Arthropod Structure & Development 38 (2009) 101–110

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Arthropod Structure & Development

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Antennal pathways in the central nervous system of a blood-sucking bug,

Rhodnius prolixus
Romina B. Barrozo a, b,1, Louise Couton b, c,1, Claudio R. Lazzari a, *, Teresita C. Insausti a,
Sebastian A. Minoli b, Nadine Fresquet a, Jean-Pierre Rospars b, c, Sylvia Anton b
a
Institut de Recherche sur la Biologie de l’Insecte, Faculté des Sciences et Techniques, Université François Rabelais, Avenue Monge, Parc Grandmont, 37200 Tours, France
b
INRA, UMR 1272 Physiologie de l’Insecte, F-78000 Versailles, France
c
INRA, Unité Mathématiques et Informatique Appliquées, Centre de Recherches de Jouy-en-Josas, Domaine de Vilvert, 78352 Jouy-en-Josas cedex, France

a r t i c l e i n f o a b s t r a c t

Article history: The haematophagous bug Rhodnius prolixus has been a model system in insect physiology for a long time.
Received 26 March 2008 Recently, several studies have been devoted to its sensory systems, including olfaction. However, few
Received in revised form 30 June 2008 data are available on the basic organisation of the nervous system in this species. By means of neuronal
Accepted 18 August 2008
backfills, histology, confocal microscopy and three-dimensional reconstruction methods, we have char-
acterized the projection patterns of antennal sensory neurons within the central nervous system of this
Keywords:
disease-vector insect. We established the first partial three-dimensional map of the antennal lobe (AL) of
Antennal lobe
a hemipteran insect. The ALs of this species are relatively diffuse structures, which nevertheless show
Three-dimensional reconstruction
Quantitative analysis a glomerular organisation. Based on computer reconstruction of the AL, 22 glomeruli with a radius of 8–
Disease-vector insect 25 mm could be identified. No obvious sexual dimorphism of the glomerular architecture was observed.
Antennal projections Antennal afferents project not only into the deutocerebrum, but also some fibres descend through the
ventral nerve cord to ganglia belonging to the abdominal segments.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2003; Carlsson et al., 2005). Thus, three-dimensional maps of

Olfaction is a crucial sense and all organisms have developed
different means to detect and recognize chemical signals to gain
information about the presence of food, mates, oviposition sites,
predators, etc. Incoming olfactory information from the external
world is represented in the brain as a spatio-temporal neural code
that allows the animal to discriminate complex chemical stimuli
and to produce adapted behavioural responses. Olfactory receptor
neurons detect volatile molecules in the periphery and extend
axons into the brain where they transmit the information to
second-order neurons within the antennal lobe (AL) of the insect
brain. The ALs are composed of functional units named glomeruli.
Knowing the exact arrangement of glomeruli within the AL is
a prerequisite for understanding how odour quality is encoded.
Functional studies in moths, fruit flies, and bees – among others –,
using e.g. optical imaging methods, have shown that odours are
encoded as specific spatio-temporal glomerular activation patterns,
often with several glomeruli responding to a given chemical
component (e.g. Joerges et al., 1997; Meijerink et al., 2003; Wang
* Corresponding author. Tel.: þ33 (0) 2 47 36 73 89; fax: þ33 (0) 2 47 36 69 66.
E-mail address: claudio.lazzari@univ-tours.fr (C.R. Lazzari).
1
Both authors have equally contributed.
the AL glomeruli have been established in various model species
and are used as a basis to interpret data from functional studies
(Rospars and Chambille, 1989; Stocker et al., 1990; Galizia et al.,
1999; Laissue et al., 1999; Rospars and Hildebrand, 2000; Chiang
et al., 2001; Sadek et al., 2002; Berg et al., 2002; Smid et al., 2003;
Greiner et al., 2004; Ignell et al., 2005; Huetteroth and Schachtner,
2005; Masante-Roca et al., 2005; Skiri et al., 2005; Ghaninia et al.,
2007; Zube et al., 2008). Besides, comparative studies of AL
glomeruli across species might be useful to study how the various
taxon-specific arrangements of odorant-processing structures have
evolved.
