Morphology and ultrastructure of the venom apparatus in the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The venom apparatus of the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) was studied with light and electron microscope and was subjected to the electrophoretic and immunohistochemical analyses. Typically its venom apparatus consists of an unbranched venom gland and a venom reservoir, which is associated with a Dufour gland. The venom gland is lined by a series of secretory units. Each secretory unit comprises a secretory cell and a duct cell. The secretory cell is associated with an end apparatus to collect its secretions into the gland lumen. Secretory cells in the venom gland are characterized by extensive rough endoplasmic reticulum and numerous electron-dense vesicles in the distal and middle parts. They also exhibit several secretory granules and vacuoles. The venom reservoir presents three distinct regions: an external layer, composed by numerous fine muscle fibers; an internal layer, represented by epithelial cell with large nucleus; and an intima portion, represented by thin and uniform organization. The morphological aspect of numerous well-developed organelles responsible for protein generation observed is in agreement with the electrophoretic and immunohistochemical results which reveal that the rich proteinaceous components are present in the venom gland and venom reservoir. The venom proteins are first mainly produced in the secretory unit of venom gland, then drained to the lumen through the end apparatus, and are finally collected and stored in the venom reservoir.     

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.     

The intramandibular gland of leaf-cutting ants (Atta sexdens rubropilosa Forel 1908)

The eusociality developed in Hymenoptera and Isoptera is driven by an efficient interaction between exocrine glands and jointed appendages, both in close interaction with the environment. In this context, the mandible of ants plays an important role, since, in addition to being the main jointed appendage, it possess glandular functions. As an example we might name the two glands associated with the mandible: the mandibular and the intramandibular glands. The intramandibular gland is found inside the mandible and consists of a hypertrophied secretory epithelium and secretory cells in the mandible's lumen. The secretion of the secretory epithelium is liberated through intracuticular ducts that open at the base of hairs at the mandible's surface. The secretion of the intramandibular gland (epithelium and secretory cells) reacted positively to tests for the detection of polysaccharides and proteins, thus suggesting that it consists of glycoproteins. The ultrastructure of the secretory epithelium presents variations related to the developmental stage of the individual, showing a large number of ribosomes and microvilli close to the cuticle in young individuals, while in the older specimens it was possible to note the formation of an intracellular reservoir. These variations of secretory epithelium, as also the interaction between the cellular groups inside the mandible, are important information about this gland in leaf-cutting ants.     

The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle

The venom gland of queens of Apis mellifera was examined through light and transmission electron microscopy and subjected to electrophoretic analyses. Virgin queens exhibited prismatic secretory cells containing large amounts of rough endoplasmic reticulum with dilated cisternae, open secretory spaces, numerous vacuoles and granules scattered in the cytoplasm, and spherical nuclei with numerous nucleoli. The secretion produced was non-refringent under polarized light and the electrophoretic analysis of glandular extracts revealed five main protein bands. In mated queens, the venom gland exhibited a high degree of degeneration. Its secretion was refringent under polarized light and one of the main bands was absent in the electrophoretic pattern obtained. The morphological aspects observed are in agreement with the function of this gland in queens, given that virgin queens use venom in battles for the dominance of the colony, a situation that occurs as soon as they emerge, while fertilized queens rarely use venom.     

Labral gland in soldiers of the neotropical termite Cornitermes cumulans (Isoptera: Termitidae: Syntermitinae)

Cornitermes cumulans is a termite species of the subfamily Syntermitinae with a nasute mandibulate soldier caste, members of which are morphologically equipped with structures and organs specialized for the defence of the colony. We investigated the labrum of soldiers of C. cumulans and described the labral gland, an exocrine structure present in this appendage. The labrum of C. cumulans soldiers presented two distinct regions, the hyaline tip and the proximal region connected to the head. The hyaline tip exhibited a thick cuticle composed of a loose endocuticle involving an epithelium of class 1 cells, which synthesize the glandular product into a subcuticular space that function as a reservoir prior to release the final secretion. The proximal region of the labrum had an epithelium composed of class 1 and class 3 cells, which released the secretion onto both ventral and dorsal surfaces. The ultrastructure showed abundant smooth endoplasmic reticulum and glycogen in the class 1 cells, whereas the class 3 cells had many electron-lucent vesicles that varied in size. We associated the labral gland with the production of toxic substances that may act inside enemy wounds made by the soldiers’ mandibles. Other possible functions for the labral secretion are also discussed.     

Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae)

The sternal gland is considered the only source of trail pheromones in termites. The morphology of the sternal gland was investigated in workers of Coptotermes gestroi using transmission and scanning electron microscopy. The results showed a small bilobed gland at the anterior part of the fifth abdominal sternite. The cuticular surface of the sternal gland showed a V-shaped structure with two peg sensilla in elevated socket and various campaniform sensilla. Pores and cuticular scale-like protuberances also occur in the glandular area. The ultrastructure showed a gland composed of class 1 cells and two different types of class 3 cells distinguished by location, different size and electron-density of secretory vesicles. Small class 3 cells (type 1) of the anterior lobe are inserted among class 1 cells and have weakly electron-dense vesicles associated with mitochondria, glycogen and smooth endoplasmic reticulum. The class 3 cells (type 2) of posterior lobe showed many round electron-lucent vesicles of secretion, abundant free ribosomes and a well-developed Golgi apparatus. Each class 3 cell is connected to the cuticle by a cuticular duct constituted by the receiving canal and the conducting canal. The secretion of class 1 cells is stored in an inner subcuticular reservoir that is delimited by the microvilli of these cells. This inner reservoir is large and crossed by the campaniform sensilla and ducts of two types of class 3 cells that open outside of the insect body. An exterior reservoir also is present between the fourth and fifth sternite. The complex structure of the sternal gland suggests multicomponents for the trail pheromone in the worker of C. gestroi.     

Ultrastructural aspects of the mandibular gland of Melipona bicolor Lepeletier, 1836 (Hymenoptera: Apidae, Meliponi) in the castes

The mandibular gland in Melipona bicolor workers and queens was studied by scanning and transmission electron microscopy. There is no difference in the gland anatomy between the castes, but the transmission electron microscopy showed variation of the cellular ultrastructure according to the secretory phase of the gland in both castes. Smooth endoplasmic reticulum was abundant in the secretory cells of physogastric queens, indicating that these cells produce lipid secretion that is stored in granules with multi-lamellar bodies. Mitochondrial variations during the cell secretory cycle indicates their participation in the lipid synthesis. After secretion, release in the reservoir lumen through the collecting canals, the secretory cells contain many myelinic bodies, indicative of cellular regression.     

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.     

Fine structure of Mytella falcata (Bivalvia) gill filaments

Bivalve filter feeders are sessile animals that live in constant contact with water and its pollutants. Their gill is an organ highly exposed to these conditions due to its large surface and its involvement in gas exchanges and feeding. The bivalve Mytella falcata is found in estuaries of Latin America, on the Atlantic as well as the Pacific Coast. It is commonly consumed, and sometimes is the only source of protein of low-income communities. In this study, gill filaments of M. falcata were characterized using histology, histochemistry and transmission electron microscopy for future comparative studies among animals exposed to environmental pollutants. Gill filaments may be divided into abfrontal, intermediate and frontal zones. Filaments are interconnected by ciliary discs. In the center of filaments, haemocytes circulate through a haemolymph vessel internally lined by an endothelium and supported by an acellular connective tissue rich in polysaccharides and collagen. The abfrontal zone contains cuboidal cells, while the intermediate zone consists of a simple squamous epithelium. The frontal zone is composed of five columnar cell types: one absorptive, mainly characterized by the presence of pinocytic vesicles in the apical region of the cell; one secretory, rarely observed; and three ciliated with abundant mitochondria. All cells lining the filament exhibit numerous microvilli and seem to absorb substances from the environment. PAS staining was observed in mucous cells in the frontal and abfrontal zones. Bromophenol blue allowed the distinction of haemocytes and detection of a glycoprotein secretion in the secretory cells of the frontal region. The characteristics of M. falcata gill filaments observed in this study were very similar to those of other bivalves, especially other Mytilidae, and are suitable for histopathological studies on the effect of water-soluble pollutants.     

