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Morphology, Histology, and Fine Structure
Morphology of the Spermathecae of Leptoglossus
zonatus (Heteroptera: Coreidae)
E. A. Souza,
1,2
L.C.O. Lisboa,
1
V. A. Arau´ jo,
1
and J. E. Serra˜o
3
1
Universidade Federal de Vic¸osa, Campus Rio Paranaı´ba, 38810-000, Rio Paranaı´ba, MG, Brazil (edmilson.souza@ufv.br; luciane.
lisboa@ufv.br; viniciusalbano@ufv.br),
2
Corresponding author, e-mail: edmilson.souza@ufv.br, and
3
Departamento de Biologia
Geral, Universidade Federal de Vic¸osa, 36570-000, Vic¸ osa, MG, Brazil (jeserrao@ufv.br)
Received 21 November 2014; Accepted 28 September 2015
Abstract
The spermathecae of Heteroptera exhibit great morphological variety. The characteristics of spermathecal mor-
phology may contribute greatly toward the taxonomy and reproductive physiology of these insects. This study
describes the spermathecal morphology of the agricultural pest, Leptoglossus zonatus (Dallas). The sperma-
theca of L. zonatus is distinguishable into three parts—the reservoir (distal portion), muscular duct (middle re-
gion), and enlarged duct (proximal end). The spherical reservoir has a cuticle-lined lumen, followed by a layer
of flattened cells, below which lies another layer of cells with secretory characteristics. The muscular duct is
characterized by a flange where the insertion of the muscle fibers is seen. The proximal duct is enlarged with a
compartmentalized lumen. The morphological features of the spermatheca of L. zonatus differ from those
described for other Heteroptera.
Key words: reproduction, secretory cell, sperm, Hemiptera
The spermatheca is an accessory organ of the reproductive tract of
the female insect, which plays an important role in the storage of
sperm from the time of mating until fertilization of the oocytes oc-
curs (Hitchcock 1956,Camargo and Melo 1970,Eberhard 1996).
The sperms are stored in spermathecae with secretion produced by
the gland or by spermathecal epithelium. Therefore, the spermathe-
cae create an environment responsible for the maintenance of viabil-
ity of sperm for long periods (Davey 1965,Ruttner and Koeniger
1971,Adams 2001).
A typical insect spermatheca consists of a reservoir, where the
sperm is stored, associated with a muscular duct (Chapman 2013).
However, the morphology of the spermathecae varies among in-
sects, ranging from a simple tube (Vavrinova 1988) to complex or-
gans divided into different chambers, which may or not have
accessory glands (Dalai 1975,Pendergrast 1957,Matsuda 1976,
Vavrinova 1988,Kocorek and Danielczoc-Demska 2002,Martins
and Serra˜o 2002,Martins et al. 2008,Souza et al. 2008,Candan
2008).
In some insects, the spermathecae have accessory glands as indi-
vidual organs. Their secretion contributes toward the maintenance
of sperm viability. These accessory glands release secretions rich in
carbohydrates and glycoproteins into the lumen of the reservoir and
create an environment appropriate for the sperm (Cruz-Landim and
Serra˜o 2002,Klenk et al. 2004,Souza et al. 2008,Chapman 2013).
However, in some species the accessory glands are not present as
individual organs; instead the cells of the reservoir wall themselves
are secretory in function (Stacconi and Romani 2011,Chapman
2013).
Morphological studies of the representatives of Heteroptera
show some variation in their spermatheca (Pendergrast 1957,
Vavrinova 1988,Pluot-Sigwalt and Lis 2008), which makes them
important in insect systematic and behavioral studies (Pendergrast
1957,Vavrinova 1988,Adams 2001,Pluot-Sigwalt and Lis 2008,
Candan 2008,Stacconi and Romani 2011). Although these studies
have provided important data on spermathecal morphology in those
species, detailed information of the spermathecae in other species is
still required.
Within Heteroptera, Leptoglossus zonatus (Dallas) (Coreidae) is
an important pest of maize (Sawazaki et al. 1989), passion fruit
(Rodrigues Netto and Guilhem 1996), and pomegranate (Raga et al.
1995) cultures, and the characterization of their spermathecae may
contribute to the comprehension of the reproductive physiology of
this insect pest.
