Morphostructural analysis of the male reproductive system and DNA barcoding in Balclutha brevis Lindberg 1954 (Homoptera,Cicadellidae)

Balclutha brevis Lindberg 1954 is an allochthonous leafhopper infesting an invasive grass, Pennisetum setaceum, in Sicily and in mainland Europe; therefore, this species could compete with populations of native species, thus contributing to the loss of biodiversity. Considering the ecological implications of B. brevis, investigations on all its biological aspects represent, therefore, a premise for further studies in applied sciences. Based on the lacking ultrastructural data about the reproductive systems of the Auchenorrhyncha, we carried out morphostructural investigations on the male reproductive system of B. brevis. Further, a first report of DNA barcoding analysis (amplification and sequencing of Cytochrome Oxidase I gene) has also been performed to characterize B. brevis compared to other congeneric species. From a morphological point of view, the male reproductive system of B. brevis has an organization comparable to the general anatomical features of most of the Auchenorrhyncha species; however, comparing our data with those concerning the different groups of Cicadomorpha, some considerations are discussed. As for the histological and ultrastructural investigations, our results show a secretory activity of the various examined structures, mainly in the lateral ejaculatory ducts and in the accessory glands. The latter, in particular, show morphostructural differences comparing the distal tract to the proximal one; moreover, the histochemical techniques showed the possible presence of a lipid component in the peculiar cytoplasmic granules found in the gland cells. The significance of these findings in the accessory glands is discussed. Finally, the ultrastructural features found in the seminal vesicles are different from those of the lateral ejaculatory ducts and are indicative of the different roles played by these structures in the organization of the spermatozoa bundles.     

Ultrastructure of the female reproductive apparatus of the egg parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera,Platygastridae)

The growing interest in Leptoglossus occidentalis, the conifer seed bug pest accidentally introduced into Europe in the 1990s, led us to investigate the female reproductive structures of the hymenopteran platygastrid Gryon pennsylvanicum, which is its candidate antagonist for biological control programmes. Our study revealed a genital apparatus with some characteristic features, such as an unusual length of the oviduct (divided into a long proximal and a short distal tract), the absence of accessory glands and the presence of a spermatheca provided with a small spermathecal gland. The ultrastructural investigation revealed that the shorter part of the common oviduct is involved in ion uptake whereas the longer part has two cell types with secretory function: the former with dense bodies and the latter with granular particles. The secretory contents of both are released into the oviduct lumen. The granular particles are formed in a complex of modified endoplasmic reticulum and appear as virus-like particles.     

Ultrastructure of the seminal vesicle and sperm storage in Panorpidae (Insecta: Mecoptera)

The histology and ultrastructure of the seminal vesicle, male accessory gland and the epididymis in Panorpa and Sinopanorpa were observed using light microscopy and scanning and transmission electron microscopy. The seminal vesicle consists of a mono-layered elongated columnar epithelium, which contains abundant electron-dense granules and secretory vesicles, and a small central lumen. In the apical region of the epithelium of the seminal vesicle, the intense secretory activity seems to be effected by means of merocrine mechanisms. The epithelium of the accessory gland is rich in rough endoplasmic reticulum and Golgi complex, and secretes seminal fluid into the lumen via both apocrine and merocrine mechanisms. The seminal vesicle is similar to the accessory gland in the epithelium structure and their secretory activity, mainly serving a secretory function rather than storing sperms. Instead, the sperms are stored in the epididymis, whose epithelium secretes nutrients into the large lumen by merocrine mechanisms. The secretory activity and function of the seminal vesicle are briefly discussed.     

