Journal of Cell and Tissue Research Vol. 15(2) 4929-4942 (2015)
(Available online at www.tcrjournals.com)
ISSN: 0973-0028; E-ISSN: 0974-0910
Original Article
ULTRASTRUCTURE AND HISTOPATHOLOGICAL CHANGES IN
TESTES AND ACCESSORY GLANDS IN CALLOSOBRUCHUS
MACULATUS (F) (COLEOPTERA, BRUCHIDAE) TRANSPIRED
BY SOLAR RADIATION
KHALED, A. S., MOHAMED, M. I., ABDEL
FATTAH, H. M., HUSSEIN, M. A.,
?
SALEM, D. A. M. AND FAWKI, S.
Entomology Department, Faculty of Science, Ain Shams University, Abbasiya 11566, Cairo, Egypt.
E. mail: [email protected], Cell: 01221001066
Received: March 26, 2015; Revised: April 15, 2015; Accepted: May 2, 2015
Abstract: The ultrastructure and histopathology in the testes and accessory glands were studied
in normal adult males and in those developed from solar energy treated pupae (53 ºC for 15
minutes) of Callosobruchus maculatus. The adult male reproductive system consists of two
pairs of testes, two vasa deferentia, vesicular seminales, an ejaculatory duct and four pairs of
accessory glands. Testes are formed of numerous testicular follicles containing germ cells at
different stages of development. The transformation process of spermatid to sperm involves
several morphological reorganization of the cells. Cross sections of elongating sperm tail of
mature spermatozoa revealed the existence of two equal mitochondria derivatives containing
crystalline material and the axial filament comprises 9+9+2 microtubules. C. maculatus has
four pairs of accessory glands (one pair of lateral gland, the mesadenia and three pairs of
median glands the ectadenia). The accessory glands consist of glandular cells with a well
developed rough endoplasmic reticulum and Golgi apparatus. Variable morphological appearance
of the accessory glands secretion was present in the gland lumen. The testicular follicles of
treated pupae showed structural abnormalities. The cyst cells have a severe vacuolation,
degeneration of germ cells, malformation and tearing of sperm bundles. Accessory glands were
the most obvious signs of damage. The three different parts of the accessory glands (muscle
layer, gland epithelium and lumen) changed as a result of solar effect. Deterioration of muscular
layer, cytoplasmic organelles were very poor and the glandular lumen was filled with vacuoles
and malformed granules. The damage was more pronounced in the gonads of (F) progeny of
the treated generation.
Key words: Callosobruchus maculatus, Male reproductive organs, Ultrastructure, Solar energy.
INTRODUCTION
Cowpea is highly susceptible infested with different
species of beetles of family Bruchidae. This family
is wide spread in many regions of Asia, Africa and
central and South America except Antarctica [1]. The
two main bruchids found in Egypt are the pulse beetle
Callosobruchus chinensis and the cowpea beetle
Callosobruchus maculatus [1,2]. C. maculatus is a
field-to-store pest of several grain legumes that
causes a substantial quantitative and qualitative lose
by larval feeding which makes the seeds unfit either
for planting or human consumption [3,4].
Several methods have been used to protected storedproducts from pest infestation including chemical
substitutes (plant extracts), exploitation of controlled
atmospheres, integration of physical methods such
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as heating [5,6] , cooling [7] and biological agents
such as parasitoids [8]. Another prospective
controlling method is utilization of solar energy.
Heating produced from solar radiation has been
demonstrated to have a potential role in
disinfestations of store insect pests [5,6]. Several
studies investigated the utilization of solar heating
in post – harvest cowpea weevil control [6,9-12].
All studies demonstrated the potential of solar in
suppression of C. maculatus population in stored
grains. Moreover, solar heating has many advantage
as it is a safe technology, free for everyone, easy to
get and not expensive.
study is to examine the ultrastructural changes and
histopathological alteration in the testes and
accessory glands of male C. maculatus induced by
the solar energy.
Reproductive system and sperm formation in insects
is an extremely complex process. Postmeiotic
maturation of sperm in insects involves a highly
ordered sequence of developmental changes.
Spermiogenesis and organization of the tail complex
in insect spermatozoa has been the subject of many
investigations. The majority of insect spermatozoa
have two central tubules, nine doublets and nine
single tubules in their axoneme as found in S. bullata,
D. melanogaster and D. radicum [13,14],
Rhynchophorus ferrugineus [15,16] and C.
maculatus [17] respectively.
The classic spermatozoa of coleopteran are
characterized by enormous accessory bodies and
large and almost fully crystallized mitochondrial
derivatives in the tail region. The head region is
formed by an acrosome (three-layered) and nucleus
[16,18-22].
