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.     

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.     

Morphological characterization of the anuran integument of the Proceratophrys and Odontophrynus genera (Amphibia, Anuran, Leptodactylidae)

The morphological characteristics of the leptodactylid integument of Proceratophrys and Odontophrynus genera were investigated by means of stereoscopic, low vacuum scanning electron and light microscopy. The integument surface of Proceratophrys boiei, Proceratophrys laticeps and Proceratophrys appendiculata exhibited several projections, while the integument of Odontophrynus americanus had rounded elevations with smooth profile. Light microscopic observations showed the basic integument morphology for all anurans, i.e., an epidermis and a dermis, which is subdivided into a spongious layer and a compact layer. The epidermis is formed by basal, intermediary and cornified layers. However, in Proceratophrys genus the cornified layer had an irregular outline, while in O. americanus the external surface was smooth. In the spongious dermis, mucous and venom exocrine glands were observed, but in O. americanus an exclusive glandular type with apocrine secretory pattern was identified. The integument morphology showed peculiar characteristics that may be helpful for genus distinction. Thus, morphological methods may be considered as an efficient means to characterize and to differentiate anuran genera.     

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus)

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-β-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.     

Morphology of Bufo ictericus integument (Amphibia, Bufonidae)

Bufo ictericus integument was investigated by stereoscopic, low vacuum scanning electron microscopy, transmission electron microscopy and light microscopy. The studies revealed, that the dorsal integument surface is rougher than ventral. Three types of projections are visualized: larger rounded verrucae, smaller conical cornified tubercles, and conical short spines. Prominent verrucae are observed on the dorsal surface, being flatter on the ventral surface. The tubercles are visualized only on the dorsal surface. The verrucae are separated by grooves that may contribute spreading and retention of the glandular secretion upon the integument. The pattern of the epidermal grooves is also important for water distribution, protecting the animal against desiccation. The epidermis is composed of a stratified epithelium with intraepithelial blood vessels, where keratinocytes predominate, but flask cells, and Merkel cells also occur. In the spongious dermis, cutaneous glands are visualized. The compact dermis is a series of alternating layers of bundles of collagenous fibers, and between spongious and compact dermis there are basophilic areas that correspond to Eberth-Katschenko layer. The dorsal and the ventral surfaces of B. ictericus are morphologically distinct. The integument structure is related to the physiology of each surface and represents an adaptation to habitat, reflecting a lifestyle of the animal.     

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.     

Volume-weighted mean volume of the submandibular gland acini in male and female diabetic rats

Mouth dryness is one of the complications of diabetes mellitus but there has been little work on morphological changes of the salivary glands. In the present study, the effects of diabetes mellitus on the serous and mucous acini of submandibular gland of male and female rats, 4 and 12 weeks after diabetes induction were studied. Male and female rats were divided into experimental and control subgroups. Diabetes was induced to experimental rats by streptozotocin. At the end of 4 and 12 weeks, the submandibular glands were removed, random sections obtained and volume-weighted mean acini volume was estimated by point-sampled intercepts method. The data revealed that volume reduction occurred only in serous acini in both male and female rats 12 weeks after diabetes induction and the others remained unchanged. The present research using stereological methods demonstrates that diabetes make some morphological changes in serous acini, the main exocrine part of the rat submandibular gland.     

AFM study of the extracellular connective tissue matrix in patients with pelvic organ prolapse

The method of atomic force microscopy (AFM) is used for the first time for morphological investigation of pathological changes in the extracellular matrix of skin connective tissue upon the prolapse of pelvic organs (common disorder among women). Skin samples of patients with clinically proven pelvic-organ prolapse and of patients that do not have any connective tissue related disease (control group) are investigated via AFM. The AFM study reveals that the extracellular matrix of the skin connective tissues from patients with pelvic-organ prolapse diverges from the normal in various organization levels including both micro- and nanotexture (packing of collagen fibers and fibrils, respectively). The results of AFM study of the normal and pathologically changed skin connective tissues are in good agreement with the data of clinical morphological analysis, which indicates the potential of AFM as an independent diagnostic tool.     

