Glomerular representation of plant volatiles and sex pheromone components in the antennal lobe of the female Spodoptera littoralis
1 Division of Chemical Ecology, Department of Ecology, Lund University,
SE-223 62 Lund, Sweden
2 Division of Chemical Ecology, Department of Crop Science, Swedish
University of Agricultural Sciences, PO Box 44, SE-230 53 Alnarp,
Sweden
3 Unité de Biométrie and Unité de Phytopharmacie,
Institut National de la Recherche Agronomique, F-78026 Versailles Cedex,
France
* Author for correspondence (e-mail: sylvia.anton{at}vv.slu.se )
Accepted 27 February 2002
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Summary |
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Key words: plant volatile, pheromone component, moth, Spodoptera littoralis, antennal lobe interneurone, olfactory processing, glomerular map
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Introduction |
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In contrast to the voluminous literature dealing with the central
processing of sex pheromone in male moths, the number of corresponding studies
on the central processing of plant volatiles is relatively limited.
Plant-derived stimuli are, however, no less important for phytophagous insects
than is the sex pheromone. Whereas host plant odours induce landing responses
and egg laying by females (Guerin et al.,
1983; Barata and Araujo,
2001
), odours emitted by naturally resistant plants or by
previously infested host plants often deter females as they signal poor
nutritional qualities, likely high intraspecific competition and potential
risk of predation or parasitism
(Langenheim, 1994
;
Turlings et al., 1995
). The
noctuid moth, Spodoptera littoralis (Boisd.), has been well studied
with respect to plant volatile-guided behaviour and detection of compounds
involved by ORNs on the antennae. Several plant-derived components have been
identified as oviposition deterrents, natural host plant odours or
herbivore-induced volatiles, and ORNs responding specifically to these
components have been described (Anderson et al.,
1993
,
1995
;
Jönsson and Anderson,
1999
). Whereas sex pheromone-related information is mostly
processed in the MGC in male ALs, plant volatile information is known to be
processed in the so-called ordinary glomeruli that are present in both male
and female moths (Anton and Hansson,
1994
,
1995
); but how specifically
plant volatile receptors project into the AL and how this information is
subsequently integrated is hardly known.
Using Ca2+ imaging techniques, studies on S. littoralis
(Hansson et al., 2000) as well
as on other species (Joerges et al.,
1997
; Galizia et al.,
2000
; Galizia and Menzel,
2001
) suggest that individual plant compounds can be represented
in identifiable single glomeruli or specific groups of glomeruli. Anton and
Hansson (1994
) described
arborisation patterns of plant odour-sensitive PNs that vary in their
specificity, but accurate identification of the innervated glomeruli demanded
an atlas of the AL glomeruli in S. littoralis, which was not
available at that time.
In addition to presenting a three-dimensional map of the glomeruli within the anterior half of the AL of female S. littoralis, the aim of the present work was to investigate whether the dendritic arborisations of plant odour-sensitive PNs convey distinct patterns, representing particular compounds or groups of plant-derived compounds. We also aimed to study the physiological integration of olfactory information in comparison with what is already known from previous studies carried out at the receptor-neurone level in this species.
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Materials and methods |
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Stimulation
The antenna ipsilateral to the recording site was ventilated by a steady
stream of charcoal-filtered and moistened air that passed through a glass tube
at a velocity of approximately 0.5 m s-1. Stimuli were presented by
inserting a Pasteur pipette, containing a piece of filter paper that carried
the stimulus, in the glass tube 20 cm upstream from the end of the tubing
where the antenna was placed. A 0.5 s air pulse (4 ml s-1) was then
sent through the Pasteur pipette by means of a stimulation device (Syntech).
The stimuli were presented randomly at 10 s intervals.