Ultrastructural observation on genesis and morphology of cortical granules in Macrobrachium nipponense (Crustacea,Caridea)

Cortical granules are secretory vesicles in oocytes that develop from the Golgi complex. In the freshwater shrimp, Macrobrachium nipponense, mitochondria participates in the formation of cortical granules. We investigated the structural changes of mitochondria and the distribution cortical granules in different stages of oocyte development. Transmission electron microscopy provided evidence for the involvement of mitochondria and a particular spiral lamellar organization and an electron-lucent area in internal cortical granules. The ooplasm provided material for the cortical granules in early oocyte development. We demonstrated that mitochondria play a role in coalescence and maturation of cortical granules in this species. Additionally, a concept of cortical granules regarded as a functional integration is put forward. The genesis of shrimp cortical granules exhibited a particular pathway of maturation. The outer shape and inner organization considering different taxa suggested general as well as specific features of the development of cortical granules.     

Ultrastructural study of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata (Stal, 1854) (Euhemiptera: Cercopidae)

Spittlebugs are insects that suck sap from plants and regurgitate saliva containing toxic enzymes into the leaves. As a consequence, the conductive channels are blocked resulting in dry leaves, thus giving a burned aspect to the plantation. This work performed ultrastructural analyses of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata, since these organs produce the enzymes that are injected into the plants, thus being responsible for the economic losses in the production of sugarcane. Three kinds of secretory cells are found in principal gland, forming the lobules I-IV. The main differences among these cells relate to size, morphology and electron density of the secretory vesicles. The accessory glands contain different secretory vesicles to those in the principal gland. Muscular cells are found around the entire gland. The different secretory vesicles found in both principal and accessory glands indicate that the gland produces different substances or that the secretion in the interior of cells passes through a maturation process.     

Ultrastructure of the mature testicular vesicle of Pseudonannolene tocaiensis Fontanetti, 1996 (Diplopoda, Pseudonannolenidae)

The mature testicular vesicles of the species Pseudonannolene tocaiensis have three portions: a peripheral portion; a central portion that is typically secretory and between these two an intermediary portion, a lumen filled with spermatozoa. Both secretory and peripheral portions present the same type of cells. The ultrastructural analysis of the peripheral portion suggests it is involved in hormone synthesis. The secretory portion probably contributes to the production of spermatic fluid, compensating for the absence of male accessory glands in this species.     

Structure and ultrastructure of spermatozoa of Chrysomya megacephala (Diptera: Calliphoridae)

The spermatozoa of Chrysomya megacephala are similar to those described for other Brachycera. In this species, the spermatozoa are long and thin, measuring about 590 μm in length, of which the head region measures approximately 60 μm. The head includes a monolayered acrosome with electron-lucid material, and the shape of the nucleus, in cross-sections, varies from circular to oval with completely condensed chromatin. The centriole was observed in the zone of flagellar implantation, below the “peg” region. In the region of overlap, the followings structures are observed: nucleus, centriolar adjunct, mitochondrial derivatives and axoneme. The two mitochondrial derivatives are of different lengths but similar diameter. The axoneme is of a conventional insectan type with a 9 + 9 + 2 microtubular arrangement, with accessory tubules flanked by the electron-dense intertubular material. The male internal reproductive tract consists of testis, vas deferens, seminal vesicle, accessory glands and ejaculatory duct.     

Histochemistry of skin glands of Trachycephalus aff. venulosus Laurenti, 1768 (Anura, Hylidae)

The skin of the adult tree frog, Trachycephalus aff. venulosus, was investigated by light microscopy. Histochemistry was carried out using various stain techniques and showed the basic integument morphology of the skin with squamous epithelium in the epidermis and connective tissue in the dermis, subdivided into spongy and compact layers. The integument observed contains two types of gland; the mucous glands and granular glands. The mucous glands are small and located in the upper layer of the stratum spongiosum of the connective tissue and contain neutral and acid sulfated glycoproteins. The granular glands are large and form a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. Histochemical analyses demonstrated the presence of collagen proteins in both types of gland. An unusual finding was the different densities of the granules in the granular glands. The morphological characteristics of the integument in Trachycephalus aff. venulosus was also compared with others species of Anura.     

