Accepted Manuscript
Title: Salivary System in Leaf-Cutting Ants (Atta sexdens
rubropilosa Forel, 1908) castes: a confocal study
Authors: Jônatas Bussador do Amaral, Gláucia Maria
Machado-Santelli
PII:
DOI:
Reference:
S0968-4328(08)00092-9
doi:10.1016/j.micron.2008.04.006
JMIC 1265
To appear in:
Received date:
Revised date:
Accepted date:
20-3-2008
12-4-2008
15-4-2008
Please cite this article as: Amaral, J.B., Machado-Santelli, G.M., Salivary System in
Leaf-Cutting Ants (Atta sexdens rubropilosa Forel, 1908) castes: a confocal study,
Micron (2007), doi:10.1016/j.micron.2008.04.006
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* Manuscript
Salivary System in Leaf-Cutting Ants (Atta sexdens rubropilosa Forel, 1908) castes:
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a confocal study
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Jônatas Bussador do Amaral * and Gláucia Maria Machado-Santelli
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Universidade de São Paulo, Instituto de Ciências Biomédicas,
Departamento de Biologia Celular e do Desenvolvimento
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Av. Prof. Lineu Prestes, 1524-Cep: 05508-901,
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São Paulo/SP- Brazil
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*E-mail [email protected]
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Telephone number: 55(11) 3091-7250
Fax number: 55(11) 3091-7402
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Abstract
The salivary system in ants is not limited to digestory functions, but also has important role
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in the communication. The glands which compose the salivary complex are: the postpharyngeal, hypopharyngeal, mandibular, and thoracic salivary gland, showing peculiar
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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
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among the glands of Atta sexdens rubropilosa workers, males and queens focused on the
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organization of microfilaments and microtubules in these ants salivary system. Although
the post-pharyngeal gland appeared to be more developed in queens, there were no
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significant 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
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collector duct seemed to be strong, being surrounded by a microtubules arrangement. The
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use of a laser scanning confocal microscopy with immunofluorescence whole mounting
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preparations revealed itself an efficient instrument for the understanding of the internal
morphology of insects.
Keywords: Cytoskeleton, 3-D reconstruction, post-pharyngeal gland, hypopharyngeal
gland, mandibular gland, thoracic salivary gland
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Introduction
Ants have the most developed eusociality among the Hymenoptera, showing highly
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complex behavioral performances and division of labor (Wilson, 1980). The basis of this
social behavior is the interaction between individuals and environment, mediated by
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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
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interact directly or indirectly with the whole body of insect (Billen, 1991). The glands of
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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
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is not limited to digestion, but may also be related to the communication, differentiation,
and recognition of individuals (Hölldobler and Wilson, 1990; Cruz-Landim and Abdalla,
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2002).
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The glands which compose the salivary complex are: the post-pharyngeal, the
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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 and 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 canaliculus, one per each cell (do
Amaral and Caetano, 2006).
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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
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found close the reservoir which is connected to the conductor canaliculi (Pavon and
Camargo-Mathias, 2005). These canaliculi are positioned inside each mandible and they
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open on trullem surface.
The thoracic salivary gland has a secretory portion located inside the mesothorax
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and the prothorax. This gland is composed by a branched secretory portion and an
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excretory portion which delivery the secretion stored inside the distal secretory tubules to
the oral cavity (Rocha and Caetano, 2004).
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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
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hypopharyngeal and post-pharyngeal glands, synthesis of saliva for the thoracic salivary
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glands, individual recognition inside the colony for the post-pharyngeal glands, and
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synthesis of alarm pheromones for the mandibular glands (Caetano et al., 2002a).
The present work compares the morphology of these four glands, accessed through
whole-mount preparation of correspondent tissues from three different castes of leafcutting ants and analyzed through 3D reconstructions from slices gathered using laser
scanning
confocal
microscopy.
The
analyzed
structures
were
evidenced
by
immunofluorescent reactions using antibodies against alpha- and beta-tubulin and the
staining of F-actin with phalloidin-FITC (Fluorescein).
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Materials and Methods
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Workers, males and queens of leaf-cutting ants were collected in the campus of the
Universidade de São Paulo. Salivary glands were dissected and fixed in 3.4%
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formaldehyde. The fixed glands were permeated with 0.1% Triton X-100 for 20 minutes
and by RNAase (10 mg/mL) for 30 minutes. The glands were then incubated overnight
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with a mix of monoclonal antibodies anti alpha-tubulin and beta-tubulin (Sigma Aldrich)
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(1:100) inside a wet chamber. After 3x washes with PBSA, preparations were incubated for
1h with the anti-mouse CY5 (Molecular Probes) (1:100). The glands were stained with
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Phalloidin-FITC (Sigma Aldrich) (7,5µM) for 40 minutes. The preparations were washed in
phosphate buffered saline (PBSA) in every step of each reaction. The nuclei were
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counterstained using 5 L of propidium iodide (PI) (10mL). The material was mounted
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between slide and cover slips with anti-fading solution (Vectashield, Vector). All glands
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viewed as whole-mount preparations using laser scanning confocal microscope (Zeiss LSM
510) and the fluorescent 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 m. 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, reflection and shadow data. Different modules from LSM 510 3D
software (Carl Zeiss-Germany) were used in the confocal analyses, including slices
projection, orthogonal sections and animations.
