitive, at least to
certain odorants.
It has been suggested that odour quality is encoded in a spatial
across-glomeruli (and thus across-OSN) code in the AL, e.g. [33],
and that this code can be visualised by different functional imaging
techniques [15,16]. Studies in different species have shown that
similarity of neural responses is correlated with perceptual similarity
of odorants [38,39,40]. The more similar the combinatorial activity
patterns the more difficult it is to discriminate between the odorants.
Hence, our results indicate that A. urticae may have a higher ability to
perceptually distinguish odours of a host plant from non-host plants
and also 6-alc from 6-ald than P. c-album. However, if glomerular
activity patterns observed in butterflies can be related to behaviour
needs to be examined in future studies. As bath application with a
Ca2+
-sensitive dye is believed to mainly reflect input activity in the
AL [46,47] it would be very interesting to investigate how olfactory
information is processed and finally shaped in the PNs. It is likely
that differences between species may be further enhanced due to
shaping by the AL network. For example, the level of neuroactive
substances in the AL may vary considerably, even during
development of an individual [48,49]. Therefore, we may assume
that larger differences between species may have evolved concerning
release and signalling with transmitters and modulators in the
AL than the responsiveness of the ORs.
In conclusion, odour-evoked activity in the ALs of two species of
nymphalid butterflies with diverging host plant range was
combinatorial across glomeruli and mostly odour unique. This
means that the butterflies not only have the ability to detect but it
also indicates that they may be able to discriminate between
Figure 5. Correlation between chemical structure and response. A Pairwise correlations between compounds with the same functional
group but different chain length. In each group (alcohols, aldehydes or ketones) in a graph the chain length of the compounds differ by 1, 2 or 3
carbon atoms, respectively. Bars capped by different letters differ significantly within the chemical group (Kruskal Wallis test followed by Dunn’s
Multiple Comparison Test). A box plot shows the median correlation index of a pair of odorants (horizontal line), the 25th and 75th percentile (lower
and upper margin of the box) together with the minimum and maximum values (whiskers). The colour coding depicts the median correlation index
according to the scale. A. urticae (n = 10) and P. c-album (n = 15). B Correlation of responses to two compounds (6-alc and 6-ald) with the same chain
length but different functional group (* p,0.05; Mann-Whitney-U test).
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plant-related odorants. Even though responses were often similar
across the species, the more specialised of the tested species, A.
urticae, responded more discriminatively to certain host plant-related
odours than P. c-album. Future studies will hopefully reveal if
our findings have a behavioural relevance. We have, however,
demonstrated that the butterflies have a neural substrate for
detection and discrimination of plant-related volatiles and that there
are species-specific differences that may be evolutionarily important.
Materials and Methods
Animals
Two species of butterflies belonging to the family Nymphalidae
were used in the experiments. These were the Comma Butterfly,
Polygonia c-album (Lepidoptera: Nymphalidae; Linneus), and the
Small Tortoise Shell, Aglais urticae (Lepidoptera: Nymphalidae;
Linneus). The parental generation of P. c-album and A. urticae were
caught in the vicinity of Stockholm, Sweden and their offspring
was used. No permits were required in accordance with the
Swedish principle of public access to the wilderness. Both mated
and virgin females were used of P. c-album whereas only virgin
females were used of A. urticae. All animals were between 3–5
weeks old when tested and the mated females were tested at least 2
weeks after mating. We did not detect any difference in response
between mated and virgin P. c-album when we tested them
separately, which justified pooling of data in the subsequent
analyses. All animals were kept separated in a low temperature
(9uC) chamber (12/12-h light/dark cycle) until start of experiment
Figure 6. Dose-response correlations. Responses to hexan-1-ol, hexan-1-al and to geraniol at different doses in P. c-album (n = 14, 8 and 9,
respectively) and A. urticae (n = 11, 8 and 7, respectively). The x-axes depict the doses; 22–2 means 4.861022
–4.86102
mg (6-alc and 6-ald) or
5.261022
–5.26102
mg (Ger). Response values are normalised to the strongest response in each animal. The glomerulus with the strongest activity at
each dose is compared with the response to the solvent in that particular glomerulus. A response to an odorant (grey bar) significantly differing from
a response to the solvent (white bar) is depicted with asterisks above the bars (two-tailed Wilcoxon matched-pairs signed rank test; * p,0.05,
** p,0.01, *** p,0.001). A box plot shows the median normalised response to an odorant (horizontal line), the 25th and 75th percentile (lower and
upper margin of the box) together with the minimum and maximum values (whiskers).
