Ultrastructure of Male Reproductive System of

MICROSCOPY RESEARCH AND TECHNIQUE 78:643–653 (2015)

Ultrastructure of Male Reproductive System of Eurydema ventrale Kolenati 1846 (Heteroptera: Pentatomidae) € NURCAN OZYURT, SELAMI CANDAN,* AND ZEKIYE SULUDERE Department of Biology, Gazi University, Science Faculty, Ankara 06500, Turkey

KEY WORDS

heteroptera; morphology; histology; ultrastructure; spermatogenesis

ABSTRACT The male reproductive system of Eurydema ventrale Kolenati 1846 is studied morphologically and histologically by using light, scanning and transmission electron microscopes. The reproductive system of the male E.ventrale consists of a pair of testis, a pair of vas deferens, a pair of seminal vesicles, accessory glands, a bulbus ejaculatorius, a pair of ectodermal sacs, and a ductus ejaculatorius. The testicular follicles have three different development zones (growth zone, maturation zone, and differentiation zone). The testes are connected to the seminal vesicles by the vas deferens that is a specialized in sperm storage. Sperm have an elongated head and a tail (flagellum) with an axonema and two mitochondrial derivatives. Vas deferens and seminal vesicles are fine, long, and cylindrical. The seminal vesicle is connected with bulbus ejaculatorius, which is balloon shaped and surrounded with accessory glands. The bulbus ejaculatorius is continuous with ductus ejaculatorius which is connected to the aedeagus. Microsc. Res. Tech. 78:643–653, 2015. V 2015 Wiley Periodicals, Inc. C

INTRODUCTION Heteroptera is a large group of insects consist of 75 families, and approximately, 90,000 species found in all climates from tropical to Arctic. It is economically an important group as it includes both useful (predators) and harmful insects (pests) (Schuh and Slater, 1995). Heteroptera order including Pentatomidae family is generally phytophagous. Many of them are concidered agricultural pests, because they can create large population that feed on grops damaging production. Also, Eurydema species are significant importance. Eurydema ventrale is the one that causes the most damage to vegetable crops from the Brassicaceae family (Stankovic´, 1963, 1964). Despite the importance of Pentatomidae which biological control agents, few reports exist on the internal morphology of the reproductive tract of these organisms, especially with reference to male reproductive system. In most insects, including the Heteroptera, male reproductive system is formed by one pair of testes in which each containing a series of testicular tubes or follicles linked to a seminal vesicle by vasa deferentia, accessory glands, and a ductus ejaculatorius. However, modifications and adaptations in the male reproductive systems can be found (Chapman, 1998). Secretions of the male accessory glands from the spermatophore contribute to the seminal fluid which nourishes the spermatozoa during transport to the female. These secretions are also involved in the activation of spermatozoa and may alter female behavior. The bulbus ejaculatorius is continuous with that of the ductus ejaculatorius. In most insects, ductus ejaculatorius is single, located medially and has a cuticular cover, demonstrating its ectodermic origin (Chapman, 1998; Nijhout, 1994; Pendergrast, 1956). C V

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The testis in Heteroptera comprises a simple follicle, and the follicle wall is a thin epithelium, sometimes consisting of two layers of cells with a basal lamina (Chapman, 1998). Four regions usually constitute the testis follicle: germarium, zone of growth, zone of maturation, and zone of transformation, where the sperm develops in successive stages of maturation in the process of spermiogenesis (Chapman, 1998). Mature sperm of most insects consist of head and flagellum regions. The greater part of the head region is occupied by the nucleus. In front of the nucleus, acrosome is present, and behind the nucleus, axial filament, or axoneme, arises (Chapman, 1998). Flagellum has the axoneme or microtubule-based cytoskeleton of the flagellum; one or two mitochondrial derivatives; and one, or more commonly two, accessory bodies that represent extensions of the centriole adjunct that forms a collar at the base of the flagellum (Nardi et al., 2013). Most insects present a number of accessory glands, which open into the vasa deferentia or the ductus ejaculatorius (Chapman, 1998; Happ, 1992; Lemos et al., 2005). The substances secreted of male accessory glands exert their effects at all phases of the reproductive biology of the mated female, from the moment that sperm is deposited in the reproductive tract to egg laying (Gillott, 2003). These effects are related to sperm protection, storage and activation, sperm competition, female behavior (reduction in attractiveness), *Correspondence to: Selami Candan, Science Faculty, Department of Biology, Gazi University, Ankara, 06500, Turkey. E-mail: [email protected] REVIEW EDITOR: Prof. Alberto Diaspro Received 27 January 2015; accepted in revised form 14 April 2015 DOI 10.1002/jemt.22514 Published online 2 June 2015 in Wiley Online Library (wileyonlinelibrary.com).