Eight plant volatiles and two sex pheromone components were used as
stimuli. The eight behaviourally relevant plant compounds used were: (1)
-humulene (1 ng); (2) ß-caryophyllene (10 ng); (3) geraniol (100
ng); (4) eugenol (100 ng); (5) benzaldehyde (1 µg); (6) (±)-linalool
(10 ng); (7) (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (1 ng);
and (8)
-farnesene (10 ng). The two sex pheromone components were: (9)
(Z,E)-9, 11-tetradecadienyl acetate (Z,E-9, 11-14:OAc) (10
µg); and (10) (Z,E)-9, 12-tetradecadienyl acetate (Z,E-9,
12-14:OAc) (10 µg). Although the behavioural significance of sex pheromone
in the life of female S. littoralis is not clear, the plant compounds
used are known to be emitted naturally from the preferred host plant of the
moth, cotton (1-3), oviposition deterrents (4,5) or inducible plant volatiles
that signal lower quality and increased resistance of a given plant (6-8)
(Anderson et al., 1995
;
Jönsson and Anderson,
1999
). The above doses for sex pheromone components were the same
as those used by Anton and Hansson
(1994
) and are only slightly
higher than the threshold in peripheral receptor neurones
(Ljungberg et al., 1993
). As
for
-farnesene, linalool and
(E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, we chose doses that
elicit ORN response (Jönsson and
Anderson, 1999
). For the other five stimuli we determined
threshold levels by using three different doses during more than 100
preliminary tests and chose a final amount above threshold for the study. Nine
of the compounds were dissolved in hexane and one (
-humulene) was
dissolved in paraffin oil. Solvent (10 µl) containing the given amount of
chemicals was applied to pieces (5x15 mm) of filter paper in Pasteur
pipettes and were renewed every day. A Pasteur pipette containing a clean
filter paper or a filter paper with solvent was used as control. Responses to
solvent blanks were not different from the mere filter paper blank. Purity of
the chemicals was more than 95 % (except
-farnesene, 85 %). Chemicals
were purchased from Sigma (St Louis, MO, USA). Not all neurones were exposed
to all ten compounds. The first half of the work was carried out using only
seven compounds, where
-farnesene and the two sex pheromone components
were not included. Whenever a contact could be kept long enough, each stimulus
was tested at least twice during the course of an experiment.
Intracellular recording and staining techniques
Standard intracellular recording and staining techniques were used
(Christensen and Hildebrand,
1987; Kanzaki et al.,
1989
). Using a micromanipulator, a glass recording electrode, with
the tip filled with 1 % Neurobiotin in 0.25 mol l-1 KCl, was
inserted into the AL. The electrode was placed close to the centre of the AL
and most successful recordings were obtained with the electrode situated close
to the surface. When intracellular contact was established, the ipsilateral
antenna was stimulated and the activity of the neurone before, during and
after stimulation was observed on a Tektronix digital oscilloscope. The
physiological data were stored on video tape for further analysis.
Physiologically characterised neurones were stained with Neurobiotin by passing 0.5-1 nA of constant depolarising current through the recording electrode for 2-10 min. Brains were dissected, fixed in 4 % buffered formaldehyde solution overnight at room temperature, incubated in Alexa NeutrAvidin 488 (Molecular Probes) at 4° C and viewed as whole mounts in a laser scanning confocal microscope (Leica TCS NT). After confocal imaging, selected brains were embedded in Spurr's resin and sectioned at 10 µm. Serial sections were photographed on Fuji Sensia 400 colour slide film, and the neurones were reconstructed from the slides.
Analysis of responses
Action potentials were counted manually from the storage oscilloscope. The
number of action potentials counted during a 600 ms period after the stimulus
had reached the antenna minus the number of action potentials counted during
the preceding 600 ms (representing the spontaneous activity of the neurone)
was noted as the net number of action potentials. The net number of action
potentials produced in response to the blank stimulus (caused by mechanical
stimulation through the applied air pulse) was subtracted from the net number
of action potentials produced in response to an odour stimulus, to quantify
the response to a specific stimulus in one neurone. A 10-50 % increase in the
number of action potentials were considered a weak response (+). An increase
of more than 50 % up to 200 % in the action potential number was considered an
intermediate response (++). An increase of more than 200 % was considered a
strong response (+++).
Neuroanatomy
Brains were stained with a synapsin antibody
(Klagges et al., 1996) and the
ALs were mapped and used as a reference for locating the particular glomeruli
innervated by stained neurones in the intracellular experiments. For this
purpose, brains of female moths were dissected and fixed in 4 %
paraformaldehyde in 0.1 mol l-1 phosphate buffer, incubated 5 days
in the primary mouse antibody against synapsin (1:50) at room temperature,
followed by incubation in the secondary antibody, rabbit- anti-mouse
conjugated with FITC (Sigma, 1:150) for 4 days at 4°C. Brains were then
cleared and mounted in 80 % glycerol and optically sectioned using the
confocal microscope.
The AL glomeruli were reconstructed using Imaris 2.7 software (Bitplane), running on a Silicon Graphics computer work station. Right and left ALs from six animals were reconstructed and compared. Glomeruli occupying the anterior aspect of the AL were unmistakably visible in all preparations, whereas posteriorly located ones were less clearly discernible. Unequivocally recognisable glomeruli were reconstructed by manually delineating the borders of each glomerulus in every optical section. A three-dimensional map of the anterior half of the AL was thus created. Of the six maps, the one that had the largest number of clearly delineated glomeruli was chosen as reference.