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and were fed with sugar water ad libitum. Animals were moved to
room temperature 24 hrs prior to the start of the experiment.
Immunostaining and AL reconstruction
Brains of butterflies were dissected in phosphate-buffered saline
with 0.25% Triton-X (PBS-Tx) and fixed overnight at 4uC in 4%
paraformaldehyde in 0.1 M sodium phosphate buffer. After
careful rinsing in PBS-Tx the brains were preincubated overnight
in 5% normal goat serum in PBS-Tx. Brain tissue was incubated
for 48-72 hours in mouse monoclonal anti-synapsin (antiSYNORF1,
1:10; Developmental Studies Hybridoma Bank, Iowa
City, IA.). For detection of antiserum we used a Cy3-tagged
secondary antibody (1:100; Jackson ImmunoResearch, West
Grove, PA).
Brains were dehydrated in a series of increasing ethanol
concentration (50–100%) and finally cleared and mounted in
methyl salicylate. The preparation was scanned with a Zeiss LSM
510 META (Zeiss, Jena, Germany) confocal laser scanning
microscope and images were obtained at optical section thickness
of 0.27–0.31 mm with a 40x oil objective. Three-dimensional
surface reconstructions of confocal stacks were generated using the
segmentation tool in AMIRA 4.1 software (Mercury Computer
Systems GmbH, Berlin, Germany) and edited for contrast and
brightness in Adobe Photoshop CS4 Extended (v. 11.0, San Jose,
CA).
Preparation and staining
Animal preparation was similar to previous studies in moths
[13,45,50]. Briefly, animals were placed in a 1000 ml pipette tip
with the tip cut open to fit the head. The protruding head at the
narrow end was fixed in this position with dental wax. Labial palps
and proboscis were removed to reduce movements during the
experiments. A window was cut in the head capsule between the
compound eyes and the tissue covering the brain was removed to
uncover the antennal lobes. The membrane-permeant fluorescent
calcium indicator (Calcium Green-2 AM, Molecular Probes) was
dissolved in physiological saline [51] with 20% Pluronic F-127
(Molecular Probes) to a final concentration of 30 mM. A drop of
this dye solution was bath applied to the exposed brain and the
preparation was incubated for about 60 min at 4uC. The brain
was subsequently rinsed several times with physiological saline to
remove excessive dye. Bath application with Calcium Green-2 AM
potentially stains different types of cells in the AL. However,
odour-evoked responses are supposed to originate mainly from
OSNs [46,47].
Optical imaging
The imaging set-up consisted of an air-cooled 12-bit slow-scan
CCD camera (Olympus U-CMAD3) mounted to an upright
microscope (Olympus BX51WI) equipped with a water immersion
objective (Olympus, 20x/0.95). Calcium green-2 AM was excited
at 475 nm (500 nm SP; xenon arc lamp, Polychrome V, Till
Photonics) and fluorescence was detected at 490/515 nm (DCLP/
LP). The set-up was controlled by the software Tillvision 4.0 (Till
Photonics). Four-fold symmetrical binning resulted in image sizes
of 3446260 pixels with one pixel corresponding to an area of
1.25 mm61.25 mm.
Odour stimuli
We used a setup of 18 odorants (Sigma-Aldrich) belonging to
different structural groups. These were: a) aromatics: methyl salicylate
(Mes), CAS-#: 119-36-8, phenyl acetaldehyde (Paa), CAS-#: 122-78-
1; b) alcohols: hexan-1-ol (6-alc), CAS-#: 111-27-3, octan-1-ol (8-alc),
CAS-#: 111-87-5, nonan-1-ol (9-alc), CAS-#: 143-08-8; c)
aldehydes: hexanal (6-ald), CAS-#: 66-25-1, octanal (8-ald), CAS#:
124-13-0, nonanal (9-ald), CAS-#: 124-19-6; d) ketones: 2hexanone
(6-ket), CAS-#: 591-78-6, 2-octanone (8-ket), CAS-#:
111-13-7, 2-nonanone (9-ket), CAS-#: 821-55-6; e) acids: hexanoic
acid (6-aci), CAS-#: 142-62-1, octanoic acid (8-aci), CAS-#: 124-
07-2, nonanoic acid (9-aci), CAS-#: 112-05-0; f) terpenes: geraniol
(Ger), CAS-#: 106-24-1, (6)-linalool (Lin), CAS-#: 78-70-6, bcaryophyllene
(Car), CAS-#: 87-44-5 and g) acetates: Z3-hexenyl
acetate (z3ha), CAS-#: 3681-71-8. Purity of the compounds was
.97% (except 9-alc: 95%, Paa: 90%, Ger: 96%. All odorants were
diluted in mineral oil (Mol, Sigma-Aldrich) to a ratio of 1:100 (vol/
vol). For the dose-response experiment odorants were diluted in
decadic steps (from 1:100000 to 1:10). These dilations correspond to
4.861022
–4.86102
mg of 6-alc and 6-ald and mg 5.261022
–
5.26102
mg of Ger when 6 ml were applied to a filter paper.