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fecundity, ovulation, oviposition, and protection of laid eggs (Chen, 1984; Davey, 1958; Friedel and Gillot, 1977; Freitas et al., 2010; Fuchs et al., 1969; Gillott, 2003; Pickford et al., 1969; ). In this study, the characteristics features of male reproductive organ of E. ventrale from Pentatomidae family has been described by light, scanning, and transmission electron microscopies, similarities and dissimilarities among different species have been discussed. MATERIAL AND METHODS Materials Fifteen adult males of E. ventrale were collected in June–July, 2013, Kazan, Ankara, Turkey. Tissue Preparation for Light Microscopy The males were dissected under a light microscope in 0.1 M, pH 7.2 phosphate buffer to which 3% of sucrose was added to remove the genital system. The gross morphology of the male reproductive systems was examined and photographed with a Leica EZ4D stereomicroscope. For the histological analysis, samples were fixed in Bouin’s for 24 h. After that, the tissues were washed, dehydrated in a grade series of ethanol solutions and finally embedded in paraffin. Paraffin sections were stained with hematoxylin–eosin (H & E) and Mallory’s Triple stain for light microscopic examination. The sections were viewed and photographed by using Olympus BX51 microscope. Tissue Preparation for Scanning Electron Microscopy For scanning electron microscope (SEM), tissues were dehydrated through a graded ethanol series and dried by critical point (Polaron CPD 7501) apparatus. Samples were then mounted on stubs, gold-coated in a Polaron SC 502 sputter coater and observed with a JEOL JSM 6060 LV scanning electron microscope in 3–5 kV. Photographs were taken by digitally in SEM. Tissue Preparation for Transmission Electron Microscopy For the transmission electron microscopy (TEM), tissue samples were fixed 2.5% gluteraldehyde in phosphate buffer. Following 2-h postfixation in 1% OsO4 in the same buffer, the material was embedded in Araldit. Semithin sections, mounted on glass slides, were stained with 1% methylene blue and observed with a light microscope. Ultrathin sections were cut by using a Leica EM UC6 ultramicrotome and stained with uranyl acetate and lead citrate according to Reynolds (1963). The sections were taken on to the shiny side of the copper grid. Ultrathin sections were observed under the transmission electron microscope (JEOL JEM 1400), and photographs were taken by digitally in TEM. RESULTS Light, SEM, and TEM Observations The male reproductive system of E. ventrale consists of two paired ovoid, red testes in the dorsal region of the abdominal cavity (Figs. 1a and 1b). Testes are

seven tubular follicles that contain the developing sperm. Testes are lined by a layer of cells (peritoneal sheath) and a noncellular layer (tunica propria) which enclose testis follicles. Within each follicle, the developing sperm are in successive stages of maturation divided in cysts (Figs. 1c–1e). Within the testicular follicles of E. ventrale, there are three development zones, the growth zone (a), where groups of spermatogonia become separated from the germarium and form into spherical clusters. These groups of cells become enclosed by several cells which form the wall of the sperm cyst (Figs. 1c and 1d). Spermatogonia increase and allowing the occurrence of mitosis and differentiation into spermatocytes (Fig. 1e). The spermatocytes of the testicular follicles of E. ventrale are occurred sperm head and tail region. Golgi complex, mitochondria, the euchromatin, and heterochromatin regions in the nucleus were observed in sperm head region. The meitotic divisions are synchronous within a given cyst, and the cytoplasmic bridges that result from incomplete cytokinesis in these divisions (Figs. 1e, 1f, 2a, and 2b). The maturation zone (b), where two meiotic divisions occur and the differentiation of the spermatids occurs within the cysts (surrounded by a somatic cell), and inside these cysts, all spermatids are in the same stage of maturation (Figs. 2c–2f). The process of differentiation is characterized by cell elongation, including flagellum development and cytoplasmic sloughing. Mitochondria in flagellum become more prominent from the previous zone (Figs. 3a and 3b). The differentiation zone (c) where spermatids enlarge and change shape thus forming spermatozoa. The spermatozoa are contained in cysts that are grouped together in bundles and from which the spermatozoa are liberated (Figs. 3c and 3d). The spermatozoa of E. ventrale have two easily distinguishable regions: spindle-shaped the head and long, cylindrical structure flagellum (Figs. 3e and 3f). The head is formed by the acrosome, nucleus, and golgi complex. The nucleus is dense and elongated. The greater part of the head region is occupied by the nucleus (Figs. 3e and 3f). Each follicle opens into an efferent duct followed by the vas deferens duct, which connects each testis to a long cylindrical seminal vesicle (Figs. 4a and 4b). The testes and vasa deferentia were covered with a capsule with pigmented granules. Histologically, the vasa deferentia and seminal vesicles are formed by a simple epithelium and basal membrane (Fig. 4c). The spermatids are differentiated and produce the spermatozoa inside the vasa deferentia, which are liberated from each cyst grouped together in bundles (Figs. 4a and 4b). Cross and longitudinal sections of the sperm flagellum and head in lumen of vas deferens were seen (Figs. 4c and 4d). The tail consists of the axonemal complex by two mitochondrial derivatives reticular appandage, and centriolar adjunct was observed near the axonema (Figs. 5a and 5b). An accessory sheath separates the axonema (axial filament) and the mitochondrial derivatives (Fig. 5c). The axonema consist of the following: (a) central tubules, (b) nine microtubule doublets, and (c) nine accessory tubules located in spaces between consecutive doublets (Fig. 5d). The matrix of the derivative is occupied by a paracrystalline material (Fig. 5b). The vesicles insert on the Microscopy Research and Technique

