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Invertebrate Reproduction & Development
ISSN: 0792-4259 (Print) 2157-0272 (Online) Journal homepage: https://www.tandfonline.com/loi/tinv20
Structure of the male reproductive accessory
glands of Pterostichus nigrita (Coleoptera:
Carabidae), their role in spermatophore formation
Stephanie Krüger, Hans-Joerg Ferenz, Marvin Randall & Alan N. Hodgson
To cite this article: Stephanie Krüger, Hans-Joerg Ferenz, Marvin Randall & Alan N. Hodgson
(2014) Structure of the male reproductive accessory glands of Pterostichus�nigrita (Coleoptera:
Carabidae), their role in spermatophore formation, Invertebrate Reproduction & Development, 58:2,
75-88, DOI: 10.1080/07924259.2013.822835
To link to this article: https://doi.org/10.1080/07924259.2013.822835
Published online: 09 Aug 2013.
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Citing articles: 3 View citing articles
Structure of the male reproductive accessory glands of Pterostichus nigrita (Coleoptera:
Carabidae), their role in spermatophore formation
Stephanie Krüger
a
, Hans-Joerg Ferenz
a
, Marvin Randall
b
and Alan N. Hodgson
c
*
a
Institute of Biology, Martin-Luther-University, Halle-Wittenberg, Germany;
b
Electron Microscope Unit, Rhodes University,
Grahamstown, South Africa;
c
Department of Zoology & Entomology, Rhodes University, Grahamstown, South Africa
(Received 14 May 2013; accepted 1 July 2013)
Accessory gland secretions of male insects have many important functions including the formation of spermatophores.
We used light and electron microscopy to investigate the structure of the accessory glands and posterior vasa deferentia
of the carabid beetle Pterostichus nigrita to try to determine where spermatophore material is produced. Each accessory
gland and posterior vas deferens had an outer layer of longitudinal muscle, beneath which was a layer of connective
tissue and a thin band of circular muscle, all of which surrounded a layer of epithelial cells lining the lumen of the
ducts. Based on the ultrastructure of the epithelial cells, and their secretory products, we identified two epithelial cell
types in each region (distal and proximal) of the accessory glands and four types in the posterior vas deferens. Most
secretory products, which stained positively for proteins and some mucins, were released into the lumen of the ducts by
apocrine secretion. The accessory glands produced one type of secretory product whereas in posterior vasa deferentia,
four types of secretory products were found layered in the lumen. Our results suggest that most of the structural material
used to construct a spermatophore is produced by the cells of the posterior vasa deferentia.
Keywords: vas deferens; secretory cells; spermatophore formation; ultrastructure; histochemistry; Insecta
Introduction
In addition to producing material that forms
spermatophores in many species of insect (Leopold
1976; Chen 1984; Colonello and Hartfelder 2005), the
secretions from male accessory glands have other
important functions including sperm activation (Leopold
1976; Davey 1985; Chen 1984), sperm inactivation
(Harshman and Prout 1994), modification of sperm
bundles (Viscuso et al. 2001), contributions to mating
plugs (Leopold 1976; Colonello and Hartfelder 2005),
and effects on many aspects of female reproductive
physiology and behavior (Leopold 1976; Ramalingam
and Craig 1978; Chen 1984; Gillott 1988, 2003; Wolfner
1997; Simmons 2001; Radhakrishnan et al. 2009).
Therefore, the accessory glands of males play a pivotal
role in insect reproduction.
Studies on the structure of the accessory glands of
male insects have shown that the glands not only vary in
size, shape, and number between species, but also in the
morphology of their epithelial cells and types of
secretions within the glands (Odhiambo 1969a,b; De
Loof and Lagasse 1972; Tongu et al. 1972; Leopold
1976; Riemann and Thorson 1979; Lai-Fook 1982; Chen
1984; Davey 1985; Happ 1992; Dallai et al. 1999;
Sukontason et al. 2009). Although secretory product
variability exists between species, most produce a
secretion rich in proteins and peptides (Leopold 1976;
Davey 1985; Happ 1992; Wolfner 1997; Gillott 2003;
Marchini et al. 2009). Surprisingly, little is known about
the secretions and tissue structure of the accessory glands
of Coleoptera, especially at a fine structural level. To
date, information is available from four species only,
Leptinotarsa decemlineata (Chrysomelidae) (De Loof
and Lagasse 1972), Tenebrio molitor (Tenebrionidae)
(Gadzama et al. 1977; Dailey et al. 1980; Grimes and
Happ 1980; Happ and Happ 1982), Acanthoscelides
obtectus (Bruchidae) (Huignard 1975; Cassier and
Huignard 1979), and Bruchidius atrolineatus (Glitho and
Huignard 1990).
Pterostichus nigrita (Paykull 1790) is a carabid
beetle with a wide temperate distribution in Europe and
Asia (Angus et al. 2008). Like other members of the
tribe Pterostichini (Takami 2002, 2007; Sasakawa 2006,
2007; Takami and Sota 2007), male P. nigrita deposit a
spermatophore, of unknown composition, containing
sperm bundles (spermiozeugmata) that were formed in
the vas deferens (Ferenz 1986; Hodgson et al. 2012), in
the bursa copulatrix of the female during copulation
(Schneider and Ferenz 2012). The spermatophore may
be formed by secretions from a pair of accessory glands
that join the vasa deferentia which in turn open into the
male’s ejaculatory duct (Ferenz 1986; Hodgson et al.