In addition, we used extracts of seven different plants: Betula
pendula (N), Betula pubescens (P), Cirsium arvense (N*), Lamium album
(N), Malva moschata (N*), Ribes nigrum (P) and Urtica dioica P, A).
Abbreviations in brackets denote non-host plants (N) or natural
host plants of P. c-album (P) and A. urticae (A), respectively. The
plant species denoted with an asterisk are host plants to related
nymphalid species [22]. Intact leaves of plants (total area 40 cm2
)
were removed from the stems and placed in 200 ml glass beakers
containing ether. The leaves were handled carefully to reduce
damage. The solution was left to evaporate and subsequently
filtered and dissolved in cyclohexane. The same extracts were used
to test responses in all animals.
Odorant stimulation
Six ml of the diluted odorants or plant extracts were applied
onto a circular piece of filter paper (diameter: 12 mm); 6 ml of
either mineral oil (single compounds) or cyclohexane (extracts)
served as control stimuli. Filter papers were inserted into glass
Pasteur pipettes and were renewed every day. A humidified and
charcoal-filtered continuous air stream (1 l/min) was ventilating
the antenna ipsilateral to the recorded AL through a glass tube
(5 mm inside diameter). The glass tube ended ,10 mm from the
distal part of the antenna. An empty Pasteur pipette was inserted
through a small hole in the glass tube, blowing an air stream of 0.1
l/min. Another air stream (0.1 l/min) was blown through the
odour-laden pipette by a computer-triggered puffer device
(Syntech, Hilversum, The Netherlands) during 2 s (starting at
frame 8) into the continuous stream of air. During stimulation, the
air stream was switched from the empty pipette to the odour-laden
one in order to minimize the influence of added air volume. One
odorant stimulation experiment lasted 10 s and was recorded with
a sampling rate of 4 Hz corresponding to 40 frames. The time
course was as follows: 2 s clean airstream (frame 1–8), 2 s stimulus
airstream (frame 9–16), and 6 s clean airstream (frame 17–40). We
used at least 60 sec interstimulus periods to reduce adaptation.
Analysis and statistics
With the software Till-Vision we constructed false-colour coded
images of relative changes of fluorescence intensity during the peak
time of activity. By visual inspection of activity maps elicited by all
odorants we drew circular regions of interest (ROI, 20 pixels
diameter corresponding to 25 mm, which is roughly 50% of the
diameter of an average glomerulus) round the centres of activity.
This resulted in 14–17 ROIs in all animals. An additional ROI
was drawn in an area with minimal activity, which served as a
control. In an earlier study in moths it was demonstrated that
activity foci correspond to individual glomeruli [45]. The mean
pixel value within a ROI was calculated for each time-point (40
Odour Maps in the Butterfly Brain
PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e24025
frames) in a sequence and exported to Microsoft Excel. In Excel
we first made a temporal median filtering of data over three
consecutive frames. Secondly we calculated the relative fluorescence
(dF/F) where F was defined as the mean value of frames 2–
7. To correct for bleaching we subtracted the values of the control
ROI from the values of all other ROIs for each recording. A
response was finally defined as the mean of frames 12–15 (peak of
activity). This means that for every odorant we have a response
profile with 14–17 response values that correspond to the relative
changes in fluorescence intensity in the ROIs.
Correlation values were calculated based on the response
profiles (above) with JMP 8 (SAS Institute Inc.). Statistical analyses
were made using Prism 5 (GraphPad Software, Inc.), XLSTAT
(Addinsoft), and PAST (Hammer and Harper).
Acknowledgments
The monoclonal antibody developed by E. Buchner was obtained from the
Developmental Studies Hybridoma Bank developed under the auspices of
the NICHD and maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA.
Author Contributions
Conceived and designed the experiments: MAC NJ SB-K. Performed the
experiments: MAC AS. Analyzed the data: SB-K MAC AS. Contributed
reagents/materials/analysis tools: BSH RM. Wrote the paper: MAC.
Original idea: MAC BSH NJ.
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