ULTRASTRUCTURE OF MALE REPRODUCTIVE SYSTEM OF EURYDEMA VENTRALE

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Fig. 1. (a, b) Light micrograph of general view of male reproductive organs in E. ventrale. Testes (T), seminal vesicle (Vs), accessory glands (Ag), ejaculatory bulb (B), ejaculatory duct (D), ectodermal sac (Es) and aedeagus (A). (c, d) Semithin section and SEM micrograph of differentiation spermatogonia (Sg) in the cyst (Cy), and spermato-

cytes (Sp), tunica propria (Tp), peritoneal sheath (Ps). (e) TEM micrographs of spermatocytes, spermatogonia, peritoneal sheath and tracheoles (Tr). (f) Nucleus formation (Nu), cytoplasmic bridges during cell division in spermatocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

anterior medial portion of the bulbus ejaculatorius, which is long, fusiform and has a very complex construction.

Ejaculatory sacs are located on both sides of the bulbus ejaculatorius. Ectodermal sac is surrounded by a single layer of epithelial cells that are filled with

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Fig. 2. (a, b). TEM micrographs of spermatocyte. Golgi complex (G), and mitochondrial nebenkern (Nb) derivatives formation. (c, d) Semithin section and SEM micrograph of the differentiation of spermatocytes (Sp), and spermatids (St) (x400). (e, f) Light and SEM

micrographs of spermatids (St) in the maturation zone (x400). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

electron-dense secretions (Figs. 6a and 6c). In epithelial cells, many rough endoplasmic reticulum and mitochondria were observed (Fig. 6d). Ejaculatory bulb is

partly formed by an epithelium, which is continuous with that of the single, median, funnel-shape ductus ejaculatorius, that is covered by irregularly shaped Microscopy Research and Technique


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Fig. 3. (a, b) TEM micrographs of spermatids in testis follicles. (c, d) Light and SEM micrographs of head and flagellum of the spermatozoon (Sz). (e, f) TEM micrographs of longitudinal sections of the spermatozoon. Revealing lamellar arrengement of the material inside of the nucleus. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

accessory glands (Figs. 6a and 6b). The ductus ejaculatorius transports sperm and secretions of the accessory glands. The ductus ejaculatorius, which is continuous with the aedeagus, is ectodermal in origin and is covered Microscopy Research and Technique

with cuticle. The glandular epithelium of accessory glands in E. ventrale consists of a single layer of epitelial cells (Fig. 7a). In the SEM images, structure of accessory glands is tubular shape and its outer surface is covered

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Fig. 4. (a, b) Light and SEM micrographs of spermatozoa (*) in the tubular vas deferens (Vd) connected to the testis (x400). (c, d) TEM micrographs of spermatozoa inside vas deferens and longitudinal and cross section of the flagellums in spermatozoa. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with tracheoles (Fig. 7b). In the TEM images, cytoplasm of epitelial cells of accessory glands has ribosomes that may be free or bound to the endoplasmic reticulum membranes, golgi comlex, mitochondria many of them large and secretory granules (Figs. 7c and 7d). DISCUSSION Morphological character studies of the male reproductive system have been made in several insect species including the Heteroptera, resulting in considerable progress with histological, ultrastructural and cytochemical studies of the structures that make up this organ (Bahadur, 1975; Bairati, 1968; Forbes and Do-Van-Quy, 1965; Ferreira et al., 2004; Freitas et al., 2007a, 2007b, 2010; Lemos et al., 2005; Louis and € Kumar, 1971; Mikheyev, 2004; Ozyurt et al. 2013a, 2013b, 2014; Wheeler and Krutzsch, 1992). The morphology of the adult male reproductive system of E. ventrale is similar to that observed in other Heteroptera, in being associated with the third abdominal segment,

with a pair of testes, two vas deferens, two seminal vesicles, a pair of accessory glands, a pair of ectodermal sac, one bulbus and ductus ejaculatorius (Adams, 2001; Bonhag and Wick, 1953; Chapman, 1998; Davis, 1956; Freitas et al., 2010; Karakaya et al., 2012; Lemos et al., € 2005; Nijhout, 1998; Ozyurt et al., 2013a, 2013b, 2014; Rodrigues Agna et al., 2008). However, the testes of some species of heteroptera may be fused into a single median mass testes (Nijhout, 1998). The male reproductive morphology of Nezara viridula (Linnaeus, 1758) (Pendergrast, 1956; Ramamurty, 1969), Chrysocoris stolli Wolf (Singh, 1968), Perillus bioculatus (F.) (Adams, 2001), Oebalus poecilus (Dallas, 1851), (Santos et al., 2003), Podisus nigrispinus (Dallas) (Lemos et al., 2005) and (Rodrigues Agnaet al., 2008), Triatoma brasiliensis Neiva, 1911 and T. melanica Neiva and Lent, 1941 (Freitas et al., 2010) have been the subject of detailed studies, with an emphasis function. The testis of E. ventrale is the basic component of the reproductive system suspended in the body cavity Microscopy Research and Technique