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.     

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.     

First evidence of an intimate symbiotic association between fungi and larvae in basal attine ants

The most conspicuous ants in all of tropical America are those that belong to the tribe Attini which cultivate fungus. The objective of the present study is to verify the alterations that occur in the cuticle of the worker larvae from Myrmicocrypta, Mycetarotes and Trachymyrmex with the purpose of trying to establish the degree of this association. Attine ants from the Atta genus were used as a control group. The analysis of histological sections showed results about the distribution of the cells and tissues on the epidermis of A. sexdens rubropilosa ant larvae and Mycetarotes parallelus, Trachymyrmex fuscus and Myrmicocrypta sp. A cuticle covering the ants was observed in the larvae of all the species, and this is formed by a simple cubic epithelium, whose cells possibly change its shape to prismatic, depending on their secretory activity. Just under this epithelium large adipose cells with reserve granules in their cytoplasm were found. The presence of a space filled by granulose and acellular material was also observed, indicating that the larvae were in a molt period. The presence of fungal hyphae is observed both on the external side of the basal attine larvae as well as emitting projections to the interior of the cuticle reaching the epithelium and the adipocyte cells. Data obtained in the present study demonstrated that the fungus deposited on the surface of immature ants from attine basal species maintain a close relationship with them, once the fungus hyphae have the ability to disorganize the cuticle lamellas, penetrating the interior of the insect cells through the emission of prolongations transporting the cuticle and epithelium barriers and making substances exchanges between larvae and fungus.     

Scanning and transmission electron microscopy and X-ray analysisof leaf salt glands of Limoniastrum guyonianum Boiss. under NaCl salinity

Leaf salt glands of Limoniastrum guyonianum were examined by scanning and transmission electron microscopes and energy dispersive X-ray analysis (EDAX) system, after growing for three months on sandy soil with or without 300 mM NaCl. Results showed that salt glands were irregularly scattered on both leaf sides and sunk under the epidermal level. Salt excretion occurred in both conditions and is mainly composed of calcium and magnesium in control plants, and essentially sodium and chloride in plants subjected to salt treatment. A salt gland is comprised of collecting, accumulating, and central compartments, and is made up of total thirty-two cells. The collecting cells were characterized by large central vacuoles. Accumulating cells contain numerous, large, and unshaped vacuoles and rudimentary chloroplasts. The central compartment was comprised of four basal cells and each one is surmounted by an apical cell. The basal cells are granulated, containing large nucleus, numerous mitochondria, endoplasmic reticulum, ribosomes, polyribosomes, and small vacuoles or vesicles. Equally, the apical cells are rich in organelles. Application of 300 mM NaCl to the culture medium increased vacuoles number and size, and organelles density especially the mitochondria which suggests energy requirement for ions transport. The reduction in size and number of vacuoles toward the interior of salt glands of treated plants and the fusion of the smallest ones with the plasma membrane substantiate the implication of such vacuoles in salt excretion process. The current study which is the first report on L. guyonianum salt gland has provided an in-depth understanding on structure–function relationship in the multicellular salt glands.     

Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings.     

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.     

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.     