Every AL with a stained neurone was similarly treated and its
three-dimensional map was compared with the reference map to locate the
specific glomerulus in which the stained neurone arborised. The orientation of
the obtained maps was visually decided depending on the known position of the
lateral cluster of cell bodies (Anton and
Hansson, 1994), the position of the antennal nerve entrance, the
location of landmark glomeruli (see Results) that occupied fixed places in all
specimens, and the symmetrical relationship between right and left ALs in each
preparation.
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Results |
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The remaining reconstructed glomeruli deviated to some extent in size, shape or position between ALs. 23 of these glomeruli were clearly recognised in the specimen chosen as a model and in another AL. In the remaining four ALs, only 22 glomeruli were detected. To facilitate identification, we assigned them to three groups of glomeruli: the first consisted of the two rows adjacent to the entrance of the antennal nerve (dark green, Fig. 1); the second was composed of the next two rows, lying in the middle of AL (yellow, Fig. 1); and the third included the remaining glomeruli in the medioventral part of the AL (pink, Fig. 1). We numbered the glomeruli in the model AL serially, counting every row from the lateral side and moving toward the median aspect of the brain (Fig. 1). The smallest and the largest diameters of the reconstructed glomeruli, including the landmark ones, were 35 and 75 µm, respectively.
Physiological characteristics of olfactory interneurones
Physiological responses of 153 AL interneurones in 146 S.
littoralis females were recorded. 17 neurones, in the brains of 12
animals, did not respond to any of the tested odorants. The remaining 136
interneurones (in 134 animals) responded to at least one compound. Responses
in PNs and local interneurones (LNs) were characterised by a sudden increase
in action potential frequency after the onset of the stimulus with different
delays in individual neurones, and sometimes by inhibition after the end of
the stimulus, before the level of spontaneous activity was re-established
(Fig. 2). Neurone responses
never started with initial inhibition and neither were purely inhibitory
responses found. Repeated stimulation with the same odour during the time
course of an experiment resulted in similar responses
(Fig. 5B). PNs and LNs could
not be easily distinguished on purely physiological grounds (see Figs
4,
5). Action potential amplitudes
varied between 10 and 45 mV.
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Specificity of the responses of AL interneurones to the 10 odorants varied
widely. 43 neurones responded to only one compound, whereas 31, 22 and 16
neurones responded to two, three and four compounds, respectively. The
remaining 24 neurones responded to several (5-8) compounds
(Fig. 3A). None of the examined
cells responded to more than eight odorants. Responses to -humulene
were most frequently obtained (38 % of the tested neurones), while responses
to most other compounds occurred in approximately 30 % of the tested neurones.
The lowest response frequency was observed for
-farnesene. A response
was obtained only in 21 % of the neurones when
-farnesene was applied
(Fig. 3B).
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Profiles of responses to different odorants are shown in Tables
1,2,3,4.
Neurones responding specifically to each of the ten tested compounds were
found. Among the 43 specific neurones, 14 responded to -humulene
(numbers 1-10, 118, 121, 126 and 127) and six neurones responded to geraniol
(numbers 14-18 and 120). Only one neurone (number 128) responded specifically
to Z,E-9, 11-14:OAc, while 2-5 specific neurones were found for the
remaining compounds. Of the 153 tested neurones, an inhibitory response was
observed only in one (number 46). This neurone, inhibited by
ß-caryophyllene and eugenol, exhibited no response to other tested
compounds (Table 2).
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Morphological characteristics
Of 136 attempted fillings, eight LNs and 19 PNs were successfully stained.
In 15 specimens, more than one neurone was accidentally filled
(Table 3), and these were not
considered in the analysis. Five of the eight stained LNs had their cell
bodies in the lateral cell body group
(Table 4) and had widespread
arborisations with varying densities throughout the AL
(Fig. 4), whereas the cell
bodies of the remaining three LNs could not be located.
PNs had their cell bodies in the lateral or medial cell body clusters and
arborised in one glomerulus each (Table
5). In six preparations, the PN axons were completely stained.
According to the nomenclature of Homberg et al.
(1988), all of those neurones
were of the PIa subtype. Their axons left the AL via the inner
antennocerebral tract (IACT). Two of those completely stained PNs (numbers 112
and 116) were reconstructed from 10µm frontal sections. Projecting
via the IACT, the axons of both PNs branched in the calyces of
mushroom bodies, and in the lateral protocerebrum
(Fig. 5).