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Fig. 5. (a) TEM micrographs of transverse section of sperm flagellum. Centriolar adjunct (Ca), Reticular appandage (Ra), axonema (Ax). (b) TEM micrographs of mitochondrial derivatives with of crystalloids (*) and axonema (Ax). (c) TEM micrographs of transverse sec-

tion of sperm tail and head regions. (d) TEM micrographs of detail of the flagellar axoneme (Ax), surrounded by microtubules and mitochondrial derivative (Md).

by fat body. Each testis is composed of testicular lobes which usually contain seven seminiferous tubules. These tubule numbers are commonly found within Heteroptera and typical for pentatomids as in the E. ventrale. The number of the follicles varies widely between species and has taxonomic importance (Matsuda, 1976; Davis, 1956; Pendergrast, 1956; Kumar, 1969a; Kumar, 1969b; Lemos et al., 2005; Jahnke et al., 2006; Pap acˇek and Sold an, 2008; Rodrigues Agna et al., 2008; Wieczorek and Swiatek, 2009; Frei€ tas et al., 2010; Karakaya et al., 2012; Ozyurt et al., 2013a, 2013b, 2014). Testes in E. ventrale, Perillus bioculatus (Fabricius) (Pentatomidae: Asopinae) and A.amygdali (Germar) € (Adams, 2001; Ozyurt et al., 2014) consists of seven sperm tubes, but in Nezara viridula (Linnaeus),

Podisus nigrispinus (Dallas), Dolycoris baccarum (Linnaeus), Graphosoma lineatum (Linnaeus) (Pentatomidae), and Aphelocheirus aestivalis Fabricius (Heteroptera: Aphelocheiridae) 4–6 sperm tubes are observed (Lemos et al., 2005; Ramamurty, 1969; € Ozyurt et al., 2013a, 2013b). In Adparaproba gabrieli Carvalho (Heteroptera: Miridae), two follicles are observed (Uceli et al., 2011). The sperm tubes of E. ventrale as in other heteropterans are covered by a peritoneal sheath and a tunica propria (Chapman, 1998; € Davis, 1956; Karakaya et al., 2012; Ozyurt et al., 2013a, 2013b, 2014; Pires et al., 2007; Rodrigues Agna et al., 2008). The testes contain sperm cells at different stages of spermatogenesis across the sperm tubes. In E. ventrale, the spermatids found in the distal and medial


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Fig. 6. (a, b) Light and SEM micrographs of the ejaculatory bulb (B) and duct (D), ectodermal sacs (Es), accessory glands (Ag) (x100). (c, d) TEM micrographs of longitudinal section through the ectodermal sac. The gland lumen (Lu) is filled with electron dense secretions.

At higher magnification, mitochondria (M), dense rough endoplasmic reticulum (RER). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

regions in the testes are encapsulated by a somatic cell, forming the cyst, where they complete the differentiation process. In the proximal region of the testes, near to insertion of the seminal vesicle, these cells are free. The mechanisms of spermatogenesis in E. ventrale, including sperm differentiation, are rather similar in other Heteroptera (Bowen, 1922; Chapman, 1998; Davis, 1956; Engelmann, 1970; Jamieson et al., € 1999; Karakaya et al., 2012; Lemos et al., 2005; Ozyurt et al., 2013a, 2013b, 2014; Pires et al., 2007; Rodrigues Agna et al., 2008). In most insects, it was observed that the number of spermatozoa per bundle is variable. For Virkki (1969), archaic orders of insects have a greater number of sperm per bundle than recent (derived) orders. The most recent or specialized groups tend to have a smaller number of sperm per bundle. In E.ventrale, the number of sperm per cyst varies, but it is common to find a maximum number of 36 cells per cyst. In Chrysomya megacephala (Diptera: Calliphoridae)

(Name et al., 2010), Sarcophaga bullata (Diptera: Sarcophagidae) (Warner, 1971), 127 cells and Lucilia cuprina (Diptera: Calliphoridae) and Lucilia eximia (Diptera: Calliphoridae) (Name et al., 2012), 256 cells were observed per cyst. Except for Apis mellifera (Hymenoptera: Apidae) and Hypanthidium foveolatum (Hymenoptera: Megachilidae) (Gracielle et al., 2009), which have around 200 and 28 follicles per testis, respectively, other Apidae studied have three or four follicles per testis (Brito et al., 2010; Ferreira et al., 2004; Fiorillo et al., 2009; Lima et al., 2006). In the Anthidiini H. foveolatum (Gracielle et al., 2009) and in the Apidae Meliponini, up to 128 spermatozoa per cyst are observed (Cruz-Landim, 2001; Lino-Neto et al., 2008; Zama et al., 2001). Therefore, when compared with other groups, this feature is consistent with the current taxonomic and phylogenetic position of these species within insect orders. Once fully developed, the sperm cells travel from the testes to the seminal vesicle via the vas deferens. Microscopy Research and Technique