Triatomines (Heteroptera: Reduviidae) are haematophagous
insects, vectors of the flagellated parasite Trypanosoma cruzi, the
agent responsible for Chagas disease. This trypanosomiasis
constitutes one of the most serious sanitary problems in Latin
America, with an important social and economical impact in the
region (WHO, 2003). Rhodnius prolixus is, in addition, a classical
model in insect physiology: in the past few years, a considerable
amount of information has been published on different aspects of
its sensory physiology and behaviour. This contrasts with the scarce
information available on neuroanatomical aspects. Since the
description of the prothoracic and terminal ganglia by Wiggles-
worth (1959), few data on the organisation of its central nervous

1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.asd.2008.08.004
102 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
system (CNS) have been added (Ramirez Pérez, 1969; Barth, 1976;
Insausti, 1994).
Host-seeking, aggregation and sexual behaviour of triatomine
insects have been investigated and several sensory modalities (such
as thermo-, hygro-, mechano-, and chemoreception) have shown to
play a role in these behavioural contexts (Barrozo and Lazzari,
2004a,b, 2006; Barrozo et al., 2003; Crespo and Manrique, 2007;
Flores and Lazzari, 1996; Lazzari and Núñez, 1989; Lorenzo Fig-
ueiras et al., 1994; Lorenzo Figueiras and Lazzari, 1998; Manrique
and Lazzari, 1994, 1995; Manrique et al., 2006). Concerning the
olfactory system, antennal sensilla were shown to respond to
a wide diversity of odours (Mayer, 1968; Bernard, 1974; Taneja and
Guerin, 1997; Guerenstein and Guerin, 2001; Diehl et al., 2003) and
the behavioural significance of many of these odours was also
demonstrated (Guerenstein and Guerin, 2001; Barrozo and Lazzari,
2004b).
Understanding how olfactory information is processed in the
triatomine brain becomes essential to gain more insight into their
biology. At present, no data are available on the structure of the AL
of Hemipteran insects. The aim of this work was to characterize the
target zones of antennal sensory neurons and to establish the
three-dimensional structure of the ALs of these bugs by means of
computational methods. We established a partial three-dimen-
sional glomerular map of the AL of R. prolixus. This map will be used
as a tool for future functional studies, whose aim will be to
understand the antennal lobe representation of host stimuli
perceived by antennal receptor neurons of different modalities.
2. Materials and methods
2.1. Animals
R. prolixus bugs were reared in the laboratory at 28  C, 60%
relative humidity, under a 12:12 h L:D illumination regime. Larvae
were fed once a week on heparinised sheep blood using an artificial
feeder (Núñez and Lazzari, 1990). Following the imaginal moult, 2–
10 day-old male and female adults were used for dissection.
2.2. Histology
Osmium-ethyl gallate staining was carried out for a global
visualisation of the brain anatomy, according to Leise and Mulloney
(1986). The head capsules were dissected in PBST buffer and brains
were immediately fixed in 2% glutaraldehyde and 1% para-
formaldehyde overnight at room temperature. Brains were then
washed several times in fresh buffer and incubated overnight on
a rotator in 1% osmium tetroxide, at 4  C in the dark. Then they
were washed again in buffer at 4  C and subsequently transferred to
a supersaturated ethyl gallate solution for 6–8 h, at 4  C in dark-
ness. The brains were rinsed and dehydrated in an ascending series
of ethanol and propylene oxide, embedded in Fluka Durcupan,
sectioned at 10 mm with a glass knife on a microtome (RM2055,
Leica Jung) and photographed on an Olympus BX50 microscope.
Photomicrographs shown in Fig. 2 were adjusted for brightness and
contrast by using Adobe Photoshop 9.0.2.
2.3. Anterograde fills of antennal nerves and microscopy
We carried out anterograde backfills from the antenna; this
staining procedure allowed a good visualization of glomeruli in R.
prolixus. Two staining techniques were used: cobalt and neuro-
biotin backfills. In both cases, the animals were dorsally immobi-
lised using double-sided tape and AralditeÒ adhesive on glass
slides. One or both antennae were cut at the pedicel and a drop of
either 1% neurobiotin (NeurobiotinÔ Tracer, Vector Laboratories,
Burlingame, USA) in 0.25 M KCl or 2.5% CoCl2 was added.
Subsequently, the cut end of the antenna and the drop were
covered with VaselineÒ to avoid evaporation. Living animals were
then placed in Petri dishes containing a moist paper tissue and
maintained for 48 h at 4  C to allow the neurobiotin or CoCl2 to
diffuse through the antennal nerve. Subsequently, the whole
nervous system was dissected in insect saline solution (Case, 1957).