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Glomeruli innervated by 14 of the 19 PNs could be individually identified.
Among the remaining five PNs, three innervated glomeruli in the central area
of the AL (light green, Fig. 1)
and two arborised in ventromedial glomeruli (pink,
Fig. 1). PNs arborising in the
same glomerulus responded to different sets of stimuli, e.g. the three PNs
innervating glomerulus C, among others
(Fig. 6). These three
generalist cells had only the response to linalool in common; otherwise they
differed in their response profiles. Conversely, the two neurones (108 and
112) were very similar in their response profiles: each of them responded to
the same three out of four compounds. Nevertheless, they arborised in two
different and distantly located glomeruli
(Fig. 7). Two neurones specific
for -humulene (118 and 121) innervated two different glomeruli; one of
them had its arborisation in a glomerulus in the middle part of the mapped
region, whereas the other innervated a different glomerulus residing in the
medioventral area of the AL (Table
5). Neurones with completely different response profiles, such as
113 and 114, arborised in two adjacent glomeruli
(Fig. 8). Projection patterns
for neurones responding to each of the 10 compounds are compiled in
Fig. 9: glomerulus C and
glomerulus 11 were more often innervated by PNs responding to the tested
compounds than other glomeruli. Each of them harboured the arborisations of
neurones responding to seven different stimuli. Glomerulus 13, which lies
adjacent to glomerulus C, was innervated by only one neurone specific to
linalool.
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Discussion |
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Physiological response of interneurones
Both PNs and LNs examined in the present study varied in their specificity,
from specific neurones that respond to single compounds, to less specific ones
that respond to two or three compounds, to generalists that responded to a
variety of up to eight compounds. Plant odour-sensitive ORNs in insects have
originally been considered as generalists, responding to a broad spectrum of
odours (Visser, 1986).
However, highly specific ORNs for single plant odours have been described in
several species, including S. littoralis
(Dickens, 1990
;
Todd and Baker, 1993
;
Anderson et al., 1995
;
Hansson et al., 1999
). In our
study, more than a quarter of the investigated interneurones were highly
specific and about 20% responded to only two compounds. Considerable numbers
of interneurones that respond specifically to plant volatiles and sex
pheromone components have been demonstrated earlier in the female, as well as
in the male S. littoralis (Anton and Hansson,
1994
,
1995
). The specificity of
receptor neurones observed in both sexes of the insect
(Ljungberg et al., 1993
;
Anderson et al., 1995
) seems,
therefore, to be remarkably preserved at the AL interneuronal level. However,
information stemming from different specific receptor neurones definitely
converge on more integrative interneurones. Neither generalist nor specific
receptor neurones in S. littoralis that respond to plant-derived
compounds respond to any of the sex pheromone components
(Anderson et al., 1995
;
Jönsson and Anderson,
1999
). Many of the generalist interneurones described in the
present study did, however, respond to plant volatiles and pheromone
components. These neurones must, therefore, receive input from both types of
receptor neurones. It is worth noting that the variation in specificity among
the investigated interneurones was observed for both PNs and LNs. The
discrimination between LNs and PNs was based on the morphological features of
the successfully stained neurones. However, for the unstained neurones, we
were unable to discriminate between LNs and PNs, as their physiological
characteristics did not differ clearly.
The highly specific interneurones found in this study indicate that not all
specific ORN types might yet have been studied on the antenna. ORNs responding
to -farnesene, together with other compounds, have been described, but
the presence of ORNs tuned to either
-farnesene only or benzaldehyde
only has not been reported in studies in the periphery
(Anderson et al., 1995
;
Jönsson and Anderson,
1999
). However, two specific interneurones that respond only to
benzaldehyde, and two others that respond only to
-farnesene were
found. Receptor neurones specific for
-farnesene and for benzaldehyde
are, therefore, very likely to exist on the antennae of female S.
littoralis, unless specificity of central neurones is obtained through
properties of the AL circuitry.
Characteristics of local interneurones and pheromone sensitive
projection neurones
The entirely stained LNs exhibited different degrees of complexity. Some
cells innervated more glomeruli than others, but in all cases large numbers of
glomeruli were innervated by one LN. In an earlier study, Anton and Hansson
(1994) described
multiglomerular LNs with homogenous arborisations throughout the AL and
oligoglomerular arborisations whose branches invade relatively few glomeruli.
In addition to these two types, we found also multiglomerular LNs with
heterogeneous arborisations that were asymmetrically distributed within the
glomeruli of the AL.