Cytochemical characterization of Triatoma infestans and Panstrongylus megistus salivary gland cells (Hemiptera, Reduviidae, Triatominae)

The Triatominae subfamily has medical sanitary importance, since these insects are vectors of Trypanosoma cruzi, etiologic agent of Chagas Disease, and Trypanosoma rangeli, which develops in the salivary glands and it is frequently found in mixed infections with T. cruzi. Triatomines of Triatoma and Panstrongylus genera possess a salivary gland complex composed of a pair with three well differentiated units: the anterior (D1), median (D2) and posterior (D3). Saliva is secreted during blood meal and antagonizes hemostatic, inflammatory and immunological systems imposed by the host, which facilitate the hematophagy. In order to identify nuclear structures, we studied interphase nuclei of salivary gland cells of adult insects, males and females, of Triatoma infestans and Panstrongylus megistus. The glands were removed from insects, fixed in acetic acid (45%) and lactic acid (50%), squashed and submitted to different cytochemical methods: lacto-acetic orcein, silver ion impregnation, Feulgen reaction, Toluidine Blue, Variant method of critical electrolyte concentration and C-banding. The preparations were examined with a Zeiss Jenaval photomicroscope and photographed. The results evidenced predominance of binucleated cells in D1 and D2 glands and mononucleated ones in D3. In all salivary glands were observed bulky and polyploid nucleus, a clear association between nucleolar and heterochromatic corpuscles, cytoplasmatic metachromasy and many pre-secretion vesicles in cytoplasm. Such characteristics were associated with intense synthesis activity to produce the saliva. Species were mainly differentiated by a larger heterochromatic corpuscle observed only in T. infestans (called as chromocenter), while P. megistus showed a predominance of euchromatin, with some heterochromatic corpuscles just in males. Females of both species showed a smaller quantity of heterochromatin, which could be related to the high metabolism because of the oviposition.     

Morphology of the male reproductive system and spermiogenesis in Hypanthidium foveolatum (Alfken, 1930) (Hymenoptera: Apidae: Megachilinae)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.     

Biochemical and cytochemical studies of the enzymatic activity of the venom glands of workers of honey bee Apis mellifera L. (Hymenoptera,Apidae)

Biochemical studies revealed that the activity of some hydrolytic enzymes from the venom glands of honey bee Apis mellifera was higher in workers of 14 days of age than in those of 40 days. Among these enzymes, the highest activity was recorded for acid phosphatase, which was cytochemically detected throughout the length of the secretory filament and surrounding the canaliculi of the distal region of the reservoir. The acid phosphatase was considered to be a typical secretion product, since it was present in the cytoplasm as well as in the canaliculi of the secretory cells.     

Ultrastructural and cytochemical characterization of T1 and T2 secretory bodies from the tegument of Echinostoma paraensei

Trematodes are lined by a syncytial layer that is named the tegument and contains small mitochondria and two different kinds of secretory inclusions. The structure and size of these bodies differs among genera and species. In a previous study, we observed many secretory bodies in the tegument of Echinostoma paraensei and named these bodies the T1 and T2 secretory bodies. No previous studies analyzed the secretory bodies of trematodes from the genus Echinostoma. Thus, the aim of this work was to use electron microscopy and cytochemistry to characterize these secretory bodies and to provide a detailed ultrastructural and morphological picture of these bodies, which are found in the tegument of E. paraensei. After ultrastructural cytochemistry analysis, we showed that both the T1 and T2 secretory bodies of E. paraensei were formed by glycoconjugates. 3D reconstruction confirmed the ovoid form of T1 secretory bodies and the biconcave and thin form of T2 secretory bodies.     

Ovary and oocyte maturation of the tick Amblyomma brasiliense Aragão, 1908 (Acari: Ixodidae)

This study describes the ovary anatomy and dynamics of oocytes maturation process of Amblyomma brasiliense ticks. The ovary is of panoistic type lacking nurse and follicular cells. This organ consists of a single continuous tubular structure comprising a lumen delimited by the ovarian wall. Oocytes of this tick species are classified into five stages (I–V) and described based on cytoplasm appearance, presence of germ vesicle, yolk granules aspects, and chorium deposition. Oocytes of various sizes and at different developmental stages remain attached to the ovary by a cellular pedicel until completing stage V. Then they are released into the ovary lumen and from there into the exterior.     