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Fig. 7. (a, b) Light and SEM micrographs sections of the accessory glands (x400). (c) TEM micrographs of the accessory glands. The secretory granules (Sg) in cytoplasm of the epithelial cells. Nucleus (Nu), Lumen (Lu). (d) At higher magnification; The cytoplasm of the

secretory cells shows numerous secretory granules (Sg). Golgi apparatus (G), mitochondria (M) and nucleus (Nu). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

However, in Notonecta glauca (Linnaeus, 1758) (Heteroptera: Notonectidae), the testicular follicles are directly connected to the seminal ducts (vasa deferentia laterals) (Pap acˇek and Sold an, 2008). In E. ventrale, as in other heteropterans, a portion of each vas deferens and seminal vesicle is dilated to form a tubular chamber. Similar ductal structure has been observed in vasa deferentia and seminal vesicles of, D. baccarum, G. lineatum, N. glauca and A. amygdali € (Ozyurt et al., 2013a, 2013b, 2014; Pap acˇek and Sold an, 2008). As within other Heteroptera, the seminal vesicles of E. ventrale connect to balloon-shaped bulbus ejaculatorius and are covered by irregularly € shaped accessory glands (Ozyurt et al., 2013a, 2013b, 2014). The morphological knowledge of insect male reproductive accessory glands of insects is extensive (Pap acˇek and Sold an, 2008). These structures vary considerably in size, shape, number, and embryological € origin (Leopold, 1976; Chapman, 1998; Ozyurt et al.,

2013a, 2013b). In most insects, their main function is the formation of seminal fluid and a spermatophore, which is vital for the transfer of sperm. Also, the accessory glands are responsible for spermatophore production and produce some active peptides (Chen, 1984; Kaulenas, 1992; Freitas et al., 2010; Uceli et al., 2011). Male E. ventrale has a pair of accessory glands, which open into the vas deferens and the ductus ejaculatorius. These accessory glands are similar to those in D. baccarum, G. lineatum and A. amygdali (Heteroptera: € Pentatomidae) (Ozyurt et al., 2013a, 2013b, 2014). However, Triatoma rubrofasciata (De Geer) and A. gabrieli Carvalho have only four accessory glands, and T. brasiliensis Neiva as well as T. melanica Neiva and Lent (Hemiptera: Reduviidae) have eight accessory glands. The bulbus ejaculatorius join to form a common ductus ejaculatorius (Bushrow et al., 2006). The terminal portion of the ductus ejaculatorius may be sclerotized to form the intromittent organ, the


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aedeagus. It differentiates from a pair of ectodermal lobes associated with the ninth abdominal segment and is often concealed within a genital chamber (Klowden, 2007). Insect sperms are morphologically similar to those of vertebrates, containing a head region and a long flagellum that is used for locomotion. Within the head are a haploid nucleus and the acrosomal complex at the tip that arises from the golgi apparatus during differentiation. (Klowden, 2007). Flagellum has the axoneme or microtubule-based cytoskeleton of the flagellum; one or two mitochondrial derivatives; and one, or more commonly two, accessory bodies that represent extensions of the centriole adjunct that forms a collar at the base of the flagellum (Nardi et al., 2013). The nucleus is attached to the flagellum by a very electron-dense structure identified as the centriolar adjunct (Jamieson, 1987; Jamieson et al., 1999). In the Hemiptera, the centriolar adjunct in true bugs, treehoppers and cicadas is elongate and made up of homogeneous and electron-dense material (Chawanji et al., 2005, 2006; Ara ujo et al., 2011). The accessory bodies, originating from the pericentriolar matrix, occur in only some families in Auchenorrhyncha (Jamieson, 1987). Moreover, in the suborder Heteroptera, the accessory bodies are completely absent (Mercati et al., 2009), but are well developed and wing shaped in the more derived suborder Sternorrhyncha, including the families Aphididae and Psyllidae (Vitale et al., 2011). The centriolar adjunct in E. ventrale, is relatively small, anterior to the mid-piece and connecting the nucleus with the mitochondrial derivatives of the flagellum. It is assumed that the centriolar adjunct is a transient organelle (Fawcett, 1981) and its role is to nourish the developing axoneme, in contrast to the attachment function usually attributed to it (Jamieson, 1987), or organize the rearrangement of chromatin in the spermatid and to direct the migration of the nuclear region from the place where the flagellum extends (Wang and Zhong, 1993), or to fasten the sperm head and tail together (Baccetti, 1998). In E. ventrale analyzed, the spermatozoa were stored along the vasa deferentia. The testes and vasa deferentia were covered with a capsule with pigmented granules, besides the granules were observed also in P. nigrispinus, P. distinctus, B. tabidus, and S. cincticeps except T. marginata (Heteroptera: Pentatomidae), which does not have pigment granules in the capsule (Ara ujo et al., 2011). As with other Pentatomidae, the ductus ejaculatorius of E. ventrale is single, located medially and has a cuticular cover demonstrating its ectodermic origin. It begins at the base of the accessory glands and later joins to form the common ductus ejaculatorius (Bushrow et al., 2006). The terminal portion of the ductus ejaculatorius may be sclerotized to form the intromittent organ, the aedeagus. It differentiates from a pair of ectodermal lobes associated with the ninth abdominal segment and is often concealed within a genital chamber (Klowden, 2007). The ductus ejaculatorius of Oncopeltus (Heteroptera) is also extremely complex, being specialized for the erection of the penis (Chapman, 1998). In conclusion, the morphological studies of the male reproductive system and spermatozoa in Heteroptera demonstrate the diversity of information provided by