In cobalt preparations, cobalt chloride was precipitated using
a freshly prepared solution of ammonium sulphide in saline. The
preparations were then fixed in glacial acetic acid/ethanol/formalin
fixative and silver intensified by means of Timm’s procedure (Bacon
and Altman, 1977). The nervous system of the bugs was then
dehydrated, cleared in methylsalicylate and observed under the
microscope as a whole mount.
Brains and ganglia backfilled with neurobiotin were fixed in 4%
paraformaldehyde in Millonig’s buffer at 4  C for 12 h, dehydrated
and rehydrated in ethanol and propylene oxide (Ignell et al., 2000).
Neurobiotin was then visualised through incubation in Oregon
Green–avidin (Oregon GreenÒ 488 conjugate A6374, Molecular
Probes, OR, USA), with 0.2 % Triton X and 1% BSA added to the
incubation solution, for 12 h at 4  C. The preparations were then
cleared and mounted in Vectashield medium (VectashieldÒ
Mounting Medium, Vector laboratories, ABCYS, France) and whole
mounts were optically sectioned with a laser scanning confocal
microscope (SP2 AOBS, Leica Microsystems Heidelberg, Germany)
equipped with an argon/krypton laser. For scanning HC PL APO CS
10.0  0.40 dry and HCX PL APO CS 40.0  1.25 oil objectives were
used. The whole mounts were scanned every 1 mm from ventral to
dorsal for detailed scanning of the AL, and every 3 mm for overview
scanning of the brain and ganglia. Forty-four out of 61 attempted
neurobiotin fills resulted in stainings in one or both ALs. From
those, 25 brains showing both ALs well stained were analysed and
compared as confocal image stacks. Stack dimensions were
512  512 pixels, by 40–90 optical sections. Finally, the four most
completely stained brains (two females and two males) were
chosen for detailed 3D analysis.
2.4. Three-dimensional computer analysis
For the successive steps of the analysis: segmentation, recon-
struction and standardisation (Chambille et al., 1980), we used
a custom-made program (K. Kiêu, L. Couton and J.-P. Rospars)
developed in the Matlab language (The MathWorks, Natick, MA,
USA). The confocal image stack of each lobe was manually
segmented, i.e. the outlines of all visible glomeruli and those of the
AL itself were drawn on each optical section. Outlines belonging to
the same glomerulus were attributed a ‘glomerulus identification
number’. Based on the coordinates of the outlines, 3D surface
reconstructions of glomeruli were then computed. In order to
account for the refractive index mismatch, the z dimension of
reconstructions was corrected by a factor of 1.6 in all cases where an
air objective was used.
The glomerular organisation of lobes was compared within and
between individuals, by simultaneously rotating the 3D surface
models and by visually comparing shapes and positions of
glomeruli at different levels throughout the AL.
The antennal nerve (AN) and some particular glomeruli were
localised in all reconstructions and used as landmarks. Those
glomeruli neighbouring the landmarks could be paired. Putative
homologous glomeruli were then attributed the same number and
colour in all lobes.
The volumes of the AL and of the glomeruli were calculated from
their outline coordinates on each section multiplied by the slice
thickness. For each glomerulus, the radius of the sphere having the
same volume was used as a measure of the size. To compare the
sizes of pairs of glomeruli from two different ALs, we plotted them,
with one AL on the x-axis and the other AL on the y-axis, and
 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
calculated their Pearson’s coefficient of correlation. In these
comparisons, if the sizes of paired glomeruli were equal, the points
would lie on a line and the coefficient of correlation would be one;
alternatively, if the pairs were random, the points would be scat-
tered and the coefficient of correlation would be zero or close to
zero.
3. Results
3.1. General anatomy of the CNS
R. prolixus, like other triatomines, has a tubular head, with the
pharyngeal pump muscles located anteriorly and the brain located
posteriorly within the cephalic capsule (Insausti, 1994). The largest
part of the brain consists of the protocerebrum and the optic lobes.
The deuto- and the tritocerebrum of R. prolixus lie in a ventral and
anterior position with respect to the protocerebrum (Fig. 1A). The
tritocerebrum is small and lies medial to the ALs on each side of the
oesophagus (Fig. 1A). The suboesophageal ganglion (SOG) is located
posterior to the deutocerebrum. The brain and the SOG are closely
connected by a thick circumoesophageal connective and thus
appear as a single mass (Fig. 1A). A pair of long, thick inter-
ganglionic connectives runs from the SOG to the prothoracic and
from the prothoracic to the posterior ganglion. The prothoracic
ganglion is located near the prosternum, and the posterior ganglion
near the mesosternum. These ganglia are clearly separated from
each other, and their connectives fuse medially. The posterior
ganglion is formed by the fusion of the mesothoracic, metathoracic
and abdominal ganglia (Fig. 1A).