Morphology of the male reproductive system and spermatozoa in Centris Fabricius, 1804 (Hymenoptera: Apidae, Centridini)

The genera Centris and Epicharis constitute the Centridini and are widely distributed in the Neotropical region. Centris is also found in the southern portion of the Neartic region, although both genera are more abundant in the humid tropical regions. To describe the structure of the male reproductive system and spermatozoa, light and transmission electron microscopy were used. The male reproductive system of Centris sp. is formed by a pair of testes, a pair of deferent ducts, a pair of seminal vesicles, a pair of accessory glands and an ejaculatory duct connected to the external genitalia, the aedeagus. In this species, testes and the pre-vesicular deferens ducts as well as the seminal vesicles are encapsulated in a single conjunctive capsule, the scrotal membrane. Each testis consists of four testicular follicles, made up of cysts with up to 64 germinative cells. Histologically, the seminal vesicles are formed by a simple cylindrical epithelium, basal membrane and muscular tunic. The spermatozoa of Centris analis, C. fuscata, C. tarsata and Centris sp. are morphologically similar. They have two easily distinguishable regions: the head and flagellum. The head is formed by the two-layer acrosome, the linear nucleus and the flagellum, the centriole adjunct, the axoneme of pattern 9+9+2 microtubules, two asymmetric mitochondrial derivatives and two accessory bodies. These Centris species share various morphological characteristics of the male reproductive system and spermatozoa with the other bees previously described, indicating that several characteristics are synapomorphic for the family Apidae. Studies on the morphology of the male reproductive system and spermatozoa in Hymenoptera have demonstrated the diversity of the information provided by these reproductive structures, which can be used in taxonomy studies and the phylogeny of this important group of insects.     

Ultrastructural studies of the mandibular glands of the minima, media and soldier ants of Atta sexdens rubropilosa (Forel 1908) (Hymenoptera: Formicidae)

The mandibular glands of Hymenoptera are structures associated with the mandibles and constitute part of the salivary glands system. Histological studies in workers of Atta sexdens rubropilosa revealed that this gland contains two portions: a secretory and a storage portion or reservoir. Both portions are connected by means of canaliculi. The object of the present work was the study of the ultratructure of the mandibular glands of minima, media and soldier ant of A. s. rubropilosa by TEM techniques. The glands, in the three castes studied, possess a reservoir, constituted by a simple pavementous epithelium surrounded by the cuticular intima and the secretory portion is constituted by cells of rounded shape. The secretory cells, mainly of minima and soldier, were rich in smooth endoplasmic reticulum. The media worker and soldier presented a large number of mitochondria, of varying shape. Well-developed Golgi complexes were also present in the soldiers. The secretory cells in minima, media and soldier were provided with collecting intracellular canaliculi, which were linked to the reservoir through the extracellular portion. The cytoplasm of the canaliculi-forming cell was poor in organelles. In the individuals of the three castes of A. s. rubropilosa, the presence of lipid secretion granules suggested, beyond the other functions, also a possible pheromonal action. The different roles executed by the different insect castes are directly dependent on the glandular products and, consequently, on the secretory cellular characteristics.     

Structural and ultrastructural characterization of male reproductive tracts and spermatozoa in fig wasps of the genus Pegoscapus (Hymenoptera, Chalcidoidea)