Sun exposure technique: Exposure to solar heat
was conducted using an obtuse-base-angle box heater
described by Mekasha et al. [6] with some
modifications. The box was constructed with 1 mm
thick galvanized metal sheet. The upper open side
of the box was 51 cm × 20 cm, length by width and
the perpendicular height was 23 cm with obtuse-base
angle (about 120º). The box was covered externally
with black polystyrene sheet to increase the
capability to sun rays absorption. In the middle of
the box a glass plate was introduced to help in raising
the inner temperature during the exposure time.
Black polystyrene sheet was held on the ground for
about 10-15 minutes to collect the sun rays on the
experimental spot and the box put on it during the
exposure time. The temperature and relative
humidity inside and outside the box were recorded
during experiment using thermometer and
hygrometer.
In most insects, the male genital system also consists
of accessory glands of mesodermal or ectodermal
origin, which open into the deferent or ejaculatory
ducts. The accessory gland are of various size, shape,
number and embryological origin [23-26]. The
secretory cells of these accessory glands are arranged
as a mono layered epithelium and contribution to
the seminal fluid and activation of the spermatozoa
[27,28].
Exposure pupal stage of cowpea weevil to solar
heat: Three pairs of newly emerged adults (0 day)
from a laboratory colony were introduced into vials
contain 15 gm of cowpea seeds. The females were
allowed to lay eggs for 24 hours then all insects were
carefully removed from the vials. The infested seeds
were then held under controlled conditions of
temperature (27±2ºC) and relative humidity (60±5%)
until treatment.
Inspite of varios studied the link between
reproduction and environmental signals is poorly
understood at the physiological, genetic and
molecular levels (27,29,30). Up to the authors’
knowledge, information on the effect of solar
radiation on the male reproductive system of C.
maculates is not available. The aim of the present
Pupal stage was exposed to sun heat about 53 oC in
an obtuse-base- angle box heater for 10, 15 and 20
minutes respectively. For each treatment control
group was run at the same time of the experiment
with 0- min exposure. After exposure to sun heat all
vials were kept at controlled temperature (27±2ºC)
and relative humidity (60±5%) until adult
MATERIALS AND METHODS
Origin of population: The strain of cowpea weevil,
C. maculatus (F) was obtained from the plant
protection Research Institute, Dokki, Giza, Egypt,
where the colony is maintained on cowpea seeds
(Vigna unguiculata L.) at temperature of 27 ± 2oC
and relative humidity of 60 ± 5%.
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Khaled et al.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7a
Fig. 7b
Figs.1-7: photographs & sections of normal male C. maculates: Fig 1: Photograph of the internal reproductive organs
(20X), Fig.2: Sagittal section of the testis (400X), Fig.3: Electron micrograph of interstitial cell (8000X), Fig.4,5: Electron
micrograph of spermatids (15000 & 20000X), Fig. 6: Electron micrograph of sperm bundle (6000X), Figs.7a,b: Transverse
section of sperm (30000, 100000X). (T=Testis, CEJ =Common ejaculatory duct, MT=Muscular tube, GA=Genital aperture,
M=Mesadenia, E= Ectadenia, Pm=Peritoneal membrane, TF=Testicular follicles, SG=Spermatogonia, SC=Spermatocytes ,
SD= Spermatids, PS= Presperm, SB=sperm bundle, IC=interstitial cell, V=vacuoles, N= Nucleus, CH=Chromatin,
CY=Cytoplasm , R=Ribosomes, RER=rough endoplasmic reticulum, AX=Axoneme, SP=sperm, MS=Membranous sheath,
MD=Mitochondria derivative, AB=Accessory bodies, ELM= Electron lucid material , PM= Plasma membrane ).
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emergence. The malformed adults developed from
solar energy treated pupae at 53 oC for 15 minutes
were subjected to ultrastructural studies.
Electron microscopy studies: For electron
microscopy, the males were dissected in 2.5%
gluteraldehyde fixation. The testes and accessory
glands were immediately removed and placed in
fresh ice-cold 2.5% gluteraldehyd in 0.1M sodium
cacodylate and 0.17M sucrose at PH 7.4 for 2hrs. at
4ºC. The testes and accessory glands were post-fixed
in 1% osmium tetraoxide in 0.1 sodium cacodylate
and 0.17M sucrose at 4ºC for 90 min. They were
then embedded in pure Epon 812 resin, sectioned
with RMC ultramicrotome, stained in 30% uranyl
acetate in methanol for 30 min. followed by aqueous
lead citrate for 7 min. and viewed in a Sumy Electron
Optics PEM 100, 75 KV Transmission Electron
Microscope at the Medical Military Academy.
Electron micrographs were taken at several
magnifications.