these reproductive structures and their validity in the elucidation of taxonomy and phylogenetic issues remaining in this important group of insects. REFERENCES Adams TS. 2001. Morphology of the internal reproductive system of the male and female two-spotted stink bug, perillus bioculatus (F.) (heteroptera: pentatomidae) and the transfer of products during mating. Invertebr Reprod Dev 39: 45–53. Ara ujo VA, Lino-Neto J, Sousa Ramalho DE Zanuncio F., Serr~ ao JCJE 2011. Ultrastructure and heteromorphism of spermatozoa in five species of bugs (pentatomidea: heteroptera). Micron 42: 560–567. Baccetti B. 1998. Spermatozoa. In: Harrison FW, Locke M, editors. Microscopic anatomy of invertebrates 11C: Insecta. New York: Wiley-Liss. pp. 843–894. Bahadur J. 1975. Histology of the male reproductive organs of a bug, halys dentata F. (hemiptera, pentatomidae). Zool Pooloniae 24: 311–318. Bairati A. 1968. Structure and ultrastructure of the male reproductive system in Drosophila melanogaster. The genital duct and accessory glands. Monitore Zool Ital 2: 105–182. Bonhag PF, Wick JR. 1953. The fuctional anatomy of the male and female reproductive systems of the milkweed bug, oncopeltus fasciatus (dallas). J Morphol 93:177–284. Bowen RH. 1922. Studies on insect spermatogenesis. 2. The components of the spermatid and their role in the formation of sperm in hemiptera. J Morphol 37:79–172. Brito P, Zama U, Dolder H, Lino-Neto J. 2010. New characteristics of the male reproductive system in the meliponini bee, friesella schrottkyi (hymenoptera: apidae): Histological and physiological development during sexual maturation. Apidologie 41:203–215. Bushrow ES, Fuller CL, Cowan DP, Byrd CA. 2006. Anatomy of the male reproductive system and sperm morphology in the caterpillar-hunting wasp ancistrocerus antilope (hymenoptera, vespidae). Invertebr Biol 125:354–362. Chapman RF. 1998. Reproductive system: male. In: Chapman RF, editor. The insects: structure and function. Cambridge: Cambridge University Press. pp. 268–294. Chawanji AS, Hodgson AN, Villet MH. 2005. Sperm morphology in four species of african platylieurine cicadas (hemiptera: cicadomorpha: cicadidae). Tissue Cell 37:257–267. Chawanji AS, Hodgson AN, Villet MH. 2006. Sperm morphology in five species of cicadettine cicadas (hemiptera: cicadomorpha: cicadidae). Tissue Cell 38:373–388. Chen PS. 1984. The functional morphology and biochemistry of insect male accessory glands and their secretions. Annu Rev Entomol 29: 233–255. Cruz-Landim C. 2001. Organization of the cysts in bees (hymenoptera, apidae) testis: Number of spermatozoa per cyst. Iheringea S er Zool 91:183–189. Davey KG. 1958. The migration of spermatozoa in the female of rhodnius prolixus. Stal J Exp Biol 35:694–701. Davey KG. 1985. The male reproductive tract. In: Kerkut GA, Gilbert LI editors. Comprehensive insect physiology, biochemistry and pharmacology. Oxford: Permagon Press. pp. 1–14. Davis NT. 1956. The morphology and functional anatomy of the male and female reproductive systems of cimex lectularius L. (heteroptera, cimicidae). Ann Entomol Soc Am 49:466–493. Engelmann F. 1970. Gonadal development the physiology of insect reproduction. 1st ed. New York: Pergamon. pp. 41–56. Fawcett DW. 1981. The cell, 2nd ed. Philadelphia: W. B. Saunders Company. pp. 862. Ferreira A, Abdalla FC, Kerr WE, Cruz-Landim C. 2004. Systematics morphology and physiology. Comparative anatomy of the male reproductive internal organs of 51 species of bees. Neotrop Entomol 33:569–576. Forbes J, Do-Van-Quy D. 1965. The anatomy and histology of the male reproductive system of the legionary ant, Neivamyrmex harrisi (Haldeman) (Hymenoptera, Formicidae). N Y Entomol Soc 73: 95–111. Freitas SPC, Gonçalves TCM, Serr~ ao JE, Santos-Mallet JR. 2007a. Fine structure of the male accessory glands of triatoma rubrofasciata (de geer, 1773) (hemiptera, triatominae). Microsc Res Tech 70: 355–360. Freitas SPC, Santos-Mallet JR, Serr~ ao JE, Lorosa ES, Gonçalves TCM. 2007b. Morphometry of the testis follicles in triatoma rubrofasciata (de geer, 1773) (hemiptera, triatominae). Anim Biol 57: 393–400.