The three Pegoscapus species present the same internal reproductive tract features comprising testes with a single testicular tubule, seminal vesicles, vasa deferentia, accessory glands and an ejaculatory duct. The seminal vesicle shows two morphologically distinct portions although they do not resemble the separate chambers found in other Chalcidoidea. The anterior portion of the seminal vesicle shows a prominent epithelium and stores the mature spermatozoa, while the posterior region is formed by a thicker muscular sheath that participates on ejaculation. The sexual maturation in Pegoscapus is achieved at emergence, when the testicular degeneration occurs. The spermatozoa of Pegoscapus reveal a basic structure similar to that of other Chalcidoidea. In Pegoscapus sp1. and Pegoscapus sp2. they present the same features, whereas Pegoscapus tonduzi comprises some different characteristics. It measures approximately 160 μm in Pegoscapus sp1. and Pegoscapus sp2., while in P. tonduzi the spermatozoa measure about 360 μm. The extracellular sheath thickness is another difference among the species. While Pegoscapus sp1. and Pegoscapus sp2. show a thick extracellular sheath, in P. tonduzi this sheath is very thin resulting in a large space intervening between the extracellular sheath and the nucleus. Despite these differences, the three species analyzed share some characteristics that allow the establishment of an identity to the spermatozoon of the genus Pegoscapus: the seminal vesicle not divided in chambers; the absence of acrosomal structures in the spermatozoa; the length of the extracellular sheath; the central microtubules being the firsts to terminate in the sequence of microtubular cutoff at the final axonemal portion.     

Microanatomy and ultrastructure of outer mantle epidermis of the cuttlefish,Sepia esculenta (Cephalopoda: Sepiidae)

This study describes the ultrastructural characteristics of external epidermis of mantle of Sepia esculenta using light and electron microscopy. The epidermis was thicker on the ventral surface than on the dorsal surface, with a higher secretory cell distribution on the ventral surface than on the dorsal surface. The epidermis was a single layer composed of epithelial cells, secretory cells, ciliated cells and neuroglial cells. Epithelial cells were columnar with well-developed microvilli on the free surface, and the microvilli were covered with glycocalyx. The epithelial cells were connected to the neighboring cells by tight junctions and membrane interdigitations of the apico-frontal surface. Well-developed microfilaments were arranged in a vertical direction in the cortical cytoplasm. The secretory cells were categorized into three types (A, B and C) in accordance with the light microscopical characteristics and ultrastructures of the secretory granules. The distribution of these cells was in the following order: Type A > Type B > Type C. SEM observation revealed that the secretory pore size of the Type A secretory cells was approximately 8.6 μm × 12.2 μm. Cytoplasm displayed a red color as the result of Masson's trichrome stain and H–E stain, and contained polygonal granules of approximately 1.2 μm² with a high electron density. The secretory pore size of the Type B secretory cells was approximately 10.1 μm × 12.1 μm. As the results of AB–PAS (pH 2.5) and AF–AB (pH 2.5) reactions, the cytoplasm displayed a red color. The cells contained membrane bounded secretory granules with very low electron density. The secretory pore of the Type C secretory cells was circular shape, and approximately 5.5 μm × 5.5 μm. Cytoplasm was found to be homogeneous under H–E stain and Masson's trichrome stain, and displayed a red color. As the result of AB–PAS (pH 2.5) reaction, the cytoplasm displayed a red color. The electron density of the secretory substance was the highest among the three types of secretory cells. The ciliated cells had a ciliary tuft on the free surface and were distributed throughout the mantle with the exception of the adhesive organs. Neuroglial cells were connected to the basal membrane, epithelial cells, secretory cells and nerve fibers through cytoplasmic process, and contained neurosecretory granules with high electron density within the cytoplasm.     

Morphology and histology of the digestive system of the vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae)

The vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae) is an important economic pest that is a serious threat to wheat in Northwest China, causing great losses to agricultural production by transmitting wheat blue dwarf (WBD) in a persistent circulative manner. Studies on morphology and ultrastructure of the digestive system were made using light, scanning and transmission electron microscopy. The gross morphology of the alimentary canal consists of esophagus (foregut), midgut, hindgut, and special filter chamber. The esophagus, a narrow and slender tube, runs through the whole thorax extending into the filter chamber and connecting with the anterior midgut. The midgut consists of three major regions, anterior, middle and posterior midgut. The anterior midgut is composed of exceptionally thick epithelial cells surrounding a large lumen. Numerous well-developed microvilli occur at the basal region of the epithelium. A large number of electron-lucent and lipid-like vesicles are observed under the microvilli. The posterior midgut is smaller than the anterior midgut in diameter. Numerous different concentric circular bodies are characteristic of the posterior midgut. The hindgut arises from the posterior midgut at the boundary of the filter chamber. It extends downward toward the anus where the hindgut enlarges to form the rectum. The rectum is formed by various cells typical of epithelium, whose nuclei are relatively smaller than those of the midgut. Relatively large muscle cells are present at the well-defined and thick basement membrane. There are two pairs of Malpighian tubules. Numerous mitochondria and lamellar rough endoplasmic reticulum in the cells of the anterior segment of the Malpighian tubules; the sub-anterior segment of the tubules resemble the distal segment which are wave-like, amount vesicles in the cells; numerous tightly packed large vesicles contain two types of brochosomes in the inflated segment of the Malpighian tubules. The salivary system of P. striatus contains one pair of salivary glands, which are made up of the principal and accessory gland. The principal glands are divided into anterior, mid- and posterior lobes. About nineteen acini are observed in the principal glands of P. striatus, and vary in structure and colour. Five cellular types are found in the principal glands by transmission electron microscopy, which are rich in secretory granules, modified in size, electron-dense and shape. Rough endoplasmic reticulum in the shape of vesicular and lamellar, mitochondria and Golgi complexes are observed. The accessory gland has only an oval or a rod-shaped acinus.     

Acute and subchronic exposure of diplopods to substrate containing sewage mud: Tissular responses of the midgut

Several discussions regarding the agricultural use of sewage mud have occurred; however, its use has been questioned due to the risks of soil contamination mainly by metals. Diplopods are saprophagous invertebrates, agile in colonizing several layers of soil. Due to the habit of this group, several researchers have proposed their use in ecotoxicological analyses. This study aimed to expose these invertebrates to substrate containing sewage mud of a STS (Sewage Treatment Station) from São Paulo State, Brazil and analyzed its toxic potential by morphological analysis of the midgut of the diplopod Rhinocricus padbergi, region of the digestive tube where digestion and nutrient absorption occur. The animals were exposed in mud concentrations at 1%, 10% and 50% mixed with soil from the collection site for periods of 7 (acute exposure), 15 (intermediary exposure) and 90 (subchronic exposure) days in each treatment. The animals from the control group did not present alterations at all the exposition times. As an acute response (7 days), it was observed in the animals exposed to mud at 1% and 10% an increase in the epithelial renovation and in the liberation of secretory vesicles of glycoprotein content. In the animals exposed to mud at 50%, the main alteration observed was the increase in the number of cytoplasmatic granules in the fat body cells; the epithelium seemed to be in reorganization process in all the observed individuals, suggesting an intense epithelial renovation. In the intermediary period of exposure (15 days), in the animals exposed to mud at 1% it was observed an increase in the number of haemocytes among the cells of the fat body, distributed both separately and grouped; the animals exposed to mud at 10% presented the same evidence of epithelial renovation observed in the animals exposed for 7 days at a concentration of 50% of mud; it was also observed an increase in the number of haemocytes, which reacted strongly to the PAS technique. Cytoplasmatic granules in the hepatic cells, fat body cells and haemocytes were also observed in great number, being that great part presented calcium in their constitution. In the animals exposed to mud at 50%, the alteration observed was the presence of numerous secretory vesicles of the apocrine type through all the epithelia of vacuolated aspect; hepatic cells with cytoplasmatic granules were also observed. As subchronic response (90 days), it was observed that all the fat body cells were completely taken by cytoplasmatic granules, into which it was, detected the presence of calcium; such granules were also observed in the apical region of the principal epithelial cells. These results suggest the existence of toxic agents in the analyzed mud sample, whose different concentrations trigger responses that occur in different manners, intensities and velocities that might prevent the entrance and action of toxic agents in the organism of the studied invertebrate.