RESULT
Morphology of male reproductive organs: The
internal reproductive organs of male C. maculatus
consist of two pairs of translucent testes. Each pair
of testes is located on each side of the gut. The testis
is a rounded sac and it is connected posteriorly to a
short vas deferens. The vas deferentia of the two
testes on each side open into a common vas deferens,
which receives at its cephalic end a bilobed accessory
gland, the mesadenia. The vas deferens
communicates with the swollen bifurcation of the
ejaculatory duct of its side. The latter receives three
accessory glands, the ectadenia. The lateral swollen
ejaculatory ducts join to form a common ejaculatory
duct, a long muscular tube which opens to the
exterior through the male genital aperture (Fig. 1).
Accessory glands: The male C. maculatus has four
pairs of accessory glands; one pair of lateral gland,
the mesadenia and three pairs of median glands, the
ectadenia. The mesadenia are the largest gland. The
ectadenia are tubular glands and it consists of two
pairs of anterior or superior glands and a pair of
posterior of inferior glands. At emergence, the
accessory glands of normal males are full of milky
white secretion (Fig. 1).
HISTOLOGY AND ULTRASTRUCTURE OF
MALE REPRODUCTIVE SYSTEM:
Testis: The testes are enclosed in a transparent nonpigmented membrane, the peritoneal membrane. The
testicular follicles are composed of aggregations of
small round or oval sacs (Fig. 2). The testicular
follicles are filled up with germinal cells in different
stages of development. These include spermatogonia,
spermatocytes, spermatids, presperm and spermatozoa. The testes of the normal male are almost
occupied by mature sperm and presperm bundles and
spermatids in various stages of spermiogenesis. Cysts
containing spermatogonia or spermatocytes are
relatively few in normal testes (Fig. 2). Electron
micrographs show that cysts are formed by the
grouping of germ cells of the same stage, in spherical
clusters which are embedded and enclosed by
interstitial tissues, the cyst cells. The nuclei of the
cyst cells have polymorphic shape with irregular
outlines. The cytoplasm of the cyst cell contains
round mitochondria, membranes of rough endoplasmic reticulum and is rich with free ribosomes and
aggregation of vacuoles of various sizes (Fig. 3).
The spermatogonia are spherical cell. They have dark
stained, round or oval nuclei (Fig. 2). The spermatocytes are generally larger than spermatogonia but
with less intensely stained nuclei (Fig. 2). Young
spermatids are relatively small in size with
intensively stainable round nuclei which are smaller
than those of the spermatocytes (Fig. 2). Spermatids
at different stages of spermatogenesis could be
distinguished in light microscope (Fig. 2). In the
electron micrograph the nucleus migrates to the
anterior tip of the cell and the cytoplasm is displaced
posteriorly. Small quantities of electron dense
material are located in the cytoplasm. The axial
filament (axoneme) appears behind the spermatid
nucleus (Fig. 4). Another morphologic nucleus
transformation changes process was observed in
electron microscope preparation where, chromatin
condenses forming an elongated dark mass in the
nucleus (Fig. 5).
Sperm: Sperm are differentiated from the presperm
by the great elongation of the tail. Within one bundle,
sperm are aligned roughly parallel to one another
and oriented in the same direction (Fig. 2). In electron
micrograph, sperm in one bundle appear to be held
closely together by an electron opaque material and
are surrounded by a membranous sheath (Fig. 6). In
cross sections individual sperm enclosed by
membranous sheath (Figs. 7a,b).
4932
Khaled et al.
The nucleus of the sperm is surrounded by a very
thin layer of cytoplasm. The head tapers anteriorly
where acrosome is present. Two unequal
mitochondrial derivatives are located behind the
nucleus. They are filled with dark granular substance
and have no distinct cristae. The axoneme flanked
by two dark crescent-shape accessory bodies, runs
along the middle line behind the two mitochondrial
derivatives. All components are spaced by an
electron lucid material and are enclosed in the plasma
membrane of the cell. In the sections of the sperm
tail, three distinct elements can be identified: the
axoneme, two mitochondrial derivatives and two
accessory bodies (Figs. 7a,b).
The axoneme in the sperm of C. maculatus is formed
of 20 fine microtubules, which are arranged as a
wheel like pattern of (9+ 9+ 2). Two central
microtubules, encircled by 9 doublets which, in its
turn, are surrounded by 9 singlets. The 2 central and
the 9 peripheral singlets have circular profile whereas
the pairs of the 9 doublets are ellipsoidal. The two
central tubules are of equal diameter and they are
connected to the (A) subfibre of the doublets by
spoke like projections or radical, links. The right
subfibre (A) is smaller than the left subfibre (B) of
the doublets. The adjacent doublets are linked
together by interdoublet links. Each of (A) and (B)
subfibre had a peripheral arm which extend outwards
forming interbridges between the 9 singlets and join
to them via the peripheral arm of the (B) fibre.
Peripheral to and spaced between the doublets are
the 9 singlets. The singlets have equal diameter.
