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Salivary system in leaf-cutting ants (Atta sexdens rubropilosa Forel, 1908) castes:

A confocal study
Jonatas Bussador do Amaral *, Gla ucia Maria Machado-Santelli
University of Sao Paulo, Institute of Biomedical Sciences, Department of Cell and Developmental Biology, Av. Prof. Lineu Prestes, 1524-Cep: 05508901, Sao Paulo/SP, Brazil
1. Introduction
Ants have the most developed eusociality among the Hyme-
noptera, showing highly complex behavioral performances and
division of labor (Wilson, 1980). The basis of this social behavior is
the interaction between individuals and environment, mediated
by pheromones and by visual, mechanical and auditory stimuli
(Caetano et al., 2002a). Due to the synthesis of pheromones,
lubricants, and digestive enzymes, the exocrine glands interact
directly or indirectly with the whole body of insect (Billen, 1991).
The glands of the salivary complex are associated with the oral
cavity and its appendixes, constituting an important part of the
exocrine system. In spite of their location, the function of these
glands is not only limited to digestion, but may also be related to
the communication, differentiation, and recognition of individuals
(Ho lldobler and Wilson, 1990; Cruz-Landim and Abdalla, 2002).
The glands which compose the salivary complex are: the post-
pharyngeal, the hypopharyngeal, the mandibular, and the thoracic
salivary glands (Toledo, 1967; Gama, 1985). The post-pharyngeal
glands are located laterally nearby the posterior region of the
pharynx and consist of two lateral expansions. These glands
contain a single layer of secretory cells arranged in dactyliform
shape structures surrounding the lumen, which also acts as a
reservoir (Caetano et al., 2002b; Eelen et al., 2006).
The former salivary gland in formicidae is the hypopharyngeal
gland, which consists of a pair of spherical aggregates of secretory
cells. This gland is located adjacently the anterior region of the
pharynx and is supported by the pharyngeal plate. Its secretion is
released directly into the oral cavity through individual canalicu-
lus, one per each cell (do Amaral and Caetano, 2005).
The mandibular gland is located laterally inside the cephalic
cavity, consists of a sac-shaped reservoir inserted at the basal
region of each mandible. The secretory cells are found close to the
reservoir which is connected to the conductor canaliculi (Pavon
and Camargo-Mathias, 2005). These canaliculi are positioned
inside each mandible and they open on trullem surface.
The thoracic salivary gland has a secretory portion located
inside the mesothorax and the prothorax. This gland is composed
of a branched secretory portion and an excretory portion which
delivers the secretion stored inside the distal secretory tubules to
the oral cavity (Rocha and Caetano, 2004).
The function of these glands in the different castes of leaf-
cutting ants (Atta sexdens rubropilosa) remains barely studied.
Some authors suggest digestive roles for the hypopharyngeal and
post-pharyngeal glands, synthesis of saliva for the thoracic salivary
glands, individual recognition inside the colony for the post-
pharyngeal glands, and synthesis of alarm pheromones for the
mandibular glands (Caetano et al., 2002a).
Micron 39 (2008) 12221227
A R T I C L E I N F O
Article history:
Received 20 March 2008
Received in revised form 12 April 2008
Accepted 15 April 2008
Keywords:
Cytoskeleton
3D reconstruction
Post-pharyngeal gland
Hypopharyngeal gland
Mandibular gland
Thoracic salivary gland
A B S T R A C T
The salivary system in ants is not only limited to digestory functions, but also has important role in the
communication. The glands which compose the salivary complex are: the post-pharyngeal,
hypopharyngeal, mandibular, and thoracic salivary gland, showing peculiar features which may vary
according to the castes the individuals belong to and according to the functions they develop. The present
study compared the morphological differences among the glands of Atta sexdens rubropilosa workers,
males and queens focused on the organization of microlaments and microtubules in these ants salivary
system. Although the post-pharyngeal gland appeared to be more developed in queens, there were no
signicant gland differences among the analyzed castes. In what regards to the secretory units of the
hypopharyngeal and mandibular glands, the association of F-actin with the collector duct seemed to be
strong, being surrounded by a microtubules arrangement. The use of a laser scanning confocal
microscopy with immunouorescence whole mounting preparations revealed itself an efcient
instrument for the understanding of the internal morphology of insects.
2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +55 11 3091 7250; fax. +55 11 3091 7402.
E-mail address: jbamaral@icb.usp.br (J.B. do Amaral).