*Corresponding author. Email: A.Hodgson@ru.ac.za
Invertebrate Reproduction & Development, 2014
Vol. 58, No. 2, 75–88, http://dx.doi.org/10.1080/07924259.2013.822835
Ó2013 Taylor & Francis
2012). However, we have observed that the posterior
region of the vas deferens (a region not differentiated
from the accessory glands in previous papers; Ferenz
1986; Hodgson et al. 2012) is also glandular and
therefore, may play a role in spermatophore formation.
As a first step towards understanding the production
and role of spermatophores in P. nigrita, we report the
results of a structural study of the male accessory glands
and secretions, and compare findings with those
published for other insects.
Materials and methods
Adult male Pterostichus nigrita were collected using
pit-fall traps from woodland on the outskirts of Halle
(Germany) in April and May (2011 and 2012) when
beetles are reproductively active. The reproductive
systems of several males were dissected in cold (4 °C)
Ringer’s solution. The accessory glands and posterior
section of the vasa deferentia were prepared for light and
transmission electron microscopy (TEM). In addition, the
secretion from glands was prepared for protein analysis
by gel electrophoresis.
Light microscopy, histochemistry
For histology and histochemistry, the entire glandular
region of three male reproductive ducts (accessory glands
and vasa deferentia) were fixed in 4% paraformaldehyde
in 0.1 M Sørensen’s buffer (pH 7.4) for 2 h at room
temperature, whilst others were fixed in aqueous Bouin’s
for at least 24 h. The paraformaldehyde-fixed tissues
were first washed in Sørensen’s buffer before
dehydration in a graded ethanol series (30–100%) and
embedded in Paraplast via xylene. The buffer step was
not included during the processing of the Bouin’sfixed
material. Sections (about 8 μm thickness) were stained in
alcian blue-periodic acid Schiff (for differentiating
between acid and neutral mucins), alcian blue (pH 2.5)
for acidic mucins, aldehyde fuscin (for sulphated
mucins), and mercuric bromophenol blue for the detec-
tion of proteins. In addition, semi-thin (about 1 μm thick)
resin sections were stained in toluidine blue (see below
for preparation methods of resin-embedded tissue).
Images were captured using an Olympus BX40
microscope and DP72 digital camera. All measurements
were made using AnalySIS version 3 (Soft Imaging
System GmbH).
Transmission electron microscopy (TEM)
Because accessory glands of insects may have distinct
secretory regions/zones with different cells types (e.g.
Tongu et al. 1972; Dapples et al. 1974; Ramalingam and
Craig 1978; Riemann and Thorson 1979), we fixed small
portions of tissue for TEM from two regions of the
glands, hereinafter referred to as the distal and proximal
(furthest and closest to joining the vas deferens,
respectively) regions (Figure 1). In addition, we also
fixed tissues from the posterior vas deferens because this
region of the male reproductive system had a glandular
appearance (Figure 1). Tissues from four males were
fixed using three methods (Table 1). After primary and
secondary fixation, tissues were washed in the respective
buffers, dehydrated through a graded ethanol series, and
embedded in Araldite via propylene oxide. Ultrathin
sections (silver/gold interference) cut using a diamond
knife and RMC 7 ultramicrotome were stained with lead
citrate and uranyl acetate, and examined using a JEOL
1010 (at 80 kV) fitted with a Megaview II camera
(University of Halle-Wittenberg) or JEOL JEM2100F (at
200 kV) fitted with a Veleta camera (University of
Pretoria).
Protein electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis (SDS-PAGE) was performed according to the method
of Laemmli (1970) using 12% (w/v) and 7.5% (w/v)
acrylamide separating gel, and a 4% (w/v) stacking gel
both containing 0.1% (w/v) SDS. The contents of three
male accessory gland/posterior vas deferens were
obtained by opening the glands and allowing the con-
tents to flow out into 1 M Tris-HCl buffer (pH 6.8). The
secretion and buffer were then mixed, heated at 95 °C
for 4 min, cooled down on crushed ice, and spun down
prior to loading in the gel slots. Electrophoresis was
carried out at a constant voltage (200 V/ 60 mA) for
80 min using a tris glycine buffer (pH 8.3) containing
Figure 1. Part of the male reproductive system of
Pterostichus nigrita showing the upper section of the single
ejaculatory duct (ed), posterior region of glandular vas deferens
(pv), a small section of the non-glandular region of vas
deferens (vd), where the accessory gland joins the vas deferens
(arrow), and the proximal (2) and distal (1) regions of the
accessory gland. Scale bar = 2 mm.
76 S. Krüger et al.
0.01% (w/v) SDS. The gel was removed from the
electrophoresis unit (Mini-Protean II-System, BioRad)
stained with Coomassie brilliant blue R-250, and
de-stained in 30:10:70 methanolic: acetic acid: water.
The protein standards were BioRad SDS-PAGE
molecular weight standards (catalog number 161–0304)
in the low (21.500–97.400 Da) and high range (45.000–
200.000 Da).
Results
Gross morphology of the glands
The glandular region of the posterior male reproductive
system of P. nigrita consists of a pair of S-shaped tubes
each about 8 mm long and 0.5 mm in maximum diameter
that open into a single ejaculatory duct (Figure 1). The
anterior half of these tubes we presume to be accessory
glands, and the posterior portion a glandular region of
the vas deferens. When mature, the entire structure has a
white appearance (Figure 1) because it contains a white
viscous secretion.