Arising from the right side of each singlet one arm
which is connected to the fore mentioned peripheral
arm of the (B) subfibre. On the left side, each singlet
has 2 side arms which join the interbridges between
the singlets (Fig. 7b).
The accessory glands: The accessory glands of C.
maculatus consist of two symmetrical groups, the
mesadenia and ectadenia (Fig. 1).
The lateral gland (Mesadenia): Light microscopic
study revealed that the gland is an elongated bilobed
tube. A basement membrane surrounds the gland and
within this, is situated one layer of columnar
epithelial cells. The cytoplasm is granular in nature
with large round or elongated nuclei. The lumen
contains a homogeneous secretion (Fig. 8).
Electron micrograph revealed that the lateral gland
is composed from outside to inside of the following:
A basic longitudinal muscular layer that is formed
of groups of fibrils. The muscles surrounding the
gland are enveloped by a basement membrane. The
gland epithelium is formed of one layer of the
epithelial cells. The cell has cubic shape with nondistinct boundaries in-between them. The cells have
a very round nuclei containing heterochromatin (Fig.
9). The cytoplasm of the epithelial cells is rich in
organelles containing an extensive rough
endoplasmic reticulum, free ribosomes and several
Golgi elements. The endoplasmic reticulum is
constituted of fine tubules; it forms ribbons which
encircle the secretory granules (Fig. 10). The rough
endoplasmic reticulum is highly organized and
located around the nucleus in vesiculated form which
has uniformly-spaced parallel cisternae (Fig. 10). In
the epithelial regions secretory droplets appear
throughout the cell. The Golgi vesicles or
dictyosomes are several in numbers formed of
aggregation of membranous vacuoles and vesicles
(Figs. 9,10). The plasma membrane at the apical
region appears to have several infoldings along with
large microvilli which are abundantly scattered in
the luminal surface (Fig. 11). Two distinct types of
granules can be distinguished inside the cells, type
(1) granule is the most common and constitutes
homogeneous dense masses (Fig. 11) and granules
of type (2) that are relatively small. The secretory
granules are frequently attached at the plasmic
membrane at the apex of the cells and in between
the bases of microvilli. Granules seem to be secreted
by exocytosis which involves the rupturing of plasma
membrane enclosing the secretory granules and
release of the granules in the gland lumen. The lumen
of gland is distended by dense materials which are
granules, containing condensed materials of
variables sizes. Vacuoles of variable size are also
crowded in the lumen (Fig. 12).
The median gland (Ectadenia): The median gland
consists of a single layer of cuboidal epithelial cells.
The cytoplasm is granular and nuclei are round or
ellipsoid, located towards the base. The epithelial
cells rest on a basement membrane which is
surrounded by a circular muscular layer. The lumen
contains a homogeneous secretion (Fig. 8).
Ultrastructural study revealed that the median gland
is composed from outside to inside of following
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J. Cell Tissue Research
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14
Fig. 15
Figs. 8-15: Sections of normal male C. maculates: Fig.8: Longitudinal section of the accessory glands(200X), Fig.s. 9,10,11,
12: Electron micrograph of the lateral gland (8000, 3000, 3000 & 2000X), Figs.13,14,15: Electron micrograph of the
median gland (4000, 8000 & 30000X): (LG=lateral gland, MG=Median gland, IG=Inferior gland, EC=Epithelial cells,
BM=Basement membrane, LML=Longitudinal muscular layer, N=Nucleus, L=Lumen, R=Ribosomes, RER=Rough
endoplasmic reticulum, SD=Secretory droplets, GV=Golgi vesicles, MV=Microvilli, SG1=Secretory granules type 1,
SG2=Secretory granules type 2 , SV=Secretory vacuoles , Spt=Septa, CML=Circular muscular layer, CEC=Cuboidal epithelial
cells , CY=Cytoplasm , GE=Golgi elements, CH=Chromatin)
4934
Khaled et al.
structure. A continuous layer of circular muscles,
which is formed of a group of muscles fibres and
enveloped by a basement membrane (Figs. 13,14).
The gland is composed of one row of cuboidal
epithelial cells which constitute the secretory region.
The epithelial cells rest on a basement membrane.
At the apices of the cells, the plasma membrane is
drawn into long oblique microvilli which are
scattered in the luminal surface. The cell components
are the nucleus, the rough endoplasmic reticulum,
free ribosomes, mitochondria and a variety of
granules in the cytoplasm. The nucleus is basely
located and the chromatin is dispersed in the
nucleoplasm (Fig. 14). The secretory granules of type
(1) aggregate in between the bases of the microvilli
to be released in the gland lumen as the plasma
membrane is ruptured. They seem to adhere in groups
localized near the periphery of the gland lumen and
before they are dispersed in the lumen (Fig. 14).