Contents lists available at ScienceDirect
Micron
j ournal homepage: www. el sevi er. com/ l ocat e/ mi cron
0968-4328/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2008.04.006
The present work compares the morphology of these four
glands, accessed through whole mount preparation of correspon-
dent tissues from three different castes of leaf-cutting ants and
analyzed through 3D reconstructions from slices gathered using
laser scanning confocal microscopy. The analyzed structures were
evidenced by immunouorescent reactions using antibodies
against alpha- and beta-tubulin and the staining of F-actin with
phalloidin-FITC (Fluorescein).
2. Materials and methods
Workers, males and queens of leaf-cutting ants were collected
in the campus of the Universidade de Sao Paulo. Salivary glands
were dissected and xed in 3.4% formaldehyde. The xed glands
were permeated with 0.1% Triton X-100 for 20 min and by RNAase
(10 mg/mL) for 30 min. The glands were then incubated overnight
with a mix of monoclonal antibodies anti alpha- and beta-tubulin
(SigmaAldrich) (1:100) inside a wet chamber. After 3 washes
with PBSA, preparations were incubated for 1 h with the anti-
mouse CY5 (Molecular Probes) (1:100). The glands were stained
with Phalloidin-FITC (SigmaAldrich) (7.5 mM) for 40 min. The
preparations were washed in phosphate buffered saline (PBSA) in
every step of each reaction. The nuclei were counterstained using
5 mL of propidium iodide (PI) (10 mg/mL). The material was
mounted between slide and cover slips with anti-fading solution
(Vectashield, Vector). All glands viewed as whole mount prepara-
tions using laser scanning confocal microscope (Zeiss LSM510) and
the uorescent images were obtained using argon lasers (at 458,
488, and 514 nm), Helium-Neon1 (at 543 nm), and Helium-Neon2
(at 633 nm). Optical sections were obtained at appropriate
sectioning ranges of the Z-axis, between 0.3 and 0.7 mm. The
Imaris-Irix 3.1.3 software (Bitplane, Switzerland) was used for
analysis and 3D reconstruction running on interactive module
based on a wireframe composed by thousand of triangles, with
color, reection and shadowdata. Different modules fromLSM510
3D software (Carl Zeiss, Germany) were used in the confocal
analyses, including slices projection, orthogonal sections and
animations.
3. Results
Whole mount preparations of hypopharyngeal, post-pharyn-
geal, mandibular and thoracic salivary glands from specimens of
each caste were analyzed by confocal microscopy. The overall
organization of each castes salivary gland was established from
their respective optical sections and representative projections of
these sections are shown in Fig. 1. Hypopharingeal glands
presented a similar morphology in 3 castes, consisting of spherical
arranged secretory cells as previously described by do Amaral and
Caetano (2005). However these cells are more numerous in queen
than in male or worker (Fig. 1AC). By focusing the analysis on
individual cells it was evidenced dense component of F-actin act
that enhanced the serpentine shape of collector ducts around the
propidiumiodide stained nucleus. The collector duct is continuous
with the canaliculus in extracellular space, nevertheless the
microlaments in canaliculus is poorly evidenced (Fig. 1A and
Movie 1 in Supplementary material) and all these canaliculi
converge from each secretory cells to a common pharyngeal plate
opening (Fig. 1B).
Post-pharyngeal gland is also associated with pharynx. Its
morphology is quite different from that of hypopharyngeal gland,
presenting dactyliform expansions formed by secretory cells
showing diffuse staining for F-actin in their cytoplasm. The
comparative analysis of this gland among the castes revealed
similar organization. Queen has much more digitiform expansions
when compared to males and workers and consequently larger
luminal secretion store capacity (Fig. 1DF and Fig. 1 in Supple-
mentary material).
Mandibular gland is inside the cephalic cavity, being visualized
mainly due to its huge reservoir composed by small atten cells
slightly stained by FITC-phalloidin. Associated with the reservoir
are secretory units whose cells present collector ducts rich in F-
actin, similar to those of hypopharingeal gland (Fig. 1GI).
The thoracic salivary glands presented more complex organiza-
tion, consisting of a distal secretory portion and a conductor
proximal portion, being a common morphology in the three castes
of ants (Fig. 1JL). The image of a whole gland of workers evidences
very well the distal branched rope-like structure with the secretory
cells arranged in a single layer surrounding the collector ducts.
These ducts were well stained by the FITC-phalloidin, evidencing
their blind end. As showed in orthogonal sections of confocal
images the gland proximal portion is constituted by a number of
rope-like ducts wrapping the central main proximal duct (Fig. 1L
and Movie 23 in Supplementary material). 3D reconstructions of
confocal slices suggest that this rope-like structures coils toward
the distal extremity and suddenly distend from the proximal
region to be inserted into a ramication in the distal portion of
gland. This surrounded main duct has larger diameter, suggesting a
reservoir function. The whole gland is connected to the oral cavity
by delicate thin duct (Fig. 1J).