When viewed in cross-section, each accessory gland
and posterior vas deferens consists of an outer layer of
well-developed, longitudinal muscle, beneath which is a
layer of connective tissue and a thin band of circular
muscle that surround a single layer of epithelial cells,
which line the lumen of the duct (Figures 2(A), 4(A),
and 5(A) and (B)). As the focus of this paper was the
structure of the cells that produce the male reproductive
secretions, the ultrastructure of the muscle and
connective tissue (which contains collagen fibers) is not
described. Based on the ultrastructure of the epithelial
cells, and their secretory products, we identified two cell
types in each region (distal and proximal) of the
accessory gland, and four cell types in the posterior vas
deferens.
Structure of the gland cells and secretion
The best fixation of cells was obtained by methods 1
and 3 (see Table 1), and all images presented are from
cells fixed by these methods. With method 2, which used
microtubule stabilizing buffer, all mitochondria of the
epithelial cells were damaged, although other cell
structures appeared to be well preserved.
Distal region of the accessory gland
In the distal region of the gland, the lumen was filled
with numerous homogeneously electron-dense droplets
ranging in diameter from about 0.2–0.6 μm within a
more homogenously fine granular matrix (Figure 2(A)
and (H)). The epithelium consisted of two types of cell,
each type grouped in one region of the epithelium
(Figure 2(A)). Cell type 1, the most common cell type
that was also found in the proximal region of the gland
as well as the posterior vas deferens, was approximately
columnar (30 μm5μm) in shape (Figure 2(B)). In the
mid-region of these cells, the plasma membranes of
adjacent cells were adpressed to each other (Figure 2(G))
and apically they were joined by demosomes and septate
junctions (not illustrated; such junctions were found
between all epithelial cells). In glands full of secretion,
the basal plasma membranes of the cell were highly
folded forming a lacunar system adjacent to the basal
lamina and between adjacent cells (Figure 2(B)). This
folding was not apparent in glands containing little
secretion (Figure 2(C)). In the cytoplasm of the basal
regions of such cells, fibrillar structures were present
(Figure 2(D) and (E)). The basal nuclei (about
5μm3μm) of type 1 cells had an abundance of
euchromatin, with heterochromatin visible in a few areas
(Figure 2(B) and (F)). The cytoplasm of the cells
contained rough endoplasmic reticulum, which was
mainly peripheral and adjacent to the plasma membrane,
a few small mitochondria (about 0.75 μm0.25 μm),
and in glands full of secretion, an abundance of
homogenously electron-dense, spherical, secretory
droplets (maximum diameter about 0.6 μm) (Figure 2(B),
(F) and (G)). A few droplets containing concentric
whorls were also observed (Figure 2(F) inset). The
electron-dense droplets appear to be released from
cytoplasmic extensions of the apical region of the type 1
cells by an apocrine mechanism (Figure 2(H)). In addi-
tion to the electron-dense droplets, the apical region of
cell type 1 contained vacuoles with fine granular material
and some small electron-dense granules (Figures 2(H)
and 3(A)). These appear to be released by merocrine
secretion (Figures 2(H) and 3(A)).
The second cell type (about 20 μm5μm) (Figure 3
(A) and (B)) in the apical region of the gland had a basal
Table 1. Fixation methods used for transmission electron microscopy.
Primary fixation Secondary fixation
Method 1 4% paraformaldehyde in 0.1 M sodium phosphate
buffer at 4 °C, overnight
1.5% osmium tetroxide in 0.1 M sodium
phosphate buffer for 2 h
Method 2 4% glutaraldehyde in microtubule stabilization
buffer at room temperature (20 °C), 4 h
2% osmium tetroxide in 0.1 M sodium
phosphate buffer, 2 h
Method 3 2.5% glutaraldehyde in 0.1 M Sørensen buffer
(pH 7.2) with 2% sucrose at 4 °C, 4 h
1.5% osmium tetroxide in 0.1 M Sørensen
buffer for 2 h
Invertebrate Reproduction & Development 77
nucleus about 3 μm1.5 μm in size (Figure 3(D)). The
basal plasma membranes of these cells were also highly
folded and separated from each other by large intercellu-
lar spaces (Figure 3(D)). Unlike cell type 1, the basal
regions of type 2 cells contained pockets of a granular
electron-dense material that we presume to be glycogen
Figure 2. Light (A) and transmission electron (B–H) microscope images of the distal region of the accessory gland of Pterostichus
nigrita. (A) Semi-thin transverse section stained in toluidine blue showing longitudinal muscle (lm) and circular muscle (cm)
surrounding two types (1 and 2) of epithelial cells, and lumen of the duct filled with secretion (se). (B) Basal region of type 1
secretory cells filled with secretory droplets (sd). Note the basal nucleus (n), folding of basal plasma membrane, and lacunar spaces
(arrows) between cells. (C) Basal region of type 1 cells that only had a few secretory droplets. Arrow indicates basal lamina between
cells and muscle (mu). (D) Basal region of type 1 cells with microfibrilar structures (arrowed) in the cytoplasm. (E) Microfibrillar
structures (arrowed) seen in cross-section. (F) Higher magnification of the nuclear region of a type 1 cell with secretory granules of
differing electron density. Inset: Secretory droplet with internal concentric layers. (G) Mid-region of a type 1 cell showing rough
endoplasmic reticulum (rer) adjacent to adpressed plasma membranes (arrow) of adjacent cells. (H) Apical region of a type 1 cell
showing release of spherical electron-dense secretory droplets (sd) and fine granular material (
⁄
) with some electron-dense spherical
granules (arrowed) into the lumen (lu) of the duct. m, mitochondrion; n, nucleus; and rer, rough endoplasmic reticulum.