Round flakes are present in the lumen (Fig. 15).
These flakes are permanent elements of glandular
cells, they are little opaque under electron microscope and are considered as secretion type (2) in these
median glands. The flakes appear to be poured out
in the gland lumen as they pass through the ruptured
plasma membrane in between the bases of microvilli.
The secretion in the gland lumen is formed of
peripheral aggregation of dense granules which may
be broken to disperse in the gland lumen among the
flaky globules secretion type (2) (Figs. 14,15).
The inferior gland: The inferior median gland
consists of a single layer of cuboidal epithelial cells
that rest on a basement membrane which is
surrounded by a circular muscular layer. The
cytoplasm of epithelium is granular and has apical
flattened nuclei. The lumen contains a homogeneous
secretion (Fig. 8). Ultrastructural study revealed that
the inferior gland is composed from outside to the
inside of the following structures A continuous
circular muscular layer which enveloped by a
basement membrane (Fig. 16). The gland is
composed of one layer of cuboidal epithelial cells.
The epithelial cells rest on a basement membrane.
The prominent components of the cell are the nuclei
which are flattened and heterochromatic.
Mitochondria are round, Golgi elements are little
developed. Dark granules aggregated at the bases of
the microvilli. The plasma membrane at the luminal
surface infolding forms short oblique microvilli. The
gland lumen is filled with non-granulated
homogeneous secretion (Fig. 16).
EFFECT OF SOLAR ENERGY ON THE MALE
REPRODUCTIVE SYSTEM:
The internal reproductive organs of male C.
maculatus developed in solar energy treated pupae
(F) are markedly reduced in size as compared to that
of the normal ones. The accessory glands of the (F)
male appear translucent, almost devoid of secretory
material which fills the lumen of these glands and
make them opaque in normal male (Fig. 17).
Testis: Severe damage of the epithelial septa was
observed in the testes. Most of the septa were
completely destroyed leaving vacuolated areas in
the organ. The testes of the (F) males were almost
occupied by spermatogonia, spermatocytes, spermatids, presperm and mature sperm bundles. Most of
gonial cells showed morphological changes than
normal. Some groups of gonial cells were lyzed and
replaced by empty areas of tissue (Fig. 18). The cyst
interstitial cells were degenerated leaving vacuolated
areas in the testes and polymorphic nuclei were
deeply invaginated in the nuclear membrane (Fig.19).
Sperm: Sperms of the (F) male exhibit a variety of
abnormalities. Deteriorated sperm bundles were
observed within the vacuolated cyst cells (Fig. 20).
Sperm bundles appear to have no regular outlines as
in normal. The sperm bundles irregularity distributed
in lyzed cyst cell in an unusual manner. Sometimes
sperms could be observed not enclosed within a
relatively thin plasma membrane. The abnormal
multiple-unit sperm are generally observed, a sperm
with axoneme and nucleus contain pairs of
malformed accessory bodies and a pair of distorted
mitochondria derivatives (Figs. 21a,b,c).
Accessory glands: The lateral gland (Mesadenia):
The lateral gland of (F) male consists essentially of
the same elements as normal (Fig. 22). Electron
micrograph revealed lysis of the epithelial cells layer.
The chromatin of nucleus is very poor. Irregular
invaginations in the nuclear membrane are
noticeable. The cytoplasmic organelles are very poor
occupied by large area of vacuolization. The rough
endoplasmic reticulums which surround the nucleus
in normal male are not noticeable. The plasma
membrane at the luminal surface is free of microvilli.
Secretary granules are not detected in the cytoplasm
of the cells. The glandular lumen is condensed with
secretory vacuoles and few dark secretory granules
comparable to those in normal (Figs. 23a,b,c,d).
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J. Cell Tissue Research
The median gland (Ectadenia): The median gland
of (F) male consists essentially of the same elements
as normal (Fig. 22). Ultrastructural study revealed
that the circular muscular layer is not noticeable. The
cytoplasm is completely devoid of any organelles
and occupied by vacuoles. The plasma membrane at
the luminal surface is free of microvilli. Secretory
granules are not detected in the cytoplasm of the
cells. The glandular lumen is condensed with
deformed flakes of variable sizes and few dark
secretory granules aggregate in the vacuolated sac
or scattered freely in the lumen (Fig. 24a,b).
The inferior gland: The inferior gland of (F) male
consists essentially of the same elements as normal
(Fig. 22). Electron micrograph revealed that
deterioration of microfibriles of circular muscular
layer. The cytoplasmic organelles are very poor,
except of few mitochondria and much reduced rough
endoplasmic reticulum. The cytoplasm is filled with
large vacuoles. The chromatin of nucleus is very
poor. The plasma membrane at the luminal surface has
few and very little microvilli. The glandular lumen is
filled with vacuoles of variable sizes which spread along
the non-granulated homogeneous secretions as
compared to that of normal ones (Fig. 25).