Effects of long-term intubation on vocal fold mucosa in dogs

A canine model was used to study effects of long-term intubation on vocal fold mucosa. Dogs' larynges were removed 5 weeks after a 7-day intubation period and were compared with control tissue. Intubation effects on vocal fold mucosa were highly variable. Most severe damage was observed posteriorly, at the presumed location of direct tube-mucosa contact. Effects judged to be less severe but still significant were noted in tissue anterior to this site. Morphometric analysis of the layers of the intubated mucosa revealed significant differences in epithelium, connective tissue, and glands, as compared with control tissue. Differences were also observed for blood vessels and nerves. Of particular clinical importance was evidence of damage along membranous, as well as cartilaginous, portions of the true vocal fold, and of damaged connective tissue and cartilage underlying epithelium which appeared normal. Implications of the findings for recovery from intubation, and for voice, are discussed.     

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.     

Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy

Collagen structural organization plays an important role in the mechanical property of the vertebrate integument. Bufo ictericus and Rana catesbeiana integument was investigated by light microscopy and laser confocal microscopy. Collagenous elements of the dermis were statistical analyzed. The integument is formed by the keratinized squamous stratified epidermis supported by the dermis that is subdivided into the spongious layer with a loose arrangement, and the compact layer formed by collagenous fibers arranged compactly in a criss-crossed manner. Thick collagenous columns have a perpendicular trajectory, and are formed by the assembling of alternating collagenous lamellae in both animals. Short intercolumns of collagenous fibrils connecting collagenous lamellae obliquely or transversally are observed in R. catesbeiana dorsal integument. The present study provides evidences that B. ictericus and R. catesbeiana integument has well-organized compact dermis, constituted by collagenous lamellae in a plywood manner. The integument organization is in contrast to the literature in some aspects. This dermal arrangement is important to the biomechanical property of both anuran integuments.     

Salivary system in leaf-cutting ants (Atta sexdens rubropilosa Forel, 1908) castes: a confocal study

The salivary system in ants is not only limited to digestory functions, but also has important role in the communication. The glands which compose the salivary complex are: the post-pharyngeal, hypopharyngeal, mandibular, and thoracic salivary gland, showing peculiar features which may vary according to the castes the individuals belong to and according to the functions they develop. The present study compared the morphological differences among the glands of Atta sexdens rubropilosa workers, males and queens focused on the organization of microfilaments and microtubules in these ants salivary system. Although the post-pharyngeal gland appeared to be more developed in queens, there were no significant gland differences among the analyzed castes. In what regards to the secretory units of the hypopharyngeal and mandibular glands, the association of F-actin with the collector duct seemed to be strong, being surrounded by a microtubules arrangement. The use of a laser scanning confocal microscopy with immunofluorescence whole mounting preparations revealed itself an efficient instrument for the understanding of the internal morphology of insects.     

Effects of chronic ozone (O3) exposure on vocal-fold mucosa in bonnet monkeys

Effects of ambient ozone (O₃) on the respiratory tract have been frequently reported. However, little is known regarding possible effects of chronic exposure to O₃ on the larynx or on vocal-fold mucosa. In this study, six bonnet monkeys were exposed to 1 ppm O₃ 8 h per day, 5 days per week, for 3 months. Three of the monkeys' larynges were examined 4 h after exposure, and three were examined 9 months after exposure. Tissues were subjected to histological staining and morphometric quantification. Results revealed increased thickness of epithelial and connective tissue components of vocal-fold mucosa in the 4-h group and connective tissue changes that persisted in mucosa examined 9 months after exposure. Additional changes indicative of hyperplasia and disorganization of epithelial and connective tissue, as well as disruption of glands and vessels, were also noted in tissues examined at both postexposure intervals. Implications of the data for the larynx and voice quality in humans exposed to similar environmental toxins are discussed.     