Scale bars: A = 100 μm; B = 5 μm; C, D, E, F, G, H = 1 μm; Inset = 0.25 μm.
78 S. Krüger et al.
Figure 3. Transmission electron microscope images of the distal region of the accessory gland of Pterostichus nigrita. (A) Adjacent
apical regions of type 1 (1) and 2 (2) cells. Note the release of granular secretion (arrow) from the type 1 cells and the microvilli
(mv) of the type 2 cells. (B) Apical region of type 2 cells showing abundance of mitochondria (m) and elongate microvilli (mv). (C)
Higher magnification of the apical region of a type 2 cell showing mitochondria (m) with electron-dense matrix, small secretory
vesicles (v), and site of fusion of vesicles with plasma membrane (arrows). (D) Basal region of type 2 cells showing intercellular
spaces (is) and pockets of glycogen (gly). (E) Microvilli (mv) of type 2 cells and associated fine granular secretion (
⁄
). (F) Higher
magnification of glycogen (
⁄
) pocket. n, nucleus.
Scale bars: A, B, D, E = 1 μm; C, F = 0.5 μm.
Invertebrate Reproduction & Development 79
(Figure 3(D) and (F)). The cytoplasm of type 2 cells
appeared more electron-dense when compared to type 1
cells (Figure 3(A)), contained many free ribosomes (not
illustrated), as well as an abundance of mitochondria
each with an electron-dense matrix (Figure 3(B) and
(C)). Apically, cell type 2 had microvilli about 2 μm long
that were often associated with a fine granular secretion
(Figure 3(A) and (E)). Small vesicles (about 80 nm
diameter) were observed close to, and possibly fusing
with, the apical plasma membrane (Figure 3(C)).
Proximal region of accessory gland
The proximal region of the examined accessory glands
prior to the merger with the vas deferens had a very
small lumen (Figure 4(A)) that contained secretion
comprising spherical to irregularly-shaped globules of
varying electron-density, as well as homogeneous
electron-dense secretion (Figure 4(B) and (C)). The
epithelium consisted of cell types 1 and 2, both of which
were larger than those in the distal region of the gland.
Cell type 1 was about 100 μm5μm, and near the
apical region the plasma membranes of adjacent cells
that were interdigitated (Figure 4(D)). Within the
cytoplasm, the electron-dense secretory droplets, most of
which were spherical, were distributed mainly in the
apical and basal regions of the cell, with an area of cyto-
plasm between that contained rough endoplasmic reticu-
lum but was largely devoid of the droplets (Figure 4(E)).
In the basal region, the cytoplasm contained the nucleus,
Figure 4. Light (A) and transmission electron (B–G) microscope images of the proximal regions of the accessory gland of
Pterostichus nigrita. (A) Semi-thin toluidine blue stained transverse section showing the outer longitudinal muscle (lm) and circular
muscle (cm) layers surrounding the columnar epithelial cells (e). (B) Part of the lumen of the accessory gland containing secretion (
⁄
)
with a homogeneous appearance, and apical region of some type 1 cells with secretion droplets (sd). (C) Part of the lumen of the
accessory gland containing heteromorphic secretory globules. (D) Apical region of type 1 secretory cells with spherical secretory
droplets (sd). Note the interdigitations of adjacent plasma membranes (arrows). (E) Mid-region of type 1 secretory cells showing area
of cytoplasm (between arrows) largely devoid of secretory droplets. (F) Example of a small Golgi body (g) in the basal region of a
type 1 cell. (G) Basal region of type 1 cells with nucleus (n) and swollen cisternae of rough endoplasmic reticulum (rer).
Scale bars: A = 100 μm; B, D, G = 2 μm; C = 1 μm; E = 5 μm; F = 0.25 μm.
80 S. Krüger et al.
Figure 5. Light (A, B) and transmission electron (C–K) microscope images of the posterior region of the vas deferens of
Pterostichus nigrita. (A), (B) Semi-thin toluidine blue transverse sections showing a less active duct (A) with very little secretion,
and a more active duct (B) full of different secretory products (1–4). (C),(D) Appearance of the secretory products in the lumen of a
full duct; (1) homogeneous less electron-dense secretion, (2) more electron-dense secretion; (3) irregularly-shaped electron-dense
secretory material, (4) electron-dense spheres in a homogeneous matrix. (E) Apical region of type 1 cells of a less active gland, with
secretory droplets (sd) and secretion (arrow) in the lumen of the duct. (F) Mid-region of more active type 1 cells with abundant
rough endoplasmic reticulum (rer) with swollen cisternae and electron-dense granules in the cytoplasm. (G) Apical region of a less
active type 3 cell with a few secretory droplets (
⁄
) with a heterogeneous content. (H) Apical region of more active type 3 cells in
which there is a greater abundance of secretory droplets (sd) in the cytoplasm. Note also the lumen of the duct filled with secretion
(se) and the release of secretory granules by apocrine secretion (arrow). (I) Mid-region of very active type 3 cells, with secretory
droplets (sd), abundant rough endoplasmic reticulum (rer), and putative Golgi body (gb). Also present are some multivesicular
secretory bodies (mvb). (J) Basal region of more active type 3 cells showing nuclei (n) and secretory droplets (sd). (K) An example
of the well-developed rough endoplasmic reticulum from the basal region of a type 3 cell.