ULTRASTRUCTURE OF MALE REPRODUCTIVE SYSTEM OF EURYDEMA VENTRALE Freitas SPC, Gonçalves TCM, Serr~ ao JE, Costa J, Santos-Mallet JR. 2010. Male reproductive system structure and accessory glands ultrastructure of two species of triatoma (hemiptera, reduviidae, triatominae). Micron 41:518–525. Friedel T, Gillot C. 1977. Contribution of male-produced proteins to vitellogenesis in melanoplus sanguinipes. J Insect Physiol 23: 145–151. Fiorillo BS, Zama U, Lino-Neto J, B ao SN. 2009. Structural and ultrastructural studies of male reproductive tract and spermatozoa in xylocopa frontalis (hymenoptera apidae). Acta Zool 89:1463– 1470. Fuchs MS., Craig GBJr., Despommier DD 1969. The protein nature of the substance inducing female monogamy in aedes aegypti. J Insect Physiol 15:701–709. Fuhrer J. 2003. Agroecosystem responses to combinations of elevated co2, ozone, and global climate change. Agric Ecosyst Environ 97:1–20. Gillott C. 2003. Male accessory gland secretions: Modulations of female reproductive physiology and behaviour. Ann Rev Entomol 48:163–184. Gracielle IMS, Fiorillo BS, Lino-Neto J, B ao SN. 2009. Morphology of the male reproductive system and spermiogenesis in hypanthidium foveolatum (alfken, 1930) (hymenoptera: apidae, megachilinae). Micron 40:719–723. Happ GM. 1992. Maturation of the male reproductive system and its endocrine regulation. Annu Rev Entomol 37:303–320. Jahnke SM, Redaelli LR, Diefenbach LMG. 2006. Internal reproductive organs of Cosmoclopius nigroannulatus (Hemiptera: Reduviidae). Braz J Biol 66: 509–512. Jamieson BGM. 1987. The ultrastructure and phylogeny of insect spermatozoa. Cambridge: Cambridge University Press. pp. 320. Jamieson BGM, Dallai R, Afzelius BA. 1999. Insects: Their spermatozoa and phylogeny. Enfield. NH: Science Publishers. Inc. pp. 555 € Karakaya G, Ozyurt N, Candan S, Suludere Z. 2012. Structure of the male reproductive system in coreus marginatus (linneaus, 1758) (hemiptera: coreidae). Turk Entomol Derg 36:193–204. Kaulenas MS. 1992. Insect Accessory Reproductive Structures. Function, Structure and Development. Springer-Verlag. New York, NY, USA. pp. 224. Klowden MJ. 2007. Physiological systems in insects. USA: Academic Press. pp. 181–223. Kumar R. 1969a. Morphology and relationships of the pentatomoidea (heteroptera): III. Natalicolinae and some tessaratomidae of uncertain position. Ann Entomol Soc Amer 62:681–695. Kumar R. 1969b. Morphology and relationships of the pentatomoidea (heteroptera) IV. Oncomerinae (tessaratomidae). Aust J Sci 17: 553–606. Lemos WP, Serr~ ao JE, Ramalho FS, Zanuncio JC, Lacerda MC. 2005. Effect of diet on male reproductive tract of podisus nigrispinus (dallas) (heteroptera, pentatomidae). Braz J Biol 65:91–96. Leopold RA. 1976. The role of male accessory glands in insect reproduction. Annu Rev Entomol 21: 199–221. Lima MAP, Lino-Neto J, Campos LAO. 2006. Sexual maturation in melipona mondury males (apidae: meliponini). Braz J Morphol Sci 23:369–375. Lino-Neto J, Ara ujo VA, Dolder H. 2008. Inviability of the spermatids with little cytoplasm in bees (hymenoptera, apidae). Sociobiology 51:163–172. Louis D, Kumar R. 1971. Morphology and histology of the mushroomshaped gland in some Dictyoptera. Ann Entomol Soc Am 64: 977– 982. Matsuda R. 1976. Morphology and evolution of the insect abdomen. Oxford: Pergamon Press. pp. 534. Mercati D, Giusti F, Dallai R. 2009. A novel membrane specialization in the sperm tail of bug insect (heteroptera). J Morphol 270:825–833. Mikheyev AS. 2004. Male accessory gland size and the evolutionary transition from single to multiple mating in the fungus-gardening ants. J Insect Sci. 4: 1–5. Name KPO, Pujol-Luz JR, B ao SN. 2010. Structure and ultrastructure of spermatozoa of chrysomya megacephala (diptera: calliphoridae). Micron 41:853–860. Name KP, Barros-Cordeiro KB, Jos e Filho BG, Wolff M, Pujol-Luz JR, B ao SN. 2012. Structure and ultrastructure of spermatozoa and spermiogenesis in three species of lucilia robineau-desvoidy, 1830 (diptera: calliphoridae). J Morphol 273:160–172.