The aim of this paper is to describe the anatomy, histology, and
histochemistry of the spermathecae of L. zonatus.
Materials and Methods
Ten adult females of L. zonatus were collected with entomological
nets from the maize crops in Rio Paranaı´ba, state of Minas Gerais,
V
CThe Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
All rights reserved. For Permissions, please email: journals.permissions@oup.com 1
Annals of the Entomological Society of America, 2015, 1–6
doi: 10.1093/aesa/sav097
Research article
Annals of the Entomological Society of America Advance Access published October 21, 2015
Brazil. The insects were transferred to the laboratory and cryo-
anesthetized. The insects were dissected in saline solution by remov-
ing the dorsal tergites, and female reproductive system was isolated
for subsequent separation of the spermatheca and then transferred
to the Zamboni fixative solution (Stefanini et al. 1967).
Scanning Electron Microscopy
Some spermathecae of L. zonatus were washed in 0.1 M phos-
phate buffer saline ph 7.2 (PBS), dehydrated in graded ethanol
series (70, 80, 90, 95, 100%), transferred to hexamethyldisilazane
for 10 min, and air dried (Nation 1983). Then the samples were
fixed onto aluminum stubs, gold covered (20 nm thick), and exam-
ined under a LEO VP 1430 scanning electron microscope in the
Nucleus of Microscopy and Microanalysis at Federal University of
Vic¸osa.
Light Microscopy
Another set of spermathecae were washed in PBS, dehydrated
in a graded ethanol series (70, 80, 90, 95%), and embedded in
historesin (Leica). Slices 3 mm thick were stained with hematoxyline
and eosin. Some sections were subjected to the following histochem-
ical tests: mercuric-bromophenol blue for the detection of total
protein, and (Periodic acid-Schiff) for neutral polysaccharides and
glycoconjugates (Pearse 1985). The sections were analyzed using a
light microscope.
Results
The L. zonatus spermathecae showed three well-defined regions:
distal, middle, and proximal (Fig. 1A). The distal region is expanded
and is referred to as the reservoir (Fig. 1A). The middle region is a
short narrow tube characterized by muscle fibers embedded in a
flange structure at the transition to the proximal region (Fig. 1A, B),
which is formed by an enlarged duct that opens into the common
oviduct (Fig. 1A).
The lumen of the spermathecal reservoir was lined by a cuticle
with pores as well as two layers of morphologically different cells
(Fig. 2A, B). The cells in contact with the cuticle are flattened with
acidophilic cytoplasm, and nuclei are rich in decondensed chromatin
(Fig. 2A, B). Some cytoplasmic regions were positive for PAS
(Fig. 2C, D) and mercuric-bromophenol tests (Fig. 2E). The outer
cell layer showed characteristics of secretory epithelium, which were
globular, having a large nucleus with decondensed chromatin and
acidophilic cytoplasm containing intracellular canaliculi (end appa-
ratus; Fig. 2A, B). The cytoplasm near the intracellular canaliculi
showed strong positive reaction for PAS (Fig. 2C, D) and proteins,
except in small portions of the basal region, which presented a stri-
ated appearance (Fig. 2E, F). Some secretory cells of the reservoir re-
vealed cytoplasm containing vacuoles positive for PAS (Fig. 3A) but
negative for proteins (Fig. 3B). The secretory cells were found on a
thin basal membrane (Figs. 2C and 3A).
The middle region of the spermatheca showed a narrow cuticle-
lined lumen, with a single layer of cuboidal cells (Fig. 3C, D).
Externally, there were well developed longitudinal muscles fibers,
which connected close to the proximal region in a cuticular flange
(Fig. 3C–E). The histochemical tests showed positive reaction for
the basal membrane alone (Fig. 3D). The epithelial cells associated
with the muscle showed strong staining for proteins (Fig. 3E).
The proximal region of the spermatheca was characterized by a
dilated duct (Fig. 4A). This region was coated by a single layer of
columnar cells with acidophilic cytoplasm and a central nucleus
with decondensed chromatin (Fig. 4A, B). In this region of the sper-
matheca, the subcuticular space was filled with acidophilic material
(Fig. 4B). The cytoplasm of the epithelial cells and the subcuticular
space were weakly positive for PAS (Fig. 4C), but strongly positive
for proteins (Fig. 4D).