Scale bars: A, B = 100 μm; C, D, H = 5 μm; E, F, G, I, J = 2 μm; K = 1 μm.
Invertebrate Reproduction & Development 81
abundant rough endoplasmic reticulum with swollen cis-
ternae, and one or two small Golgi bodies (Figure 4(F)
and (G)). Spherical secretory droplets were in close
proximity to these organelles. The structure of cell type
2 was as described for the distal region of the gland and
therefore not illustrated.
Posterior vas deferens
The posterior regions of vasa deferentia that contained a
small amount of secretion only (Figure 5(A)), and those
filled with secretion (Figure 5(B)), were examined.
We refer to the empty and filled vasa deferentia as less
active, and more active, respectively. In the less active
posterior vas deferens, the secretion in the lumen when
viewed with the TEM was similar in appearance (and
therefore not illustrated) to that in the distal region of the
accessory gland, although some globules with a
heterogeneous appearance were present (Figure 5(E)). In
posterior vasa deferentia full of secretion, four types of
secretory product (based on appearance and staining in
toluidine blue) were found in the lumen. The first type
was homogenous in appearance, stained light blue with
toluidine blue (Figure 5(B); 1), and was not very
Figure 6. Transmission electron microscope images of the posterior region of the vas deferens of Pterostichus nigrita. (A) Apical
region of a type 4 cell containing spherical secretory vesicles (v) with a fine granular content and electron-dense periphery. Some of
the vesicles (arrow) are being released into the lumen (l) of the posterior vas deferens that is full of secretion. (B) Basal region of a
type 4 cell showing nucleus (n) surrounded by spherical secretory vesicles (v) and some rough endoplasmic reticulum (rer). (C) Basal
region of a less active type 4 cell showing close association of secretory vesicles (v) and rough endoplasmic reticulum (rer). (D)
Basal region of a type 4 cell that was full of secretory vesicles. Note the extensive infolding of the plasma membrane (arrows). (E)
Apical region of a type 5 cell showing microvilli (mv) and secretory vesicles (v) with coarse granular content. Arrow points to
vesicle being secreted into lumen (l) of posterior vas deferens. (F) Apical region of a type 5 cell showing apocrine secretion
of vesicles (arrow) between microvilli (mv) into lumen (l) of posterior vas deferens. (G) Basal region of a type 5 cell showing
granular vesicles in close proximity to rough endoplasmic reticulum (rer) and nucleus (n).
Scale bars: A, B, C, D, F, G= 2 μm; E = 1 μm.
82 S. Krüger et al.
electron-dense (Figure 5(C); 1). The second type stained
magenta with toluidine blue was slightly more
electron-dense and not as homogeneous in appearance
when compared to secretion 1 (Figure 5(B) and (C); 2).
The third type (Figure 5(B)–(D); 3) stained dark blue in
toluidine blue, and consisted of irregularly shaped
electron-dense globules of variable dimensions. Finally,
the fourth type (Figure 5(B) and (D); 4) also stained dark
blue in toluidine blue, but consisted of electron-dense
granules within a more homogenous electron lucent
matrix.
The epithelium of the posterior region of the vas
deferens contained four types of secretory cell. Type 1
cells were similar in structure and size (about
100 μm5μm) to those found in the accessory glands.
Each cell had a basal nucleus and the cytoplasm
contained a few small mitochondria and numerous
spherical electron-dense droplets (0.2–0.6 μm diameter)
mainly located in the apical (Figure 5(E)) and basal
regions of the cells (not illustrated but see Figures 2(B)
and 4(D) and (E)). The apical regions of these cells
protruded into the lumen of the duct (Figure 5(E)), and
like type 1 cells in the accessory gland the cytoplasmic
droplets were released by apocrine secretion (see Figure 2
(H)). The cytoplasm of the mid-regions of type 1 cells
was mainly devoid of secretory droplets but contained
abundant rough endoplasmic reticulum often with
swollen cisternae (Figure 5(F)). The second type of
secretory cell (designated cell type 3) contained secretory
droplets with a heterogeneous appearance (Figure 5(G)).
In the less active posterior vasa deferentia, the secretory
droplets which were up to 2 μm in diameter with a rela-
tively uniform electron-dense appearance (Figure 5(G))
occupied about 50% of the cytoplasm. In the more active
posterior vas deferens, the secretory droplets (up to 2 μm
in diameter) of cell type 3 consisted of globules of
electron-dense material surrounded by a more electron-
lucent region (Figure 5(H), (I) and (J)). The droplets
now occupied most of the cytoplasm (Figure 5(H)). In
addition to the secretory droplets, type 3 cells had a
basal nucleus, an abundance of well-developed rough
endoplasmic reticulum that often had swollen
cisternae (Figure 5(H)–(K)), a few small Golgi bodies
(Figure 5(I)), and multivesicular bodies up to 1.8 μm
Figure 7. Light microscope images of the accessory gland (A, B) and posterior vas deferens (C, D, E). (A) Longitudunal section of
the distal region of an accessory gland stained in bromophenol blue, showing positive staining of type 1 cells, but not type 2. Arrows
indicate muscle layers. (B) Transverse section through the posterior vas deferens showing positive variable staining in bromophenol
blue of epithelial cells and secretion (s) in the duct lumen. (C) Transverse section through the posterior vas deferens stained in PAS
showing weak staining of cells and secretion in the lumen of the duct (s). (D) Oblique section of the junction between the proximal
region of the accessory gland (p) and posterior vas deferens (pvd) stained in aldehyde fuchsin. Note the positive staining of the type
4 cells (4) and some of the secretion (s). (E) Transverse section of the posterior vas deferens stained in aldehyde fuchsin showing
positive staining of type 4 cells (4) and part of the secretion (s).