PNs responding to the major sex pheromone component arborised in three
different anteromedially located glomeruli. Interestingly, these three
glomeruli did not include any of the glomeruli that were shown to receive
inputs from receptor neurones tuned to the same pheromone compound in female
S. littoralis (Ochieng' et al.,
1995). The relationship between axonal arborisations of receptor
neurones and the dendritic branches of PNs that display identical response
characteristics to female pheromones is particularly well studied in male
moths. Receptor neurones on the male antennae often project specifically to
different MGC compartments in the AL
(Hansson et al., 1992
;
Ochieng' et al., 1995
;
Todd et al., 1995
;
Berg et al., 1998
). In some
moths, e.g. Heliothis virescens, PNs seem to innervate precisely the
same glomerulus or subunit of the MGC to which physiologically identical ORNs
send their axonal branches (Vickers et
al., 1998
). However, in several other noctuid species, the
correspondence between the PN dendrites and the axonal terminals of
corresponding ORNs is limited (Hansson et
al., 1994
; Anton and Hansson,
1995
; Wu et al.,
1996
; Anton and Hansson,
1999
).
Characteristics of projection neurones sensitive to plant odours
The axonal arborisation patterns of ORNs specific to plant-derived
compounds in female S. littoralis are unknown. However, the
projection patterns of plant odour-sensitive PNs investigated in the present
study were not restricted to a particular glomerulus or group of glomeruli in
a restricted area of the AL that depended on the response spectrum. Each of
the 19 stained PNs responded to one or more of the plant-derived compounds and
they innervated in all at least 13 different glomeruli scattered throughout
the whole anterior aspect of the AL. This finding can be interpreted in the
framework of the hypothesis of `across-glomeruli pattern'
(Rospars, 1983;
Rospars and Fort, 1994
):
according to this hypothesis, a large number of odours can be discriminated by
a few glomeruli because a small set of interconnected, anatomically identified
and functionally unspecific glomeruli can give rise to a large number of
different patterns of activity, each pattern characterising a specific odour
(e.g. n such glomeruli in two states can give rise to
2n patterns). This is also consistent with what has been
recently revealed in S. littoralis using optical imaging techniques
(Hansson et al., 2000
;
Carlsson et al., 2001
;
Meijerink et al., 2001
).
Similar results have also been reported in a number of other insect and
vertebrate species, where an odour molecule elicits activity in several
glomeruli and each glomerulus participates in the evoked pattern of several
odours (for a review, see Galizia and
Menzel, 2001
). Molecular studies on Drosophila have shown
that the number of expressed odour receptor genes
(Clyne et al., 1999
; Vosshall
et al., 1999
,
2000
;
Gao et al., 2000
) is
comparable with the number of glomeruli in the AL of the fly
(Laissue et al., 1999
). Each
receptor neurone is believed to express a single receptor gene and the axons
of ORNs expressing the same gene all converge onto one or two glomeruli in the
AL. However, what is clear from our observations, is that not only one or two,
but several glomeruli are innervated by PNs responding to a single compound,
emphasising that one compound might bind to several odour receptor types and
that LNs might play an important role as mediators between ORNs and PNs in the
AL.
It has recently been shown that specific glomeruli in the female moth,
Manduca sexta have characteristic limited molecular receptive ranges,
harbouring the dendritic arborisations of output neurones that respond only to
one compound or chemically related compounds
(King et al., 2000). In the
present study, successfully stained PNs that arborised in the same glomerulus
had wider response spectra. However, their response profiles suggest that a
single glomerulus in the AL of S. littoralis could be innervated by
output neurones responding to compounds that are neither behaviourally nor
chemically related. PNs responding to compounds of different behavioural
roles, e.g. oviposition deterrents, host plant odours and sex pheromones, or
compounds with different functional groups, e.g. alcohols, aldehydes and
sesquiterpenes, arborised in the same glomerulus. This is consistent with what
has been found in the honeybee, where glomeruli are preferentially activated
by one functional group over another, but can also be activated by other
functional groups, even though the level of activation is weaker
(Sachse et al., 1999
;
Galizia and Menzel, 2001
).
In conclusion, our work shows that a single plant volatile compound is not represented within a single glomerulus in the AL of the female S. littoralis. Similarly, neither compounds with the same functional group nor those that play the same behavioural role are represented in one glomerulus or one group of adjacent glomeruli. We, thus, expect complex patterns of glomeruli to be involved in the processing of olfactory information concerning both plant-derived compounds and pheromones in this species.
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Acknowledgments |
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References |
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