3D reconstruction and scanning electron microscopy of salivary glands of the millipede Rhinocricus padbergi (Verhoef, 1938) (Diplopoda: Spirobolida)

The three-dimensional technique applied to the salivary gland of the millipides Rhinocricus padbergi showed that it is a single structure, dorsolaterally located to the animal gut and composed of two distinct portion: an acinar and a tubular one. The last one opening onto the oral cavity. This work shows that the salivary gland in R. padbergi, although is a single body, it displays two excretory ducts (one in each side of the anterior end of the gut) which has the function of carrying out the secretion produced toward to the oral cavity, contrary to the anterior data registered by other authors who suggested that the salivary gland in diplopod animals would be a paired structures.     

Immunolocalization of an osteopontin-like protein in dense granules of Toxoplasma gondii tachyzoites and its association with the parasitophorous vacuole

Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional glycophosphoprotein, secreted by different cell types, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated in this work by immunofluorescence and immunoelectron microscopy approaches the localization of an OPN-like protein in dense granules of extracellular T. gondii tachyzoites. Western blotting and RT-PCR confirmed this protein expression by the parasites. Our results also showed, after macrophage invasion, an intense positive labeling for OPN-like protein at the sub-apical portion of tachyzoites, the site of dense granules secretion, and the localization of this protein at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we speculate that this protein participates during the parasite interaction process with host cells and parasitophorous vacuole formation.     

Microscopical methods promote the understanding of the integument biology of Rhinella ornata

In Rhinella ornata, the integument of different body regions was investigated using low-vacuum scanning electron and light microscopy through histochemical and immunohistochemical methods, and revealed the basic structure found in other anurans. Keratinocytes formed the keratinized squamous stratified epithelium, and flask cells occurred among the epidermal superficial layer. Just below the epidermis, the dermis was subdivided into a spongious dermis and a compact dermis. Mixed and granular glands were located in the spongious dermis, and myoepithelial cells surrounded their secretory portions. The Eberth-Katschenko (EK) layer occurred as basophilic areas between the spongious and compact dermis and throughout the spongious dermis. A series of alternating layers of bundles of collagenous fibers characterized the compact dermis, being firmly attached to a thin hypodermis. Regarding the morphological features, each integument region revealed distinct structural aspects. The dorsal integument was rougher than ventral, having conical tubercles and spines upon the verrucae. In the ventral and subgular regions, the slightly elevations are visualized, tubercles were absent but some spines occurred. The pelvic integument had a non-keratinized epidermis with an irregular profile and small poorly developed elevations. In addition, the EK layer was absent, and the presence of numerous blood vessels suggests its participation in water absorption and distribution. In the parotoid gland, the epidermis was similar to the dorsal integument; except that the reticular dermis was present, and contained three exocrine glandular types. Expression of cytokeratin was evident in the basal and intermediary layer, but absent in the cornified layer. Flask cells showed strong cytokeratin labeling. Consequently, the integument had the basic morphology, but exhibits regional characteristics, which may be related to the physiology of each surface.     

Structure of the sexually dimorphic gland of Cycloramphus fuliginosus (Amphibia, Anura, Cycloramphidae)

Cycloramphus fuliginosus males (Amphibia, Leptodactylidae) have discoid glandular elevations on the abdominal inferior body region which are considered a characteristic of genus. In this work, this sexually dimorphic gland of C. fuliginosus was investigated by low vacuum scanning electron microscopy, light microscopy, and transmission electron microscopy. In this cycloramphid, the covered integument exhibits the basic structure: the epidermis, a keratinized squamous stratified epithelium with flask cells, and the dermis. The dermis is subdivided into spongious and compact dermal layers. In the iliac gland region, an aggregate of small mucous glands and larger syncytial tubuloalveolar glands occur in the spongious dermis, and they do not go beyond the Eberth-Katschenko layer limit. The adenocytes of the mucous gland produce neutral glycoproteins, in contrast to the larger tubuloalveolar glands that elaborate a proteinaceous secretion. Myoepithelial cells surround the alveoli, and play an important role in the secretion extrusion. The final secretion, elaborated by the iliac gland, is a mixture of mucus and protein. Both secretions are important to the cycloramphid biology, and may act as pheromone and/or as chemical parental care.