DISCUSSION
.
The current study of the internal reproductive organs
in male C. maculatus resulted from solar energy
treated pupae showed that the sizes of testes and
accessory glands are markedly reduced as compared
to the normal. It has been reported that different
abiotic factors such as heat stress, starvation,
chemical stress and radiation had physiological and
morphological changes on insect’s reproductive
organs [31-36]. These morphological changes
include reduction or enlargement of different parts
of both male and female reproductive organs. In
yellow dung fly, Scathophaga stercoraria testis
length decreased with increasing developmental
temperatures. In contrast, sperm length increased or
showed a negative quadratic relationship with
increasing temperatures [37].
The gonads and accessory glands resulted from solar
energy treated pupa consist basically of the same
elements as normal. However, these elements may,
exhibit some histological and ultrastructural changes.
The testicular follicles in C. maculatus are composed
of aggregations of oval or round sacs which open
directly in the vas deference. The germarium in the
testis of C. maculatus is followed by a series of sperm
cysts in various stages of development. Testes of
normal males are mostly occupied by mature sperm,
presperm and to lesser extent by spermatids but
spermatogonia and spermatocytes were relatively
scarce. This suggests that complete spermatogenesis
in C. maculatus takes place during larval and pupal
stages as in many other insect species having a short
life span [38-40]. In contrast to normal, the testis of
(F) male C. maculatus is almost filled with different
stages of spermatogenesis. Some of these gonial cells
degenerate and a few of them continue developing
to reach the stage of spermatid, or to the mature
sperm. Retardation, degeneration of spermatogenesis
occurs in sterile males which were induced by
irradiation [41-43] or hybrid sterile males [44,45] .
The transformation process of spermatids to sperm
involves several morphological reorganization of the
cells. The changes are occurred by eccentric
displacement of nucleus and the caudal displacement
of the cytoplasm. These structural changes of nuclear
development have been described for other beetles
[21,46]. In sperm the nucleus elongates, the tail
develops, the two mitochondria derivatives, which
extend in front of the axial filament (axoneme). This
later is flanked by two crescent shaped dark accessory bodies. Baccetti et al. [18] found that accessory
bodies in Tenebrio sperm, exhibit an intense UT pase
and AT pase activities which suggest their
involvement in supplying the energy required for the
maintenance of the large waves of sperm movement.
The mitochondrial derivatives are considered as the
“power plant”, supply the flagellum with energy.
In spermatids and spermatozoa of Tenebrio molitor,
[18] found that cristae of the mitochondrial
derivatives are rich in cytochromo-C-oxidase.
However, the authors found no ATPase activity
associated with the mitochondrial derivatives. The
presence of accessory bodies and mitochondria
derivatives comparable to those found in the sperm
of C. maculatus have been reported in several other
insects such as the Lepidopteran Plodia
interpunctela [41] and the Coleopteran Tenebrio
molitor [18], Anthonomus grandis [47] and S.
zeamaisand S. oryzae [22].
4936
Khaled et al.
Fig. 16
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 21a
Fig. 21b
Fig. 21c
Fig.16: Electron micrograph of the inferior gland of normal male C. maculatus (4000X). Figs. 17- 21: Sections of male C.
maculatus resulting from solar energy treated pupa. Fig. 17: Photograph of the internal reproductive organs (20X), Fig. 18:
Sagittal section of the testis (400X). Fig.19: Electron micrograph of testis (400X), showing malformed interstitial cells.
Fig. 20: Electron micrograph of sperm bundle (4000x), Fig. 21: (a,b&c) Transverse sections of sperm bundle
(15000,,30000,&,40000X). HS=Homogeneous secretion, MV=Microvilli, T= Testis, CEG= Common ejaculatory duct,
MT=Muscular tube, GA= Genital aperture, M= Mesadenia , E=Ectadenia , Pm=Peritoneal membrane , TF=Testicular follicles,
SG=Spermatogonia, SC=Spermatocytes, SD=Spermatids, Sp= malformed Sperm, IC=malformed interstitial cells,
N=deteriorated nucleus, CML=Circular muscular layer, CH= Chromatin, arrows=deteriorated sperm bundle,
MD=Mitochondria derivative, AB=Accessory bodies , AX=Axoneme, ELM=Electron lucid material & PM=Plasma
membrane.
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J. Cell Tissue Research
The microtubules of the axoneme are considered as
a framework, or cytoskeleton which processes the
shaping of the cell and redistributes its contents [48].