Scale bars = 100 μm.
Invertebrate Reproduction & Development 83
diameter (Figure 5(I)). Apically, the secretory droplets
were released into the lumen of the duct by apocrine
secretion (Figure 5(H)). In the more active posterior
vas deferentia, the lumen of the duct adjacent to cell
types 1 and 3 was full of secretory products 3 and 4
(Figure 5(B)).
The third type of gland cell (designated cell type 4)
was approximately columnar in shape and of similar
dimensions to the cell type 1. Each cell had a basal
nucleus, about 5 μm4μm in size, with a distinct
nucleolus about 0.5 μm in diameter, and small patches of
heterochromatin (Figure 6(B)). In the more active
posterior vasa deferentia, the cytoplasm of the cells was
packed with spherical (up to 1.6 μm diameter) vesicles
with a fine granular content and thin electron-dense
periphery (Figure 6(A) and (B)). In the basal region of
these cells, the vesicles were closely associated with
rough endoplasmic reticulum (Figure 6(B) and (C)).
Release of the vesicles into the lumen of the posterior
vas deferens appears to be by holocrine secretion
(Figure 6(A)). The basal plasma membrane of type 4
cells that were full of secretory vesicles was highly
folded and joined the basal lamina by hemidesmosomes
(Figure 6(D)). By contrast, in those type 4 cells that had
a few secretory vesicles only, the basal plasma
membrane was not folded (Figure 6(C)). Secretory
products 1 and 2 in the duct lumen (Figure 5(B)) were
in close proximity to type 4 cells.
The fourth type of gland in the posterior vas deferens
(designated cell type 5 and also columnar in shape) had
a cytoplasm containing spherical vesicles (about 1.5 μm
diameter) with either a coarse granular or mosaic
appearance (Figure 6(E)–(G)). Unlike the other gland
cells of the posterior vas deferens, type 5 cells possessed
well-developed apical microvilli (Figure 6(E) and (F)).
The vesicles when released into the lumen of the duct of
the posterior vas deferens by apocrine secretion formed a
fine granular secretion (Figure 6(E) and (F)).
Histochemistry and protein gel electrophoresis
Four of the cell types (1, 3, 4, and 5) from the accessory
glands and posterior vasa deferentia, and most of the
secretion in the lumen of these regions of the male
reproductive system stained positively for protein with
bromophenol blue, although the intensity of the staining
was variable (Figure 7(A) and (B)). By contrast, cell
type 2 did not stain positively with bromophenol blue
(Figure 7(A); 2). The results of SDS-Page showed the
that secretion within the accessory glands and posterior
vas deferentia contained proteins with a wide range of
molecular weights, with bands detectable below 45 kDa,
between 45 and 66 kDa as well as 66 and 97 kDa and
faint bands at about 100 and 116 kDa (Figure 8).
All of the epithelial cells and some of the components
of the secretion stained weakly with PAS indicating the
presence of some neutral mucins (Figure 7(C)). Cells in
the accessory gland did not stain in aldehyde-fuchsin,
however, in the posterior vas deferens, the epithelial cells
where type 4 gland cells were located, and secretion
product 2, stained positively for sulphated mucins
(Figure 7(D) and (E)).
Neither the epithelial cells nor the secretions stained
in alcian blue, suggesting the absence of acidic mucins.
Discussion
The number of accessory glands in male Pterostichus
nigrita is similar to that of Zygogramma exclamationis
and Leptinotarsa decemlineata (Chrysomelidae), Popillia
japonica (Scarabaeidae) and several Cleridae, all
possessing a single pair of simple tubules (Anderson
1950; De Loof and Lagasse 1972; Gerber et al. 1978;
Opitz 2003) that join the posterior region of the vasa
deferentia. These beetles have the simplest male duct
morphology of the Coleoptera (Kaulenas 1992) in
contrast to other beetle species which can have two,
three, or five pairs of glands (Gerber et al. 1971;
Huignard 1975; Gadzama et al. 1977; Happ et al. 1977;
Singh 1978; Cassier and Huignard 1979; Glitho and
Huignard 1990).
Each accessory gland and posterior vas deferens of
P. nigrita, consist of a lumen surrounded by a single
layer of columnar epithelial cells with basal nuclei.
These cells in turn are surrounded by a relatively thin
layer of circular muscle and connective tissue, and a
thicker sheath of longitudinal muscle. This arrangement
is typical of the reproductive ducts of many male insects
116
97
66
45
116
97
66
45
Figure 8. SDS-PAGE of protein contents of male accessory
glands and posterior deferentia. (A) 7.5% gel, left lane
molecular weight standards; (B) 12.5% gel, right lane molecular
weight standards) (standards: 116 kD β- galactosidase; 97 kD
phosphorylase b; 66 kD bovine serum albumin; 45 kD
ovalbumin).