The images of FITC-phalloidin stained salivary glands showed
high concentration of microlaments nearby the plasma mem-
brane region next the canaliculi and collector duct wall (Fig. 2AD
and Fig. 2 in Supplementary material). The microlaments seemto
irradiate from the collector duct wall surface into the cytoplasm
when observed in higher magnication (Fig. 2D).
The cuticle, widely present in the pharyngeal plate, showed red
autouorescence in contrast with FITC-phalloidin stained F-actin
(Fig. 2A, B and E). Pharyngeal plates can also be observed in light
microscopy preparation (Fig. 1 in Supplementary material).
Microtubules distribution was similar in every secretory cell
analyzed, showing a spread pattern all over the cytoplasm and
been concentrated around the nucleus and beneath the plasma
membrane (Figs. 2FGand 3). High concentrations of microtubules
were also observed surrounding the canaliculi and collector duct
(Fig. 3B and C). The reservoir cells presented a rich microtubule net
distributed along all the cytoplasm (Fig. 3A and C).
4. Discussion
The glands of the salivary system can have two types of
arrangements: cells disposed in monolayer (post-pharyngeal and
thoracic salivary glands) or in spherical units (hypopharyngeal and
mandibular glands). Noirot and Quennedey (1991) suggest a
classication of exocrine glands based on their morphology and on
the mechanism by which the secretion is released. According to
this classication, the thoracic salivary glands and the post-
pharyngeal glands belong to class-1. Likewise, the mandibular and
hypopharyngeal glands should be classied as class-3. In
consequence of their ectodermic origin, all the glands of the
salivary system are associated with cuticle, which is located at the
apical extremity of the secretory cells (in class-1 glands), or
associated with the duct and reservoirs (class-3 glands) (Billen,
1991; Ross and Mathews, 1991; Cruz-Landim and Abdalla, 2002)
(see also Fig. 2 in Supplementary material).
The strong association of microlaments with the plasma
membrane nearby the cuticle in glands of salivary system of ants
was observed for the rst time. The role of these laments in the
glandular arrangement has not yet been described in ants. These
laments are also associated to the microvilli close to the cuticle,
J.B. do Amaral, G.M. Machado-Santelli / Micron 39 (2008) 12221227 1223
with the specic stain being found next to the ducts of all glands of
the salivary system. The present results agree with those of
Riparbelli et al. (1993). By analyzing the microlaments organiza-
tion in salivary glands of Drosophila melanogaster, they described
their association to the microvilli of the apical region and to some
basolateral domains. A small amount of F-actin was found in the
post-pharyngeal gland when compared to the other glands. This
aspect would reect the scarce microvilli associated to plasma
membrane beneath the cuticle.
The collector ducts of class-3 gland were strongly stained by the
FITC-phalloidin. During the embryogenesis, the collector duct
originates from invagination of the apical surface membrane of
glandular cells. This process would result in microlament
concentration around the duct. Couble et al. (1984) suggest that
Fig. 1. Projections of laser scanning confocal microscope images of hypopharyngeal glands (AC), post-pharyngeal glands (DF), mandibular glands (GI) and thoracic salivary
glands (J and L) of the different castes of leaf-cutting ants. F-actin in green (FITC-phalloidin) nuclei in red (propidium iodide) and in (I), the reservoir is showed in gray (DIC
image was merged with uorescence channels). Worker (A, D, G, J and L), Male (B, E and H) and Queen (Fig. C, F and I). Cc = canaliculi, N = nucleus, Pp = pharingeal plate,
R = reservoir, Sc = secretory cell, D = duct, Md = main duct. (*) = epithelial cells of main duct.
J.B. do Amaral, G.M. Machado-Santelli / Micron 39 (2008) 12221227 1224
the microlaments in silk gland cells of Bombyx mori would
participate of the secretory process by contracting of the gland.
Since no muscle cell was found associated to the secretory units of
the mandibular and hypopharyngeal glands, the high concentra-
tion of F-actin around the reservoir could be related to contraction
processes, and thus help the release of secretion.