The lumen of the dilated duct was subdivided into small com-
partments (Figs. 3E and 4A) and the wall that compartmentalized
the lumen was negative for PAS and strongly positive for proteins
(Fig. 4E, F), while the content of these compartments was positive
for proteins and PAS (Fig. 4E, F).
Fig. 1. Scanning electron micrograph of the spermatheca of L. zonatus.(A) The spermatheca is divided into three regions: the distal region is the reservoir (r); the
middle region is formed by a muscular duct (md); and the proximal region is formed by an expanded duct (d). tr, trachea. Scale bar: 300 mm. (B) Details of the mid-
dle region of the spermatheca. md, muscular duct; f, flange. Scale bar: 100 mm.
2Annals of the Entomological Society of America, 2015, Vol. 0, No. 0
The Figure 5 shows the schematic design of the spermatheca of
L. zonatus.
Discussion
The division of the spermathecae of L. zonatus into reservoir, mus-
cular duct, and enlarged proximal duct is similar to that described
for other Pentatomomorpha (Pendergrast 1957).
The spermathecal reservoir contains class III secretory cells,
based on the classification of Noirot and Quennedey (1991), charac-
terized by the presence of intracellular canaliculi or end apparatus,
which correspond to the collecting cells. According to the secretory
model suggested by the type III gland cells, their products are re-
leased into the end apparatus and transported via the collecting cells
(Noirot and Quennedey 1991). Our findings suggest a similar secre-
tory pattern because the cuticle lining the reservoir lumen has some
pores, which may be the openings of the collecting cells. Type III
glandular cells were also found in the spermatheca of Heteroptera
species such as Podisus nigrispinus (Rodrigues et al. 2008) and
Murgantia histrionica (Stacconi and Romani 2011).
The histochemical tests of the type III cells suggest that their se-
cretion is rich in carbohydrates and glycoconjugates. The function
of the secretory cells of the spermathecal reservoir appears similar to
that of the spermathecal accessory glands found in some insects
(Cruz-Landim and Serra˜o 2002,Martins and Serra˜ o 2002,Souza
et al. 2008). Thus, because the accessory gland is absent in the sper-
mathecae of L. zonatus we suggest that the secretory epithelium of
the reservoir is responsible for the synthesis of the compounds that
probably contributes to the maintenance of the viability of the
stored spermatozoa.
The striated aspect of the basal region of the epithelium of the
spermathecal reservoir that is negative for the protein test may be
due to the presence of the infoldings of the basal plasma membrane
resulting in a basal labyrinth, which plays an important role in the
transport of substances (Alberts et al. 2010). A similar striated as-
pect is reported in the secretory cells of insects under low resolution,
which have been confirmed to be cell plasma membrane infoldings
using high-resolution electron microscopy (Serra˜o et al. 2008,Hora
et al. 2010,Martinez et al. 2014).
The middle region of the spermatheca of Heteroptera is morpho-
logically variable (Pendergrast 1957,Vavrinova 1988,Kocorek and
Fig. 2. Histological and histochemistry sections of the spermathecal reservoir of L. zonatus.(A) Section of the reservoir that shows the presence of cuticle (ct) and
two cell types: class III secretory cells (sc) and epithelial cells associated to the cuticle. Scale bar: 30 mm. (B) Details of the spermathecal reservoir. The class III se-
cretory cells have a large nucleus (n) with decondensed chromatin and intracellular canaliculi (ca). The epithelial cells (ec) with decondensed chromatin nucleus
are smaller. The reservoir cuticle has several pores (p). Scale bar: 30 mm. (C) Histological section of the reservoir showing the cells and the basal lamina of the res-
ervoir which are positive for the PAS test. Scale bar: 30 mm. (D) Details of the two cell types found in the reservoir. The class III secretory cells show cytoplasm
positive for PAS test near the intracellular canaliculi (ca); and the epithelial cells (ec) show positive cytoplasm homogeneously. Scale bar: 20 mm. (E) Secretory
(sc) and epithelial (ec) cells positive for the mercuric-bromophenol blue test. Scale bar: 30 mm. (F) Details of the class III secretory cells (sc) showing the striated
(arrows) aspect of the basal region. Scale bar: 15 mm.