84 S. Krüger et al.
(Dapples et al. 1974; Lai-Fook 1982; Gillott 1988;
Glitho and Huignard 1990; Kaulenas 1992; Gillott
1996). The accessory glands of Coleoptera can be either
mesodermally or ectodermally derived (Kaulenas 1992).
The position of the glands of P.nigrita with respect to
the vasa deferentia, and lack of cuticle, suggests that the
accessory glands are mesadenia.
The number of morphological types of secretory cell
in tubular accessory glands of male insects is highly
variable. Those of L. decemlineata (De Loof and Lagasse
1972), Periplaneta americana (Adiyodi and Adiyodi
1974), Tenebrio molitor (Gadzama et al. 1977; Happ
et al. 1982), and Frankiniella occidentalis (Dallai et al.
1997) have one type only. By contrast, the tubular acces-
sory glands of other male insects can be divided into dis-
tinct regions, each region with a different type of
secretory cell and/or secretion (Ramalingam and Craig
1978; Riemann and Thorson 1979; Lai-Fook 1982;
Marchini et al. 2009). Two morphological types of
epithelial cells were found in the male accessory glands
of P. nigrita, one with a cytoplasm full of secretory drop-
lets and an apical plasma membrane lacking microvilli
(type 1 cells), and a second type (type 2 cells) lacking
secretory droplets but with well-developed microvilli.
Whereas type 1 cells are undoubtedly secretory, releasing
their electron-dense droplets into the lumen of the gland
by apocrine secretion, the function of type 2 cells is
uncertain. However, because these cells contain well-
developed rough endoplasmic reticulum, an abundance of
mitochondria, glycogen, and have small vesicles that
appear to fuse with the apical plasma membrane, it sug-
gests that they are also biosynthetically active and secre-
tory. Similar small secretory vesicles were found in some
of the accessory gland cells of Schistocerca gregaria
(Odhiambo 1969a) and Achroia grisella (Fernandez and
Cruz-Landim 2005). In contrast to the accessory glands,
four types of secretory cell were identified in the epithe-
lium of the posterior vas deferens of P. nigrita. This
greater gland cell diversity means that this region of the
male reproductive tract is more like the accessory glands
of Culex,Drosophila,Locusta,Schistocerca, and the
bean-shaped glands of Tenebrio (Gadzama et al. 1977).
Morphological features typical of the epithelial cell
types of insect accessory glands and the posterior vas
deferens of P. nigrita are the basal nucleus, infoldings of
the basal cell membrane (Freitas et al. 2010) that may
form a basal lacunar system (Glitho and Huignard 1990),
interdigitations of the lateral regions of the cell
membranes, septate junctions, an abundance of well-
developed rough endoplasmic reticulum and secretory
droplets that vary in appearance between cell types (e.g.
Bairati 1968; Adiyodi and Adiyodi 1974; Dapples et al.
1974; Gadzama et al. 1977; Ramalingam and Craig
1978; Cassier and Huignard 1979; Riemann and Thorson
1979; Grimes and Happ 1980; Couche and Gillot 1990;
Kaulenas 1992; Dallai et al. 1997, 2012; Cruz Landim
and Dallacqua 2005; Fernandez and Cruz-Landim 2005;
Freitas et al. 2007; Marchini et al. 2009; Freitas et al.
2010; this study). Freitas et al. (2007) suggested that
basal infolding of the plasma membranes of cells
indicated that they are absorbing substances from the
hemolymph. The well-developed rough endoplasmic
reticulum is a feature of a cell with very active protein
synthesis. Well-developed Golgi bodies are also a com-
mon feature of biosynthetically active gland cells of male
insects (e.g. Bairati 1968; Odhiambo 1969a; Gadzama
et al. 1977; Riemann and Thorson 1979; Dailey et al.
1980; Glitho and Huignard 1990; Kaulenas 1992; Dallai
et al. 1997; Fernandez and Cruz-Landim 2005; Marchini
and Del Bene 2006; Marchini et al. 2009; Radhakrishnan
et al. 2009). However, in the gland cells of some species
of male insect, the Golgi bodies are scarce and poorly
developed (Dapples et al. 1974; Cassier and Huignard
1979) which is also the case in P. nigrita.InLocusta
and Melanoplus (both Orthoptera), for example, Golgi
bodies only become well developed with male
maturation. In our study, all beetles were reproductively
mature, possessing spermatozoa and sperm bundles in
the seminal vesicle region of their vasa deferentia. The
reason for the scarcity of Golgi bodies in the accessory
gland cells of P. nigrita and some other insects is
unknown. Finally, some authors have reported the
presence of filamentous and fibrillar structures in the
epithelial cells of accessory glands (e.g. Bairati 1968; De
Loof and Lagasse 1972). Similar structures were found
in the type 1 cells of P. nigrita. To date the function of
such structures has not been established.
The epithelial cells of some insect accessory glands
all bear microvilli (e.g. Odhiambo 1969a; Adiyodi and
Adiyodi 1974; Dapples et al. 1974; Gadzama et al.