Microtubules play an important role in the secretory of cell
machinery, actively participating in the processes of exocytose and
endocytosis. Sasaki and Tashiro (1976) described two different
arrangements of the microtubules in glands of insects: randomly
oriented or on the luminal surface. The distribution of micro-
tubules surrounding the collector duct and beneath the plasma
Fig. 2. The association of hypopharyngeal gland and the pharyngeal plate can be observed in (A) by merging DIC channel with uorescence image of F-actin (green). The
morphology and association of collector ducts of hypopharyngeal glands with nuclei are showed in (B) by shadow projection reconstruction (Imaris, Bitplane) and in higher
magnication in (D). Mandibular gland cells are showed in (C) evidencing the collector ducts morphology. In (EG) confocal images of secretory cells in the thoracic salivary
gland (queen) submitted to immunouorescent reactions with anti-tubulin inblue, F-actin in green (FITC-phalloidin) and nuclei in red (propidiumiodide). Ducts wall showed
a layer of microlaments concentrated in the plasma membrane region next to the cuticle (autouorescent in red in E double arrows). The microtubules are widely
distributed in the cytoplasm and concentrated around the nucleus (arrow in F). (G) The three channels merged. (A, C and D) = Worker; (B) = Male. Cd = collector duct,
Mu = muscle, Sc = secretory cells, N = nucleus, Pp = pharingeal plate, D = ducto.
J.B. do Amaral, G.M. Machado-Santelli / Micron 39 (2008) 12221227 1225
Fig. 3. Images of the mandibular (A, B and C) post-pharingeal (D) and hypopharyngeal glands (E and F) of queen showing microtubules stained in blue (immunouorescent
reactions with anti tubulin and Cy-5 anti mouse secondary antibody), F-actin in green (FITC-phalloidin) and nuclei in red (propidium iodide). In (A) the mandibular glands
optical sections were 3D reconstructed by shadow projection module (Imaris, Bitplane) evidencing its spatial organization. They are similarly organized in secretory cells of
hypopharyngeal and mandibular glands where the microtubules concentrate (arrows) around the canaliculus and collector ducts (arrows in B and C). (C1) (insert) is an
orthogonal section of the canaliculus showing the microtubule component around microlaments. The microtubules concentrated beneath the plasma membrane and
nearby the nucleus (arrows in E and F). Cc = canaliculi, Cd = collector duct, N = nucleus, R = reservoir, Sc = secretory cell.
J.B. do Amaral, G.M. Machado-Santelli / Micron 39 (2008) 12221227 1226
membrane, would suggest that they are playing structural role in
these cells. The amount and arrangement of microtubules in the
mandibular reservoir cells reinforce their possible structural
function. This function was already proposed for microtubules
by Riparbelli et al. (1993) when they analyzed the salivary gland
cytoskeleton of D. melanogaster.
The greatest volume as well as the high number of glandular
projections suggests that post-pharyngeal glands are different in
queens when compared with workers and males. These
characteristics were already described in other species of ants
such as Monomorium pharaonis (Eelen et al., 2006) and Lasius
niger (Niculita et al., 2007). It is difcult to establish the func-
tions of this gland, since it has been associated with synthesis of
lipids (Caetano et al., 2002b) and with synthesis of cuticle
hydrocarbon (Soroker et al., 1994). The queen hypertrophy
would be associated with caste-specic functions (Niculita et al.,
2007).
Regarding other glands of the salivary system, no morpholo-
gical differences were detected among the different castes.
Glandular variations between castes could occur in species of
the order Hymenoptera. Cruz-Landim and Abdalla (2002)
described the atrophy of the hypopharyngeal gland in queens of
Apis mellifera, this gland being signicantly smaller when
compared to the gland of workers that produce royal jelly. Gama
(1985) described salivary glands histological sections of ant castes
and reported only difference in size. Our results failed to show
signicant differences in the organization of gland cytoskeleton
and the general morphology. These results do not exclude the
possibility of differences at the intracellular (Pavon and Camargo-
Mathias, 2005) or molecular levels.
Laser scanning confocal microscopy of whole mounting
preparations yielded valuable images of salivary glands. This
technique associated to immunouorescence of cytoskeleton
elements enables us to describe the 3D organization of the
different types of salivary glands. The approach chosen in this
study led to better understanding of spatial interaction of
microtubules, microlaments with collector duct and providing
a basis for further molecular and physiological studies.
Acknowledgements
This paper is dedicated to Dr. Lurdes Foresti de Almeida Toledo,
who was the rst to describe the morphology of salivary glands in
Atta sexdens rubropilosa, 40 years ago.
The authors would like to thank Roberto Cabado Modia Jr. for
the aid with the artwork and Dr. Fa bio Siviero and Dr. Paula
Rezende Teixeira for helpful comments.
Financial support to this research was provided by CNPq,
FAPESP and by Secretaria da Educacao do Estado de Sao Paulo.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.micron.2008.04.006.
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