The axoneme which is believed to be the motile
element of the sperm arises behind the nucleus in
the sperm head and extends longitudinally flanked
by the accessory bodies along the sperm tail. The
axoneme, two accessory bodies and two
mitochondria derivatives are the distinct elements
in the sperm tail in C. maculatus. This pattern similar
to most Coleoptera [22]. However, the flagellum of
Coelomeralanio and Cerotomaarcuata (Coleoptera:
Chrysomelidae) have a single accessory body [21,49]
.The axoneme of sperm in both normal and (F) male
has (9)+ 9+ 2 configuration; with two central tubules
encircled by 9 doublets which in their turn are
encircled by 9 singlets. This concentric wheel-like
pattern of axoneme microtubules is comparable to
that described in most pterygota [50,51], in the
Lepidopteran, Heliothis virescens and Heliothis
subflexa [45], in Anticarsi agemmatalis
(Lepidoptera: Noctuidae) [52].This pattern was also
described in Tenebrio molitor (Coleoptera:
Tenebrionidae) [18], Anthonomus grandis [47], C.
maculates [7,53] , Trogoderma granarium [43] and
Rhynchophorus ferrugineus [6,54] , S. zeamaisand,
S. oryzae [22] and in Halictidae (Hymenoptera:
Apoidea) [55]. Links connect the components in the
centre or inner circle to those in the outer circle.
Links are illustrated in electron micrographs of
axoneme in Tenebrio molitor [18]. In insects, ATPase
and UTPase activities have been detected in the
coarse fibres between the accessory tubules.
Degenerating and rupturing sperm bundles were
observed mostly in light or electron microscope
preparations of (F) male. The lack or scarcity of the
irregularity of bundles outlines, rupture and
degeneration of bundles and interlocking of the
plasma membranes of adjacent sperms within the
same bundle in (F) male may reflect a probable
interference with or reduction of sperm activity. In
the sterile insects produced by irradiation treatment
[56] or by interspecific crosses [57] and [17]
abnormalities such as the presence of number of
double, multiple-unit sperm cells rupture of bundles
and dispersion of sperm were observed in (F) male.
Extragonial cells of testis of (F) male C. maculatus
have suffered of damage. The epithelial septa in
between follicles showed extensive vacuolation and
degeneration. The cyst cells may supply nutrient to
the developing sperm and in Popillia (Coleoptera),
the sperm at one stage have their heads embedded in
the cyst-cells, this perhaps facilitating the transfer
of nutrient [58] or the interstitial cells in Heteroptera,
Oncopletus [59], are considered as trophocytes
which are involved in the transfer of nutrients to the
sperm cysts. Trophic tissues degeneration and
vacuolization were observed among testicular cysts
of sterile insects produced by irradiation [56].
Inhibition of spermiogenesis of eupyrene
spermastozoa also occurred by elevated, sublethal
temperatures during pupal adult development of the
bertha armyworm [60].
The male C. maculatus has four pairs of accessory
glands; mesadenia (one pair of lateral glands) and
ectadenia (two pairs of median glands and one pair
of inferior gland). In most insects, the male genital
system consists of accessory glands of mesodermal
or ectodermal origin. Among insects, the accessory
glands vary notably in size, shape, number, and
embryological origin [23-26,29,61,62]. In the
mesadenia and ectadenia, the secretory epithelium
forms a single layer in each of the normal and (F)
male. In normal male C. maculatus, the secretory
epithelium of the two types of the accessory gland
are provided with all ultrastructural characteristics
appropriate to the synthesis and secretion of proteins,
as has been demonstrated in other insects [63,64].
Among the functions of the secretions of these glands
is the contribution to the seminal fluid and activation
of the spermatozoa [27,28,61,65].
The rough endoplasmic reticulum is highly
developed and consists of stacks of large flattened
cisterne covered with ribosomes and occupying most
of the cell in normal male C. maculatus, while in the
(F) male the rough endoplasmic reticulum is not
developed in the mesadenia and inconspicuous in the
ectadenia. The rough endoplasmic reticulum is
developed in cells actively engaged in protein synthesis
[48]. Marchini et al. [66] found a large amount of rough
endoplasmic reticulum in the cytoplasm of the male
accessory glands of the medfly Ceratitis capitata,
which is in accordance with the presence of proteins
in the secretions. In Vespula vulgaris rough endoplasmic reticulum remains present in considerable
quantity, indicates a high-secretory activity [67].
4938
Khaled et al.