Hyaluronan in the epidermal and the dermal extracellular matrix: its role in cutaneous hydric balance and integrity of anuran integument

New researches have revealed that hyaluronan (HA) is not a passive molecule. HA has being pointed out to participate in many processes, such as cell interactions, proliferating and migrating cell events and function as hydrate agent. The present study was undertaken to localize HA in Bufo ictericus integument providing a better understating for the role of cutaneous HA. Paraffinized sections were stained with haematoxylin-eosin and with 1% Alcian blue 8GX at pH 1.0 and 2.5. Alcianophilic reaction was visualized in both spongious dermis and Eberth-Katschenko layer. The mucus cells of mucus glands were also stained with AB methods. Besides these histological techniques, the localization of HA was performed using the FITC-labeled HA probe labeled with fluorescein isothiocyanate. In the extracellular matrix of spongious dermis, the reaction for HA was evident, being less intense in hypodermis and in pericellular keratinocyte matrix of the cornified tubercles regions. Thus, since HA was localized in the pericellular epidermal matrix and in the spongious dermis of anuran integument, it plays an important role in hydric balance, and is essential for integument integrity and functionality.     

Multistage Segmentation of Prostate Cancer Tissues Using Sample Entropy Texture Analysis

In this study, a multistage segmentation technique is proposed that identifies cancerous cells in prostate tissue samples. The benign areas of the tissue are distinguished from the cancerous regions using the texture of glands. The texture is modeled based on wavelet packet features along with sample entropy values. In a multistage segmentation process, the mean-shift algorithm is applied on the pre-processed images to perform a coarse segmentation of the tissue. Wavelet packets are employed in the second stage to obtain fine details of the structured shape of glands. Finally, the texture of the gland is modeled by the sample entropy values, which identifies epithelial regions from stroma patches. Although there are three stages of the proposed algorithm, the computation is fast as wavelet packet features and sample entropy values perform robust modeling for the required regions of interest. A comparative analysis with other state-of-the-art texture segmentation techniques is presented and dice ratios are computed for the comparison. It has been observed that our algorithm not only outperforms other techniques, but, by introducing sample entropy features, identification of cancerous regions of tissues is achieved with 90% classification accuracy, which shows the robustness of the proposed algorithm.     

Distribution of chondroitin sulfate in human endometrium

Sulfated glycosaminoglycan (GAG) composition was characterized in the human endometrium during proliferative and secretory phases of the menstrual cycle. Sulfated GAGs were analyzed in endometrium tissue using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed that CS was the main sulfated GAG species detected, accompanied by small amounts of heparan sulfate and dermatan sulfate. CS was distributed overall the connective stroma, around arteriole vessels and glands, and there was no important difference in the immunostaining between the proliferative and secretory endometrium phases. Our findings extend previous observations on the GAG composition in the human endometrium providing new information regarding the tissue distribution and location of endometrial CS.     

New morphological data on fat bodies of semi-engorged females of Amblyomma cajennense (Acari: Ixodidae)

The present ultramorphological, histological and ultrastructural study on the fat body of semi-engorged females of Amblyomma cajennense revealed that this tissue is diffuse and consists of strands of cells surrounding the tracheal trunks. Morphometric analysis showed that the cellular and nuclear areas of round-shaped trophocytes are larger than those of cuboidal trophocytes, indicating that the arrangement of the former provides more contact area with the haemolymph. In this species, the fat body is found right underneath the integument and around organs. It consists of two cell types that despite distinct morphological characteristics and locations in the tissue, present the same histological features. In this study, these cells were termed cuboidal trophocytes when arranged as strands of cells and present in larger numbers, and round-shaped trophocytes when lying on these strands and observed in fewer numbers. Histological observations revealed that both types of trophocytes have one nucleus in their cytoplasm and also exhibit numerous vacuoles of different sizes and contents. Ultrastructural examination revealed that the organelles more frequently observed were the vesicular and lamellar rough endoplasmic reticulum, and mitochondria with tubular crests, indicating that they might be involved in lipid synthesis. Smooth endoplasmic reticulum was not observed. Cuboidal trophocytes arranged in strands, despite being closely associated, do not exhibit fused plasma membranes. Rather, the fusion of basal lamina of two neighboring cells is occasionally observed, acting as a selective permeability barrier. Here, a new terminology for tick fat body is proposed. It is based on fat body location (parietal, when located right underneath the integument instead of peripheral; and perivisceral, when located around organs instead of central) terminologies previously suggest by Obenchain and Oliver and for the cells constituting them, cuboidal trophocytes when arranged as strands, and round-shaped trophocytes when lying on these strands. Nephrocytes were not observed in semi-engorged females of A. cajennense.     

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.     

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.