The deutocerebrum is organized in two parts: the ALs and the
antennal motor and mechanosensory centre (AMMC) also called
dorsal lobe in some insects (Fig. 1A; Homberg et al., 1989). Both
neuropils receive sensory afferents originating from the antenna.
The ALs are situated next to the oesophagus (Figs. 1A and 2A). A
thick antennal nerve innervates the ipsilateral AL laterally to
antero-laterally (Fig. 2B and C), first subdividing in two main
branches (b1 and b2 in Fig. 3), before further dividing into many
groups of fibres with a small diameter (1–1.5 mm) which terminate
in vague globular structures (Figs. 1–3). One cluster of cell bodies of
AL neurons is found in the periphery of the ALs. Fig. 2C shows the
axonal fibres emerging from the cluster situated in an antero-
lateral position. A large number of afferent fibres (with a larger
diameter than axons innervating the AL, i.e. 1.7–2.3 mm) bypass the
ALs to join the AMMC located posteriorly, and adjacent to the AL.
This neuropil is a less well-defined structure and becomes visible
because of the high density of receptor neuron terminals (Fig. 1A).
Interestingly, antennal backfills revealed fibres that also extend to
the posterior ganglia of the nervous system (Fig. 1). A branch of
descending fibres arborises in the SOG (data not shown) and in the
prothoracic ganglion (Fig. 1). Finally, a few fibres end in the
posterior ganglion (Fig. 1B). Axons innervating both the prothoracic
and the posterior ganglia have large diameters (ca. 4.2 mm).
3.2. The antennal lobe
The AL has an ellipsoid shape, flattened in the dorso-ventral axis
(Fig. 2B). It is organised, as in other insects, in glomerular units
surrounding a central fibre core (Fig. 2C). Fig. 3 shows an example of
an anterograde labelling of antennal afferent neurons revealing the
innervation patterns of AL glomeruli. In certain regions, fibres are
more densely packed than in others, thus well-delineating the
limits of glomeruli through dense innervation originating from the
AN (see glomeruli 1–3 in Fig. 3A and glomerulus 8 in Fig. 3C). In
some cases, it is possible to follow an antennal nerve branch
entering the lobe and outlining the associated glomerulus (Fig. 3B).
Other glomeruli show a sparse and diffuse innervation from
103
afferent sensory neurons. In these glomeruli, very few receptor
neuron terminals were stained and glomerular outlines could not
be clearly defined (Fig. 3D and E). In the dorsal-most part of the AL,
stainings are sparse and it was not always possible to delineate
glomerular outlines. In the centre of the ALs, corresponding to the
central fibre core, the afferent innervation is lacking (Fig. 3E and F).
The size of the AL in males and females of R. prolixus is
191  7 mm medio-laterally (x-axis), 234  6 mm antero-posteriorly
(y-axis), and 110  9 mm (z-axis) ventro-dorsally. We were able to
identify glomeruli up to a mean ventro-dorsal depth of 85  9 mm
(corresponding approximately to 77% of the total depth). Beyond
this depth the structure becomes highly diffuse, making the
delineation of glomerular outlines difficult.
3.3. The AL map: Number and size of glomeruli
The 3D reconstructions of the ALs allowed us to recognise pairs
of glomeruli between the ALs of the same individual and different
individuals and finally to attribute the same number and colour
code to putative homologous glomeruli. Fourteen to 23 glomeruli
could be identified in the eight ALs investigated (Table 1). The
number of identified glomeruli varied from one individual to
another in both males and females of R. prolixus. The largest
number of glomeruli was found in male 1 (Table 1), where 22
glomerular units were recognised in the left lobe and 18 in the right
lobe. In the other individuals the number of glomeruli varied from
14 to 18 (Table 1). Eighty-two percent of all glomeruli were iden-
tified as present in at least three of the four animals analysed. The
visual comparison of the 3D maps revealed no obvious difference
between sexes (Fig. 4).