1977; Glitho and Huignard 1990; Marchini et al. 2003;
Fernandez and Cruz-Landim 2005; Marchini and Del
Bene 2006; Freitas et al. 2007; Freitas et al. 2010; Dallai
et al. 2012). In some species, the presence of microvilli
can be related to reproductive activity. For example, in
the beetle Bruchidius atrolineatus, Glitho and Huignard
(1990) observed that the cells of the accessory glands of
diapausing males do not possess microvilli, whereas
those of sexually active males do. These authors suggest
that in cells with microvilli, secretions are released by
exocytosis, and Freitas et al. (2010) have proposed that
microvilli function to increase secretion surface area. By
contrast, Fernandez and Cruz-Landim (2005) suggested
that in A. grisella, the microvilli of the accessory gland
cells play a role in the partial resorption of some
secretory substances. Only two cell types were found to
possess microvilli in P. nigrita, type 2 from the acces-
sory glands and type 5 from the posterior vas deferens.
As type 5 cells showed evidence of apocrine secretion,
the role of microvilli in this cell type is unclear.
Invertebrate Reproduction & Development 85
The mode of secretion of male insect accessory gland
cells studied to date is apocrine or merocrine (Dapples
et al. 1974; Gadzama et al. 1977; Ramalingham and Craig
1978; Lai-Fook 1982; Chen 1984; Davey 1985; Kaulenas
1992; Freitas et al. 2007) and this was so for most of the
secretory cell types in P. nigrita. One cell type in the
posterior vas deferens, however, showed evidence of
holocrine secretion. The appearance of the secretion in the
lumen of the accessory glands of P. nigrita was similar
throughout the gland, and in addition was similar in
appearance to that in the 1st order accessory glands of
Bolivarius siculus (Orthoptera) (Marchini et al. 2009). In
T. molitor, secretion from the tubular accessory glands is
mixed with the fluid and sperm from the seminal vesicles
in the interior of the spermatophore and forms part of the
seminal plasma (Grimes and Happ 1980; Black et al.
1982; Happ 1992). It is possible that the secretion from
the accessory glands of P. nigrita contributes to the
spermatophore in a similar way.
By contrast to the accessory glands, the lumen of the
posterior vas deferens of P. nigrita contained several
different types of secretion. In Orthoptera, the spermato-
phore is formed by secretions from different accessory
gland tubules (Kaulenas 1992; Marchini et al. 2009),
each tubule producing different proteins. In T. molitor,
the spermatophore is produced by a pair of bean-shaped
accessory glands, each gland possessing seven types of
epithelial cell organized as homogeneous populations
(Dailey et al. 1980). Each cell type produces a specific
product that contributes to a layered secretory plug
which contains spermatophorins used in the construction
of the spermatophore (Happ 1992). In P. nigrita, the
gland cells of the posterior vas deferens appear to fulfill
this function. Cells producing the same secretion are
grouped together and the secretion they produce is
arranged as layers in the lumen of the duct. This
suggests that most of the structural material of the
spermatophore is formed in the posterior region of the
vas deferens. The role of the vas deferens in spermato-
phore production has also been reported for the beetle
Lytta nuttalli (Gerber et al. 1971). By contrast, in insects
with more than one pair of accessory gland, different
glands produce the different secretions that form the
spermatophore (Glitho and Huignard 1990).
Numerous histochemical and biochemical studies
have shown that the secretions of male accessory glands
are composed mainly of proteins and mucopolysaccha-
rides (e.g. Odhiambo 1969b; De Loof and Lagasse 1972;
Adiyodi and Adiyodi 1974; Huignard 1975; Gadzama
et al. 1977; Cassier and Huignard 1979; Davey 1985;
Gillott 2003; Cruz-Landim and Dallacqua 2005;
Fernandez and Cruz-Landim 2005). It is therefore, not
surprising that the secretion in the lumen of the acces-
sory glands and posterior vasa deferentia of P. nigrita
stained positively for proteins and mucosubstances (but
not for acidic mucins which was also found by De
Loof and Lagasse 1972 in the Colorado beetle).
Electrophoretograms have revealed that the proteins in
male accessory gland secretions have molecular weights
of 10 to >100 kDa (Happ et al. 1982; Happ 1992; Cruz
Landim and Dellacqua 2005) although a few typically
dominate e.g. 72 kDa protein LHP1 (Gillott 1996). Our
preliminary analysis of the secretions of the glands in
P. nigrita also revealed proteins with molecular weights
mainly within this range.
In conclusion, we suggest that based on our morpho-
logical observations, during copulation, the secretions
from the accessory glands of male P. nigrita mix with
the spermiozeugmata from the anterior vasa deferentia,
and are then surrounded by secretions produced by the
glandular epithelial cells of the posterior vasa deferentia
to form the spermatophores.
Acknowledgments
This study was funded by grants to from Rhodes University,
the National Research Foundation and Institute of Biology,
University of Halle-Wittenberg to ANH, and the DAAD-
German Academic Exchange Service to SK. We would like to
thank Prof. Dr. G. Moritz for use of the Jeol 1100 TEM in the
Institute of Biology, University of Halle-Wittenberg, and to Mr
C. van der Merwe and Mr A. Hall of the Laboratory for
Microscopy and Microanalysis, University of Pretoria, for use
of the Jeol JEM 2100F TEM. We thank Eva Schladitz for
assistance with the protein gel electrophoresis. ANH would
also like to thank Prof. M.G. Bentley for handling the editorial
work on this paper.
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