653

Nardi JB, Delgado JA, Collantes F, Miller LA, Bee CM, Kathirithamby J. 2013. Sperm cells of a primitive strepsipteran. Insects 4:463–475. Nijhout HF. 1998. Reproduction. In: Nijhout, HF editor. Insect hormone. Princeton: Princeton University. pp. 142–159. Nijhout H. F. 1994. Insect Hormones. Princeton University Press. Princeton: New Jersey, USA. pp. 280. € Ozyurt N, Candan S, Suludere Z. 2013a. The morphology and histology of the male reproductive system in Dolycoris baccarum Linnaeus 1758 (heteroptera: pentatomidae) light and scanning electron micoscope studies. Micron 44:101–106. € Ozyurt N, Candan S, Suludere Z, Amutkan D. 2013b. Reproductive system in Graphosoma lineatum (Heteroptera: Pentatomidae) based on optical and scanning electron microscopy. Journal of Entomology and Zoology Studies 1(4): 40–46. € Ozyurt N, Candan S, Suludere Z. 2014. The morphology and histology of the male reproductive system in Apodiphus amygdali (Germar, 1817) (Heteroptera: Pentatomidae). Life: The Excitement of Biology 2(1): 31–41. Pap acˇek M, Sold an T. 2008. Structure and development of the reproductive system in Aphelocheirus aestivalis (Hemiptera: Heteroptera: Nepomorpha: Aphelocheiridae). Acta Ent Mus Nat Pra 48(2): 339–338. Pickford R, Ewen AB, Gillott C. 1969. Male accessory gland substance: An egglaying stimulant in melanoplus sanguinipes (orthoptera, acrididae). Can J Zool 47:1199–1203. Pires EM, Ferreira PSF, Guedes RNC, Serrao JE. 2007. Morphology of the phytophagous bug platyscytus decempunctatus (carvalho) (heteroptera: miridae). Neotrop Entomol 36: 510–513. Ramamurty PS. 1969. Histological studies of the internal organs of reproduction in Nezara viridula Fabr. Pentatomidae: Heteroptera, Insecta. Zool Anzeiger 183: 119–139. Reynolds ES. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J Cell Biol 17:208–212. Rodrigues ARS, Serrao JE, Teixeira VW, Torres JB, Teixeira AA. 2008. Spermatogenesis, changes in reproductive structures, and time constraint associated with insemination in podisus nigrispinus. J Insect Physiol 54:1543–1551. Santos RSS, Redaelli LR, Diefenbach LMG, Romanowski P, Prando HF. 2003. Characterization of the imaginal reproductive diapause of Oebalus poecilus (Hemiptera: Pentatomidae). Braz J Biol 63(4): 695–703. Schuh RT, Slater JA. 1995. True bugs of the world (Hemiptera: Heteroptera): Classification and Natural History. Cornell University Press: Ithaca, NY, USA and London, England, UK. pp. 336. Stankovic´ A. 1963. Tok pigmentacije i hibernacije vrste eurydema ventrale kol. Letopis Naucˇnih Radova Poljoprivrednog Fakulteta Novi Sad, Br 7:58–65. Stankovic´ A. 1964. Broj generacija i nosivost eurydema ventrale kol. U SR srbiji. Zastita Bilja, Br. 80:403–409. Uceli LF, Pirovani VD, Vicente NMF, Pikart TG, Ferreira PSF, Serro JE. 2011. Morphology of the reproductive and digestive tracts of Adparaproba gabrieli (Heteroptera: Miridae). Int J Trop Insect Sci 31(4): 219–224. Vitale DGM, Brundo MV, Viscuso R. 2011. Morphological and ultrastructural organization of the male genital apparatus of some aphididae (ınsecta, homoptera). Tissue Cell 43:271–282. Wang ZS, Zhong XC. 1993. Origin and function of the centriolar adjunct in spermatids of insects. Acta Entomol Sinica 36:419–422. Warner FD. 1971. Spermatid differentiation in the blowfly sarcophaga bullata with particular reference to flagellar morphogenesis. J Ultrastruct Res 35:210–232. Wheeler DE, Krutzsch PH. 1992. Internal reproductive system in adult males of the genus Camponotus (Hymenoptera, Formicidae, Formicinae). J Morphol 211: 307–317. Wieczorek K, Swiatek P. 2009. Comparative study of the structure of the reproductive system of dwarfish males of Glyphina betulae and Anoecia corni (Hemiptera, Aphididae). ZoolAnzeiger 248(3): 153– 159. Zama U, Lino-Neto J, Dolder H. 2001. Ultrastructure of spermatozoa in plebeia (plebeia) droryana friese (hymenoptera: apidae, meliponina). J Hym Res 10:261–270.