Male 1 and female 2 served as references because they showed
the most complete ALs. Glomeruli 1–3 have a characteristic posi-
tion and shape; they are the most antero-ventrally located
glomeruli and are situated close to the AN (Figs. 3 and 4). They are
present in all animals and contain very dense afferent arborisations.
Being easily recognisable, they served as landmarks. Glomerulus 1,
the biggest of the three, is in-between the other two, anterior to
glomerulus 2 and posterior to glomerulus 3. Arranged around these
three, a group of six glomeruli – numbers 5, 6 and 9 to12 – forms
a circle of glomeruli of different sizes (Figs. 3 and 4). In close vicinity
to glomeruli 1–3, glomeruli 6 and 11 become visible at medial
positions. Glomerulus 5 emerges antero-dorsally to 1–3, and
glomerulus 9 appears dorso-laterally. Lastly, and completing the
circle, glomeruli 10 and 12 could be found postero-dorsally.
Glomeruli 21 and 22 lie dorsally to this group. One big glomerulus,
4, extends centrally. Numbers 7 and 8 are situated medially,
neighbouring glomerulus 4. Another large glomerulus, number 18,
is positioned dorso-medially to 4, 7 and 8 (Figs. 3 and 4). Glomer-
ulus 23 is located laterally to glomerulus 4. Glomeruli 15, 16 and 19,
keep a lateral position with respect to the antennal nerve branch b2
(Fig. 4). In addition, glomeruli, 13, 14 and 20 are found in the
proximity of 15, 16 and 19. However, glomeruli 13 and 14 were only
identified in male 1 and glomerulus 20 only in female 1. Similarly,
glomerulus 17 was only found in two lobes, out of eight lobes
analysed, keeping the most anterior position in the AL (Fig. 4,
Table 1).
In both lobes of each brain (intraindividual comparison) 82% of
glomeruli were matched in male 1 (i.e. 18 of 22 glomeruli were
recognised as present in both ALs) vs. 69% in male 2 (i.e. 11 of 16
glomeruli were present in both ALs) (Table 1). In female 1, 93% of
glomeruli were matched (i.e. 14 glomeruli of 15 were found in both
lobes), and 78% in female 2 (i.e. 12 glomeruli of 18 were identified in
both ALs). On the other hand, the comparison of the two males
(intrasexual) led us to 82 % of matching (i.e. from a total of 22
glomeruli, 18 were present in both males) and 62% of matching for
females (i.e. 13 glomeruli were found in at least one lobe of the two
104 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110

Fig. 1. Anterograde fills of the antennal nerve (AN) of R. prolixus. A, ventral view of the CNS showing the afferent innervations throughout the antennal lobe (AL), the antennal motor
mechanosensory centre (AMMC), the prothoracic and posterior ganglia by means of cobalt backfills carried out on the ipsilateral antenna. Insets show neurobiotin fills of both
antennae, visualising projections within the ALs and the AMMCs and a scheme of the CNS of the bug showing the position of the ALs in the brain, the suboesophageal ganglion
(SOG), the prothoracic ganglion (PG) and the posterior ganglion (PoG). B shows a higher magnification of cobalt-stained PG and PoG, and insets show the same views of prothoracic
staining (top) and staining in the posterior ganglion (bottom) but following a neurobiotin–avidin staining procedure. OL: optic lobes; P: protocerebrum; T: tritocerebrum; OE:
oesophagus. Scale bars: 100 mm.

females analysed from a total of 21 identifiable glomeruli of
females). Finally, the intersexual comparison of all eight lobes,
revealed 87% of matching between both sexes, meaning that 20
glomeruli out of 23 were identified in at least one AL of each sex.
3.4. Quantitative analysis
The visual determination of pairs between ALs was confirmed
by a quantitative analysis based on the size of glomeruli,
measured as the glomerular radius. First, we compared the radii
of putative homologous glomeruli belonging to the left and right
lobes of each individual (intraindividual comparison). For each
pair of glomeruli, the radius in one lobe was plotted against the
radius in the second lobe (Fig. 5A). The points were distributed
around a line, although some deviations did occur. The coeffi-
cients of correlation were significantly different from zero for the
intraindividual comparison (Table 2), meaning that identified
glomeruli of right and left ALs had similar sizes. The same
procedure applied to the intrasexual comparison (Fig. 5B and
Table 2) showed that males 1 and 2 exhibited equivalent
glomerular sizes, and similarly in the case of females. Finally, the
intersexual comparison revealed that the glomeruli described in
both sexes presented similar dimensions, denoting the absence of
a sexual dimorphism (Fig. 5C and Table 2).