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Results
Whole mount preparations of hypopharyngeal, post-pharyngeal, mandibular and
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thoracic salivary glands from specimens of each caste were analyzed by confocal
microscopy. The overall organization of each castes salivary gland was established from
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their respective optical sections and representative projections of these sections are shown
in figure 1. Hypopharingeal glands presented a similar morphology in 3 castes, consisting
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of spherical arranged secretory cells as previously described by do Amaral and Caetano
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(2006). 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-
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actin act that enhanced the serpentine shape of collector ducts around the propidium iodide
(PI) stained nucleus. The collector duct is continuous with the canaliculus in extracellular
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space, nevertheless the microfilaments in canaliculus is poorly evidenced (Fig. 1A and
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Mov. 1 in supplementary material) and all these canaliculi converge from each secretory
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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. 1D-F and Fig. 1 in supplementary material).
Mandibular gland is inside the cephalic cavity, being visualized mainly due to its
huge reservoir composed by small flatten cells slightly stained by FITC-phalloidin.
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Associated with the reservoir are secretory units whose cells present collector ducts rich in
F-actin, similar to those of hypopharingeal gland (Fig. 1G-I ).
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The thoracic salivary glands presented more complex organization, consisting of a
distal secretory portion and a conductor proximal portion, being a common morphology in
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the three castes of ants (Fig. 1J-L). 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
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layer surrounding the collector ducts. These ducts were well stained by the FITC-
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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
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the central main proximal duct (Fig. 1L and Mov. 2-3 in supplementary material). 3D
reconstructions of confocal slices suggest that this rope-like structures coils toward the
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distal extremity and suddenly distend from the proximal region to be inserted into a
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ramification in the distal portion of gland. This surrounded main duct has larger diameter,
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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 microfilaments nearby the plasma membrane region next the canaliculi and collector
duct wall (Fig. 2A-D and Fig. 2 in supplementary material). The microfilaments seem to
irradiate from the collector duct wall surface into the cytoplasm when observed in higher
magnification (Fig. 2D).
The cuticle, widely present in the pharyngeal plate, showed red autofluorescence 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).
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Microtubules distribution was similar in every secretory cell analyzed, showing a
spread pattern all over the cytoplasm and been concentrated around the nucleus and
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beneath the plasma membrane (Fig. 2F-G and 3). High concentrations of microtubules
were also observed surrounding the canaliculi and collector duct (Fig. 3B and C). The
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reservoir cells presented a rich microtubule net distributed along all the cytoplasm (Fig 3A
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Discussion
The glands of the salivary system can have two types of arrangements: cells disposed
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in monolayer (post-pharyngeal and thoracic salivary glands) or in spherical units
(hypopharyngeal and mandibular glands). Noirot and Quennedey (1991) suggest a
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classification of exocrine glands based on their morphology and on the mechanism by
which the secretion is released. According to this classification, the thoracic salivary glands
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and the post-pharyngeal glands belong to class-1. Likewise, the mandibular and
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hypopharyngeal glands should be classified as class-3. In consequence of their ectodermic
origin, all the glands of the salivary system are associated with cuticle, which is located at
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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
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Abdalla, 2002) (see also Fig. 2 in supplementary material)
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The strong association of microfilaments with the plasma membrane nearby the
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cuticle in glands of salivary system of ants was observed for the first time. The role of these
filaments in the glandular arrangement has not yet been described in ants. These filaments
are also associated to the microvilli close to the cuticle, with the specific 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 microfilaments organization 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 postpharyngeal gland when compared to the other glands. This aspect would reflect the scarce
microvilli associated to plasma membrane beneath the cuticle.
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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
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surface membrane of glandular cells. This process would result in microfilament
concentration around the duct. Couble et al. (1984) suggest that the microfilaments in silk
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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
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and hypopharyngeal glands, the high concentration of F-actin around the reservoir could be
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related to contraction processes, and thus help the release of secretion
Microtubules play an important role in the secretory of cell machinery, actively
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participating in the processes of exocytose and endocytosis. Sasaki and Tashiro (1976)
described two different arrangements of the microtubules in glands of insects: randomly
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oriented or on the luminal surface. The distribution of microtubules surrounding the
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collector duct and beneath the plasma membrane, would suggest that they are playing
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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 Drosophila 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
difficult to establish the functions 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.,
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1994). The queen hypertrophy would be associated with caste-specific functions (Niculita
et al., 2007).
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Regarding other glands of the salivary system, no morphological differences were
detected among the different castes. Glandular variations between castes could occur in
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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 significantly
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smaller when compared to the gland of workers that produce royal jelly. Gama (1985)
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described salivary glands histological sections of ant castes and reported only difference in
size. Our results failed to show significant differences in the organization of gland
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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.