Fig. 22
Fig. 23a
Fig. 23b
Fig. 23c
Fig. 23d
Fig. 24a
Fig. 24b
Fig. 25
Figs. 22-25: Sections of male C. maculatus resulting from solar energy treated pupa. Fig. 22: Longitudinal section of the
accessory glands (400X), Figs. 23a-d: Electron micrograph of lateral gland (4000, 10000, 10000 & 4000X), Fig. 24a,b:
Electron micrograph of median gland (3000, 8000X), Fig. 25: Electron micrograph of the inferior gland (8000X). (LG=lateral
gland, MG=Median gland, IG=Inferior gland, HS=Homogeneous secretion, EC=lysis of Epithelial cells, CY=Vacuolated
cytoplasm, V= Vacuoles, Bb= Brush border free of microvilli, N=Nucleus, L=Lumen, SG1=secretory granules type1,
SV=Secretory vacuole, Spt= Septa, Bb= Brush border free of microvilli, CML=Circular muscular layer, CH=Chromatin,
MF=Microfibriles, PM=Plasma membrane, RER=Rough Endoplasmic Reticulum, m=Mitochondria, MV=Deterioration of
microvilli)
4939
J. Cell Tissue Research
The cytoplasm in accessory glands of normal male
C. maculatus is rich with ribosomes while that of
(F) male is poor. Ribosomes are organelles that are
composed of ribonucleic acids and proteins and have
a role in protein synthesis [48]. In (F) male C.
maculatus, the observed reduction in the rough
endoplasmic reticulum and ribosomes suggests the
reduced biosynthetic capacity of proteins.
Golgi elements are numerous and distributed
throughout the cytoplasm of the accessory glands in
normal male C. maculatus, while they are not
noticeable in (F) male. Golgi vesicles receive the
protein synthesized at the rough endoplasmic
reticulum [48]. The abundance of Golgi vesicles in
accessory glands of normal male C. maculatus
suggest a period of high activity in the synthesis of
secretory granules. The accessory gland of Achroia
grisella consisted of glandular cells with a welldeveloped rough endoplasmic reticulum and Golgi
apparatus [68].
et al. [48] anticipated movement of microvilli may
help mixing of the secretions within the gland lumen.
The secretory cells of Achroia grisellahad poorly
developed luminal microvilli [68]. The lumen of the
mesadenia in normal male C. maculatus has secretory dark granules and vacuoles of different sizes.
In the ectadenia, the secretion in the gland lumen is
formed of peripheral aggregates of dense granules
which are dispersed in the gland lumen among
electron-lucent flaky globules. Histochemical and
ultrastructural studies suggested that the materials
in the secretions within the epithelial cells and in
the lumen are proteins [27,63,70-72]. The secretion
in the lumen organized in globules was essentially
proteinaceous for most insects [65,73,74].
In normal male C. maculatus, the mesadenia secrete
small and relatively large dark granules. The granules
attach themselves to the plasma membrane. The
plasma membrane ruptures and the secretory
granules are poured into the lumen of the gland, a
process which resembles exocytosis [48]. The
ectadenia of normal male C. maculatus have dark
granules and electron opaque flaky secretions. They
are aggregate at the bases of the microvilli to be
released in the gland lumen by exocytosis [48].
Similarly, in Acanthoscelides obtectus [69].
In the (F) male C. maculatus no ultrastructural sign
or indication that active production of secretion is
going on within the mesadenia. The dark secretory
granules are absent in the glandular epithelium. The
glandular lumen is occupied by deformed vacuoles.
The microvilli at the luminal surface disappeared.
In the ectadenia of the (F) male C. maculatus, the
dark granules are lacking and the glandular lumen
contains only malformed flaky secretions and
vacuoles. Microvilli of the luminal surface of the
mesadenia and ectadenia are completely reduced in
the (F) male C. maculatus. Hihara [76] postulated
that the microvilli in the accessory gland secretion
of the sterile male Drosophila melanogaster are
probably defective. In some insects, the accessory
gland secretion exerts physiological and biochemical
control of the female reproductive physiology
[27,30]. In Drosophila, the secretory product of the
accessory glands is transferred to the female tract
during mating where it forms a plug and modifies
the behavior and physiology of the female [77-80].
The effect of temperature on the reproductive system,
especially on the male accessory gland in
Drosophila, including structure of gland of cell size
and cell number, that changed when flies were
exposed to different temperature [81].
At the apices of the secretory cells of the accessory
glands of normal male C. maculatus, the plasma
membrane is drawn into microvilli which have higher
density in the mesadenia than in the ectadenia. The
microvilli and the infoldings of the plasma membrane
increase the area of secretory surface. De Robertis
Prospective controlling method is utilization of solar
energy. Heating produced from solar radiation has
been demonstrated to have a potential role in
disinfestations by stored grain pests. Moreover, solar
heating has many advantage as it is a safe technology,
free for everyone, easy to get and not expensive.
Mitochondria are distributed throughout the
cytoplasm of the glandular epithelium of normal
male C. maculatus and are not seen in the accessory
glands of the (F) male. The abundance of
mitochondria in the epithelium and their
accumulation near the luminal surface may reflect
the increasing demands for energy in the cells for
synthesis of the secretion, transport and release into
the gland lumen.
4940
Khaled et al.
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