 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
Fig. 2. Histological sections of the brain of R. prolixus stained with osmium-ethyl
gallate. A, frontal section of the brain showing the position of the antennal lobes (ALs)
relative to other neuropils of the brain (e.g. protocerebral lobe, PL, mushroom bodies,
MB, and optic lobes, OL) and the position of the oesophagus. B, sagittal view of the AL
showing its flattened shape in the dorso-ventral axis, and the antennal nerve (AN)
partially bypassing the AL ventrally. C, frontal section of the AL showing the central
fibre core (FC) formed by the axons of AL neurons whose cell bodies are positioned
medially constituting a cluster (CB). d: dorsal, v: ventral, a: anterior, p: posterior, l:
lateral, m: medial. Scale bars: 100 mm.
4. Discussion
4.1. Identification and number of glomeruli
In the present study, using a qualitative and quantitative
neuroanatomical approach, we were able to establish the projec-
tion pattern of the antennal receptor neurons in the central nervous
system of the haematophagous bug R. prolixus. We identified
105
a maximal number of 22 glomeruli in R. prolixus within a volume
representing ca. 80% of the total AL. The remaining 20% of all ALs
analysed (corresponding to the dorsal region) appeared too diffuse
to delineate glomeruli. However, if glomeruli with the same size as
those described above are present in the remaining 20% of the AL
volume, it can be hypothesized that the AL contains a total of about
28 glomeruli. However, it is also possible that the ALs are aglo-
merular in their dorsal part.
We are convinced that the AL of R. prolixus has a conserved
glomerular pattern. Notwithstanding the intrinsic biological vari-
ability and technical limitations (see below), our results revealed
87% of matching between glomeruli of the eight lobes analysed.
Moreover, glomeruli that were identified from their similar posi-
tions in different lobes were also shown to have the similar sizes
(Fig. 5 and Table 2). The fact that the size of all glomeruli, including
those for which size was not a matching criterion, followed the
same global trend (Fig. 5) provided an additional confirmation of
the stable organisation of the lobes, as previously reported in other
species (Rospars and Chambille, 1989).
In other insects, the number of glomeruli is species-specific and
ranges from about 50 to 60 in some dipteran species, around 60 in
moths to 160 in honeybees, and 215–460 in ants (e.g. Rospars, 1983;
Galizia et al., 1999; Laissue et al., 1999; Berg et al., 2002; Smid et al.,
2003; Ignell et al., 2005; Fishilevich and Vosshall, 2005; Ghaninia
et al., 2007; Nishikawa et al., 2008; Zube et al., 2008). More than
1000 small glomeruli constitute the AL of locusts and some social
wasps, which are arranged in a multiglomerular layer and are not
individually identifiable (Hanström, 1928; Ernst et al., 1977; Ignell
et al., 2001). On the other hand, there are a few groups of insects
(probably primarily anosmic) including Thysanura, Ephemenoptera
and Odonota in which no glomeruli have been found and the ALs
are reduced (Strausfeld et al., 1998). In another hemimetabolous
insect, the carrot psyllid (Homoptera), no discrete glomeruli in the
AL were distinguished (Kristoffersen, 2006), whereas slightly
heterogeneous structures in the AL of aphids suggest the presence
of poorly defined glomeruli in Homoptera (Minoli, Tagu, Anton,
pers. comm.). As an example of a heteropteran species, the stink
bug Euschistus heros also displays a vague AL structure with poorly
defined glomeruli (Kristoffersen, 2006). Given the anatomy of the
related species and similar results found with different staining
techniques (see below), we believe that the relatively diffuse
morphology of the ALs of R. prolixus represents a typical charac-
teristic of this species.
The number of glomeruli present in an insect species is obvi-
ously not correlated with its body size, e.g. very small parasitic
wasps have as many glomeruli as the much bigger honeybee
(Galizia et al., 1999; Smid et al., 2003), but rather with the number
of odours they can discriminate. Highly social insects like ants and
honeybees, which use a large variety of chemical cues, have rela-
tively large numbers of glomeruli (Galizia et al., 1999; Kleineidam
et al., 2005). The low number of glomeruli in R. prolixus might
reflect a limited variety of olfactory cues used, even if each of them
is of great importance to elicit behaviour.