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Laser scanning confocal microscopy of whole mounting preparations yielded
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valuable images of salivary glands. This technique associated to immunofluorescence of
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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, microfilaments 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 first 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 Fábio Siviero and Dr Paula Rezende Teixeira for helpful comments.
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Financial support to this research was provided by CNPq, FAPESP and by Secretaria da
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Educação do Estado de São Paulo.
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Gama V., 1985. O sistema salivar de Camponotus (Myrmothrix) rufipes (Fabricius, 1775),
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(Hymenoptera: Formicidae). Revista Brasileira de Biologia 45, 317–359
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Hölldobler B., Wilson E. O. 1990. The ants. Springer, Berlin Heidelberg New York
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glands among castes of the black ant Lasius niger. Arthropod Structure & Development 36,
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Noirot C., Quennedey, A., 1991. Glands, gland cells, glandular units: Some comments on
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Pavon, L. F., Camargo-Mathias, M. I., 2005. Ultrastructural studies of the mandibular
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(Hymenoptera: Formicidae). Micron, 36, 449-460
Riparbelli, M. G., Callaini, G., Dallai, R., 1993. Spatial organization of microtubules and
microfilaments in larval and adult salivary glands of Drosophila melanogaster. Tissue and
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Rocha T., Caetano F. H., 2004. Ultrastructure of the thoracic salivary gland of Polistes
versicolor (Olivier, 1791) (Hymenoptera, Vespidae). Brazillian Journal Morphological
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Sciences 21, 59-64
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Ross G. R., Mathews, R. W., 1991. The function and evolution of exocrine glands. Cornell
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University Press, London
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Sasaki S., Tashiro, Y., 1976. Studies on the posterior silk gland of the silkworm Bombyx
mori. VI. Distribution of microtubules in the posterior silk gland cells. Journal of Cell
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Biology 71, 565-574
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Soroker V., Vienne C., Hefetz A., 1994. The postpharyngeal gland as a "Gestalt" organ for
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Wilson, E. O., 1980. Caste and division of labor in leaf-cutter ants (Hymenotera:
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Legends of figures
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Figure 1
Projections of laser scanning confocal microscope images of hypopharyngeal glands (A-C),
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post-pharyngeal glands (D-F), mandibular glands (G-I) and thoracic salivary glands (Fig. J
and L) of the different castes of leaf-cutting ants. F-actin in green (FITC-phalloidin) nuclei
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in red (propidium iodide) and in figure I, the reservoir is showed in gray (DIC image was
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merged with fluorescence 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=
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secretory cell, D= duct, Md= main duct, (*)=epithelial cells of main duct
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Figure 2
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The association of hypopharyngeal gland and the pharyngeal plate can be observed in
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figure A by merging DIC channel with fluorescence image of F-actin (green). The
morphology and association of collector ducts of hypopharyngeal glands with nuclei are
showed in figure B by shadow projection reconstruction (Imaris, Bitplane) and in higher
magnification in (D). Mandibular gland cells are showed in figure C evidencing the
collector ducts morphology. In figure E-G, confocal images of secretory cells in the
thoracic salivary gland (queen) submitted to immunofluorescent reactions with anti-tubulin
in blue, F-actin in green (FITC-phalloidin) and nuclei in red (propidium iodide). Ducts wall
showed a layer of microfilaments concentrated in the plasma membrane region next to the
cuticle (autofluorescent in red in E double arrows). The microtubules are widely distributed
in the cytoplasm and concentrated around the nucleus (arrow in F). Figure G the three
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channels merged. Figures A, C and D= Worker; B= Male. Cd= collector duct, Mu= muscle,
Sc= secretory cells, N= nucleus, Pp=pharingeal plate, D= ducto
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Figure 3
Images of the mandibular (A, B and C) post-pharingeal (D) and hypopharyngeal glands (E
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and F) of queen showing microtubules stained in blue (immunofluorescent reactions with
anti tubulin and Cy-5 anti mouse secondary antibody), F-actin in green (FITC-phalloidin)
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and nuclei in red (propidium iodide). In figure A the mandibular glands optical sections
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were 3D reconstructed by shadow projection module (Imaris, Bitplane) evidencing its
spatial organization. They are similarly organized in secretory cells of hypopharyngeal and
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mandibular glands where the microtubules concentrate (arrows) around the canaliculus and
collector ducts (arrows in B and C). Figure C1 (insert) is an orthogonal section of the
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canaliculus showing the microtubule component around microfilaments. The microtubules
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concentrated beneath the plasma membrane and nearby the nucleus (arrows in E and F).
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Cc=canaliculi, Cd= collector duct, N=nucleus, R=reservoir, Sc= secretory cell
Supplementary Material
Movie 1
Projections of laser scanning confocal microscope images of hypopharyngeal gland (work)
Movies 2 and 3
Projections of laser scanning confocal microscope images of thoracic salivary gland (work)
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Figure1
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Comparation of post-pharingeal glands (Pg) among castes. Hg= hypopharyngeal glands,
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Pp= pharyngeal plate.
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Drawing showing the formation process of the collector duct in secretory cells class-3
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showing F-Actin (green) and cuticle (brown).
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Figure 1
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Figure 2
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Figure 3
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