Annals of the Entomological Society of America, 2015, Vol. 0, No. 0 3
Danielczok-Demska 2002,Pluot-Sigwalt and Lis 2008). This region
is characterized by a muscular duct internally lined by a cuticle that
controls the release of the stored spermatozoa to fertilize the oocyte.
Besides this basic pattern, some Heteroptera species have one or two
flanges for muscle attachment, which is an important taxonomic
trait (Vavrinova 1988,Kocorek and Danielczok-Demska 2002,
Pluot-Sigwalt and Lis 2008). In L. zonatus there is one flange, which
seems to be the characteristic of Coreidae (Vavrinova 1988,Candan
2008).
The proximal region of the spermatheca of L. zonatus is an en-
larged duct. In Heteroptera this region varies from simple to com-
plex and from narrow to enlarged or expanded (Vavrinova 1988,
Kocorek and Danielczok-Demska 2002,Pluot-Sigwalt and Lis
2008). Pluot-Sigwalt and Lis (2008) have showed that the proximal
duct of the spermatheca has two types of dilations, simple dilations
of the duct wall or dilations containing folds toward the lumen
forming a complex structure. The L. zonatus spermathecal duct has
folds of the duct wall characterizing a complex structure. The cham-
bers between the folds are filled with secretion and sperm, suggest-
ing that they pass through the chambers with their secretion directed
toward the reservoir. The secretion from this region may contribute
to sperm maintenance and motility.
Although the model proposed is a hypothetical one, we consider
that, in the spermatheca of L. zonatus, during mating, the sperm
with the secretion produced by the male reproductive tract reaches
the common oviduct of the female and migrates into the enlarged
proximal duct of the spermatheca. In the proximal duct, the sperma-
tozoa move through the duct wall folds toward the spermathecal
reservoir, where the type III secretory cells release compounds for
sperm maintenance until they are required for oocyte fertilization.
Fig. 3. Histological section of the spermathecal reservoir and muscular duct of L. zonatus.(A) Secretory cells (sc) of the reservoir containing several vesicles posi-
tive for the PAS test. Scale bar: 30 mm. (B) Secretory cells (sc) of the reservoir containing several vesicles negative for the mercury-bromophenol test. Scale bar:
30 mm. (C) Spermathecal muscular duct with inner cuticle coating (ct), epithelial cells (ec), and striated muscle (m). Scale bar: 50mm. (D) Muscular duct subjected
to the PAS test positive in the basal lamina. Scale bar: 30 mm. (E) Muscular duct subjected to the mercury-bromophenol test with positive muscle fiber (m). Details
of the site of the insertion of the muscle fibers into the ring (circle). Scale bar: 200 mm.
4Annals of the Entomological Society of America, 2015, Vol. 0, No. 0
Fig. 4. Histological section of the proximal region of the spermatheca of L. zonatus.(A) Proximal region comprising a duct coated by a layer of epithelial cells (ec)
and a compartmentalized oval structure (dc). Stain: hematoxylin and eosin. Scale bar: 100 mm. (B) Details of the proximal region of the spermatheca stained with
eosin and hematoxylin showing epithelial cells (ec) and subcuticular area (s) filled with secretion. Scale bar: 50 mm. (C) Duct submitted to the PAS test, showing
the cytoplasm and secretion in the slightly positive subcuticular space (s). Scale bar: 20mm. (D) Duct subjected to the mercury-bromphenol test showing epithelial
cells (c) positive for the test and slightly positive secretion. Scale bar: 30 mm. (E) Duct submitted to the PAS test, showing the compartment wall inside the duct
negative to the test, and the secretion, positive. Scale bar: 30 mm. (F) Duct submitted to the mercury-bromophenol test showing positive secretion and compart-
ment wall inside the duct. Scale bar: 30mm.
Annals of the Entomological Society of America, 2015, Vol. 0, No. 0 5
Acknowledgments
We are grateful to Larissa Yoko Asai Carvalho for doing illustration and to
the Nucleus of Microscopy and Microanalysis of the Federal University of
Vic¸ osa for the technical assistance. This research was supported by Brazilian
research agencies National Council of Research (CNPq) and Minas Gerais
State Research Agency (FAPEMIG).
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