Although most glomeruli are sexually isomorphic, sexual
dimorphism has been reported in moths, cockroaches and bees,
where a macroglomerular complex, involved in pheromone infor-
mation processing, is identifiable in males (reviewed in Anton and
Homberg, 1999). However, we found no indication of sexual
dimorphism in glomerular size or number in R. prolixus with the
methods of analysis used.
4.2. Projections of sensory neurons to the antennal lobes
We observed some variation in the innervation pattern of
sensory neurons in the ALs of R. prolixus. Several glomeruli received
a dense innervation, whereas others only contained rather sparse
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R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
Fig. 3. Neurobiotin stained series of confocal sections (A–F) through the left antennal lobe of a male of R. prolixus from ventral to dorsal (reconstructed in Fig. 4). Glomerular outlines are delineated in red and numbered in optical sections
A–F. Arrows denote the antennal nerve branches innervating the glomeruli. Asterisks indicate foci with not well-delineated glomerulus outlines and also areas without innervation. AN: antennal nerve, b1: antennal branch 1, b2: antennal
branch 2, a: anterior, p: posterior, l: lateral, m: medial. Scale bar: 50 mm.
 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110 107

Table 1 using other staining techniques such as the monoclonal antibodies

Occurrence of the glomeruli identified in the eight antennal lobes of R. prolixus against nc82, anti-phalloidin combined with anti-synapsin or with
studied
propidium iodide, as well as Lucifer yellow staining or auto-
Glomerulus Male 1 Male 2 Female 1 Female 2 Volume fluorescence of glutaraldehyde-fixed brains. Although the best
L R L R L R L R (mm3) resolution of glomeruli was obtained with neurobiotin backfills,
1         46,545  6230 sensory neuron axon uptake of neurobiotin might vary between
2         19,367  2045 preparations and might account for ‘‘missing’’ glomeruli in certain
3         17,905  3381 ALs.
4       31,917  4232
Nevertheless we presume that some areas of the AL might
5        22,975  4963
6       12,037  1874
receive only few receptor neuron axons, whereas others are
7         12,167  1571 innervated by larger numbers of receptor neurons. If we follow the
8         5837  948 hypothesis that glomeruli are functionally specific (Wang et al.,
9         12,223  2833 2003), it might be that sparsely innervated glomeruli receive input
10         22,109  4736
from a small number of specific receptor neurons on the antenna.
11      10,117  2377
12     11,567  2968 Contrary to recent results from mosquitoes (Ghaninia et al.,
13   7731  2116 2007), no projections of afferent neurons from other appendages
14   6551  980 into the ALs were found in R. prolixus. Backfills from the rostrum
15         15,091  2114 showed fibres converging into the SOG but not into the ALs (Fer-
16       15,009  3749
17   27,971  407
reira, Lazzari and Lorenzo, pers. comm.).
18         44,907  8665
19         15,197  3947 4.3. Non-olfactory antennal nerve projections
20   19,783  3448
21     18,105  6469
Antennal afferents of R. prolixus also project to the AMMC, the
22     13,570  3622
23      9394  2364 SOG, the prothoracic and the posterior ganglia (Fig. 1). The fibres
Total 22 18 15 16 15 14 18 16 bypassing the AL might mainly be of a mechanosensory nature. An
The volume of glomeruli is expressed by the mean and the standard error. L, left AL; antennal innervation of the ALs, the AMMC and the SOG was also
R, right AL. described in other insect species, such as Lepidoptera (Camazine
and Hildebrand, 1979; Koontz and Schneider, 1987; Jørgensen et al.,
2006), cockroaches (Boeckh et al., 1970; Nishikawa et al., 1995), flies
branches. The morphology of the AL glomeruli becomes more (Boeckh et al., 1970; Stocker, 1994), mosquitoes (Ignell et al., 2005)
diffuse from ventral to dorsal. However, we cannot exclude that and honeybees (Maronde, 1991). Primary antennal projections to
incomplete antennal backfills were the origin of the sparse inner- the thoracic ganglia have so far only been described in the blowfly
vation of some of the glomeruli or even the lack of innervation. Calliphora erythrocephala (Nässel et al., 1984). However, in most
Unfortunately, glomerular outline resolution was not improved by cases, the lack of information beyond the SOG might be due to the

Fig. 4. Three-dimensional reconstruction of the ALs of a male and a female of R. prolixus sectioned at different ventrodorsal levels. A, ventral view of the left AL of male 1 (same
animal as in Fig. 3), showing the glomeruli and the antennal nerve branches, b1 and b2. In B, the first 26 optical sections of male 1 were removed; in C and D the consecutive five and
thirteen optical sections were subtracted respectively, allowing the visualisation of deeper glomeruli. E, ventral view of the left AL of female 2; in F, the first 23 optical sections were
removed and the following eight and five sections were removed in G and H, respectively. Scale bars: 50 mm.
108 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
Fig. 5. Scatter diagrams of glomerular radii. A, the radii of the left AL of male 1 are
plotted against the radii of the right side. B, radii of female 2 are plotted against
glomerular radii of female 1, each value is the average radius of the right and left pairs.
C, the glomerular radii of males 1 and 2 were averaged and plotted against the radii of
the two females, in all individuals the right and left pairs were averaged. The two
regression lines x/y and y/x are shown.
Table 2
Comparison of glomerular radii of R. prolixus antennal lobes
N r t p
Intraindividual
Male 1 18 0.85 6.4 0.000004
Male 2 13 0.84 5.2 0.00015
Female 1 14 0.78 4.3 0.0005
Female 2 15 0.65 3.1 0.004
Intrasexual
Male 1 vs. male 2 18 0.59 2.9 0.005
Female 2 vs. female 1 13 0.68 3.1 0.005
Intersexual
Males vs. females 20 0.56 2.9 0.005
The correlation coefficients r were computed on N pairs of homologs on a scatter
diagram such as Fig. 5. Three levels of comparisons were carried out, intraindividual
(both ALs of each individual), intrasexual (male 1 vs. male 2 and female 2 vs. female
1) and intersexual (males vs. females). The statistics t assesses the significance of the
correlation coefficient under the null hypothesis of r ¼ 0.
fact that previous studies mainly examined the brain and over-
looked other antennal projections. Prominent mechanosensory
afferents in R. prolixus might play a role in the control of antennal
movements during the localisation of potential food sources in
space (Wigglesworth and Gillett, 1934; Lazzari and Núñez, 1989;
Flores and Lazzari, 1996) and to precisely guide the proboscis
during the localisation of blood-vessels located under the host skin
(Ferreira et al., 2007). Descending fibres with larger diameters (as
shown in Fig. 1B) might serve to insure a rapid neuronal trans-
mission of vital sensory inputs towards centres responsible for
controlling motor activities and physiological processes.
4.4. Role of the olfactory system in triatomine bugs
As in other insects, the reception of odours in triatomine bugs
occurs in chemoreceptive units located in sensilla on the antenna
(Bernard, 1974; Catalá, 1997; Insausti et al., 1999). At least six
morphological types of chemosensory hairs have been identified in
these insects (Insausti et al., 1999), which occur in different
numbers, densities and distributions. Notably, several host-associ-
ated volatiles, which evoke responses at the reception and/or the
behavioural level in triatomines (Barrozo and Lazzari, 2004a,b;
Guerenstein and Guerin, 2001), have also been described as
components of odour blends involved in their intraspecific
communication (Cruz-López et al., 1995; Rojas et al., 2002; Fontan
et al., 2002; Guerenstein and Guerin, 2004; Manrique et al., 2006).
Therefore, the same odour compounds in different concentrations
and/or proportions would define a specific behavioural context and
evoke a characteristic behaviour in the bugs. We speculate that the
parsimonious use of chemical signals and their dependence on the
context would allow a more efficient exploitation of the sensory
system, since only few types of receptor neurons would be neces-
sary (Barrozo and Lazzari, 2004a). A low number of receptor neuron
types would then correlate well with the low number of glomeruli
observed in our study.
5. Conclusions
So far, methods for optical imaging of neural activity, and elec-
trophysiology have provided new and interesting insights into how
different odours are coded over the glomerular array of the AL. The
precise knowledge of the AL anatomy of the blood-sucking bug R.
prolixus will help to understand structure–function relationships
concerning receptor neuron input, AL output and coding mecha-
nisms of sensory information within the primary olfactory centre of
this disease-vector insect.
 R.B. Barrozo et al. / Arthropod Structure & Development 38 (2009) 101–110
Acknowledgments
The authors are deeply indebted to Christophe Gadenne, to
Nélia Varela and Rickard Ignell for critical reading of the manu-
script. This investigation received financial support from the CNRS,
the Université François Rabelais (Tours) and INRA.
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